>>WELCOME TO SYMPOSIUM
HAVE BEEN MORE DIFFICULT TO
DEVELOP AND RECENT INVESTMENT
FROM THE NIH COMMON FUND AND
RELATED FUNDING AGENCIES TO
ADDRESS THIS ISSUE HAS RESULTED
IN THE DEVELOPMENT OF THE NEXT
GENERATION TOOLS DESIGNED TO
DEVELOP RATE COMPLEX CA CAR BOW
HIGH DATE STRUCTURES AND RELATED
MICRO INTERACTIONS TODAY WE ARE
EXTREMELY LUCKY TO HAVE SUCH A
DIVERSE SET OF SPEAKERS TODAY.
21 AMAZING SCIENTISTS HAVE
JOINED REPRESENTING A DIVERSE
REPERTOIRE TO COVER SUCH GROUND
THAT THIS CROSS ROADS AND YOU
WILL NOTICE THAT THEIR TALK WILL
INTERSECT DIFFERENT PIECES OF
THESE CROSS ROADS AND EXCITING
FOR HAVING SO MANY EXPERTS ON
THIS SYMPOSIUM PANEL AND THERE'S
OVER 800 OF YOU JOINING ON-LINE
SO THERE'S A WAY JUST SOME
GROUND RULES, THERE'S A WAY TO
ASK QUESTIONS.
OUR SPEAKERS HAVE STRUCTURED
TALKS AND THERE'S TIME AT THE
END OF THEIR TALK FOR SOME
DISCUSSION AND IN ADDITION, IF
YOU ARE JOINING US IN THE
VIRTUAL PLATFORM, THERE'S SOME
WAYS THAT YOU CAN ASK QUESTIONS
AND THERE'S A POP-UP LIVE
FEEDBACK ASK A QUESTION.
TYPE YOUR QUESTION IN THERE AND
IT WILL BE DIRECTLY ASKED TO THE
SPEAKERS IN THE ROOM AND IN
ADDITION IF THEY DON'T HAVE A
CHANCE TO ASK OR ANSWER YOUR
QUESTIONS, THE SPEAKER WILL HAVE
YOUR E-MAIL ADDRESS AND E-MAIL
YOU AN ANSWER AND IT WILL IF SIL
TATE A DISCUSSION POST
SYMPOSIUM.
THANK YOU FOR JOINING.
IT'S BEEN REALLY GREAT TO PUT
THIS TOGETHER WITH MY CO
ORGANIZERS SEAN STOWWELL UP AT
THE HOSPITAL AT HARVARD MEDICAL
SCHOOL AND WE ARE REALLY VERY
GRATEFUL TO THE COMMON FUND FOR
FUNDING AND ORGANIZING AND
HELPING, ESPECIALLY Dr. PAMELA
MORENO AND THANK YOU, VERY MUCH.
WE'RE EXCITED TO GET STARTED.
VICTOR NIZET WILL START US OFF.
HE IS COMING TO US REALLY EARLY
IN THE MORNING FROM SAN DIEGO AT
THE UNIVERSITY OF CALIFORNIA IN
SAN DIEGO SO THANK YOU VICTOR
FOR WAKING UP EXTRA EARLY.
I KNOW YOU ARE AN EARLY RISER
BUT THANK YOU.
WE'RE LOOKING FORWARD OF YOUR
TALK OF BACTERIAL SEPSIS SO
THANK YOU SO MUCH AND ENJOY YOUR
TALK.
>> OK.
I'D LIKE TO THANK CATHERINE AND
SEAN FOR THE INVITATION.
AND I'M A PHYSICIAN SCIENTIST AT
UC SAN DIEGO AND WE STUDY HOST
PATHOGEN INTERACTIONS AND INNATE
IMMUNITY.
SO, I'M GOING TO TELL YOU A
LITTLE BIT ABOUT THE PLATELET,
WHICH IS MAYBE UNDER STUDIED AS
AN IMMUNE CELL COMPARED TO ITS
PROMINENT ROLE IN HEMO STAY SIS
AND THE WORK IS LARGELY LED BY
THESE TWO IN THE LAB.
JOSH SUN AND SATOSHI.
I'M GOING TO TELL YOU TWO
STORIES ABOUT TRANSLATIONAL
GLYCOBIOLOGY THAT MIGHT LEAD TO
DRUG REPURCHASING.
SO, PLATELETS, ALTHOUGH THEY
DON'T HAVE A NUCLEUS, HAVE
GRANULES AND THEY'RE BIO ACTIVE
MOL ACTUALS THAT PARTICIPATE IN
CLOTTING INFLAMMATION, BUT THERE
ARE ALSO CATIONIC PEPTIDES THAT
HAVE ANTIMICROBIAL ACTIVITIES.
AND WE HAVE BEEN STUDYING THE
WAY THAT FRESHLY ISOLATED HUMAN
PLATELETS INTERACT WITH
DIFFERENT BACTERIA AND IT'S
INTERESTING THAT THERE ARE GREAT
DIFFERENCES IN INDIVIDUAL
PATHOGENS IN THEIR
SUSCEPTIBLITY.
IF YOU RENDER AN ANIMAL, BY
GIVING THEM A ANTI PLATELET
ANTIBODY, THEY BECOME
SIGNIFICANTLY MORE SUSCEPTIBLE
TO MRSA INFECTION BUT IT HAS NO
EFFECT ON GROUP B STREP
INFECTION.
AND MIXING HUMAN PLATELETS WITH
BACTERIA, WE CAN SEE THAT MRSA
GROWTH IS INHIBITED, IN FACT THE
MRSA ARE KILLED BY THE FRESHLY
ISOLATED HUMAN PLATELETS AND THE
GROUP B STREP GROW EVEN FASTER
THAN THEY WOULD OTHERWISE WHEN
THEY ARE ENCOUNTERING THE
PLATELETS.
AND HERE YOU SEE THAT A MAIN
REASON FOR GROUP B STREP
RESISTANCE IS GLYCO BIOLOGICAL
IN NATURE.
GROUP B STREP HAS A EXO POLY
CAPSULE AND ALPHA 2/3 LINK
TERMINAL ACID IN THE CAP LAR AND
ON HUMAN CELLS THAT IS OFTEN
RECOGNIZED ITSELF TO COUNTER
REGULATE, COMPLIMENT AND OTHER
IMMUNE ACTIVATION AND YOU CAN
SEE HERE THAT THE RESISTANCE OF
THE BACTERIA TO PLATELET KILLING
DEPENDS ON THE CAPSULE BECAUSE
IF WE ELIMINATE THE CAP FULL,
THEY'RE MORE EASILY CLEARED IN
THE BLOOD AND THIS WAS FULLY
DEPENDENT ON THE PRESENCE OF
PLATELETS AND THE CAPSULE MUTANT
IS MORE SUSCEPTIBLE TO KILLING
BY HUMAN PLATELETS AND THE
RELEASE CONTAINING THE
ANTIMICROBIAL TIDES AND WE CAN
TREAT WITH A CIALDAISE LEAVING
THE CAPSULE AND CREATING THIS
SUSCEPTIBLE TO PLATELET KILLING.
ALSO, IF YOU TAKE THE PURIFIED
ANTIMICROBIAL PEPTIDES LIKE
PLATELET FACTOR 4 AND WE'VE ALSO
SEEN HUMAN PRESENT IN PLATELET
GRANULES, THE DEFICIENT MUTANT
IS MORE SUSCEPTIBLE TO KILLING
AS DOES THE SIALADATE TREATED
WILD TYPE BACTERIA.
IT'S MORE THAN JUST A DIFFERENCE
IN SUSCEPTIBLITY, WE DISCOVERED
THE ACID CAPSULE ALSO CONFERS AN
ABILITY ON THE BACTERIA TO
SUPPRESS PLATELET ACTIVATION.
SO IF YOU MEASURE PLATELET
ACTIVATION BY P SELECTION OR
GLYCO PROTEIN AND PLATELET
ACTIVATION YOU SEE THAT THE SIA
MUTANT LACKING THE CAPSULE AR
SIGH ALICK ACID -- IT'S SUPPRESSING
THE LATE LET ACTIVATION, WHICH
ARE HERE AND YOU SEE THEM
ACTIVATING AND DEGRANULATING
WITH THE SIALIC ACID MUTANT.
SO, HOW IS THIS WORKING?
WE HAVE DONE A LOT OF WORK AND
ONE OF OUR UPCOMING SPEAKERS,
AMANDA LOUIS, WAS A MAJOR
CONTRIBUTE OR TO THESE PARADIGMS
SHOWING THAT GROUP B STREP
DETACK US SIALIC ACID IS IN
INHIBITORY SIGNIFICAN SIGLEC ON THE SURFAC E
OF CELLS LIKE MACRO FAGES TO
INITIATE THROUGH THEIR INTER
CELLULAR ITE DOMAINS FOSS IF
TASE ACTIVATION THAT INHIBITS
PRO INFLAMMATORY SIGNALING
CASCADES TO IT MIMIC ONE OF THE
MECHANISMS OF HUMAN HOMEOSTATIC
IMMUNE REGULATION.
WE FOUND THAT HUMAN LATE LET'S,
THIS HADN'T BEEN STUDIED
EXTENSIVELY, EXPRESS A LOT OF
THE SAME RECEPTORS IN PARTICULAR
SIGLEC9 THAT ARE FOUND ON HUMAN
NEUTRAL FILLS AND MACRO FAGES
AND THEY ARE ABLE TO BIND TO THE
PLATELET IN A SIGNIFICANTLEC
DEPENDENT MANNER AND HERE YOU
SEE IN THIS 7.4% PLATELET
BINDING DROPPING ALL THE WAY
DOWN TO .9% WHEN YOU ELIMINATE
THE SIALIC ACID OR BLOCK IT WITH
AN ANTI BODY.
WELL, THE BINDING OF THE SEC
GLEC ON THE PLATELET INHIBITS
PLATELET ACTIVATION AND WE CAN
SEE THAT IF WE USE AN ANTI
SIGLEC ANTIBODY TO BLOCK THE
ABILITY OF THE BACTERIA, YOU GET
MORE SIGLEC -- I'M SORRY, MORE
PLATELET ACTIVATION AND WITH
MORE PLATELET ACTIVATION YOU
WILL GET MORE RELEASE OF THE
ANTIMICROBIAL PEPTIDES.
HERE YOU SEE THAT IN MIRRORING
PLATELETS WHICH HAVE SIGLEC E
WHICH IS SIMILAR TO SIGNIFICANT
ELECTRIC 9, THEREFORE, THE
ABILITY OF THE SO TO SUMMARIZE THIS FIRST
STORY, GROUP B, THROUGH IT'S
SIALIC ACID CAPSULE IS ABLE TO
BLOCK PLATELET ACTIVATION AND
ALSO INHERIT THE ACTIVITY OF THE
PLATELET PEPTIDES WHICH REN
DERSE IT ROW CYSTANCE TO
PLATELET KILLING WHEREAS MRSA IS
MORE SUSCEPTIBLE.
SO THE INCREASE ACCEPTABILITY OF
MRSA IS THE TRANSITION TO OUR
TRANSLATIONAL STORY.
WE FOUND, IN OUR PATIENT SERIES,
IN WHICH WE'RE STUDYING
ANTIBIOTIC THERAPY AND SEPSIS
THAT IF YOU LOCK AT WHAT
CORRELATES TO MORTALITY IN
PATIENTS, IT'S NOT HIGH OR WILL
HE WHITE BLOOD CELL COUNT BUT
LOW PLATELET COUND THAT IS
STATISTICALLY SIGNIFICANTLY
ASSOCIATED WITH MORTALITY IN
PATIENTS WITH A DISEASE FOR
WHICH MORTALITY IS IN THE 20%
RANGE AND HAS NOT IMPROVE I IMPROVED IN
THE LAST THREE DECADES, EVEN
WITH ADVANCES IN ICU CARE.
AND WE HIGHLIGHTED STAFF AUREUC
AND THIS EXAMPLE AND WHEREAS
IT'S RELATIVELY RESISTANCE TO
NEWT TRA FILL KILLING IN LABS
INCLUDING OUR OWN, STUDY ALL
MECHANISMS FOR THAT SO PLATELETS
MIGHT BE THE MOST IMPORTANT
IMMUNE CELL IN YOUR BLOOD FOR
STAFF.
AND, WHAT IS INTERESTING IS
THEY'RE GOING MONO A MONO AT
EACH OTHER AND THE PLATELETS
HAVE THE ABILITY TO KILL THE
STAFF, BUT THE STAPH HAS WEAPONS
TO DEPLOY AGAINST THE PLATELETS.
WE FOUND, WHEN YOU INFECT A
MOUSE WITH STAPHORIOU IT DRIVES
THE PLATE LESS DOWN AND DRIVING
THE PLATELET DOWN IS A POOR
CONSEQUENCE FOR THE HOST
IMMUNITY BECAUSE WE SHOWED AND
REITERATE HERE THAT EXPERIMENTAL
THROMBOCYTOPENIA IS LESS ABLE TO
CLEAR STAPH AND CLEAR IT IN
VIVO.
WHAT IS DRIVING THE PLATELET
COUNT DOWN, WE FOUND IT'S A
HIGHLY CONSERVED TOXIN STRESSED
BY THE MAJORITY OF STAFF STRAINS
KNOWN AS THE ALPHA TOXIN IN OUR
PATIENT COHORT, THE MORALE ALPHA
TOXIN EXPRESSION THE GREATER OF
THEIR, AND IF MAKE AN ISOGENIC
MUTE AN BACTERIA IN MRSA, WE
FOUND THE DEGREE THE
THROMBOCYTOPENA WAS LESS AND
THAT MUTANT WAS MORE KILLED BY
THE BLOOD OR PURIFIED PLATELETS.
SO, OUR LAB HAS BEEN VERY
INTERESTED IN THEY AR -- THERE ARE MEDS
THAT HUMANS TAKE THAT TARGET
PLATELETS.
PATIENTS WHO HAVE HAD HEART
ATTACKS OR STROKES AND YOU WANT
TO REDUCE THEIR RISK OF HAVING A
REPEAT EVENT, AS ASPIRIN OR MORE
SOPHISTICATED ANTI-PLATELET
MEDS.
AND WE MIXED EXEMPLARS OF ALL
THESE ANTI-PLATELET MEDS INTO
THE ASSAY OF PLATELET KILLING OF
STAFF AND FOUND THAT ONE CLASS
OF MEDICINE, THE P2Y12
INHIBITORS DRAMATICALLY
INCREASED THE EFFECTIVENESS OF
PLATELET KILLING OF STAPH AND
ROREDUCES THE TOXICITY OF THE
ALPHA TOXIN ON THE PLATELET.
YOU CAN SEE IN VITRO, STHPH,
BASICALLY VAPORIZING PLATELETS
BUT IF YOU HAVE TICAGRELOR THEIR
INTEGRITY IS INTACT IN FACE OF
THE ALPHA TOXIN.
AND IF WE TREATED ANIMALS WITH
TICAGRELOR BEFORE WE GAVE THEM
THE STAPH INFECTION, THIS GOES BY
BRILLINTA AND IS GIVEN TO
MILLIONS OF AMERICANS, YOU CAN
SEE HERE THE DEGREE IS REDUCED
AND THE ABILITY OF THE BLOOD TO
CLEAR THE PLATELETS AND TO
DEFEND AGAINST STAPH INFECTIONS
IN THE ORGANS IS IMPROVED.
AND HERE YOU SEE LESS DAMAGE.
>> Zahra: TO THE LIVER AND IN
DAMAGEGIVEN A DOSE OF LETHAL
DOSE INFECTION WE SAW
SIGNIFICANT PROTECTION AGAINST
MORTALITY IN THE ABSENCE OF
ANTIBIOTIC THERAPY, JUST BY
ADMINISTERING THIS FDA-APPROVED
ANTI-PLATELET DRUG.
SO A QUIZ THAT I GUESS IS HARD
TO GET AN ANSWER ON-LINE BUT I'M
CURIOUS HOW LONG A PLATELET LAST
IN CIRCULATION.
RED BLOOD CELLS LAST A FEW
MONTHS BUT THE ANSWER TO THIS
QUESTION IS EIGHT OR NINE DAYS.
IT TURNS OUT THERE'S A GLYCO
BIOLOGIC REGULATION TO THIS
PROCESS.
PLATELETS, JUST LIKE MOST OTHER
HUMAN CELLS ARE CAPPED WITH MANY
GLYCO PROTEINS AND THESE HAVE
TERMINAL SIALIC RESIDUE OR
RESIDUE AS THE PLATELET AGES,
THROUGH THE ACTION OF ENDOGENOUS
SOME PRODUCED BY THE PLATELET
ITSELF, THESE TERMINAL SIALIC
ACID RESIDUES ARE REMOVED,
EXPOSING AN UNDERLYING GALACTOSE
WHICH HAS PROTEIN ON THE SURFACE
OF HELP AT SIGHTS IN THE FIRST
LEPTIN DESCRIBED, AT THE NIH IN
FACT, THE ASHWELL-MORELL
RECEPTOR AND THAT'S WHERE
THEY'RE CLEARED IN THE LIFE.
LIVER.
AS IT AGES IT LOSES THESE SIALIC
ACID AND IT'S CLEARED AND
REPLACED BY NEW PLATELETS FROM
THE MARROW.
STAPH DOESN'T MAKE BUT IT
ACTIVATES THE ENDOGENOUS
ACTIVITY OF PLATELETS, CAUSING
THEM TO MAKE THEMSELVES BALD PRO
PREMATURELY AND THIS DEPENDS ON
THE ACTION OF THE ALPHA TOXIN
AND THIS IS WHAT IS BLOCKED BY
TICAGRELOR SO IN FACT THE
TICAGRELOR IS PREMATURE TICA IN
CLEARANCE OF THE PLATELETS.
THIS IS VERY IMPORTANT BECAUSE
WE ELIMINATE THE ASHWELL-MORELLE
FROM THE LIVER AND THE STAFF
INDUCES NEVER OCCURS AND THE
PLATELET COUNT STAYS HIGH AND
THE ANTI STAPH FUNCTION IS
RETAINED AND THESE KNOCK-OUT
MICE ARE RESISTANCE TO
STAFF-INDUCED SEPSIS BUT THEY'RE
USED IN REDUCING THE SEVERITY OF
INFLUENZA INFECTION AND WE
WONDERED WHETHER WE MIGHT BE
ABLE TO PINO PINOT BENEFIT THIS BY
BLOCKING THE ACTIVITY SINCE
THESE ARE NOT COMPLETELY SEC FOR
THE BACTERIA ENZYMES BUT ALSO
HAVE ACTIVITY AGAINST THE HOST
ENZYMES.
AND IN FACT, REPURCHASING
OSELTAMIVIR LED TO LESS AND
ANOTHER NON FORM SUED CAL DNA
INHIBITOR, DANA, ALSO HAD A
PROTECTIVE EFFECT IN MICE.
TO SUMMARIZE, WE CAME UP WITH
THIS OBSERVATION THAT PLATELETS
ARE ACTUALLY THE MOST IMPORTANT
CELL IN YOUR BLOOD TO DEFEND
AGAINST THE MOST COMMON SOURCE
OF BACK TO BEEMIA IN THE BLOOD
IN STAPHORIUS THAT KILL THE
STAPH THROUGH ANTIMICROBIAL
PEPTIDES AND THE STAPH IS TRYING
TO DEPLOY A TOXIN THAT CAUSE THE
PLATELETS TO GO BALD AND GET
CLEARED IN THE LIVER AND
UNDERSTANDING THIS PATHWAY
ALLOWS US TO REPURPOSE TWO
COMMON FDA APPROVE DRUGS,
TIKAGOLOR TO IMPROVE OUTCOMES
AND BEE WE'VE BEEN EXPLORING THIS IN
PATIENTS AND THERE'S ANECDOTAL
CASE REPORTS OF AT STATION OF
TICAGRELOR LEADING TO BLOOD
STREAM CLEARANCE AND RESCUE OF
THE PLATELET COUNT AND ENHANCED
ANTI-PLATELET ACTIVITY AND JUST
PUBLISHED THIS WEEK, A CLINICAL
STUDY LOOKING AT THE OLD SCHOOL
P2Y1 INHIBITOR SHOWING THAT
PATIENTS WHO IN A VERY, VERY
LARGE COHORT WHO HAPPEN TO BE ON
THAT DRUG, HAD DECREASED
INPATIENT AND 30-DAY MORTALITY
COMPARED TO OTHERS.
SO, THAT IS MY TALK.
I WANT TO THANK A LOT OF PEOPLE
INCLUDING MY GLYCOBIOLOGY
MENTORS WHICH INCLUDE JEFF ESCO
AND JAMIE MARTH.
>> EXCELLENT.
THANK YOU FOR A FANTASTIC TALK.
I AM NOT SURE, PAM, I ARE THERE
QUESTIONS?
IF NOT, I CAN ASK YOU A OR IF
OTHERS HAVE A QUESTION.
IF WE HAVE TIME FOR A QUESTION?
>> WE DI DON'T HAVE ANY YET?
I HAVE A QUESTION.
HOW LONG DOES -- DO YOU SEE
RESISTANCE TO THIS?
WILL IT EVENTUALLY BECOME THAT
YOU CAN'T DO THIS
PROPHYLACTICALLY FOREVER?
>> THOSE ARE GOOD QUESTIONS
ABOUT HOST-DIRECTED
THERAPEUTICS.
IN GENERAL IT'S A LITTLE HARDER
FOR A MICROBE TO DEVELOP
RESISTANCE TO AN IMMUNE CELL OR
A, IN THIS CASE THE PLATELET,
THAT HAS BEEN MADE MORE
RESILIENT OR ACTIVATED IN A WAY
TO RELEASE ITS ANTIMICROBIAL
ARSENAL BECAUSE IT'S
MULTIFACETED, RIGHT.
ONE OF THE CORNER STONES OF
AVOIDING ANTIBIOTIC RESIST AS
SOON AS TO USTOUSE IT SIMULTANEOUSLY TO PU LL
DIFFERENT DIFFERENT TAG AT THE
TIMING ANTIMICROBIAL AND THAT'S
WHAT A PLATELET IS RELEASING AND
A NEW TRA FILL IS RELEASING IF
IT'S DESIGNED TO ENHANCE THE
RESILIENCE AND THIS WILL BE
OCCURRING IN CONCERT WITH
ANTIBIOTICS, NO ONE WOULD BE
DEPRIVED OF EFFECTIVE.
IT WOULD BE ENGAGING THE IMMUNE
SYSTEM IN A PARTICIPANT AND
IMPROVING TREATMENT OUTCOMES.
>> EXCELLENT.
WE HAVE ONE QUESTION THAT CAME
IN FROM OUR ON-LINE VIEWING
AUDIENCE.
I WONDER WHAT TYPES OF GPS WAS
USED AND SHOW INHIBITED PLATELET
ACTIVATION AND ALL HAVE ALPHA 2,
3 LINKS.
MAYBE SOME SPECIFIC GPC OR CPS9
STRONGER?
>> THAT'S A VERY GOOD QUESTION.
WE ONLY STUDIED THREE ZERO TYPES
OF THE NINE KNOWN BUT TOGETHER
THEY REPRESENT ALMOST 70% OF THE
INVASIVE CASES TYPE 3, TYPE 1-A
AND TYPE 5.
WE SEE THAT ALL GVS ARE
RESISTANT TO PLATELETS BUT WE
ONLY KNOW THAT SIALIC ACID IS
SPECIFICALLY CONTRIBUTING TO
THAT RESISTANCE IN THOSE STRAINS
WHERE WE HAVE ISOGENIC
KNOCKOUTS.
BUT ONE THING THAT IS TRUE, GBS
IN GROUP A STREP GO AVIDLY IN
HUMAN BLOOD.
THEY HAVE NO RESISTANCE.
IT'S AMAZING HOW WE DON'T ALL
DIE WHEN WE GET THESE INFECTIONS
BECAUSE IN THE LABORATORY, OUR
BLOOD HAS NO NEUTRALIZING
ACTIVITIES AND YET, YOU KNOW,
MOST PEOPLE RESIST THESE
INFECTIONS THROUGH MORE
MECHANISMS THAN ARE PRESENT IN
THE BLOOD.
>> GREAT.
THANK YOU SO MUCH.
THAT WAS NICE THAT WE HAD TWO
QUESTIONS FROM EACH SECTION OF
YOUR TALK.
A BEAUTIFUL TALK IN TYING IT
TOGETHER SO NICELY AND THANK YOU
FOR REPURCHASING THOSE ALREADY
USED THERAPEUTICS AND IT'S
AMAZING WORK THAT YOUR GROUP IS
DOING AND THANK YOU FOR LOGGING
ON TODAY TO JOIN OUR TALK.
IT WAS REALLY NICE WORK.
THANK YOU FOR BEING OUT SO EARLY
TOO.
I APPRECIATE IT AND WE'RE GOING
TO SWITCH GEARS TO OUR NEXT
SPEAKER WHO IS LAURA KIESSLING
AND SHE'S GOING TO TALK ABOUT
OUR WORK.
A NICE WAY FROM VICTOR'S TALK.
LEPTIN IS PROBES OF BIOME.
THANK YOU FOR JOINING US.
>> THANK YOU TO THAT ORGANIZERS
FOR PUTTING THIS TOGETHER.
I'M REALLY EXCITED ABOUT TODAY'S
SYMPOSIUM.
SO, AS CATHERINE SAID, I'M GOING
TO TALK TO YOU ABOUT ELECTRIC
TINS AS PROBES AND I WANT TO
START BY
BY -- IT'S ALSO TRUE FOR
BACTERIA AND THIS IS A STREP
PNEUMONIA AND YOU CAN SEE THE
GLYCANS ON THE SURFACE AND WE'VE
JUST HEARD A BEAUTIFUL TALK
ABOUT THE ROLE OF THOSE GLYCANS
IN THE INNATE IMMUNE RESPONSE
AND WE ARE ALSO INTERESTED IN
THAT POINT AND WE'RE INTERESTED
ESPECIALLY IN THE ROLE THAT
LECTINS HAVE TO PLAY SO WHEN YOU
YOU THINK ABOUT HOW WE DECIDE
WHICH MICROBES ARE IN US AND
WHICH ONES WE'LL GET RID OF,
LECTINS ARE THE INTERFACE
BETWEEN THE MICROBES AND OUR
TISSUE.
AND WE CAN ACTUALLY LOOK AT
WHERE THE LOCATION OF LECTINS
ARE VERSUS WHERE WE SEE
INFECTIONS.
YOU CAN SEE THAT THERE'S A
REALLY NICE CORRESPONDENCE.
WE HAVE ELECTRIC KINS A LECTINS AT THE
INTERFACES WHERE WE FACE
BACTERIAL PATHOGENS BUT OF
COURSE THE SAME PLACES WE FACE
BACTERIAL PATHOGENS WE SPACE
BACTERIAL COMMENCEALS SO THERE'S
A LOT OF INTEREST IN REALLY
UNDERSTANDING HOW THESE
DIFFERENT LECTINS FUNCTION IN
THESE DIFFERENT DISEASE
PROCESSES AND ONE OF THE
SURPRISING THINGS WE FOUND WHEN
WE STARTED LOOKING THAT THE IS
JUST HOW LITTLE THERE IS KNOWN
ABOUT WHAT SPECIFIC MICROBES
THESE ELECTRIC KINS BIND.
IT CAN BLIND ON MICROBES AND
COMPONENTS THAT RESULT IN
FORMATION, FOR EXAMPLE, OF WOMEN
RAINA TACMEMBRANE -- SO THESE TWO
PROCESSES ALLOW US TO CLEAR
PATHOGENS OR MICROBES THIS
ELECTRIC KIN ENCOUNTERS AND ONE
OF THE QUESTIONS THAT WE HAD IS,
YOU KNOW, THIS IS THE GENERALLY
ACCEPTED ROLE OF LECTINS AND DO
THEY ALL FUNCTION SIMILARLY AND
WHAT ARE THEIR MICROBE
SPECIFICITIES?
SO AS I SAID BEFORE, THAT'S THE
QUESTION THAT WE WANT TO ASK
WHAT IS OUR HUMAN LECTINS
INTERACT TONE.
WE KNOW LECTINS, THE ASSOCIATION
BETWEEN LECTINS AND MICROBES
SEEMS TO HAVE SOME INFLUENCE ON
MICROBIAL BIOME COMPOSITION SO
THIS IS DATA IF YOU KNOCK OUT OR
DELETE MAN OWE BINDING LECTINS
I'VE BEEN DISCUSSION YOU SEE UP
REGULATION OF SOME MICROBES AND
SOME DOWN REGULATION OF OTHERS.
THERE'S ALSO GENOME-WIDE
SEWSIZATION DATA THAT SHOWS THAT
LECTINS CAN BE ASSOCIATED WITH
DISEASES THAT INVOLVE DIS
BIOTICS MIKE CRO HIGH CROW BIOME HIGH CROW BIOME AND WHAT
THESE ARE DOING.
WE FOUND THAT THIS LECTINS IS
HAS A WIDE VARIETY OF MICROBIAL
GLYCANS AND IT'S REALLY NICE FOR
ME BECAUSE WE DID THIS WORK AND
COLLABORATION WITH RICK CUMMINGS
AND SEAN CELL WHO ARE TWO PEOPLE
SPEAKING TODAY SO I'M EXCITED TO
HEAR THEIR TALKS.
WHEN WE LOOKED AT THE
SPECIFICITY, THIS LECTINS BINDS
TO EPITOPE THAT ARE FOUND ON
MICROBIAL GLYCANS AND WE WERE
ABLE TO GET A STRUCTURE SO WE
UNDERSTAND THE SPECIFICITY AND
IF YOU ARE INTERESTED, THERE ARE
PAPERS HIGHLIGHTING THAT.
THIS BINDS TO MICROBIAL SUGARS
AND WE WERE ALSO CURIOUS DOES IT
BIND TO COMMENCE CELLS AND WE
SHOWED BY PAINTING THESE LECTINS
ON TO SLIDES AND THIS IS A MOUSE
SMALL INTESTINE, YOU CAN SEE
THAT THE BACTERIA LIGHT UP AND
WHAT I NEGLECTED TO TELL YOU IS
THIS LEPTIN SAY
CALCIUM-DEPENDENT ELECTRIC TIN
SO WE CAN ADD BDTA AND AND WE KNOW
THIS LECTIN IS MADE BY CELLS
THAT ARE INVOLVED IN INNATE
IMMUNE DEFENSE.
THESE PENNANT CELLS THAT MAKE
ANTIMICROBIAL PEPTIDES SO WE
KNEW IT HAD THE FEATURES OF AN
INNATE IMMUNE LEPTIN SO WE
WANTED TO STUDY ITS SPECIFICITY
AND I'M GOING TO SHOW YOU A LOT
OF DATA THAT THOSE HUMAN SAMPLES
AND I WANT TO EXPLAIN WHY WE'RE
ESPECIALLY INTERESTED IN LOOKING
AT MIXED COLONIES AND THAT IS
BECAUSE WHEN WE LOOKED AT SINGLE
SPECIES MONO CULTURES, WHICH IS
HOW MANY STUDIES HAVE LEPTIN
SPECIFICITY WERE DETERMINED
RIGHT BINDING TO A SINGLE
SPECIES, WE CAN DETECT BINDING
TO A LOT OF DIFFERENT SPECIES
THAT ARE RELEVANT TO THE HUMAN
GUT AND SO HERE I JUST SHOW TWO
SPECIES WHERE WE DON'T SEE
BINDING SO THIS IS A BACK TA
ROIDY AND WE DO SEE LECTIN
BINDING TO THESE BUT WE SEE
REALLY ROBUST WINDING TO THE
OTHERS SO THAT SUGGESTS THAT IF
WE WERE TO MIX A COLONY TOGETHER
WE COULD SEE BINDING TO THESE
TWO SPECIES, NOT THESE TWO
SPECIES.
THIS IS A OVER LAY OF THE DATA I
SHOWED YOU SO THESE WERE
INDEPENDENT EXPERIMENTS THAT I'M
JUST OVER LAYING AND WHEN WE MIX
TOGETHER AND MAKE A COMMUNITY,
WOOZY THE LECTIN BINDING AND IT
DIDN'T MATTER HOW MUCH LEPTIN WE
ADD WE ONLY SEE BINDING TO THE
RUTER AND I IT'S IN COMMUNITY
AND SO WE WENT ON TO THEN STUDY
THEIR COMMUNITY ACTIVITIES.
SO THE WAY THAT WE'RE GOING TO DO
THIS IS HE WE'RE FOCUS ON THE
GUT MICROBIOME AND YOU CAN GET
PICTURE BY LOOKING AT HUMAN
STOOL SAMPLES SO WE MAKE LECTINS
AND ALL OF OUR LECTINS ARE HUMAN
LECTINS SO WE'RE THEN GOING TO
COMPARE BINDING BY A FLOW SIGH
TOM TREE.
THIS IS A LECTIN THAT IS THE
BEST STUDIED LECTIN THAT IS
AGAIN INVOLVED IN HOST DEFENSE.
AND THE OTHER REASON WE CHOSE
MAN OWE BINDING LECTIN IS
BECAUSE THEY REALLY HAVE BINDING
SPECIFICITY IN TERMS OF THEIR
SUGAR BINDINGS SO WHAT I'M
SHOWING YOU HERE IS A HEAT MAP
OF MICROBIAL GLYCAN ARRAY DATA
SO WE HAVE THESE BEAUTIFUL GUY
CAN ARRAYS WHERE YOU CAN
MEASURE, AGAIN, BINDING TO
INDIVIDUAL SUGARS ON THE ARRAY
AND THESE ARE THE MICROBIAL GUY
CAN ARRAYS WHICH WHY REALLY
PIONEERED BY THE PAULSON AND
CUMMINGS LAB SO WE USED THESE
AND THEY HAVE VERY DIFFERENT --
THE TWO LECTINS HAVE DIFFERENT
GLYCAN SPECIFICITY SO, IS THIS
MANIFESTED IN THEIR MICROBE
BINDING?
WELL, IF WE TAKE A HUMAN STOOL
SAMPLE AND LABEL HUMAN WITH ONE
COLOR AND MAN OS BINDING WITH
ANOTHER COLOR YOU CAN SEE THESE
ARE CELLS THAT BIND TO MAN OWES
BINDING AND HUMAN ENTA LECTIN
AND TO BOTH AND THERE'S VERY FEW
CELLS THAT SHOW MY COORDINATE
BINDING.
AND SO THIS SPECIFICITY THAT WE
SEE AT THE INDIVIDUAL GLYCAN
LEVEL IS MANIFESTED AT THE
CELLULAR LEVEL.
SO WHAT ORGANISMS ARE THESE
BINDINGS?
AND TO DO THAT, WE COLLABORATED
WITH THE XAVIER AND HOLL MANY
LABS SO THIS IS REALLY LED BY
CHRISTINE ISABELLA AND LYLE
McPHERSON AND MY LAB IN
CONJUNCTION WITH THESE OTHER
LABS.
SO WHEN WE DID IS SINCE WE KNEW
WE COULD IDENTIFY THESE USING
FLOW CYTOMETRY, WE DID SORTS
WHERE WE COULD SORT LECTIN-BOUND
AND LECTIN UNBOUND AND ASK WHAT
IS THE DIFFERENCE BETWEEN THE
SPECIES HERE?
AND SO THE WAY WE DID THAT IS TO
APPLY SHOTGUN SEQUENCES AND META
GENOMIC ANALYSIS TO UNDERSTAND
SPECIES ENRICHMENT.
SO I'M GOING TO SHOW SOME OF OUR
DATA AND SHOW THEM IN A FORM OF
PROBABILITY RATIOS AND
PROBABILITY RATIOS INTRINSICALLY
TAKE INTO ACCOUNT THE ABUNDANCE
OF DIFFERENT SPECIES IN THE
INITIAL INPUT.
AS YOU CAN IMAGINE, IF YOU HAVE
DONORS AND YOU ARE LOOKING AT
THE INPUT YOU DON'T WANT TO BE
BIASED BY, FOR EXAMPLE, A LOT OF
ONE SPECIES THAT THE LECTIN
BINDS SO THESE PROBABILITY
RATIOS TAKE THAT INTO ACCOUNT
AND WHAT THE DATA LOOKS LIKE ARE
THE SIZE OF THE DOT IS THE
ENRICHED FRACTION LIKE WHAT
PREPORTION OF THAT PARTICULAR
SPECIES WAS IN THE FINAL
ENRICHED FRACTION AND THE
PROBABILITY RATIO GIVES YOU A
SENSE OF HOW MUCH WAS ENRICHED
IN THE LECTIN PLUS VERSUS THE
LECTIN MINUS FRACTION.
SO, DATA LIKE THIS SUGGESTS THAT
YOU SEE SOME ENRICHMENT BUT YOU
ALSO SEE A LOT OF THAT
PARTICULAR MICROBE IN THE
NEGATIVE CONTROL AS WELL.
SO AS YOU CAN SEE IF YOU LOOK AT
THIS DATA, AND EACH OF THESE
DIFFERENT SAMPLE IS INDIVIDUAL
HUMAN DONOR AND WE HAVE IN THIS
SAMPLE SET, FIVE PEOPLE THAT ARE
LABELED HEALTHY SO THESE ARE
DONORS THAT HAVE NO DISEASE AND
THE D SAMPLES ARE PEOPLE THAT
HAVE SOME FORM OF IBD OR ULCER
COLITIS.
WE SEE THERE ISN'T VERY HIGH
DONOR SPECIFICITY IN THIS HUMAN
BINDING AND WHAT THESE BACTERIA
HAVE IN COMMON IS THEY'RE RARE.
AND SO HUMAN ENTA LECTIN BINDS
LOW A BUDDANC ABUNDANCE SPECIES BECAUSE IF
IT'S A LOW ABUNDANCE SPECIES,
RIGHT, I MIGHT HAVE IT BUT YOU
MIGHT NOT SO WE SEE THAT THIS
LECTIN BINDS AGAIN TO THESE RARE
SPECIES AND THIS WAS REALLY
INTERESTING BECAUSE, IT DOESN'T
FIT THAT KIND OF MODEL OF THIS
LECTIN INVOLVED IN DEFENSE
BECAUSE AGAIN THESE ARE RARE
SPECIES AND MANY OF THEM HAVE
BEEN OFFICIAL PROPERTIES.
ONE OF THE THINGS WE NOTED IS
THESE DATA ARE REALLY INTRIGUING
AND EVIDENCE GROWING THAT
INTELLECT ANT CAN DAMPEN IMMUNE
RESPONSES AND TWO PARTICULAR
BACTERIA AND SO WE THINK THAT
BINDING COULD SHIELD THESE
PARTICULAR BACTERIA FROM IMMUNE
CLEARANCE AND ONE THING THAT
I'LL MENTION IS OF COURSE,
HAVING A MORE I DO VERSE DIVERSE MICROBIOM E
IS ASSOCIATED WITH BENEFICIAL
HEALTH.
SO THAT SUGGESTED THAT MAYBE ALL
OF THESE LECTINS BIND REALLY
RARE INDIVIDUALS SPECIES SO WE
WANTED TO ASK IS MAN OWES
BINDING LECTIN BROAD, LIKE I
JUST SHOWED YOU.
WHEN WE DID A SIMILAR EXPERIMENT
TO THE ONE I SHOWED YOU WE GOT
DIFFERENT RESULTS WITH THIS
LECTIN.
IT BINDS TO F PRAUSNITZII AND
THIS IS VERY PREVALENT IN THE
HUMAN MICROBIOME AND ITS
ASSOCIATED WITH ROBUST HUMAN
HEALTH AND WE SAW THIS REEL IN
VIRTUALLY ALL OF THE DONORS WITH
THE EXCEPTION OF A FEW PEOPLE
THAT LACK THIS F. PRAUSNITZII.
NOW F. PRAUSNITZII, IS THIS
BINDING TO ALL OF THE ABUNDANT
SPECIES BUT REALLY F.PRAUSNITZII
IS THE ONLY THAT MBL-BOUND.
AND AS I MENTIONED BEFORE, IT'S
INTERESTING TO NOTE THAT WHEN
THAT SPECIES IS NOT THERE IT
BINDS TO A. MUCINIPHILA, IT WAS
ONLY FOUND IN SAMPLES WERE
PEOPLE THAT HAD SOME SORT OF
INFLAMMATORY DISEASE.
AND I GUESS MAYBE I'LL JUST
MENTION HERE, THIS SUGGESTS
THESE KINDS OF LECTIN-BINDING
EXPERIMENTS CAN BE USED TO
SATURDASTARTTO SORT AND UNDERSTAND AND
DIAGNOSE DIFFERENT DISEASES.
I JUST WANT TO BRIEFLY MENTION,
# H IT WOULIT WOULD HAVE ACCESS TO THE GUT
AND IN FACT, IN COLORECTAL
CANCER PATIENTS WITH BLOOD IN
THE STOOL, THEY HAVE VERY IT
FELT BUELLLY WITH THE LECTIN
BINDING THAT WE HAVE DETECTED SO
YOU COULD IMAGINE IT DOWN
REGULATING THIS BACTERIUM.
SO WE KNOW A LOT ABOUT HOW TO
PER TISH THESE HOST MICROBE
INTERACTIONS AND THAT THESE
PROTEINS HAVE WE NEED TO
UNDERSTAND THEIR TARGETS AND
I'VE JUST SHOWN YOU TWO OF THESE
MINI LECTINS AND THERE ARE SO
MANY OUT THERE THAT WE COULD USE
TO PROBE AND HOST MICRO
INTERACTIONS AND WE REALLY NEED
THESE NEW KINDS OF TOOLS FOR
PROFILING MICROBIOMES AND WE
HOPE THAT LECTINS WILL BE A PART
OF THAT.
SO, I'VE TRIED TO THANK THE
PEOPLE WHO DID THE WORK
THROUGHOUT THE TALK AND I WANT
TO JUST THANK THE ENTIRE GROUP
AND I WANT TO THANK THE NIH FOR
FUNDING THIS WORK AND AGAIN, THE
ORGANIZERS AND THANK YOU ALL FOR
YOUR ATTENTION AND I'M HAPPY TO
ANSWER QUESTIONS.
>> FANTASTIC TALK.
YOU ALWAYS IMPRESSES US WITH
SOMETHING NEW YOUR LAB IS
ENDURING ON.
QUESTIONS FROM THE AUDIENCE --
WE HAVE A QUESTION.
>> THANK YOU, CATHERINE.
HI, LAURA, THAT WAS A GREAT
TALK.
I'M WONDERING ABOUT YOUR
DIFFERENCES BETWEEN THE TWO
LECTINS AND ONE WAS MORE BROAD
AND ONE WAS MORE SPECIFIC.
SO, DOES THAT, I MEAN,
PRESUMABLY IT GIVES YOU IN SIGHT
INTO THE TYPES OF SUGARS THAT
ARE ON THE SURFACES OR ON
CERTAIN PATHOGENS VERSUS OTHERS?
I MEAN, IS MAN OWES MORE
UBIQUITOUS VERSUS KVO AND THE
OTHER SUGARS OR IS THAT NOT THE
RIGHT EXTRAPOLATION?
>> YEAH, SO, THANK YOU FOR
SAYING THAT.
I WAS GOING TO SAY, I SHOULD
HAVE ENDED MY TALK BY SAYING
THAT THIS ALSO SHOWS HOW
DIFFERENT THE FUNCTIONS OF THESE
LECTINS LIKELY ARE AND AND
THERE'S A TRANSFERASE OR GA TACK
LOW ON THE SURFACE OF THE CELLS
BECAUSE WE ACTUALLY SEE EVEN
THOUGH THEY HAVE THE SUGAR
DENSITIES AND ACCESSIBILITIES OF
THOSE SUGAR ARE REALLY IMPORTANT
AND SO, WE WOULD LIKE TO BE ABLE
TO GO IN AND DO EXACTLY WHAT YOU
ARE SAYING START TO RELATE
SPECIFICALLY SUGARS WITH THE
LECTIN BINDING BUT WE'RE NOT
QUITE THAT FAR YET IN TERMS OF
CAPTURING THE SPECIFIC SUGARS SO
THAT ALSO POINTS TO THE NEED FOR
KNEW TOOLNEW TOOLS WHICH IS SOMETHING
WE'RE WORKING ON.
>> LOOKING FORWARD TO IT.
>> GREAT QUESTION.
>> AMAZING TALK.
THE FIRST QUESTION WE HAVE FROM
THE ON-LINE AUDIENCE IS FROM
NICOLAS AND HE ASKS, HE IS FROM
VANDER BELT AND WITH SO MANY
LECTINS, WHAT WOULD BE THE BEST
PRIORITIZATION FOR STUDYING TO
PROBE MOST MICROBE INTERACTIONS.
>> YEAH, SO, WE HAVE SOME OTHER
REAL TARGETS LECTINS THAT WE'RE
LOOKING AT AND SO SOME OF THE
LECTINS THAT WERE VERY
INTERESTED IN ARE DRAMATICALLY
ALTERED IN DISEASE AND THAT IS I
THINK A GOOD PRIORITIZATION
STRATEGIES THAT ONE CAN USE.
SO, FOR EXAMPLE, I'LL JUST
MENTION THAT SPD IS THE
LECTIN -- ACTUALLY THE NUMBER
ONE PROTEIN PERTURBED IS IT'S
REALLY IMPORTANT FOR DISEASE AND
I THINK THOSE ARE ONES WE'RE
GOING AFTER FIRST.
>> THERE ARE MORE AND MORE
QUESTIONS COMING IN ON THE CHAT
BUT WE'LL MOVE ON AND I PROMISE
YOU, SHE WILL ANSWER YOU IN YOUR
REMALE-MAIL ADDRESSES.
THANK YOU SO MUCH.
KEEP THE QUESTIONS COMING IN.
THAT'S WHAT MAKES THIS MORE
INTERACTIVE AND FUN SO THANK YOU
FOR AN ENGAGING TALK.
SHE'S GOING TO TALK ABOUT NOVEL
STEP TOE COCKAL GUY CAN BINDING
ADHERENCE.
>> I WANT TO SEE WHAT EVERYONE
IS GOING TO SHARE TODAY SO I'D
LIKE TO START BY THANKING THE
MEMBERS OF THE LAB THAT DID THIS
WORK AND ESPECIALLY MEZ AND
ANIRUDH AND THE ANIMAL MODELING
AND OF COURSE FOR OUR FUNDING.
SO, IT'S AN ORAL COMMENCAL AND
IT'S CONSIDERED TO BE A OR
ALPINE HERE AND SO WHEN WE THINK
ABOUT ORAL PIONEER.
THE SURFACE IS COVERED IN A
PELICAL AND A BACTERIA ARE
THOUGHT TO BIND TO THAT PELICAL
AND EVENTUALLY WE GET THIS
COMPLEX ORAL AND THIS BIO FILM
IS ASSOCIATED WITH DENTAL
CARRIES AND FOR EXAMPLE,
2.5 BILLION ADULTS ARE THOUGHT
TO HAVE UNTREATED DENTAL CARRY
SO THIS IS A SIGNIFICANT ISSUE.
THE STREP OR ALICE CAN CAUSE
SUBACUTE -- WHEN YOU PRESSURE
TEETHAND THEVEGETATION CAN BREAK OFF AND I T
CAN GET STUCK IN A SMALLER
VESSEL AND CAUSE A STROKE.
SO, IN THAT ENVIRONMENT, IT'S
THOUGHT THANK YOU TO VICTOR TO
BRING OUR PLATELETS EARLIER,
IT'S A CRITICAL STEP IN THAT
PROPERTY SO EITHER THE BACTERIA
CAN BIND TO THE PLATELETS IN
CIRCULATION AND IT'S LIKE A
TROJAN HORSE TAKING THE BACTERIA
TO THE SITE OF DAMAGE OR CAN
BIND TO THE SITE OF DAMAGE
DIRECTLY.
AND IN BOTH THESE ENVIRONMENTS,
BINDING IS A CRITICAL STEP SO
WE'RE INTERESTED IN
UNDERSTANDING HOW THE BACTERIA
BINDS BECAUSE IF WE THINK WE CAN
WORKOUT OUT THEY BIND WE CAN
REDUCE THE BURDEN OF DISEASE AND
WE BELIEVE SOME OF THE SAME
MECHANISMS CAN BE IMPORTANT IN
BOTH ENVIRONMENTS AND IT'S
ACCIDENTAL THAT STREPTOCOCCUS
GOES INTO THE SAME BINDING
MECHANISMS WHICH NOT TO GIVE A
SPOILER, A CA A IN CARBOHYDRATES.
IT WAS RECLASSIFIED ADDITIONAL
STRAINS WERE ADDED AND IT WAS
DIVIDED INTO THREE SUB SPECIES
AND SO JUST FOR CLARITY, I'M
GOING TO TAKE ABOUT OR ALICE
TODAY.
THEY COMBINED A VARIETY OF
DIFFERENT HOST AND BACTERIAL
SURFACES AND THEY ARE NAMED FOR
THE RESPEED SO THESE MAKE UP THE
MAJORITY OF THE PROTEIN AND
EVERY OTHER AMINO ACID IS A
SERINE AND SO THIS FORMS THE
STORK THAT STRETCHES THE BINDING
DEMANDS OUT SO WHAT THEY'RE BIND
IS ACTUALLY THROUGH THE NON
REPEAT REGION SO IN THAT NON
REPEAT REGION YOU CAN HAVE
MODULAR STRUCTURES AND IT TERMS
THE RECEPTORS THAT IS BOUND SO
IN THE CASE OF ORAL
STREPTOCOCCUS, IT BINDS THROUGH
THESE PROTEINS THAT NON REPEAT
REGION CONTAINS ONE DOMAIN AND
UNIQUE DEMAIN SO THE FUNCTION OF
THE TO BLIND SALIC ACID AND IT
IT MIGHT BE FOLDING THE SIALIC
DOMAIN BUT IN OTHER SPECIES,
THESE ARE PROTEINS THAT HAVE
BEEN SHOWN TO BE IMPORTANT IN
VEGETATION FORMATION FOR
SEVERALLEN DOUGH CAR DIET IS
PATHOGENS SO IT'S NOT SURPRISING
WHEN A FEW YEARS AGO WE
IDENTIFIED A REPEAT PROTEIN THAT
WE HOLD FAP1 THAT SIGNS ACID SO
IT'S A REPEAT PROTEIN AND IT
CONTAINS ONE DOMAIN AND IT ALSO
CAN HAVE VERY WEAK STRUCTURAL
SIMILARITY TO A CA CARBOHYDRATE AND
IT'S RESURFACE TO SO WE
PREVIOUSLY PUBLISHED THAT FAP1
IS IMPORTANT IN WINDING
PLATELETS SO HERE I'M GOING TO
SHOW YOU DATA WITH SALIVA AND MY
LITTLE SCHEMATIC ON THE RIGHT IS
TO SHOW YOU THE TOOLS WE HAVE.
IT'S A VAST OVER SIMPLIFICATION.
SO IF THAT ONE DOES BIND
TERMINAL SIALIC ACID, IF WE ADD
A BINDING MODULE, THIS IS FOR A
SMALL DOMAIN THAT IS
SPECIFICALLY BIND SIALIC ACID WE
WOULD BE ABLE TO INHIBIT THE
BINDING OF ORALISE AND THAT'S
WHAT WE SEE AND IF WE MAKE A
FAP1 MUTANT IT WOULD BE IN
BINDING 2.
WE WOULD EXPECT IF WE ADD THEM
TOGETHER, WE REALLY WOULD EXPECT
TO SEE NO FURTHER REDUCTION IN
BINDING AND THAT'S ALSO THE CASE
W KNOW THAT FAP1 IS IMPORTANT
FOR BINDING SIALIC ACID ON
PLATELETS AND ON SALIVA.
OUR QUESTION IS BINDING, I MEAN,
A LOT OF PEOPLE HAVE IDENTIFIED
THE PROTEINS AND THERE'S BEEN
SOME STUDIES IN OR STRP SO IT
CONSERVED MECHANISM OF ADHESION
AND WE USE CBM 40 AND WE TOOK
FIVE ADDITIONAL ISOLATE AND
SHOWED EACH WAS REDUCED BY CDM40
AND IT'S JUST TO COMPLICATE
MATTERS, A LOT OF THESE STRAINS
CAN UNDERLINE CARBOHYDRATES.
ONLY TWO OF THE FIVE HAD FAP1 SO
WE KNEW NOW THAT FINDING SIALIC
ACID IS CONSERVED AND FAP ONE IS
NOT AND WE WANTED TO FIND AND
WHY HAVE A LOT OF TIME TO TELL
YOU ALL ABOUT IT BUT BASICALLY
WHAT WE WAS WE MADE A SURFACE
PROTEINS AND MOST STREPTOCOCCUS
HAVE BEEN DESCRIBED AND ARE
FOUND TO THE SURFACE OF THE
BACTERIA BY SORT ACE A AND WE
THAT WAS REDUCED IN BINDING SO
WE CAN DO RELATIVELY COMPARATIVE
GENOMICS TO COMPARE TRAINS THAT
HAD FAP1 AND IDENTIFY PRE TEEN
GENES AND IT WOULD BE DIFFERENT
AND WE WOULD DO STRUCTURAL
PREDICTIONS TO SEE IF THEY HAVE
A DOMAIN OR OTHER THINGS AND YOU
MIGHT WELL-KNOWN THEY'RE NOT
EASILY IDENTIFIED BY SEQUENCE
SIMILARITY.
IT'S EASIER BY STRUCTURAL
PREDICTION AND AFTER THAT, WE
IDENTIFIED WHAT WE CALLED ASAA
AND ADHESION A AND THIS HAS A
SIMILAR STRUCTURE IN A WAY BUT
ALSO IT'S QUITE DIFFERENT AND
STEED OF REPEATS WE HAVE 42
REPEATS AND THESE ARE OFTEN
FOUND DAN TEM IN SURFACE
PROTEINS AND THEY'VE NOT BEEN
STUDIED A LOT BUT WHEN ANOTHER
GROUP ARE COMMONLY EXPRESSED
SOME OF THESE IN TANDEM THEY
FORMED AFTER ALPHA ELECTION SO
WE'RE NOT ABLE THELIX AND WEBELIEVE IT MAK ES A FEW RAL AND
IN THE NON REPEAT REGION, THERE
ARE TWO UNIQUE DOMAINS IN ASAA
AND ANOTHER PART OF THAT REGION
WHICH HAS SOME STRUCTURAL
SIMILARITIES TO A FAMILY VARIOUS
ARCHITECTURE DOMAIN AND A CVM SO
PERHAPS THERE'S MORE THAN ONE
RECEPTORS.
SO THEN WE WENT ON TO LOOK AT
THE FUNCTION OF ASAA AND IF WE
KNOCK OUT ASAA WE SEE AS WE
PREDICT, A REDUCTION IN BINDING.
IF WE USE SIALIC ACID THE STRAIN
DOES NOT BIND TO AN UNDERLYING
CARBOHYDRATE AND WE SEE A
REDUCTION IN BINDING AND IF WE
THEN USE CELLS AND ASAA WE SEE
NO FURTHER REDUCTION AND WE ALSO
SEE BINDING WITH SALIVA TOO.
WE THEN WERE COMMONLY EXPRESSED
THE NON REPEAT REGION OF ASAA
AND SHOWED IT CAN INHIBIT BIND
NORTH A DOSED MANNER AND TO LOOK
AT THIS IN A MORE DIRECT WAY, WE
WERE COMMONLY EXPRESSED THE NON
REPEAT REGION OF ASAA AND BOUND
IT TO A PLATE AND THEN ADDED
PLATELETS AND I DON'T KNOW HOW
WELL YOU CAN SEE IT BUT
HOPEFULLY YOU SEE THE DOTS AND
EACH IS A PLATELET AND THEN IF
WE TREAT THE PLATELETS WE SEE A
VAST REDUCTION IN BINDING AND WE
THEN WANTED TO SHOW IT WAS JUST
A SIGLEC AS WE PRO DICTIONARY SO
THAT EXPRESS CAN ALSO BIND
PLATELETS AND ALSO GREATLY
REDUCE WITH TREATMENT AND
BECAUSE BOTH OF THESE PROTEINS
WERE GTST TAGGED WE USED THAT AS
A CONTROL.
AND SO IF WE QUANTIFY THAT DATA
YOU WITH CLEARLY SEE THAT THE
SEC GLEC-LIKE AND UNIQUE DOUGH
MINES BIND IN A SIALIC DEPENDENT
MANNER SO THEN TO LOOK AT THIS
IN THE CONTEXT OF THE BACTERIA,
WHAT MEZ DID SO SHE TAGGED THE
PROTEIN AND MADE TWO
DEELECTRICKION TO REMOVE THE
SIGLEC-LIKE AND UNIQUE DOMAINS
AND SHOWED THOSE PROTEINS WERE
STILL EXPRESSED AND ON THE
SURFACE OF THE BACTERIA, AND WE
LOOKED AT BINDING.
SHOWING YOU THEY'RE IMPORTANT
FOR BINDING SIALIC ACIDS ON THE
PLATELETS.
THE PHENOTYPE HERE ISN'T AS
STRONG AS WE SAW PREVIOUSLY AND
I THINK THAT'S BECAUSE, WE KNOW
THAT THE THREE STRAIN DOES NOT
BIND QUITE AS WELL AND WE THINK
THAT'S BECAUSE IT'S EFFECTING
THE FOLDING A LITTLE BIT.
THAT'S WHY IT LOOKS LESS.
WITH TODD KITTEN, HE WAS KIND
ENOUGH TO DO SOME RABBIT MODELS
FOR US AND WE COULD SEE ABOUT A
THREE-FOLD REDUCTION IN THE
NUMBER OF BACTERIA IN THE
VEGETATION.
THIS IS IN KEEPING WITH A MOST
DATA FROM OTHER REPEAT PROTEINS
IN FACT IT'S MORE OF A REDUCTION
THAN IS OFTEN OBSERVED.
WE THINK THE REASON WHY THIS
REDUCTION ISN'T LARGER IS
PROBABLY BECAUSE WE'RE EXPOSING
ADDITIONAL RE SCEPTER ON THE
SURFACE OF THE BACK TO BE BY
REMOVING THE FIBER.
WE SEE IN A ASAA MUTE CAN'T
INCREASED BINDING TO FIBER.
WE'RE INTERESTED WHETHER THERE
WERE ASAA IN OTHER SPECIES.
THESE COMBINE SIALIC ACID BUT
THERE ARE OTHERS WE DIDN'T KNOW
HOW THEY BIND AND SO WE FOUND
ASAAORTHOLOGS THAT CAN CAUSE.
I DON'T HAVE THE DATA YET TO
SHOW YOU, BUT WE TROY TO STUDY
WHETHER ASAA IS A MEMBER OF A
FAMILY AND BECAUSE IT'S CLOSEST
WE CHOSE NATURALLY TRANSFORMAL
WHEN WE WORK WITH THAT AND IT'S
ONE STRAIN OF MITIS THAT ENCODES
TWO POTENTIAL SIALIC
ACID-BINDING PROTEINS AND WE SEE
IT IN CODE FAP1 OR ASAA AND IN
MITIS IT'S MORE COMPLICATED SO
THIS TRANSCRIPTION WHICH A
REPEAT PROTEIN THAT'S NOT BEEN
STUDIED AND ALSO AN ASAA ORTH
OWE LOG SO ONE OF OUR UNDER
GRADS.
OVER ALL, I THINK WE'VE SHOWN
THAT SIALIC ACID BINDING IS MORE
COMPLEX THAN PREVIOUSLY
APPRECIATED SO WE WONDERED IF
THERE'S OTHER ADDITIONAL SIALIC
ACID BINDINGS AND SO THESE ARE
THE TWO THAT I'VE ALREADY TALKED
ABOUT, ASAA AND THE REPEAT
PROTEINS AND I DON'T HAVE A LOT
OF DATA FOR YOU BUT IF WE LIKE
AT GENOME WE HAVE OTHER SURPASS
PROTEINS INCLUDING THE ONE THAT
I'VE ILLUSTRATED HERE WHICH
INSTEAD OF DOVE 1542 REPEATS HAS
A TYPICAL REPEATS AND SO WE'RE
ALSO NOW BECOMING INTERESTED IN
THIS IDEA THAT A BACTERIA AND I
DON'T KNOW THAT IT'S A NOVEL
IDEA, BUT THE IDEA THAT THE
BACTERIA HAS A PORTFOLIO OF AT
ADHERENCE SO WE KNOW THERE ARE
OTHER REPEAT CONTAINING BINDING
PROTEINS THAT HAVE DIFFERENT SO
WE CAN HYPOTHESIZE THAT THEY
HAVE DIFFERENT PROTEINS AND
THERE'S SOME EVIDENCE THAT THE
PROTEINS CAN IMPORTANT IN BIO
FILM FORMATION BUT THERE ARE NOT
A LOT OF STUDIES OF THOSE
REPEATS SO ONE OF THE THINGS
WE'RE INTERESTED TO DO IS TO
MAKE IC ISOGENIC STRAINS EXPRESSING
FAP1 AND ASAA THAT HAVE THE SAME
BINDING REGION OR NO BINDING
REGION SO WE CAN STUDY THE
CONTRIBUTION OF REPEATS SO YOU
CAN IMAGINE IF A BACTERIA HAS A
PORTFOLIO OF ADHESIONS AND THEY
CAN BE DIFFERENT, IT CAN CONFER
DIFFERENT DISTANCES OUT FROM THE
SURFACE THAT YOU COMBINE THESE
DIFFERENT RECEPTORS AND
DIFFERENT CHARGE, DIFFERENT
(INAUDIBLE) AND THINGS LIKE THAT
THAT COULD CHANGE HOW THESE
BACTERIA BEHAVE.
IN CONCLUSION, I HOPE I'VE SHOWN
YOU THE FAP1 AND ASAA AND THESE
BOTH BIND SIALIC ACID AND WITH
TODD'S HELP WE SHOWED THAT ASAA
IS IMPORTANT IN FORMATION OF A
VEGETATION AND WE'VE SHOWN THAT
SIALIC ACID BINDING IS MORE
COMPLEX THAN WE PREVIOUSLY
APPRECIATED AND WE THINK THAT
ASAA IS THE FOUNDING MEMBER OF A
FAMILY OF ADHESIONS AND THE
OTHER CHARACTERIZED SIALIC EXIST
IN OUR BACTERIA AND NOW WE'RE
INTRIGUED OF WHAT THE BIOLOGICAL
CONSEQUENCES OF THESE ADHESIONS
AND I'LL TAKE ANY QUESTIONS WITH
THAT.
>> EXCELLENT TALK.
THANK YOU FOR GETTING US INTO OR
AVENUORAL CAVITY AND THE BACTERIA
EXPRESSING THESE RECOGNITION
PROTEINS OF SIALIC ACIDS SO
THANK YOU.
WHAT QUESTIONS DO WE HAVE?
ARE THERE QUESTIONS COMING IN
FROM THE CHAT?
DO OTHER PEOPLE IN THE AUDIENCE
HAVE QUESTIONS FOR Dr. KING?
>> I WAS TRYING TO FIND THE
RAISED HAND IN THE CHAT AND I
COULDN'T FIGURE OUT.
I AM REALLY CURIOUS ABOUT
BINDING TO MUCUS THROUGH THESE
ADHESIONS AND WHETHER OR NOT
YOU'VE HAD A CHANCE TO LOOK AT
THAT BECAUSE THAT WOULD BE AN
INTERESTING ROLE FOR THEM.
>> SO WE JUST HAD A PAPER
ACCEPTED WITH SARAH LINDEN IN
COPENHAGEN.
AND SHE LOOKED AT BINDING TO
MOCK 5A AND 7 ENRICHED FRACTIONS
AND WE DIDN'T SEE A TON OF
FINDINGS OF ATCC OR 01577 THE FA
APPROXIMATE 1 STRAIN EVEN THOUGH
IT DOES BIND.
THAT PAPER JUST GOT ACCEPTED SO
IT SHOULD BE OUT ANY DAY NOW.
>> YEAH, I THINK MAYBE CAT OR
EVEN A IS A GOOOR -- ITIS HOW THEY INFLUEN CE IT IS
WHETHER OR NOT YOU SEE THINGS
TOO.
>> IN HER SAMPLES, WE MAY BE
SEEING BINDING TO OTHER FACTORS
TOO AND HOW MUCH THAT LEADS TO
CLEARANCE FROM THE ORAL CAVITY
AND BINDING IN THE ORAL CAVITY
IS PROBABLY A BALANCE THERE THAT
YOU ARE SEEING CLEARANCE AND
ALSO SOME BINDINGS.
SO, YEAH, IT'S INTRIGUING AND I
THINK WE WOULD LIKE TO LOOK AT
MOST STUT HE'D.
>> SHE'S ON-LINE HERE AND SHE'S
IN THE CHAT ROOM AND IF YOU WANT
TO COMMUNICATE WITH CAT OR EVEN
A OFF LINE AS WELL AS WE'RE
DOING THIS WHO KNOWS BUT I DON'T
THINK SO.
>> AWESOME.
WELL, THANK YOU FOR AN AMAZING
TALK.
THIS IS BEEN A SUPER TREAT SO
FAR AND STARTING OUT THIS IS
GREAT.
WE'RE GOING TO SWITCH GEARS FROM
EMILY AND SHE'S COMING FROM
HARVARD UNIVERSITY AND SHE'S
GOING TO TALK TO US ABOUT HER
WORK ON ENZYME DISCOVERY IN THE
HUMAN MICROBIOME.
IF YOU FOR JOINING US AND I
APPRECIATE YOU COMING AT THE
INTERSECTION OF MICROBIOME
METABOLISM AND GLYCO SCIENCE SO
THANK YOU SO MUCH FOR JOINING
AND WE'RE LOOKING FORWARD TO
YOUR TALK.
>> THANK YOU SO MUCH CATHERINE
AND THANK YOU TO ALL THE
ORGANIZERS FOR IN INVITING ME TO
BE PART OF THIS INTERESTING
SYMPOSIUM.
AND I'M EXCITED TO BE HERE.
SO, WE ARE INTERESTED BECAUSE OF
THE RICH CHEMISTRY THAT THEY
PERFORM.
SO WE THINK THAT THESE ORGANISMS
REALLY PROVIDE SOME AMAZING
OPPORTUNITIES FOR THE DISCOVERY
OF NEW CHEM TRE CHEMISTRY AND METABOLISM
BUT WE'RE FASCINATED BY THE
BIOLOGICAL ROLES THAT IT PLAYS
IN HUMAN ASSOCIATED MICROBIAL
COMMUNITIES.
SO MICROBIAL METABOLISM EXPANDS
THE CHEMISTRY OF THE HUMAN BODY
AND MEDIATES IMPORTANT
INTERACTIONS WITH THE HOST.
BUT ARE UNDERSTANDING OF THE
BIOLOGICAL CONSEQUENCES IS STILL
LIMITED.
MOST GUT MICROBIAL METABOLIC
ACTIVITY HAVEN'T BEEN LINKED TO
GENES OR ENZYMES AND BOTH GENES
AND METABOLITES ARE
UNCHARACTERIZED AND FINALLY, WE
HAVEN'T YET MANY OF THE
MOLECULAR MECHANISMS BY WHICH
THEY EFFECT THE HEALTH OF THE
HOST AND OTHER MEMBERS OF THE
MICRO BIOME SO THESE QUESTIONS
REALLY INTRIGUE MY LAB AND OUR
GOAL IS TO USE CHEMICAL
KNOWLEDGE AND APPROACHES TO TROY
TO DO THINGS LIKE DISCOVER NEW
METABOLIC ACTIVITIES AND ENZYMES
FROM THE HUMAN MICROBIOME AND TO
DECIPHER HOW THEY INFLUENCE HOST
BIOLOGY AND OTHER MICROBES.
WE HOPE IT WILL REVEAL INSIGHTS
INTO MICROBIOME AND PAVE THE WAY
FOR THERAPEUTIC INTERVENTIONS.
MOST OF OUR WORK HAS FOCUSED ON
THE HUMAN GUT MICROBIOME SO OVER
THE LAST 10 YEARS WE'VE STUDIED
A WIDE RANGE OF MET BOL
DETECTIVE TEASE THAT HAVE
EXPANDED OUR UNDERSTANDING OF
ENZYMES FROM THESE ORGANISMS AND
REVEALED CORRELATION S WITH
HUMAN HEATH.
WE HAVE DISCOVERED HOW A GROUP
OF UNCUL TURD GUT BACTERIA
METABOLIZED CHOLESTEROL LOWERING
LEVELS OF THIS DISEASE
ASSOCIATED MOLECULE AND WE'VE
UNDISCOVERED GUT BACTERIA THAT
METABOLIZE THE PARKINSON'S
DISEASE AND REDUCING ITS BIO
AVAILABILITY AND WE'VE ALSO
DESCRIBED HOW GUT BACTERIA MAKE
THE COMPLEX DAMAGING NATURAL
PRODUCT LINKED TO COLORECTAL
CANCER BUT GIVEN THE THEME OF
TODAY'S SYMPOSIUM, I ACTUALLY
WANT TO TELL YOU ABOUT A NEW
STORY, THIS IS OUR FIRST EFFORTS
TO KIND OF MOVE BEYOND THE GUT
AND INVESTIGATE ENZYMES FROM THE
AND THIS IS ALL ALSO OF OUR
FIRST ADVENTURES INTO THE
DISCOVERY AND CHARACTERIZATION
OF CARBOHYDRATE METABOLIZING
ENZYMES.
SO THE HUMAN BIKE ROW BIOME IS
DIFFERENT THAN THE HUMAN GUT.
THE HEALTH ASSOCIATED STATE IS
TYPICALLY DOMINATED BY A SINGLE
SPECIES OF LACTOBACILLU IS AND
THEY LOWER THE PH OF THE
CERVICAL VAGINAL ENVIRONMENT BUT
THIS COMMUNITY CAN TRANSITION TO
A MORE DIVERSE GROUP OF
ANTI-ROBES AND THIS IS
ASSOCIATED WITH AN ELEVATION OF
PH AND THIS CHANGE IN COMMUNITY
COMPOSITION AS ASSOCIATED WITH
CHANGES IN THE CERVICAL VAGINAL
ENVIRONMENT AND THE DEROGATION
OF MUCUS AS WELL AS HEALTH
CONDITIONS INCLUDING BACTERIAL,
HIGHER RISK FOR PRE TERM BIRTH
AND STI ACQUISITION SO WE DON'T
UNDERSTAND WHAT PROMOTES
DOMINANCE OR THE FACTORS
FACILITATE THIS TO A DIVERSE
COMMUNITY.
THIS LACK OF KNOWLEDGE LIMITS
OUR ABILITY TO INTERVENE
THERAPEUTICALLY AND HASN'T BEEN
A MAJOR ADVANCE IN THE TREATMENT
OF BACTERIAL ADVANTAGE ENOSIS.
SO THIS EFFECTS A COULD THIS
CHANGE IF WE HAVE A BETTER
UNDERSTANDING OF VAGINAL
BACTERIA AND WE COULD IDENTIFY
METABOLIC ACTIVITIES AND ENZYMES
THAT SUPPORT VAGINAL BACTERIA IN
DIFFERENT COMMUNITIES OR THAT
ENABLE THE TRANSITION BETWEEN
THESE DIFFERENT COMMUNITIES AND
AGAIN, MOST IMPORTANTLY IF WE
UNDERSTAND THE FUNCTIONS AND THE
MECHANISMS BY WHICH HOST AND
OTHER MEMBERS OF THE COMMUNITY
CAN RELEVERAGE THAT INFORMATION
SO THERAPEUTIC DEVELOPMENT SO
TODAY, I HOPE TO GIVE THESE ARE
INTERESTING QUESTIONS TO EXPLORE
AND WE'RE STILL AT AN EARLY
STAGE BUT I HOPE THIS WILL
ILLUSTRATE HOW CHEMISTRY CAN
PLAY A KEY ROLE IN THESE EFFORTS
AND PARTICULARLY, HOW IT'S
IMPORTANT THAT WE DISCOVER AND
CHARACTERIZE ENZYMES FROM THIS
COMMUNITY.
THIS IS THE WORK OF TWO
TRAINEES, A POSTDOC IN THE LAB
AND A GRADUATE STUDENT AND WE'VE
WORKED TOGETHER ON THIS PROJECT
WITH SETH AND SETH WILL TALK
LATER ON TODAY.
AND CAROLYN MITCHELL IS ANOTHER
KEY COLLABORATOR AND WE'RE PART
OF A LARGER VAGINAL MICROBIOME
CONSORTIUM FOUNDED BY THE GATES
FOUNDATION.
THIS PROJECT BEGAN WITH AN
INTEREST IN UNDERSTANDING HOW
VAGINAL BACTERIA ACCESSED AROUND
IMPORTANT CARBON SOURCE.
GLYCO GINA ACCUMULATIO GENE ACCUMULATES AN D IT'S
AN IMPORTANT NUTRIENT SOURCE FOR
VAGINAL BACTERIA AND NOW OF
COURSE IN ORDER FOR BACTERIA TO
ACCESS GLUCOSE FROM GLYCO AGAIN
THEY NEED TO HYDRO GUYS THIS TO
SMALLER PRODUCTS AND THEY WERE
UNSUCCESSFUL AND THIS RACED THE
QUESTION OF WHAT TYPE OF ENZYMES
MIGHT BE RESPONSIBLE FOR GLYCO
GLYCOGEN BREAKDOWN IN THE
VAGINAL ENVIRONMENT.
THERE'S BEEN EVIDENCE SUPPORTING
A ROLE NOR HUMAN USING ALLY A
AND STUT HE'D CORRELATION
BETWEEN THE ABUNDANCE OF THIS
ENZYME IT WAS SUPPORTED THAT
THEY RETAINED HIGH GLYCOGEN
ACTIVITY EVEN AT PH4 HAVE
ACTIVITY SO THIS HINTED AT THE
POTENTIAL RELEVANCE OF BACTERIAL
ACTIVITY AND IN DEED THE LAST
FEW YEARS HAVE SEEN SOME REALLY
GREAT ADVANCES IN OUR
UNDERSTANDING OF GLYCOGEN
METABOLISM BY VAGINAL BACTERIA
AND IN 2019 COURT LAB PERFORMED
AN ANALYSIS OF STRAINS AND
ASSOCIATED WITH DOMINATED VERSUS
STRAINS THAT WERE PRESENT IN
MORE DIVERSE DIS BIOTICS
COMMUNITIES AND THIS ANALYSIS
SHOWED FOR THE FIRST TIME THAT
ACTUALLY A LARGE FRACTION OF AND
STRAINS IN THEIR PANEL COULD IN
DEED GROW ON GLYCOGEN CONTAINED
MEDIA AND THEY PERFORMED
INCLUDING A GLYCOGEN METABOLISM
A TYPE 1 THEY NAMED AND IT HAS A
CENTRAL DOMAIN AND HAS A SIGNAL
PEPTIDE AND DOMAIN PREDICTED
ASSOCIATION S WITH THE BACK
TERRAL S AND IS EXTRA IN
ADVANTAGE VAGINAL FLUIDS AND
THEY HAVE METABOLISMING ACTIVITY
USING THE PAGE AND THEY SEQUENCE
DETECTED SEQUENCE SO THIS
ANALYSIS IMPLICATES BOTH HUMAN
AND BACTERIAL ENZYMES AND BUT
WHILE THESE HAVE GREATLY
ADVANCED OUR UNDERSTANDING OF
ENZYMES FOR METABOLISM A MAJOR
GAP IN ALL OF THESE WAS A LACK
OF BIOCHEMICAL CHARACTERIZATION
OF THESE ENZYMES.
AND SO THESE WERE THE QUESTIONS
THAT MY GROUP HAVE STARTED THIS
PROJECT.
CAN THESE ENZYMES REALLY
BREAKDOWN GUY C GLYCO AGAIN AND GROWTH
ON GLYCOGEN.
WE HAD OUR OWN SEARCH SO WE HAD
VAGINAL BACTERIA USING THAT
SEQUENCE AS A QUERY SO WE WERE
LOOKING SPECIFICALLY FOR
PROTEINS THAT CONTAIN PREDICTED
SNAP PEPTIDES THAN AN EXTRA
CELLULAR AND WE FOUND 69
POTENTIAL AND INTERESTINGLY, WE
FOUND THESE SEQUENCES WERE
PRESENT IN MANY SPECIES BEYOND
SO IT INCLUDES GARDNER ELLA
STRAINS AND -- AND SO YOU WILL
NOTE THAT DIVERSITY IN THE
ENZYME SEQUENCES AND THE DOMAIN
ARRANGEMENTS, WHILE ALL OF THE
ENZYMES HAVE ONE PREDICTED
DOMAIN THERE'S LARGE VARIATIONS
IN THE LENGTH AND DOMAINS
PRESENT IN THESE DIFFERENT
ENZYMES AND THE PROTEINS HAVE
CARBOHYDRATE FINDINGS AND
INTERESTINGLY THE GARDNER ELLA
HAS TWO MLA DOMAINS AND SO OVER
ALL THIS INITIAL ANALYSIS
SUGGESTED THAT A VARIETY OF
VAGINAL BACTERIA MIGHT
POTENTIALLY HAVE ENZYMES CAPABLE
OF METABOLIZING GLYCOGEN.
WE WANTED TO UNDERSTAND HOW
THESE ENZYMES WORKED IN MORE
DOUGH TAIDETAIL SO WE EXPRESSED THESE
PROTEINS IN E. COLI WITHOUT
THEIR PEPTIDES AND WE BEGAN TO
STUDY THEIR ACTIVITIES.
SO, FIRST WE EXAMINED IF THEY
COULD SUPPORT BACTERIAL GROWTH
ON GLYCOGEN AND SO TO DO THIS WE
IRON YOU BATED A STRAIN THAT IS
MISSING THE POOL A GENE.
THIS IS A NATURALLY LACKS THIS
GENE.
WITH INDIVIDUAL PURIFIED ENZYMES
AND MONITORED THE EFFECTS ON
GROWTH IN GLYCOGEN CONTAINING
MEDIUM AND WE SEE THAT IN DEED,
ADD NORTH THE PURIFIED
L.CRISPATUS FROM A DIFFERENT
STRAIN ALLOWS THIS STRAIN TO
GROW WELL ON GLYCOGEN ADDING THE
PULAEN GAME DOES NOT SUPPORT
GROWTH SO WE TESTED THE OTHER
PROTEINS IN THIS AS SAY AND
FOUND THAT IT'S NOT JUST THE
L.CRISPATUS THEY CAN ALL SUPPORT
BACTERIAL GROWTH ON GLYCO AGAIN.
POTENTIALLY IMPLICATING THEM ALL
IN THIS ACTIVITY.
SO WE NEXT SOUGHT TO GAIN MORE
DETAILED INFORMATION ABOUT THE
ENZYME SPECIFICITY WITH GLYCOGEN
SO WE CHARTER THE PARAMETERS OF
THESE DIFFERENT ENZYMES OF
DIFFERENT SUBSTRATES THAT
CONTAIN DIFFERENT TYPES SO WE
LOCKED AT WE SEE SOME
INTERESTING DIFFERENCE THIS IS
SPECIFICITY SO, ALL OF THE
ENZYMES CAN DEGRADE AND SOME OF
THEM ARE UNABLE TO METABOLIZE
LINKAGES AND I ALSO WANT TO
POINT OUT THE DIFFERENCES IN
ACTIVITIES BETWEEN ENZYMES FROM
DIFFERENT ORGANISMS AND THE
GARDNER ELLA ENZYME STANDING OUT
AS BEING MUCH MORE ACTIVE THAN
ENZYMES FROM SOME OF THE OTHER
SPECIES LIKE LACK TOE BASIL IS
AND THIS IS INTERESTING TO THINK
ABOUT.
IT SUGGESTS GARNER ELLA MAY
METABOLIZE GLYCOGEN MUCH MORE
EXTENSIVELY AND GARDNER ELLA IS
KNOWN TO POSSESS OTHER TYPES OF
OFEN GYMS VERY NOTABLY THEY HAVE
SIGHAL DATES ENZYMES AND WE'LL
HEAR MORE ABOUT SOME OF THESE
GARDNER ELLA ENZYMES TODAY IN
AMANDA LOUIS' TALK.
SO WE MEASURED ALL OF THESE
ENZYME PARAMETERS AND A PH OF
5.4, WHAT ABOUT ENZYME ACTIVITY
AT LOWER PH VALUES THAT MAY
BETTER REFLECT THE PH ASSOCIATED
WITH A LACK OF BA LIS DOMINATED
COMMUNITY.
WELL HERE WE OBSERVED SOMETHING
VERY INTRIGUING SO, WHEN WE
VARIED PH AND MEASUREDDEN TIME
AND THE ENZYME GENERALLY
RETAINED MUCH MORE OF THEIR
ACTIVITIES AT LOWER PH VALUES
THAN ENZYMES FROM OTHER
ORGANISMS SO WE THINK THIS COULD
INDICATE THESE EXTRA CELLULAR
ENZYMES MIGHT BE ADAPTED IN AN
ACIDIC ENVIRONMENT AND SUGGESTS
THAT VAGINAL PH COULD HAVE AN
IMPORTANT INFLUENCE ON ENZYME
ACTIVITY AND SO NEXT WE WANTED
TO CHARACTERIZE THE SPECIFIC
BREAKDOWN PRODUCTS GENERATED BY
DIFFERENT BACTERIAL ENZYMES AND
COMPARE THAT TO THE ACTIVITY OF
THE HUMAN SAL A VARY AM A LACE.
AND SO, WE'RE CHARACTERIZING
DIFFERENT BREAKDOWN PRODUCTS BY
LCMS AND WE SEE THAT THE
PRODUCTS GENERATED BY THE
BACTERIAL ENZYMES DIFFER BETWEEN
DIFFERENT ORGANISMS AND ALSO,
DIFFER FROM THE PRODUCT
DISTRIBUTION OF THE HUMAN
ENZYMES AND HERE WE LOOK AT
PRODUCTS THAT ARE GENERATED WITH
WE INCUBATE THESE AND WE DID
THIS EXPERIMENT WITH POLULIN AND
THERE'S DISTINCT DIFFERENCE AND
WHEN USING A SUBSTRATE AND IT IS
LINKAGES WE SEE ALL BACTERIA
ENZYMES CAN METABOLIZE THIS
SUBSTRATE AND THE HUMAN ENZYME
CANNOT SO THIS IS ACTUALLY A
VERY SIGNIFICANT OBSERVATION
BECAUSE IT SUGGESTS THAT
PULLULAN'S ACTIVITY MIGHT
POTENTIALLY BE USED AS A MARKER
OF BACTERIAL ENZYMES IN COMPLEX
HUMAN MICROBIOME SAMPLES.
SO WITH THIS IN MIND, WE HAVE
STARTED TO SECRETARY QUESTION OF
WHETHER THESE BACTERIAL ENZYMES
ARE PRESENT IN CERVICAL LAVAGE
SAMPLES AND THEY HAVE A ROLE IN
HELPING US TO ACCESS THESE
SAMPLES.
WE DID JUST ATTEMPTED TO DETECT
HUMAN AM A LACE IN ASSUMER MUTE
ANTS USING ALI SA AND WE
VERIFIED WE COULD DETECT THIS
ENZYME IN OUR SAMPLE AND WE ALSO
FOUND THAT THERE'S NO CROSS
REACTIVITY WITH ANY OF OUR
PURIFIED MICROBIAL.
SO WE SEE VARYING LEVELS OF THE
HUMAN AM A LISTEN SIM.
NEXT WE WANTED TO MEASURE
GLYCOGEN DEGRADATION AND YOU
LOOKED AT THIS AT VARIOUS PHs
AND WE SEE GENERALLY THAT
THERE'S A CORRELATION BETWEEN
AMALYSE ACTIVITY AND THE LEVEL
OF HUMAN AMALYSE AND WE FOUND IN
ONE OF OUR SAMPLES HIGHLIGHTED
HERE THERE'S ENHANCED ACTIVITY
AND THIS REALLY DOESN'T SEE -- SO,
WE ARE HERE AGAIN USING LCMS FOR
THE PRODUCTION OF MULTI TRY OWES
FROM THE VAGINAL SAMPLES AND WE
OBSERVED VARIOUS LEVELS OF
PRODUCT FORMATION WHEN THESE
SAMPLES ARE INCUBATED WITH GLYCO
AGAIN BUT WE SEE A REALLY
DIFFERENT PATTERN OF METABOLISM
WHEN WE INCUBATE THESE SAL POPS
WITH PULLULAN SO THE HUMAN
ENZYME CAN'T METABOLIZE THIS
SUBSTRATE IN THE EXACT SAME
SAMPLE THAT RETAINED ACTIVITIES
AT LOW PH, WE SEE SUBSTANTIAL
PRODUCTION OF MULTO TRY OWES
FROM PULLULAN SO WE THINK AT
THIS OBSERVATION MAY DEMONSTRATE
THAT ENZYMES ARE PRESENT IN THIS
SAMPLE.
NOW THERE'S IMPORTANT CAVEATS.
IT'S A SMALL NUMBER OF SAMPLES.
WE WERE WORKING WITH SAMPLES
THAT WERE NOT SEQUENCES SO WE
DON'T KNOW WHICH ORGANISMS ARE
PRESENT THIS THESE SAMPLES SO A
GOAL WE'RE WORKING TOWARDS RIGHT
NOW IS REALLY INVESTIGATING A
LARGER NUMBER OF SAMPLES TO
REALLY TRY TO PINPOINT WHICH
ENZYMES ARE RESPONSIBLE FOR THIS
ACTIVITY.
BUT, TO START TO WRAP-UP, I HOPE
THAT THIS SHORT STORY HAS SHOWN
HOW UNDERSTANDING BACTERIAL
ENZYMES CAN BEGIN TO HELP US
GAIN A MECHANISTIC UNDERSTANDING
OF MICROBIOMES SO UNDERSTANDING
ENZYMES AND LINKING IT BACK TO
GENES, REALLY IMPROVES OUR
ABILITY TO START ANALYZING
MICROBIAL GENOME AND MIKE OWE
BIOME SEQUENCING DATA AND MAKE
ACCURATE PREDICTIONS ABOUT
SPECIFIC MICROBIAL FUNCTIONS.
BIO CHEMICALLY CHARACTERIZING
ENZYMES PROVIDE KEY INSIGHTS
INTO ENVIRONMENTAL FACTORS THAT
MIGHT INFLUENCE METABOLISM SO
THINGS LIKE PH AND FINALLY,
UNDERSTANDING ENZYMES CAN HELP
US AND EITHER THROUGH DEVELOPING
INHIBITORS OF SPECIFIC ENZYMES
OR THROUGH HELPING US SELECT AND
SO THESE TYPES OF STRATEGIES MAY
BE USEFUL IN THE SHORT TERM FOR
HELPING US STUDY SO KNOWLEDGE OF
THESE AMYLASES IN DIFFERENT
STRAINS HAVE HELPED THE SELECT
THE STRAINS FOR POTENTIAL
DEVELOPMENT OF THESE TYPES OF
PROBIOTICS INTERVENTIONS.
WHILE I FOCUSED ON ONE FAMILY OF
ENZYMES TODAY, WE'RE EXCITE
TODAY A PLOY THIS WORK FLOW TO
OTHER TYPES OF VAGINAL
BACTERIAEL ENZYMES.
SO, FROM AN EVEN BIGGER PICTURE
PERSPECTIVE, I THINK THIS WORK
HIGHLIGHTS SOME VERY IMPORTANT
PITFALLS OF MICROBIOME SEQUENCES
RELIES ON AUTOMATED ANNOTATIONS
AND IT'S IMPORTANT TO REMEMBER
THAT THESE ANNOTATIONS ARE
FREQUENTLY INCORRECT SO MORE
OVER I THINK THAT THIS STORY
PARTICULARLY HIGHLIGHTS THEIR
IMPORTANT FEATURES OF ENZYME AND
PROTEIN FUNCTION THAT CAN'T
READILY BE PREDICTED FROM
SEQUENCES DATA ALONE.
THINGS LIKE THE PH TOLERANCE OF
THE ENZYMES OR ENHANCED ACTIVITY
OF THE GARDNER ELLA ENZYME SO
THIS REALLY SPEAKS TO THE NEED
TO INTEGRATE BIO CHEMISTRY TO
MICROBIOME STUDIES.
I WILL THANK MY RESEARCH GROUP
AND OR COLLABORATORS AND I
HIGHLIGHTED THE SPECIFIC
STUDENTS WHO CONTRIBUTED TO THIS
PROJECT.
I'M GRATEFUL TO EVERYONE IN THE
LAB AND THANK THE MEMBERS OF THE
CONSORTIUM AND THE GATES
FOUNDATION FOR FUNDING AND I
WOULD BE HAPPY TO ANSWER ANY
QUESTIONS.
>> EXCELLENT TALK AND I'M GLAD
THAT WE'RE GETTING YOU INTO THE
CARBOHYDRATE PART OF GLYCAN SO
PERFECT THAT WAS AWESOME.
THANK YOU SO MUCH.
WE HAVE QUESTIONS FROM THE -- WE
HAVE ONE.
AMANDA LOUIS, IF YOU WANT TO ASK
YOU CAN ASK IF NOT I CAN ASK YOU
FOR YOU.
IT'S SUPPOSE TO YOU.
>> I CAN ASK.
GREAT TALK, EMILY.
>> THANK YOU.
>> SO I WONDER THE OLD IDEA
USING GLYCOGEN AS A PRE BIOTICS
AND IT'S A GOOD IDEA.
>> IT'S A GREAT QUESTION.
YOU HAVE TO CONSIDER THE
IMPLICATIONS OF DOING THAT IN A
COMMUNITY CONTEXT AND CERTAINLY
I THINK IF YOU HAVE A COMMUNITY
THAT IS DOMINATED BY GARDNER
ELLA WITHOUT SOME SORT OF
MANIPULATING THE ENVIRONMENT I
WOULD BE A LITTLE BIT CONCERNED
ABOUT THAT, GIVEN THAT I THINK
THAT GARDNER EARLY I ELLA IS LIKELY TO
USE THAT SUBSTRATE MORE
EFFICIENTLY THAN THE ORGANISMS
THAT YOU WOULD PERHAPS LIKE TO
ENCOURAGE TO GROW.
>> EMILY, YOU'VE ENCOURAGED LOTS
OF TALK FROM THE QUESTIONS ON
OUR ON-LINE COMMUNITY.
SO, WE HAVE A QUESTION FROM
DANIEL WHO ASKED S. THERE ANY
DATABASE TOOL WE CAN USE TO FIND
POTENTIAL BACTERIAL ENZYMES OR
GENES THAT PRODUCE CERTAIN
METABOLITES?
>> SO THERE'S A NUMBER OF TOOLS,
I THINK PARTICULARLY DEVELOPED
IN THE CONTEXT OF WANTING TO
PROBE THE HUMAN GUT MICROBIOME
SO IT DEPENDS ON WHAT YOU LOOK
FOR SO A LOT OF THE TOOLS THAT I
THINK COULD BE -- MOST FAMILIAR
WITH FOCUS IN ON THE NATURAL
PRODUCTS SPACE SO THE GROUP
THAT'S DEVELOPED SOME GREAT WEB
BASED TOOLS AND TOOLS THAT ALLOW
TO JUST IDENTIFY THINGS THAT
LOOK LIKE GENE CLUSTERS THAT
ENCODE ENZYMES IN GUT BACTERIA
AND SO IT IS SORT OF HELPED
POTENTIALLY USE GENOMIC CONTEXT
SO I'M NOT AS FAMILIAR WITH
TOOLS AVAILABLE TO HELP DISCOVER
CARBOHYDRATE ENZYMES BUT THEY'RE
OBVIOUSLY REALLY IMPORTANT AND
I'M SURE THERE'S SOME REALLY
GREAT RESOURCE THIS IS THAT
SPACE.
>> WE'RE GETTING LOTS OF
QUESTIONS ON-LINE BUT IN A TEEN NADINE HAS
A QUESTION.
>> SURE.
I JUST HAVE A MECHANIC NICE TICK
QUESTION.
FOR THE ONE THAT HAS A HIGH
ACTIVITY THAT THE DVAGINILIS AND
KNOWING WHAT YOU KNOW ABOUT THE
MECHANISM OR THE STRUCTURES
OFFER WHATEVER, IT IS OBVIOUS
WHY THAT IS SO MUCH MORE ACTIVE?
WHAT DO YOU KNOW, I GUESS,
FROM --
>> WELL, SO THE INTRIGUING THING
TO ME ABOUT THAT ENZYME IS IT'S
THE ONE THAT HAS TWO ALPHA AMO
LACE DOMAIN AND IF YOU MUTATE
JUST ONE YOU LOSE GREATER THAN
90% OF THE ACTIVITY.
IT'S DEAD IF YOU MUTATE TWO
DOMAINS BUT THE LOSS OF ACTIVITY
WHEN YOU MUTATE JUST ONE OF
THOSE DOMAINS, SPEAKS TO PERHAPS
COORDINATION BETWEEN THE TWO
DOMAINS AND SO I WONDER IF
THAT'S POSSIBLY SOMETHING THAT
IS LINKED TO THE INCREASED
DEFICIENCY OF THAT PARTICULAR
ENZYME.
>> THANK YOU.
SO EMILY THERE'S A VARIETY OF
QUESTIONS IN THE CHAT BUT DUE TO
THE INTEREST OF TIME, WE'RE
GOING TO -- I CAN E-MAIL THOSE
TO YOU AND YOU CAN READ THEM
YOURSELVES AND ALL OF THE SEE
MALES ARE-MAILS ARE ASSOCIATED WITH THE
QUESTIONS THERE SO YOU CAN
RESPOND BACK AT YOUR LEISURE.
KEEP THE QUESTIONS FROM THE
ON-LINE PART IT MAKES IT MORE
INTERACTIVE AND FUN SO THANK YOU
FOR THE PARTICIPATION AND
FANTASTIC TALK.
THANK YOU FOR THAT.
I REALLY APPRECIATE YOU TELLING
US THAT NEW WORK COMING OUT OF
YOUR LAB.
OUR NEXT SPEAKER IS DARIA
PICANTINO AND ALCOHOLISM AT THE
NIH AND SHE'S A POSTDOC TO BAL
FELLOW AND SHE'S GOING TO TALK
ABOUT HER WORK ON THE GUT
MICROBE IN ALCOHOL USE DISORDER
SO REALLY DIVERSE TALKS THIS
MORNING SO THANK YOU FOR JOINING
TODAY.
>> THANK YOU SO MUCH CATHERINE.
I'M GOING TO SHARE MY SCREEN.
SO -- GREAT.
I'M JUST GOING TO MINIMIZE HERE.
PERFECT.
SO THANK YOU CATHERINE AND THANK
YOU EVERYONE FOR HAVING ME
TODAY.
I'M REALLY EXCITED TO PRESENT
THE WORK IN THE PAST FOR YEARS
SO I'M STILL CARRYING OUT AT CPN
LAB AND IT'S PSYCHO LAB BY Dr.
LYNN AND TODAY I'M GOING TO
PRESENT OUR WORK ON THE GUT
MICROBIOME IN ALCOHOL USE
DISORDER FROM BENCH TO BED.
SO WHAT WE DO IS BASICALLY TAKE
ADVANTAGE OF THE HIGHLY
TRANSITIONAL NATURE OF THE LAB
TO PHYSICALLY DEVELOP NEW
TREATMENTS FOR ALCOHOL DISORDER
BY STUDYING THE GAP MICROBIOME
AND MA LAB LOAM.
WE START AND WE MOVE FORWARD AS
YOU CAN SEE HERE FROM NON HUMAN
PRIMATES AND WE MOVED TO HUMANS
AND THIS IS THE LAST STUDY WE
WRAPPED UP DURING THE PANDEMIC
AND WE'RE BASICALLY ANALYZING
THE DATA AS I'M SPEAKING RIGHT
NOW SO IT'S VERY EXCITING AND
I'LL SHOW SOME PRELIMINARY
RESULTS.
WHAT IS THE REASON WHY WE ARE
KIND OF FOLLOWING THIS -- WHAT
IS THE RATIONAL BEHIND THIS?
THE RATIONAL IS WE'D LIKE TO
IDENTIFY SOME COMMON MICROBIAL
ACTS OR METABOLITES BETWEEN THE
THREE SPECIES THAT HAVE A
POTENTIAL OF FUNCTION OF ALCOHOL
AND DRINKING AND THIS IDEAL
SCENARIO OBVIOUSLY COULD
REPRESENT LIKE THE STARTING
POINT TO DEVELOP A NOVEL
TREATMENT FOR ALCOHOL ABUSE
DISORDER SO THIS IS BASICALLY
WHAT WE'RE FOCUSING ON NOW.
SO, LET ME GIVE YOU A LITTLE BIT
OF BACKGROUND.
SO, WE WERE BASICALLY DOING IS
STUDYING THE EFFECTS OF
EXCESSIVE ALCOHOL DRINKING ON
THE GUT AND MICRO OWN AND IT
INCLUDES BASICALLY A COMBINATION
OF BINGE DRINKING AND HEAVY
DRINKING.
BINGE DRINKING CAN BE THOUGHT
ABOUT DRINKING TOO MUCH TOO FAST
AND THE DEFINITION IS BASICALLY
FIVE OR MORE DRINKS FOR MEN THAT
ARE CONSUMED ON ONE OCCASION AND
BY ONE OCCASION WE MEAN TWO
HOURS AND IN WOMEN IT'S FOUR OR
MORE DRINKS CONSUMED ON ONE
OCCASION SO WITHIN THESE TWO
HOURS AND ANOTHER DRINKING IS
THAT DRINKING THIS FAST AND IN
THIS MUCH -- AS I SAID THERE'S
ANOTHER COMPONENT WHICH IS HEAVY
DRINKING SO DRINKING TOO MUCH
TOO OFTEN, AGAIN, I'M GOING TO
ROW FER TO THE NIAAA DEFINITION
AND HEAVY DRINKING WHICH IS 15
OR MORE DRINKS ON AN AVERAGE PER
WEEK FOR MEN AND EIGHT OR MORE
DRINKS ON AVERAGE PER WEEK FOR
WOMEN.
SO, THIS IS A BIT OF NUMBERS.
SO, TO SHOW YOU HOW EXCESSIVE
ALCOHOL USE AFFECTS THE U.S.
POPULATION.
SO BINGE DRINKING IN THE U.S.
EFFECTS ALMOST 26% OF PEOPLE
OVER 18 YEARS OF AGE.
AS FOR THE ALCOHOL USE DISORDER
SO THE PROPER DIAGNOSIS SO BINGE
DRINK ASKING DRINKING IS A HABIT THAT HAS
CRITERIA AND 14.5 MILLION OF
INDIVIDUALS IN THE U.S. ARE
AFFECTED BY THIS DISORDER SO
IT'S A PUBLIC-HEALTH PROBLEM.
AS FOR ALCOHOL RELATED DEATHS,
THE NUMBER IS VERY HIGH SO
95,000 PEOPLE DIE FROM
ALCOHOL-RELATED CAUSES ANNUALLY.
ALCOHOL RELATED CAUSES ENCLOUD
ACUTE CAUSES AND THIS MAY BE
TRAGIC ACCIDENTS, MAYBE
SELF-INFLECTED INJURIES,
VIOLENCE, BUT WE ALSO HAVE A LOT
OF CHRONIC CAUSES AND ABOUT THE
THE MOST WELL-KNOWN IS OBVIOUSLY
LIVER SIG CIROSIS AND DYSFUNCTION OF
THE CARDIOVASCULAR SYSTEMS AND
LOTS OF CANCERS AND SO ON.
IT'S WORTH MENTIONING BECAUSE
THE NUMBER DOESN'T GIVE THE IDEA
BUT 95,000 MEANS 1/10 U.S.
CITIES AND TOYS FROM AN ALCOHOL
RELATED CAUSE AND THIS NUMBER IS
NOT HIGHER THAN OPIOID-RELATED
DEATHS SO OPIOID-RELATED DEATHS
IS HALF.
DESPITE WE'RE LIVING AN OPIOID
EPIDEMIC SO THIS IS JUST TO GIVE
AN IDEA THAT ALCOHOL IS
PREVALENT.
SO, IN TERMS OF ECONOMIC WE CAN
SEE HERE THAT EACH YEAR ALCOHOL
CAUSES 249 BILLION COST TO THE
HEALING CARHEALTH-CARE SYSTEM AND THE MAIN
COST DERIVES FROM WORK-PLACE
PRODUCTIVITY A DISRUPTION OF
WORK AND THEN OTHER COMPONENTS
ARE JUSTICE HEALTHCARE AND
BASICALLY TRAFFIC RELATED
INCIDENTS.
SO, BACK TO OUR RESEARCH.
AS I SAID BEFORE, EXCESSIVE
ALCOHOL USE SO A COMBINATION,
BINGE AND HEAVY ALCOHOL DRINKING
SO DRINKING TOO MUCH AND TOO
OFTEN IS KNOWN TO PRODUCE
CHANGES IN THE FECAL MICRO BIOME
IN RODENTS AND HUMANS.
IT DOESN'T GO IN THE SAME LINE
SO WE CAN SEE SOME STUDIES
HIGHLIGHTING AN INCREASE OR
DECREASE OF CERTAIN MICROBIAL
SPECIES AND OTHERS SHOW
DIFFERENT RESULTS, IT'S HARD TO
FIND A WAY TO PUT THE PIECES OF
THE PUZZLE TOGETHER AND THAT'S
WHY WE TRIED TO DO SOME STUDIES
OURSELF AND SO WE STARTED FROM
ROARODENTS.
THIS WAS BASICALLY OUR STARTING
POINT AND WE BASICALLY GENERATED
SOME PROMISING ALTHOUGH VERY
PRELIMINARY DATA ON ALCOHOL
BINGE-LIKE EXPOSURE IN MALE RATS
IN COLLABORATION AND NIAAA AS AN
EXPLORATORY AIM PART OF A LARGER
STUD TEE ON WILD TYPE FOR THE
BRAIN RECEPTORS WE INVESTIGATED
GUT MICROBIAL DIVERSITY WHICH
MEASURE THE DIVERSITY WITHIN AND
IN BETWEEN.
WHAT DID WE FIND?
AS WE CAN SEE HERE, LET'S FOCUS
ON THE FIGURE ON THE LEFT.
SO WE FOUND THAT THE GUT HIGH
CROW ALPHA DIVERSITY INDEX WHICH
IS AS I SAID ON THE LEFT, SHOWED
A SIGNIFICANT DECREASE IN BOTH
THE SELF-ADMINISTRATION, THE
DIVERSITY IN THE
SELF-ADMINISTRATION BY THE
RODENTS OF A SWEET SOLUTION AND
SWEET END ALCOHOL SOLUTION
COMPARED TO THE BASELINE AND
THEN WHEN WE MOVED TO THE RIGHT
PART OF THE FIGURE SO WE MOVE TO
THE GUT MICROBIAL DIVERSITY AND
MET BY AN ANALYSIS OF DISTANCES
WHAT WE SEE HERE IS THAT THERE'S
A CLEAR SEPARATION BETWEEN THE
ALCOHOL SWEET SOLUTION
SELF-ADMINISTRATION COMPARED TO
THE NON ALCOHOLIC SWEET SOLUTION
AND THE BASELINE.
WHICH ACCOUNTS FOR 22.4% OF
DEFISDEFICIENCIES.
THESE FINDINGS ARE TO HIGHLIGHT
THE IMPORTANCE AND THESE
FINDINGS POINT TO THE FACT THAT
RODENTS WHO DRINK ALCOHOL HAVE A
DES TRA MENTAL DECREASE IN BOTH
ALPHA AND BETA DIVERSITY SO WE
KNOW THAT LIKE FOR NORMAL GUT,
THERE HAS TO BE A HIGH DIVERSITY
OF BACTERIAL SPECIES SO WHAT
WE'RE SEEING HERE IS OBVIOUS LOW
A NEGATIVE CONDITION, IN WHICH
ALCOHOL REDUCES THE DIVERSITY.
SO, THEN WHAT WE DID IS WE, AS I
SAID BEFORE, WE MOVED FORWARD
WITH NON HUMAN PRIMATES AND WE
THOUGHT IT WOULD BE IMPORTANT
BECAUSE THERE ARE VERY FEW
STUDIES, THERE ARE ONLY TWO
STUDIES WHICH WERE CONDUCTED IN
MAY AND WE DECIDED TO PROCEED
WITH PRIME IT'S A FOR A SERIOUS
REASON THAT AND WE TRANSLATED
THE MODEL IN CHRONIC ALCOHOL IN
COLLABORATION WITH STUDY MADE
POSSIBLE AND WE INVESTIGATED FOR
THE FIRST TIME THE CHRONIC
EXCESSIVE ALCOHOL DRINKING WITH
CHANGES IN THE GUT MICRO WE USED
ALL OF THEM AND SO, WHAT DID WE
HAVE, SO WE HAD STUDIED THREE
GROUPS OF BABOONS SO WE HAVE
LSTL IS WHAT WE CALL THE
LONG-TERM DRINKING GROUPS SO
THIS GROUP BASICALLY CHRONICALLY
DRANK ALCOHOL EVERYDAY OF THE
WEEK SO SEVEN DAYS OUT OF SEVEN
FOR OVER 10 YEARS AND THEN WE
HAD THE SHORT TERM ALCOHOL
DRINKING GROUP THAT CHRONICALLY
DRANK ALCOHOL EVERY DAY FOR TWO
OR THROW YEARS.
I WOULD CALL IT RELATIVE SHORT
TERM BECAUSE IT'S NOT SO SHORT
TERM.
AND THEN WE HAD OUR CONTROL
GROUP THAT DRANK AND I'M GOING
TO SHOW YOU HERE AND THAT IS
BASICALLY AN ORANGE FLAVORED
DRINK AND CALORIES AS ALCOHOL
BUT NOT ALCOHOLIC AND THEY DRANK
FOR EIGHT YEARS AND WE DECIDED
TO STUDY THIS THREE GROUPS IN
TWO CONDITIONS SO WE BASICALLY
STUDIED THESE THREE GROUPS IN
THREE CONSECUTIVE DAYS OF ACTIVE
DRINKING AND THEN, THESE DAYS
WERE FOLLOWED BY THREE
CONSECUTIVE OF SHORT TERM
ACCIDENTS AND MOVING FORWARD,
SO, I'M GOING TO EXPLAIN A
LITTLE BIT WHY WE CHOSE BABOONS,
BACK BOONES IN CAPTIVITY HAVE A
CHANGE IN THEIR MICROBIOME
BECAUSE IT COMES SIMILAR TO THAT
IN HUMANS SO IT HAS THIS EFFECT
AND THE SECOND REASON IS THAT
THE GUT MICROBIOME OF BABOONS IS
THE CLOSEST TO THE HUMAN SPECIES
COMPARED TO APES AND MONKEYS SO
IT OFFERS A CLEAR ADVANTAGE AND
ANOTHER REASON IS THAT THE BACK
BOONES METABOLIZE COMPARED TO
STUDIES WHO HAVE AND THEY ALLOW
LONG-TERM EXPOSURE TO ALCOHOL IN
WELL CONTROLLED EXPERIMENTAL
CONDITIONS THAT OBVIOUSLY CANNOT
BE IN HUMAN STUDIES THIS THIS IS
ANOTHER ADVANTAGE AND IN TERMS
OF THE BALLOON MODEL WE USED,
IT'S BASED ON A VALIDATED
SCHEDULE OF REINFORCEMENT
PROCEDURE AND IT'S CONSISTENT
WITH NOT A AAA DEFINITION OF
HEAVY AND BINGE DRINKING IN
HUMANS SO IT'S HEAVY AND BINGE
DRANKING ACID DESIGNED THEM FOR.
DRINKING TOO MUCH TOO FAST AND
TOO OFTEN.
SECOND, THERE'S SPECIFICITY WAS
TESTED IN ALCOHOL VERSUS AND IF
I WAITED MEDICATION DEVELOP FOR
ALCOHOL DISORDER AND STUDIES ON
THROW DRUGS.
AND IMPORTANCE HERE IS IS ONE OF
THE THROW FDA APPROVED
MEDICATION FOR ALCOHOL USE
DISORDER AND THE OTHER
MEDICATIONS ALTHOUGH THEY'RE NOT
APPROVED BY THE FDA HAVE SHOWN
INCREDIBLE EFFICACY IN THEM
EFFECTED BY ALCOHOL USE DISORDER
SO WE CAN SEE THE CLINICAL
IMPORTANCE OF THIS POINT AND SO
IN TERMS OF THE ONE PART IS
INTERESTING TO NOTE THE BA BONES
WERE MALE AND THEY DID NOT
DIFFER IN TERMS OF SIGNIFICANT
IN TERMS OF AGE OR WEIGHT AND
ALSO WHAT I WANTED TO HIGHLIGHT
IS THAT THE LIVER ENZYME, THE
LIVER FUNCTION TEST WAS NORMAL.
MEANING THEY DID NOT SHOW ANY
ALTERATION THAT CAN MIMIC
ALCOHOL AND LIVER CONDITIONS IN
HUMANS SO THEY WERE HEALTHY BACK
BONES AND WHEN THEY DRANK IN
THEIR TWO HOUR PERIOD IN THE
TRAINED SCHEDULE O SCHEDULE OF PROCEDURE
THEY RECEIVED OVER .08 TO MAKE
SURE THEY WERE ACTUALLY BINGE
DRINKING.
SO THIS IS WHAT WE SEE, THIS IS
WHAT WE SEE IN TERMS OF GUT
MICRO BIOME.
WHAT WE SEE IN THE LEFT SIDE OF
THE FIGURE IS THE ALPHA
DIVERSITY SO GUT MICROBIAL IS
MEASURED BY THE SHANNON
DIVERSITY INDEX AND
SIGNIFICANTLY DECREASED AFTER
LONG-TERM ALCOHOL DRINKING WHEN
COMPARED TO, AS WE CAN SEE HERE,
TO THE ALCOHOL SHORT-TERM
DRINKING WHICH IS IN THE MIDDLE
IN TANG ON THE LEFT.
AND BASICALLY, WHAT WE CAN SEE
HERE IS THAT THERE ARE NO
SIGNIFICANT DIFFERENCES IN TANG
AND ALCOHOL SHORT TERM.
CONTRARILY TO OUR EXPECTATIONS,
WE THOUGHT THAT TWO OR THREE
YEARS OF DRINKING OF ALCOHOL
DRINKING WOULD BE STILL KIND OF
CONTRIBUTED TO BASICALLY
INDUCING A DECREASE IN DIVERSITY
AND WE DID NOT FIND THAT.
WHAT WE CAN SEE IN TERMS OF THE
TWO CONDITIONS WE STUDIED, THE
THREE GROUPS OF BABOONS AND
THREE DAYS OF SHORT TERM
ABSTINENCE WERE NOT SUFFICIENT
TO RESTORE THE NORMAL MICROBIOME
AND IT WOULD BE INTERESTING TO
PROLONG THE ABSTINENCE AND SEE
IF THE MICRO BUY OWN IN THE
LONG-TERM GROUP RETURNS TO
NORMAL AND WHAT WE SEE HERE ON
THE RIGHT, IS THE GUT DIVERSITY
AND WHAT WE CAN SEE AS MEASURED
BY THE BRAY-CURTIS SIMILARITY
THAT SUGGESTION THAT BASICALLY
THE ALCOHOL LONG-TERM GROUP IN
RED WHICH IS IN RED, AND THE
TANG GROUP IN BLUE, ARE
DIFFERENT.
ACCOUNTING FOR 8.3% OF THE
DIFFERENCES.
AND AGAIN THE TWO CONDITIONS SO
DRINKING AND ABSENCE DID NOT
EXHIBIT DIFFERENT COMMUNITY
STRUCTURES AND POSSIBLY AGAIN
BECAUSE THESE THREE DAYS OF
ABSENTEEISM DID NOT MAKE ANY
IMPACT ON THE STRUCTURE OF OUR
MICRO IMPACTS.
WE CAN SEE THE RESULTS OF OUR
METMET SO WE USED MODELS TO
INVESTIGATE WHICH METABOLISM
WERE IN THE GROUP FOR A
CONDITION INTERACTION AND AS WE
CAN SEE AS SHOWN IN THIS HEAT
MAP, WE FOUND 66 METABOLITES TO
BE SIGNIFICANTLY DIFFERENT IN
THE LONG-TERM ALCOHOL DRINKING
ON THE LEFT IN RED, COMPARED TO
THE SHORT-TERM ALCOHOL DRINKING
GROUP WHICH IS IN GREN IN THE
MIDDLE AND THE TANG GROUP WHICH
IS THE LAST ONE.
AGAIN, THERE WERE NO SIGNIFICANT
DIFFERENCES BETWEEN THE TWO
CONDITIONS SO THAT FOCUSES ARE
NOT SHOWING THEM HERE AND THE
TWO CONDITIONS DRINKING AND
SHORT TERM ACCIDENTS.
WHICH WERE THE METABOLITES THAT
WHY SIGNIFICANTLY DIFFERENT IN
THE LONG-TERM ALCOHOL DRINKING
GROUP COMPARED TO THE OTHER TWO?
WE SAW THAT THERE WAS AN
INCREASED IN METABOLITES RELATED
TO GMDYSBIOSIS AND BRANCH CHAIN
AMINO ACIDS, FATTY ACIDS,
CENTRAL ENERGY METABOLITES AND
PENTOSE MET TAB LIGHTS AND WE
SAW AMINO AS SITS AND AGAIN,
THESE PROBABLY MEANS THAT THE
ALCOHOL, THE LONG-TERM ALCOHOL
DRINKING GROUP HAD A GREATER
DAMAGE OF THE CAUSE COMPARED TO
THE SHORT-TERM ALCOHOL DRINKING
GROUP.
SO LAST BUT NOT LEAST THIS IS
OUR STUDY IN HUMANS AND
BASICALLY WE USED THE FINDINGS
FROM THE BABOON STUDY IN
INDIVIDUALS WITH ALCOHOL USE
DISORDER AND WHAT DID WE HAVE
HERE?
WE HAD THREE GROUPS OF
PARTICIPANTS.
WE HAD ABSTINENT INDIVIDUALS
WITH ALCOHOL USE DISORDER.
THESE INDIVIDUALS USED TO BE
HEAVY DRINKERS BUT THEY HAD
STOPPED DRINKING FOR AT LEAST
SIX WEEKS AND OF THESE SIX
WEEKS, FOUR WEEKS WERE SPENT IN
THE NIHAAA INPATIENT UNIT AND
THEY WERE DISCHARGED AND AT
LEAST TWO WEEKS WERE SPENT IN
THEIR NORMAL DAILY LIVES.
AND THE REASON WE WANTED THAT IS
BECAUSE WE WANTED THEM TO RETURN
TO THEIR NORMAL DIET, I DON'T
KNOW, PHYSICAL EXERCISE AND ANY
OTHER CONDITION.
THEN WE HAD THE CURRENTLY HEAVY
DRINKING INDIVIDUALS WITH AUD SO
THIS INDIVIDUALS WERE NOT
SEEKING TREATMENT.
THEY DRANK HEAVY AND THEY JUST
CAME TO OUR OUT PATIENT UNIT,
JUST FOR THE PURPOSE OF THE
STUDY.
AND THEN WE HAD WHAT WE CALL THE
HEALTHY CONTROLS BUT IT'S MORE
PRO PRE TO CALL THEM NON-AUD
CONTROLS IN THAT THEY DID NOT
HAVE A DIAGNOSIS OF CURRENT OR
FAST AUD AND I ALSO HAVE TO
MENTION IN REALITY, THAT ALL
THESE THREE GROUPS OF
PARTICIPANTS WERE HEALTHY AND
THAT WE EXCLUDED ANY ALCOHOL
ASSOCIATED DISEASE, WE EXCLUDED
MAJOR HEALTH PROBLEMS, MAJOR
PSYCHIATRIC PROBLEMS.
SO I WOULD SAY THEY WERE ALL
PRETTY HEALTHY.
NO DIABETES AND SO ON.
SO, THESE ARE THE -- AND
OBVIOUSLY I FORGOT TO TELL YOU
OBVIOUSLY NO INTAKE OF
PROBIOTICS, ANTIBIOTICS OR ANY
MEDICATIONS THAT CAN INFLUENCE
THE MICRO.
WE CAN SEE ALREADY THAT THERE
WERE NO SIGNIFICANT DIFFERENCES
IN TERMS OF GENDER, RACE,
ETHNICITY, AGE, AND THE THREE
GROUPS WERE COMPARABLE IN TERMS
OF SOCIAL DEMOGRAPHICS AND WHICH
WAS KIND OF IMPORTANT FOR US,
ALTHOUGH WE DIDN'T REALLY LOOK
FOR THAT.
WE WERE KIND OF LUCKY.
THIS IS WHAT WE FOUND.
SO I'M SHOWING THIS FOR THE
FIRST TIME BECAUSE AS I SAID, WE
REANALYZED IN MICROBIOME AND --
THIS WAS NOT WHAT WE EXPECTED.
AS WE CAN SEE HERE, WE HAVE THE
CURRENT DRINKER, THE PEOPLE THAT
WE HAD EXPECTED TO ACTUALLY HAVE
DETRIMENTAL DECREASE OF THE
MICROBIOME OR DIVERSITY AS SHOWN
IN THE LEFT, WELL, THEY DID NOT
HAVE THAT.
BASICALLY THEY DID NOT FOLLOW
THE RESULTS OF THE RODENTS AND
BABONE STUDIES WHERE ALCOHOL IS
IN GUT MICROBIOME DIVERSITY.
WE SEE THE OPPOSITE.
BASICALLY OUR CURRENT DRINKERS
HAVE AN ALPHA DIVERSITY
SIGNIFICANTLY HIGHER THAN THE
ABSTINENCE GROUP AND THE HEALTHY
CONTROLS.
AS I SAY, ALSO, BRAINSTORMING
WITH OUR COLLEAGUES, OUR
COLLEAGUES MICROBIOLOGIST IS
THAT A HIGHER ALPHA DIVERSITY
DOES NOT NECESSARILY MEAN A
BETTER GUT MIKE RE BIOME, A
HEALTHIER GUT MICROBIOME BECAUSE
IT COULD BE THE RESULT OF
INSULTS THAT CAUSE A DIS BIO SIS
BUT IT'S JUST THE DIRECTION OF
THE CHANGE THAT IS DIFFERENT.
IN FACT, WHAT WE CAN SEE THAT IS
THE ALPHA DIVERSITY OF HEALTHY
CONTROLS IN ABSTINENCE IS
NORMAL.
IT IS ABNORMAL IN CURRENT
DRINKERS BUT WE HAVE TO SEE WHY
THE DIRECTION IS DIFFERENT,
COMPARED TO RODENT AND BABOONS.
IN TERMS OF BETTER DIVERSITY,
WHAT WE CAN SEE HERE IN TERMS OF
IS THE SIMILARITY IS THAT
THERE'S A KIND OF OVERLAP
NUCLEAR DISTINCTION BETWEEN THE
THREE GROUPS ALTHOUGH THE
HEALTHY CONTROLS, YEAH -- I
WOULD SAY THERE'S AN OVERLAP
WITH THE TWO AXIS EXPLAINING
ONLY A COMBINED 18% AND 18.1% OF
THE BETA TA DIVERSITY.
WE HAVE NOT ANALYZED THE MA TAB
A LOAM YET BUT I WOULD LIKE TO
SAY THAT IN ORDER TO PROPERLY
UNDERSTAND WHAT HAPPENS TO THE
MICROBIOME IN HUMANS, JUST
BECAUSE OF WHAT I MENTIONED
EARLIER, THERE ARE SO MANY COM
THINGS PRESENT WHEN STUDYING
HUMANS WE'RE ACTUALLY ANALYZING
ALL THE DATA WE HAVE AS REGARDS
TO NUTRITION AS WE CAN SEE HERE
ON THE RIGHT.
BECAUSE OBVIOUSLY WE WANT TO SEE
IF THERE ARE A SIGNIFICANT
DIFFERENCES IN NUTRITION BETWEEN
OUR THREE GROUPS.
WE'RE STUDYING LEVELS OF
PHYSICAL EXERCISE, ALTHOUGH AS I
SAID BEFORE, WE EXCLUDED MAJOR
PSYCHO PATHOLOGICAL DISORDERS
AND WE'RE STILL LOOKING AT OUR
QUESTIONNAIRES THAT MAY
HIGHLIGHT DIFFERENCE AND
DEPRESSION OR ANY OTHER SIGH
LOGICAL FEATURES, AND THEN WE
OBVIOUSLY WANTED, AS YOU CAN SEE
HERE, UP HERE, THIS LADY AND
THIS SORT OF DOME, THIS IS THE
MEASURE OF THE META, THE
METABOLISM AND WHETHER THEY ARE
NOT BEHIND ANY PHYSICAL ACTIVITY
AND HERE ON THE RIGHT, WE HAVE
THIS AND WE DID IT FOR THE POEM
TO BASICALLY HAVE AN IDEA OF HOW
THE AMOUNT OF FAT IN THE BODY,
WE KNOW THAT INTRA DOMINAL FAT
AND IT'S ANOTHER IMPORTANT
FACTOR THAT WE KNOW TO BE
ASSOCIATED WITH THIS GUT
DISBIOSIS AND THIS IS SOMETHING
THAT WE'RE ALSO INVESTIGATING
THROUGH OUR PROBE METHOD AND
THIS IS BASICALLY WHAT WE'RE
TRYING DO NOW.
WITH ALL THESE ELEMENTS AND KIND
OF SEE IF IT MAKES SENSE TO HAVE
THEM AS CO VARIANTS WHILE WE
STUDY THE MICROBIOME AND WE'RE
GOING TO TRY TO CORRELATE ALL
THESE MEASURES WITH A BUY PO
BEHAVIORAL MEASURES WITH CRAVING
MEASURE IN OUR BAR SETTING AT
THE NIH.
I WANT TO THANK YOU AND FIRST OF
ALL I WANT TO ACKNOWLEDGE ALL MY
COLLEAGUES.
THIS STUDY WAS FUNDED BY JOINT
FUNDING BY THE NIDA, NIAAA AND I
COULDN'T HAVE THEM SO MANY DATA
AND I COULDN'T HAVE DONE WITH
THEM AND OBVIOUSLY WITH MY
MENTOR AND OUR COLLABORATORS AND
ANYWAY, THANK YOU SO MUCH FOR
YOUR TIME AND
>> THANK YOU FOR A GREAT TALK
THINKING ABOUT ANOTHER DYNAMIC
OF THE MICROBIOME, THAT IS
ALCOHOL.
WE'LL LOOK FOR QUESTIONS FROM
THE ONLINE QUESTIONS AND
AUDIENCE IN THE ZOOM ROOM TOO.
AND I GUESS I HAVE A QUESTION IF
NO ONE ELSE HAS ONE.
DREW, PERFECT, I WILL LET YOU
HAVE A VOICE.
>> I DID HAVE ONE QUESTION.
CURIOUS, DID YOU EVER DO
SAMPLING AT DIFFERENT POINTS IN
THE G.I. TRACT?
IT GETS ABSORBED DIFFERENTLY
PROXIMALLY OR DISTALLY.
>> UNFORTUNATELY NOT.
WE DO HAVE MEASURES OF
INTESTINAL PROBABILITY, WHEN YOU
ON A US, AS I SAID, A 4-PROBE
TEST, WHERE WE'RE TRYING TO SEE
IF PERMEABILITY CHANGES GOING
FROM THE STOMACH TO THE COLON.
BUT OBVIOUSLY WE DON'T HAVE
UNFORTUNATELY THE GUT
MICROBIOME.
THAT WOULD HAVE BEEN AMAZING.
>> WE WANT AN ACTIVE ONLINE
GROUP.
SO A QUESTION, WILL YOUR GROUP
DIFFERENTIATE COHORTS OF HIGHLY
SENSITIVE PEOPLE OR SPECTRUM,
PEOPLE WITH AUTOIMMUNE DISEASES?
SHE SAYS GREAT WORK.
>> THANK YOU SO MUCH FOR THE
QUESTION.
THIS IS VERY INTERESTING.
IF BY AUTOIMMUNE DISEASES, WE
MEAN DISEASES THAT ARE
DIAGNOSED, WE EXCLUDED AS I SAID
BEFORE ANY MAJOR DISEASE BECAUSE
THAT WOULD HAVE COMPLICATED.
WE'RE ALREADY TRYING TO
EXTRAPOLATE, TRYING TO BASICALLY
ALREADY SIGNIFY THE COVARIATE,
WHAT HAPPENS IN THE HUMAN WORLD.
SO I WOULD SAY NO.
WE EXCLUDED THOSE DISEASES
UNFORTUNATELY.
BUT OTHERWISE IT'S REALLY HARD
TO SAY WHAT IS DUE TO THAT AND
WHAT IS DUE TO ALCOHOL.
ALCOHOL DOESN'T CAUSE THE SAME
EFFECT IN HUMANS COMPARED TO
RODENTS AND BABOONS, IT'S
ALREADY VERY DIFFERENT.
>> THANK YOU.
AND THERE'S LOTS OF QUESTIONS IN
THE CHAT.
DUE TO THE INTEREST OF TIME I
WILL E-MAIL AND YOU'LL HAVE A
CHANCE TO E-MAIL THE QUESTIONER
BACK.
IT'S IMPORTANT TO GO A LITTLE
OVER TIME TO KEEP THE DISCUSSION
PERIOD GOING, THE COOL PART
ABOUT DOING THIS.
WE RECOGNIZE WE'RE OVER TIME AND
WE'RE ETCHING INTO THE LUNCH
HOUR, THAT WILL BE OKAY BECAUSE
WE ARE ONLINE.
YOU CAN GO AS YOU PLEASE.
ALSO, THESE ARE BEING RECORDED
AND ARCHIVED AT THE NIH LIBRARY,
YOU CAN VIEW THEM AFTER TODAY.
KEEP THAT IN MIND AS YOU'RE
LOOKING AT THE CLOCK.
SWITCHING GEARS, SEAN WILL
INTRODUCE THE NEXT SPEAKER.
>> NEXT MARIO FELDMAN FROM WASH
U.
TAKE IT AWAY.
>> THANK YOU VERY MUCH.
CAN YOU SEE MY SLIDES?
>> YES.
>> PERFECT.
THANK YOU.
IT'S A PLEASURE TO BE HERE,
LEARNING ABOUT THINGS I NEVER
THOUGHT I WOULD BE HEARING.
VERY COOL.
BUT TODAY I'M GOING TO TALK
ABOUT OUR WORK ON BACTERIAL
VESICLES AND DEGRADATION OF
POLYSACCHARIDES IN THE GUT.
IT'S KNOWN FOR MANY YEARS
BACTERIA AND VESICLES, OUTER
MEMBRANE CONFORMS VESICLES,
TRAPPING PROTEINS.
SO, THOSE VESICLES HAVE BEEN
PROPOSED TO HAVE A LOT OF ROLES
IN BACTERIAL PHYSIOLOGY.
THEY ARE PROPOSED TO PLAY A ROLE
IN TRANSFER, IMMUNOMODULATION,
DELIVERY OF FACTORS, DELIVERY OF
ANTI-MICROBIALS.
AS I TOLD YOU BEFORE, THIS HAS
BEEN DESCRIBED IN THE '60s,
1960s, YOU CAN SEE IN THIS
IMAGE, THEY WERE HAVING A LOT OF
INTEREST, FINDING A GOOD
MECHANISM TO UNDERSTAND HOW
THESE THINGS ARE MADE.
THAT LED TO THE CONTROVERSY IN
THE FIELD, REAL THINGS,
MICROSCOPY, TODAY 50% OF
MICROBIOLOGISTS BELIEVE THESE
ARE REAL THINGS, THE OTHER HALF
THINGS IT'S ARTIFACTS.
SO WHAT DO WE KNOW ABOUT THE
MECHANISMS OF HOW THE BACTERIAL
VESICLES ARE FORMED, HOW MUCH IS
KNOWN.
IN THE '80s, IT WAS PROPOSED
THAT THERE WAS SOME PEPTO
GLYCANS, OUTER MEMBRANE, A BASIC
MECHANISM.
IF YOU WONDER HOW WE LEARN IN 40
YEARS OF RESEARCH, UNFORTUNATELY
THE ONLY THING WE'VE LEARNED IS
TO MAKE BETTER DRAWINGS BECAUSE
IT'S THE SAME.
THIS IS FROM THE LAST REVIEW.
AS YOU CAN SEE HERE THERE'S NOT
MUCH MORE INFORMATION.
SOMEHOW OUTER MEMBRANE IS
DETACHED.
THAT'S WHERE WE ARE NOW.
SOME MECHANISMS HAVE BEEN
PROPOSED IN DIFFERENT BACTERIA,
BUT NOT UNIVERSAL.
PEOPLE THINK MAYBE THOSE
MECHANISMS ARE NOT REAL BECAUSE
ALL INVOLVE STABILIZATION.
SO DESPITE WE DON'T KNOW MUCH OF
HOW THEY ARE MADE, A LOT OF
PAPERS TALKING ABOUT ROLES OF
ALL THIS IN THIS CASE I'M GOING
TO TALK ABOUT BACTEROIDES.
IN THE MICROBIOME THESE ARE
FOUND TO HAVE DIFFERENT ROLES
FROM PROTECTION TO
POLYSACCHARIDES.
ALL THOSE PAPERS HAVE USED
VESICLES, NOBODY KNOWS HOW MANY
ARE PRODUCED, ALSO BACTERIA HAS
BEEN GROWN AT 37 DEGREES,
WHATEVER TEMPERATURE THEY ARE
USING, WE DON'T KNOW REALLY HOW
MUCH THAT REALLY REFLECTS THE
COMPOSITION.
THAT'S WHAT WE WANT TO TRY TO
SHOW YOU TODAY.
SO, BACTEROIDES ARE MEMBERS OF
THE MICROBIOME AND PRODUCE THIS,
IT'S REALLY A GOOD MODEL TO
INVESTIGATE HOW THESE THINGS ARE
MADE.
THE FIRST THING WE HAVE DONE
SEVERAL YEARS AGO WAS TO
CHARACTERIZE PROTEINS PRESENT
THERE AND COMPARE TO PROTEINS IN
THE OUTER MEMBRANE.
THE POSITION OF OUTER MEMBRANE
IS COMPLETELY DIFFERENT.
THERE'S A PROCESSER OF
SELECTION.
SOME PROTEINS CHOSEN, FOR
EXAMPLE, YOU CAN SEE SOME
PROTEINS NOT GOING TO THE OMV.
FOR EXAMPLE, THIS PROTEIN.
THIS IS TELLING US A CLEAR
PROCESS HERE.
THE SECOND THING WE REALLY FOUND
THAT WE WERE NOT EXPECTING WAS
TO FIND A LOT OF HYDRO LYTIC
ENZYMES.
THERE ARE DIFFERENT ENZYMES
INVOLVED IN DEGRADATION, TRUE
FOR BACTEROIDES.
THESE ARE SOMETHING THE BACTERIA
IS TRYING TO DO WITH PURPOSE.
WE VALIDATED THE PROTEOMIC DATA
BY WESTERN BLOT.
WE SEE MOST OF THE PROTEINS
LOCALIZE IN THE OUTER MEMBRANE.
IF YOU TAKE ANOTHER PROTEIN IN
THE ANALYSIS, ENRICHED IN OUTER
MEMBRANE, DOESN'T GO TO ALL
THIS. HERE WE HAVE ANOTHER
BATCH OF MARKERS, NIECE ARE
OMVs, ALWAYS SOMETHING IN THE
MEMBRANE BECAUSE PROTEIN HAS TO
BE IN THE MEMBRANE.
AND THEN WE HAVE PROTEINS HERE
THAT ARE IN THE OUTER MEMBRANE,
EXCLUDED FROM THE VESICLES.
AND HERE EXAMPLE OF WHAT ARE
THOSE FOUND, ALL OF THOSE ARE
LIPOPROTEINS, SUPPOSED TO BE
LOCALIZED INTO MEMBRANES.
MANY OF THESE HYDROLASES, THESE
ARE PROTEINS ENCODED USUALLY IN
ONE, AND THEY HAVE SEVERAL
HYDROLASES, A CHANNEL ALLOWS THE
POLYSACCHARIDES TO BE IMPORTED.
SO MANY PROTEINS THAT WE FIND IN
ALL THESE ARE ACTUALLY PART OF
THIS.
NEE WE A LOT OF LIPOPROTEINS.
THIS IS VERY IMPORTANT TO
CONSIDER THAT THE PULs
CONSTITUTE 20% OF GENOME OF
THESE BACTEROIDES.
HOW BACTERIA MANAGE TO DEGRADE
AS POLYSACCHARIDES.
AND ONE VERY INTERESTING WORK
PUBLISHED SHOWED THAT ONE
BACTERIUM CAN SUPPORT THE GROWTH
OF ANOTHER.
OUTER MEMBRANE VESICLES, THE
OTHER BACTERIUM HAS LACTOSE, NOW
CAN GROW.
THIS IS NOT JUST ONE EXAMPLE SO
EACH OF THESE MEMBERS OF THE
MICROBIOME CAN PRODUCE ALL THESE
THAT CAN SUPPORT THE GROWTH OF
DIFFERENT ONES DEPENDING ON THE
CONDITION.
IT'S A COLLABORATION.
AND I WOULD PROPOSE VESICLES ARE
ACTUALLY PARTICLES.
NOW I WANT TO SHOW NO DATA BY
MARIANA IN MY LAB, TWO AIMS.
VISUALIZATION OF OUTER MEMBRANE
VESICLES EMPLOYING MARKERS, CAN
WE VISUALIZE THIS?
THEY NEED OXYGEN TO GROW,
BACTEROIDES IS ANAEROBIC
CONDITIONS, WE NEED TO GIVE SOME
TIME EXPOSED TO OXYGEN TO GROW,
THAT CAUSES SOME ISSUES TO THE
METABOLISM OF THE BACTERIUM.
BUT WE MANAGED TO GET GOOD DATA
ON THAT.
I'M GOING INTO MORE IN THE ROLE
OF THE OMV IN DEGRADATION OF
POLYSACCHARIDES.
IN THE FIRST IMAGE, YOU HAVE
INULINASE, ONE PROTEIN, A MARKER
WE FOUND, PROTEOMICS, AND THE
FIRST THING YOU CAN SEE IS WHERE
THE MARKERS GO TO THE OMVs ARE
FOUND IN PARTICULAR LOCI, THE
ONES IN OUTER MEMBRANE, YOU CAN
SEE THE PROTEINS MOSTLY TO
OMVs.
IN THIS PARTICULAR SLIDE WE'RE
NOT SO HAPPY WITH OXYGEN.
ALLOW US TO SEE WHERE THIS FORM.
WE HAVE SEPARATE EXPRESSION,
WHAT HAPPENS WHEN WE CO-EXPRESS
THE TWO MARKERS?
THAT'S WHAT YOU CAN SEE HERE IN
GREEN, OUTER MEMBRANE VESICLE
MARKER, WE SEE VESICLES IN THIS
EXPERIMENT.
IN RED WE DON'T SEE ANY
VESICLES.
THAT'S OKAY.
THIS IS A MARKER OF THE
MEMBRANE.
WHAT YOU SEE IN THE IMAGE
COMBINED, BACTERIA HAS TWO
COLORS, OMVs ARE ONLY SHOWING
THE GREEN.
SO RECAPITULATING WHAT WE SAW ON
THE MASS SPEC, THIS PROTEIN
REALLY GOES TO OMVs IN THE
CELLS, STILL SHOWING PHENOTYPES.
SO THAT'S WHAT YOU EXPECT IF
OMVs ARE REAL BUT ANOTHER
POINT WOULD WHAT IF WE EXPRESS
TWO DIFFERENT MARKERS OF OMVs?
WE HAVE TWO MARKERS.
PRETTY MUCH EVERY VESICLE THERE
IS SEEN BY THE TWO COLORS.
SO INDICATING THESE DOTS WE SEE
ARE REACTING TO BOTH OMV
MARKERS.
SO REALLY THEY CO-LOCALIZE.
AND NOW WE'RE GOING TO SEE
MOVIES.
THIS IS VERY CHALLENGING BECAUSE
AGAIN OXYGEN WILL NOT ALLOW US
TO SEE A LOT.
YOU CAN SEE HERE THE VESICLES
COMING OUT WITH THE OMV MARKER,
WHERE IT'S NOT VISIBLE WITH
OUTER MEMBRANE MARKERS.
YOU CAN SEE VESICLE COMING OUT
OF THE CELL.
AND WE HAVE ANOTHER IMAGE, THIS
HAS MOVEMENT THAT WE'RE GOING TO
CORRECT.
YOU CAN SEE AS WELL HERE VESICLE
COMING OUT OF THE CELL, NOT
VISIBLE IN THE RED CHANNEL, THE
OUTER MEMBRANE MARKER.
SO, THIS IS ACTUALLY THE FIRST
TIME THAT VESICLES CAN BE SEEN
BY LIVE MICROSCOPY.
NOW I'M GOING TO SWITCH GEARS
AND WE'RE GOING TO SEE HOW --
NOW THAT WE KNOW THESE ARE REAL,
THESE THINGS WE FOUND INITIALLY
IN THE MASS SPEC ACTUALLY WE CAN
SEE THEM AS WE EXPECT FOR THE
PROCESS, WE WONDER HOW DOES ALL
THIS CHANGE THE COMPOSITION
CHANGE?
WE CULTURED BACTEROIDES, GROW IN
MINIMUM MEDIA ADDING DIFFERENT
POLYSACCHARIDES.
YOU CAN SEE FIRST ALREADY THE
COMPOSITION CHANGES LIKE THIS
ONE LEVAN, HEPARIN.
DIFFERENT PROTEINS, MANNAN, THE
PRICE HERE IS MUCIN.
MAYBE IT'S NOT A SURPRISE.
WE DON'T HAVE MANY OF THESE,
THEY ARE MORE LIKE HAVING LESS
PROTEINS.
SO, IT'S NOT A PATHOGEN.
THEY CAN GROW BUT ARE NOT SO
HAPPY.
SO THEN WE DID PROTEOMICS FOR
THESE.
I'M GOING TO TRY TO EXPLAIN WITH
ONE OF THE CASES.
WE HAVE THE COMPOSITION OF THE
OMV, FOR THE DIFFERENT ONES.
IN RED THESE ARE ENRICHED TO
THIS PARTICULAR CONDITION.
THESE PROTEINS ARE PARTICULARLY
ENRICHED IN OMVs, A BATCH OF
PROTEINS MISSING PRESENT IN ALL
THE OTHER CONDITIONS ABSENT IN
LEVAN, SHOWING THE COMPOSITION
IS DIFFERENT, A BUNCH OF
PROTEINS ARE ENRICHED IN OMVs,
WE WANT TO SEE WHERE THEY ARE,
WELL, HAVING RED HERE, THESE ARE
LIPOPROTEINS.
WHEN YOU WANT TO SEE A GOOD
MATCH WITH PREVIOUS WORK, DATA
SHOWING THESE WERE INDUCED, NOW
WE SHOW EXPRESSION, PROTEINS
ENRICHED TO OUTER MEMBRANE
VESICLES.
I'M TRYING TO SHOW ALL THE DATA
COMPARED.
AS YOU CAN SEE FOR EACH
PARTICULAR NUTRIENT THERE'S A
GROUP OF HYDROLASES ENRICHED,
MORE IN MANNOSE THAN ANY OTHER
CONDITION.
HEPARIN GLUCOSE, HYALURONIC
ACID, DIFFERENT GROUPS.
I DON'T KNOW, WE TRY TO AVOID
USING OMVs WITH MUCIN.
MOST THINGS ARE MISSING, A FEW
STUFF THERE, WHAT ALLOWS
BACTERIA TO GROW IN MUCIN, NOT
WHAT THEY REALLY WANT TO DO IN
THIS CASE.
ANOTHER POSSIBLE INTERPRETATION
WHERE MANNOSE, HEPARIN,
HYALURONIC ACID AND STARCH ARE
ALL SIMPLE POLYSACCHARIDES, THEY
HAVE TWO DIFFERENT SUGARS WHICH
FAVOR ACTION OF PARTICULAR GENE,
THE MUCIN IS MATCHING, SO REALLY
THAT'S ALSO NOT SENSING THE
SIGNAL NOT WORKING AS WELL.
AND THIS IS ANOTHER WAY TO
PRESENT IT IN WHICH HERE IS THE
PLOT SHOWING PROTEINS ENRICHED
FOR EACH, TO THE RIGHT, AND
PROTEINS ENRICHED IN THE OUTER
MEMBRANES TO THE LEFT.
RED PROTEINS ARE ALL
LIPOPROTEINS THAT CONTAIN A
SIGNAL THAT I'M GOING TO SHOW
YOU IN A SECOND, IN THE LEFT
WHAT WE GET HERE IN BLUE THESE
ARE MEMBRANE PROTEINS, IN YELLOW
THESE ARE LIPOPROTEINS THAT
DON'T HAVE THE SIGNAL THAT I'M
GOING TO SHOW YOU IN A SECOND.
ALL THESE PROTEINS IN RED NOT
ONLY HAVE THE FUNCTION OF
LIPOPROTEINS, THEY CONTAIN A
SIGNAL THAT TARGETS TO OMVs.
THERE ARE NEGATIVELY CHARGED
AMINO ACID, EVIDENCE TO REMOVE
THESE MOTIF, THESE PROTEINS WILL
NOT GO TO THE OMV ANYMORE.
PROTEINS THAT REMAIN IN THE
CELLS DON'T HAVE THAT SIGNAL.
SO CLEARLY HERE THIS IS PART OF
THE PROCESS.
VERY DIRECTED PROCESS WITH A
CLEAR GOAL.
I'M GOING TO TRY TO SUMMARIZE
WHAT I JUST TOLD YOU IN ONE
FIGURE.
WHAT WE THINK IS HAPPENING IN
THE PRESENCE OF SPECIFIC
POLYSACCHARIDES BACTERIA WILL
SENSE, FRUCTOSE, HEPARIN,
INDUCED SPECIFIC POOLS WHICH
ALSO ACTUALLY KNOWN THIS PART
HAS BEEN SHOWN BEFORE BY OTHER
GROUPS.
LIPOPROTEINS THAT ARE HERE THEY
ARE GOING INTO THE MEMBRANE,
SOMEHOW MAKE IT ACROSS THE OUTER
MEMBRANE.
EXPOSED TO THE SURFACE.
AND THEY ARE RECRUITING SPECIFIC
FOCI, WHAT YOU SEE HERE, OMB IS
GOING TO BE GENERATED, VERY
DYNAMIC, SOME IMAGES WE CAN SEE
THEY ARE MOVING AROUND.
SO THIS PROCESS OF TRANSLOCATION
IS MEDIATED BY LESS SIGNAL,
COMPOSED BY NEGATIVE CHARGE
AMINO ACIDS.
WHAT HAPPENED THEN IS THE
MECHANISM, NOW WHAT ARE THE
FACTORS THAT ARE RECRUITING TO
THE FOCI, QUESTIONS WE WANT TO
ANSWER RIGHT NOW.
VESICLES ARE FORMED.
YOU CAN SEE WITH SPECIFIC
MARKERS.
OMV, THE COMPOSITION, DIFFERENT
MARKERS IN THE CELLS.
NOW A LOT GLYCOSIDIC, DEGRADE
POLYSACCHARIDE.
ALSO YOU'RE GOING TO HAVE A TALK
LATER SHOWING PATHOGENS CAN ALSO
BENEFIT BY ACTIVITY OF THE OUTER
MEMBRANE VESICLES FROM
BACTEROIDES.
TAKEHOME MESSAGES I HOPE YOU
WILL REMEMBER AFTER THIS IS THAT
ALL THESE, THE FIRST TIME WE
HAVE IT PROVED IT'S NOT AN
ARTIFACT.
HIGHLY ORCHESTRATED PROCESS.
BACTEROIDES PACKS
GLYCOSYLHYDROLASE.
I THINK THAT BASICALLY OUR WORK
AND THE GROUP, OMVs CONTRIBUTE
TO GUT HOMEOSTASIS.
THE ONLY THING LEFT IS TO THANK
THE PEOPLE IN MY GROUP.
THE WORK IS BASED ON TWO
PREVIOUS POSTDOCS IN MY LAB.
AND OUR COLLABORATOR FOR
PROTEOMICS, AND I THANK YOU FOR
YOUR ATTENTION.
>> THANK YOU.
WE GET QUESTIONS COMING FROM THE
ONLINE GROUP.
DO YOU SEE EVIDENCE WHERE YOU
MIX BACTERIAL CULTURES OR
SPECIES WITH OTHER BACTERIA OR
HOST CELLS?
>> ACTUALLY I WOULD LIKE TO TELL
YOU NO BUT YES.
WE SEE TIMES BACTERIA WITH
VESICLES, FROM ONE BACTERIUM TO
ANOTHER SO I DON'T KNOW IF
THAT'S ARTIFACT OR REALLY
HAPPENING.
SOME PEOPLE HAVE FOUND EVIDENCE
IN OTHER BACTERIA, ONE BACTERIUM
CAN PROVIDE.
THIS IS EVEN MORE MYSTERIOUS
THAN WHAT WE'RE ALREADY DOING.
>> SETH AND WARREN?
>> MARIO, NICE TO MEET YOU.
I'M SETH.
THANKS FOR THE ACKNOWLEDGMENTS.
I'M GOING TO ASK QUESTIONS
BECAUSE I WONDERED FOR A WHILE.
I'LL ASK A QUESTION TO AN EXPERT
IN OMVs, HOW FAR DO THEY GO?
AND DO YOU THINK THEY HAVE
SPECIAL BIOPHYSICAL
CHARACTERISTICS THAT ALLOW THEM
TO TRAVEL THROUGH PERHAPS MUCUS?
>> THERE IS WORK SHOWING THAT
THEY CAN GO FAR, ONE OF THE
ADVANTAGES OF PACKING HYDROLASES
IN OMV.
WHY PUT THEM IN OMVs?
WELL, YOU ONLY CARE FOR YOU, IT
WOULD BE EFFICIENT TO GET ONLY
AT THE MEMBRANE, I CAN ONLY
CATCH WHATEVER COMES NEXT TO ME.
WHEREAS IF I PUT AN OMV THERE,
OMV CAN DIFFUSE, FIND
POLYSACCHARIDES, DEGRADE THEM,
THE SACCHARIDES, SUGARS WILL
ACTUALLY DIFFUSE MORE.
HIGHLY DIFFUSIBLE IN SOME CASES
PROBABLY OR MORE PATHOGENIC
BACTEROIDES CAN PROBABLY
PENETRATE AND REACH FARTHER
YEAH.
THAT, THAT'S ONE OF THE KNOWN
ADVANTAGES OF THE OMVs, THEY
CAN GO FAR, THEY ARE VERY SMALL,
THEY CAN GET WHERE BACTERIA
CANNOT GET.
WE TRIED TO DO EXPERIMENTS ON
ANIMALS, STILL CHALLENGING FOR
US NOW.
WE NOW CAN SEE THEM AND WE HOPE
TO BE ABLE TO GO INTO ANIMAL
STUDIES AND CAN SEE THESE OMVs
TO SEE WHERE THEY GO.
WE TRY WITH ORGANOIDS AS WELL.
NOW THAT WE HAVE THE TOOLS THESE
ARE QUESTIONS WE WANT TO ANSWER
BUT IT WILL TAKE A WHILE.
>> THANKS FOR THAT COOL SCIENCE
CUREOUTS CURIOUS WHETHER OR NOT
UP LOOKED AT METABOLITES IN THE
OMVs BECAUSE THAT COULD BE
ANOTHER WAY THEY COULD SIGNAL
AND BE VALUABLE AS CARRIERS.
>> WE HAVEN'T DONE IT.
THE MAIN REASON WHY, I THINK IT
WOULD CHANGE SO MUCH COMPOSITION
BASED ON HOW WE GROW THE
BACTERIA.
I DON'T KNOW IF THEY ARE GOING
TO REFLECT WHAT REALLY HAPPENS
IN THE ANIMAL MODELS.
OR IN THE HUMAN.
WE CAN AT LEAST START AND SEE IF
THERE'S SOMETHING DIFFERENT THAN
THE OTHER COMPONENTS.
MY CONCERN IS THIS.
SOME OF THE WORK HAS BEEN DONE
WITH ALL THESE AND IMMUNOLOGY,
RANDOM AMOUNTS, I DON'T KNOW HOW
MUCH THEIR EFFECT, NOW WE KNOW,
DEPENDING HOW YOU GROW OMVs
WITH A COMPLETELY DIFFERENT
COMPOSITION, YEAH, SOMETHING WE
CAN HOPEFULLY ADDRESS IN THE
FUTURE IN MORE REALISTIC
CONDITIONS OF GROWTH.
>> ONE MORE QUESTION FROM THE
ONLINE.
I DON'T WANT THEM TO FEEL
EXCLUDED.
WE HAVE A BUNCH OF QUESTIONS SO
PLEASE CONTINUE TO HAVE
QUESTIONS, WE CAN ANSWER OFFLINE
AS WELL.
HELEN DUKE, QUESTION IS ARE YOU
ABLE TO PROBE GENOMES
TRANSCRIPTOMES USING GENETIC
MARKERS OR CHANGE IN SPECIFIC
TRANSCRIPTIONAL ACTIVITIES?
>> IF I'M ABLE TO PROBE GENOMES?
USING EPIGENETIC MARKER OR
CHANGE SPECIFIC -- WE DON'T KNOW
YET.
WE HOPE IN THE FUTURE ONCE WE
KNOW A LITTLE BIT MORE.
THAT'S THE BIG QUESTION FOR ME.
I'M NOT A MICROBIOME PERSON, I'M
A BACTERIA PERSON.
SO NO, WE ONLY HAVE A CARGO.
WE REALLY WANT TO KNOW NOW TO
INVESTIGATE MACHINERY AND
HOPEFULLY MAY BE ABLE TO ANSWER
QUESTIONS, IF I INTERPRET THAT
CORRECTLY.
>> SOUNDS GREAT.
THANKS, MARIO.
FOR TIME'S SAKE WE'LL MOVE ON.
PLEASE PUT YOUR QUESTIONS WITH
YOUR E-MAIL ADDRESSES, THAT WILL
ALLOW SPEAKERS TO REACH OUT AND
ANSWER QUESTIONS.
WE APPRECIATE EVERYONE'S
INVOLVEMENT.
NEXT IS FIKRI AVCI, UNIVERSITY
OF GEORGIA, GLYCAN MEDIATED
IMMUNOREGULATION IN HEALTH AND
DISEASE.
>> THANK YOU FOR THE INVITATION
AND PUTTING TOGETHER THIS
FANTASTIC SYMPOSIUM TODAY.
I'M GOING TO SHARE ARE YOU TWO
OF OUR UNPUBLISHED SHORT STORIES
PERTAINING TO GLYCAN MEDIATED
IMMUNOREGULATION IN HEALTH AND
DISEASE.
FIRST WILL HAVE TO DO WITH THE
DISEASE WHICH IS GOING TO BE
BREAST CANCER, SECOND STORY,
EVEN SHORTER, TO DEAL WITH
PRELIMINARY DATA ON HOW GLYCANS
MAY INDUCE HOMEOSTASIS IN THE
GUT, GUT MICROBIOME.
SO THE FIRST STORY HAS TO DO
WITH TN ANTIGEN. ANTIGEN, ROLE
IN IMMUNE EVASION IN BREAST
CANCER.
IT IS PARTICULARLY ON MUCIN 1,
HERE YOU SEE A NUMBER OF THOSE
HYPO GLYCOSYLATION EVENTS IN THE
CANCER CELL, ANTIGEN IS
BASICALLY THIS SUGAR UNIT.
SO, THIS IS AN NCI PILOT
PROJECT, BASED ON MULTIPLE
CRITERIA, NOT POST-TRANSLATIONAL
EVENTS RESULTING IN ANTIGENS.
THE OTHER IS ACTUAL LECTIN, IN
MICE IT'S EITHER MGL 2
MACROPHAGE GALACTOSE LECTIN 2 OR
1B THAT BINDS TO TN ANTIGEN,
DEVELOP WELL ESTABLISHED,
LITERATURE DEMONSTRATES
INTERACTIONS LEAD TO REACTION.
HOMEOSTASIS OF T CELLS.
WITH THE TWO IMPORTANT PLAYERS
WE HYPOTHESIZE ABERRANT TUMOR
GLYCOSYLATION IN THIS CASE
INDUCES ACCUMULATION OF TN
INDUCED CELLS IN BREAST TUMOR
MICROENVIRONMENT, AND
INTERACTION LEADS TO TUMOR
IMMUNE EVASION.
WE USED MOUSE BREAST CANCER
MODEL, BY INJECTING CANCER CELL
LINE CELLS, EXPRESS TN ANTIGEN,
WE WANT TO LOOK AT TUMOR
MICROENVIRONMENT, WHETHER CELLS
ACCUMULATE.
WE OBSERVED B CELL POPULATION TO
OUR SURPRISE, PREDOMINANTLY OR
MAYBE ALL THESE ASSOCIATED WITH
MYELOID CELLS.
WE WERE ATTRIBUTING CELLS
IMMUNOREGULATORY FUNCTIONS,
WANTED TO SEE WHETHER THEY HAVE
REGULATORY B CELL MARKERS, CD24,
CD1D AND SEVERAL OTHER MARKERS,
THEY DO SHOW SOME MARKERS, AS
WELL.
AND WE ALSO WANTED TO LOOK AT
DISEASED B CELLS.
IN GENERAL, NOT ALWAYS,
ASSOCIATED WITH IMMUNE
SUPPRESSIVE FUNCTION.
SO WE'RE CONTINUING TO
CHARACTERIZE CELLS AS PART OF
THE CHARACTERIZATION, TRANSCRIPT
OMIC ANALYSIS OF CELLS, WE
COMPARE, BUT THEN WE KNEW THEY
WOULD BE VASTLY DIFFERENT FROM
ONE ANOTHER AS YOU CAN SEE IN
DIFFERENTIALLY EXPRESSED GENES
THOUSANDS ARE DIFFERENTIALLY
EXPRESSED.
ALMOST ALL IN THE TUMOR
MICROENVIRONMENT, SEPARATED THEM
INTO LOW AND HIGH GROUPS.
AND THEN COMPARED THOSE TWO
POPULATIONS, THAT'S WHAT YOU'RE
SEEING IN HERE.
WE WANTED TO CARRY OUT OUR
ANALYSIS, USING THOSE
POPULATIONS BECAUSE WE FELT LIKE
THEY ARE THE CLOSEST, WITH ONLY
CD301B EXPRESSION THAT WAS
DIFFERENT.
IN MANY PROCESSES AND EVENTS
WE'VE SEEN EXPRESSED GENES BUT
HAVE BEEN INTERESTED.
IN THIS VOLCANO PLOT OBSERVED
SOME INTERESTING GENES THAT ARE
ASSOCIATED WITH
IMMUNOREGULATION, EXPRESS HIGH
POPULATIONS AS OPPOSED TO LOW
POPULATIONS.
WE SHALL CONTINUING OUR ANALYSIS
BUT WE WANTED TO SEE WHETHER TO
TEST OUR HYPOTHESIS THE CELLS,
AND ACCUMULATION IN THE TUMOR
MICROENVIRONMENT, HAS ANYTHING
TO DO WITH IMMUNE EVASION AND
THEREFORE THE TUMOR GROWTH.
AND THIS IS AGAIN A PRELIMINARY
DATA, PERFORMED BY USING
TRANSGENIC MICE, IN CD301B,
VERSUS WILDTYPE MOUSE, OBSERVED
VERY SIGNIFICANT DECREASE IN
TUMOR VOLUME WHEN THE CD301B IS
NO LONGER EXPRESSED ON THOSE
CELLS IN THE TUMOR
MICROENVIRONMENT.
THESE CELLS ARE IN FACT PLAYING
THE ROLE OF IMMUNE EVASION CELL
POPULATION.
THIS IS A VERY BROAD SLIDE,
STILL THIS IS THE WORK IN
PROGRESS BUT ONE OBSERVATION
MADE US VERY EXCITED.
THE OTHER SIDE OF THE STORY HAS
TO DO WITH TN.
WE WANTED TO MODULATE TN
EXPRESSION LEVELS AND FOR THAT
WE MAPPED OUT COSMC, IMPORTANT
FOR ELONGATION OF THE
OLIGOSACCHARIDE CHAINS, AND BY
DOING THAT WE WERE ABLE TO
GENERATE WHAT WE CALL TN HIGH
CELL LINE, AS YOU CAN SEE BY
PROBING THE TN ANTIBODY OBSERVED
HIGH TN EXPRESSION ON THESE
CELLS.
WE'RE BEGINNING TO EXPLORE
DIFFERENTIAL EXPRESSION, AND
THEIR ROLES IN ACCUMULATION,
ALSO TUMOR GROWTH AND
PROGRESSION.
AND THIS IS DATA AGAIN, TN LOW
OR HIGH CELL LINES.
THERE SEEMS TO BE CORRELATION
BETWEEN THE TWO.
WE'RE ALSO -- WE'VE OBTAINED
TRANSGENIC MOUSE LINE THAT'S
EXPRESSING HUMAN MUCIN 1 WITH TN
EXPRESSION, INDUCE WITH
CARCINOGEN WE COMPARED WILDTYPE
MICE WITH TRANSGENIC MICE AND
OBSERVED NON-TRANSGENIC MICE
SURVIVED AT LOWER RATE THAN
WILDTYPE MICE.
AGAIN SUGGESTING THAT THE TN
ANTIGEN IS IMPORTANT IN CANCER
PROGRESSION.
IN THIS PARTICULAR EXPERIMENT WE
LOOKED AT ACCUMULATION OF CD19+
CELLS AND, AGAIN, LOWER SURVIVAL
AND HIGHER ACCUMULATION OF
CD301B IN THE PRESENCE WERE
ASSOCIATED ALTOGETHER.
AGAIN, THIS IS SOME UNPUBLISHED
RESULTS FOR A PROJECT WHICH
WE'RE SO FAR SHOWING THAT THERE
IS A LYMPHOCYTE POPULATION IN
THE BREAST TUMOR ENVIRONMENT BUT
I DON'T FIND BY CD301B
EXPRESSION AND THESE TN
EXPRESSION ON CANCER CELLS IS
CORRELATED WITH THESE CELLS AND
CANCER GROWTH.
WE'RE CONTINUING TO CHARACTERIZE
THESE POPULATION OF CELLS.
AND WE HAVE SO MANY QUESTIONS,
I'M GUESSING YOU DO TOO, TO
DETERMINE MECHANISMS ASSOCIATED
WITH THIS INTERACTION AND WE'RE
HOPING TO TAKE IT FROM THOSE
MECHANISTIC WORKS INTO
TRANSLATABLE TRANSLATIONAL
RESEARCH WHERE WE CAN OVERCOME
CANCER PROGRESSION THROUGH
INHIBITING THIS CD 301B
INTERACTION, IN HUMANS.
SO THAT LEADS ME TO MY SECOND
ONE-SLIDE STORY THAT HAS TO DO
WITH GLYCAN MEDIATED REGULATION
OF IMMUNE RESPONSES IN THE GUT
MICROBIOME.
HAVING SEEN TN ANTIGEN IN TUMOR
CELLS, RECRUITING IMMUNE CELLS
IN THE TUMOR MICROENVIRONMENT
AND LEADING THEM TO FURTHER
GROWTH OF THE TUMOR WE ASKED
OURSELVES WOULD THESE CELLS HAVE
ANY USE IN THE HOMEOSTASIS OF
IMMUNE SYSTEM?
AND WE LOOKED AT THE GUT
MICROBIOME AND COMPARTMENTS IN
THE GUT WHETHER CELLS ARE
INTERACTING WITH THE SYMBIOTIC
BACTERIA, WHETHER THEY INDUCE
REGULATORY TYPE AND THEREFORE
HOMEOSTASIS.
FOR THAT WE LOOKED, EXPRESSION
IN THE GUT IN COMPARTMENTS WE'VE
SEEN AN INCREASED CD301B
EXPRESSION IN SPF MICE AND
WANTED TO SEE WHETHER THEIR
PRESENCE HAD ANYTHING TO DO WITH
IMMUNE SYSTEM HOMEOSTASIS, AND
DID THAT THROUGH MODEL, DEPLETE
CD301 B+ CELLS SEE DIFFERENCE IN
SURVIVAL THAT DEMONSTRATED CELLS
ARE VERY IMPORTANT FOR
PREVENTING AUTOIMMUNE BIAS IN
THE GUT COMPARTMENTS.
WE'RE NOW LOOKING AT COMPARING
WILDTYPE VERSUS CD3 01B,
ENHANCED IN THE PRESENCE OF CD3O
1B+ CELLS OR NOT, THIS IS STILL
ONGOING WORK FOR US.
THEY SAME TIME WE'RE USING
ANTI-TN ANTIBODY, TRYING TO
IDENTIFY OR ZOOM IN TO
PARTICULAR SPECIES THAT ARE
ACTUALLY LEADING TO THIS
INTERACTION THAT LEADS TO WHAT
IS OBSERVED IN COLITIS.
WITH THAT, WE THINK THAT CD301B
MICROBIOME OR MICRO FLUORIDE
INTERACTION LEAD TO HOMEO
STAYSES, HOMEOSTASIS, YOU GET
INFLAMMATORY DISEASES AND
POTENTIALLY CANCER DUE TO THOSE
INFLAMMATORY PROCESSES.
AND THAT'S OUR OWN HYPOTHESIS.
HAVING SAID THAT, WE'RE MOVING
TO EMORY UNIVERSITY MEDICAL
SCHOOL IN A MONTH, THIS PROJECT
IS UP FOR GRABS, NOBODY IS
WORKING ON THIS.
AND I HEARD THAT THERE ARE 800
OR SO PEOPLE WATCHING THIS SO IF
ANYONE IS INTERESTED IN WORKING
ON THIS PROJECT PLEASE CONTACT
ME.
WITH THAT, I'D LIKE TO THANK
AMY IN MY LAB, HELPING WITH
BIOINFORMATICS ON THE MICROBIOME
PROJECT.
SANDRA PROVIDED MICE AND AKIKO
PROVIDED CD301B TRANSGENIC MICE.
>> WHEN YOU LOOK AT KNOCKOUT
MICE, JUST IN GENERAL, IMMUNE
RESPONSE, DO YOU SEE DIFFERENCE
IN B CELL RESPONSE,
INFLAMMATION, ANTIBODY TITERS IN
GENERAL FOLLOWING TRADITIONAL
CHALLENGES?
>> I THINK PUBLISHED WORK ON
THAT ONE, SHE HAS ACTUALLY
SHOWN, STILL NOT KNOWN THESE B
CELLS WERE ACTUALLY CD301B+,
IT'S ALWAYS BEEN ASSOCIATED WITH
MACROPHAGES.
>> RIGHT.
>> AND SHE HAS SHOWN REDUCEED
ANTIBODY MEDIATED IMMUNITY.
>> OKAY.
I DON'T SEE QUESTIONS IN THE
CHAT.
MAYBE WE'LL MOVE ON.
IF YOU HAVE QUESTIONS, PLEASE
PUT THEM INTO THE CHAT AND WITH
YOUR E-MAIL ADDRESS, PEOPLE CAN
RESPOND.
>> SHOOT ME AN E-MAIL.
>> WE APPRECIATE IT.
WONDERFUL TALK.
BEAUTIFUL WORK.
>> THANK YOU.
>> WE'RE GOING TO MOVE ON TO
DREW KIRALY FROM MOUNT SINAI TO
TALK ABOUT MANIPULATIONS, I'M
TRANSITIONING GLASSES,
MANIPULATIONS OF GUT MICROBIOME
DIVERSITY.
>> CAN EVERYONE HEAR ME AND SEE
MY SLIDES?
>> LOOKS GREAT.
>> SO THANK YOU TO THE
ORGANIZERS FOR INVITING ME AND
THANK YOU TO EVERYONE WATCHING
ONLINE, EXCITED TO TAKE PART IN
IMPRESSIVE SYMPOSIUM AND HOPE I
HAVE A CONTRIBUTION.
I WAS HOPING I WOULD HAVE SOME
INTERESTING NEW DATA AND WASN'T
SURE IT WOULD BE OUT YET SO I
GAVE A GENERIC TITLE BUT WE GOT
NEW DATA SO I'M GOING TO
TRANSITION AND SACRIFICE POLISH
ON THE TALK TO SHOW SOME REALLY
NEW AND EXCITING THINGS.
PRETTY MUCH EVERYTHING IN THE
INTRODUCTION IS UNPUBLISH AND
UNPRESENTED BEFORE BY ME.
MORE SPECIFICALLY WE'LL LOOK AT
EFFECTS OF THE GUT MICROBIOME
AND HOW IT REGULATES FENTANYL
SEEKING BEHAVIOR AND SYNAPTIC
PROTEOMICS IN THE VENTRAL
STRIATUM, IMPORTANT IN REWARD IN
OPIOID USE DISORDER.
NO CONFLICTS OF INTEREST RELATED
TO THE PRESENTATION.
MANY IN THE ROOM ARE AWARE,
OPIOID USE DISORDER IS A MAJOR
PUBLIC HEALTH PROBLEM.
ESTIMATES ARE SOMEWHAT DIFFICULT
BUT CURRENT BEST GUESS, THIS IS
A CONDITION AFFECTING MORE THAN
16 MILLION PEOPLE WORLDWIDE.
OVERDOSE IS ON THE RISE.
SOME MOST RECENT DATA FROM NIDA
ABOUT RATES OF OVERDOSE, DRUG
OVERDOSES, IN THE UNITED STATES,
WITH A NUMBER OF OVERDOSES PER
YEAR ON THE Y-AXIS.
YOU CAN SEE THIS LIGHT BLUE LINE
HERE, SYNTHETIC OPIOIDS ARE THE
METHADONES, PRIMARILY FENTANYL.
IN RECENT YEARS HAVING 50,000+
OVERDOSES DUE TO FENTANYL AND
SIMILAR OPIOIDS.
THIS IS SOMETHING THAT EARLY
DATA SUGGESTS THAT COVID
PANDEMIC HAS ACTUALLY BEEN
EXACERBATING THIS PHENOMENON,
THAT THE ISOLATION, OTHER
FACTORS, HAVE BEEN DRIVING
INCREASED OVERDOSES.
IF ANYTHING THIS IS A PROBLEM
CURRENTLY GETTING WORSE, NOT
BETTER.
I'M A PHYSICIAN-SCIENTIST, A
PSYCHIATRIST.
WHAT'S DIFFICULT FOR ME TREATING
PATIENTS, I HAVE NO GOOD WAY OF
RISK STRATIFYING FOR RELAPSE OR
UNDERSTANDING RISK OF BECOMING
DEPENDENT WHEN STARTING USE OF
OPIOIDS.
MULTIPLE GOOD TREATMENT OPTIONS,
STILL INSUFFICIENT FOR A LOT OF
PATIENTS.
PATIENTS HAVE DIFFICULT
TOLERATING, MAINTAINING, A
NUMBER OF SIDE EFFECTS.
WORKING TO IDENTIFY WAYS TO RISK
STRATIFY AND TREAT PATIENTS IS A
CRITICAL NEED FOR THE FIELD.
I WOULD ARGUE SOCIETY AT LARGE
GIVEN THE MAGNITUDE OF THE
PROBLEM.
WE'VE BEEN FOCUSING ON THE GUT
MICROBIOME.
OPIOID FITS WELL.
RECEPTORS ARE EXPRESSED HIGHLY
THROUGHOUT THE GUT.
THERE HAVE BEEN STUDIES SHOWING
HIGH DOSE LONG-TERM OPIOID USE
CAN AFFECT COMPOSITION AND
METABOLOMIC MICROBIOME.
WE'VE BEEN DOING WORK LOOKING AT
HOW METABOLITES CAN SIGNAL TO
THE BRAIN AND HAVE WAYS
IMPORTANT FOR BEHAVIOR.
WE'RE LOOKING AT COMPLEX
INTERACTIONS THAT EXIST BETWEEN
THE COMMENSAL MICROBIOME AND THE
INTESTINAL IMMUNE SYSTEM THAT
CAN DRIVE BEHAVIORS.
TODAY WE'RE GOING TO TALK ABOUT
SIMILAR MODELING, DRILLING DOWN
ON WHAT SOME MICROBIOME EFFECTS
ARE ON OPIOID USE DISORDER.
THE MODEL WE'RE USING RELATIVELY
SIMPLE, A BIT OF A BROAD
APPROACH, A LARGE MICROBIOME
KNOCKDOWN ASKING THE QUESTION OF
IF YOU HAVE A MARKEDLY REDUCED
PRESENCE OF THESE NORMAL
BACTERIA IN THE GUT HOW WILL
THAT AFFECT REGULATION OF BRAIN
AND BEHAVIOR LONG TERM.
WE'RE DOING A HOME CAGE MODEL, A
COCKTAIL THROUGH HOME CAGE
DRINKING WATER, ANTIBIOTICS THAT
ARE BROAD SPECTRUM AND POORLY
ABSORBED WITH THE HOPE THE
ANTIBIOTICS WILL GO THROUGH AND
KNOCK DOWN MICROBIOME HAVING
MINIMAL OFF-TARGET EFFECTS BY
ABSORPTION.
WE'RE USING NEOMICKEN,
BACITRACIN AND VANCOMYCIN.
WE GET GREATER THAN 90%
REDUCTION, WE'RE INTERESTED IN
BEHAVIOR AND HOW DOES THIS
REGULATE BEHAVIORAL RESPONSE,
WE'VE SPENT A GREAT DEAL OF TIME
TRYING TO ENSURE THAT THIS IS
WHERE THE ANIMALS ARE HEALTHY
AND BEHAVING RELATIVELY NORMALLY
AT BASELINE BEFORE WE GIVE
FURTHER MANIPULATION.
WE'VE IDENTIFIED COCKTAIL,
ANIMALS WILL DRINK SOLUTION
NORMALLY, NO DIFFERENCE OF
TREATMENT OF WATER CONTROLS, HOW
MUCH ANTIBIOTIC ANIMALS ARE
DRINKING.
WE SEE NORMAL BODY WEIGHT GROWTH
OVER TIME INCLUDING PERIODS UP
TO SIX WEEKS OR LONGER ON
ANTIBIOTICS.
WE'VE LOOKED AT LOCOMOTOR
ACTIVITY TO MAKE SURE ANIMALS
ARE MOVING NORMALLY AT BASELINE
AND SEE AGAIN UP TO EVEN 6 WEEKS
OF ANTIBIOTICS THERE'S NO
DIFFERENCE BETWEEN THE TWO
GROUPS.
I'M SHOWING MORPHINE BUT MOSTLY
TALKING ABOUT FENTANYLS TODAY,
NO CHANGES IN LEVELS OF
INJECTION IN SERUM OR BRAIN ARE
OPIOIDS OR METABOLITES, SO
EFFECTS ARE NOT SIMPLY DUE TO
CHANGES IN AVAILABILITY OF THE
DRUGS.
WE'VE DONE CONTROL FOR
INFLAMMATORY RESPONSE, BUT WHAT
WE'VE CONVINCED OURSELVES SOME
WORK WE PUBLISHED ALREADY, WE
HAVE THE SYSTEM WHERE WE'RE
ROBUSTLY KNOCKING DOWN
MICROBIOME, AND HAVE AN ANIMAL
RELATIVELY NORMAL AND HEALTHY AT
BASELINE.
SOME PREVIOUSLY PUBLISHED WORK
WAS TO IDENTIFY SOME EARLY
EFFECTS OF MICROBIOME ON DRUG
SEEKING BEHAVIORS RELEVANT TO
OPIOID USE DISORDER.
THIS IS A MOUSE STUDY, ANIMALS
ON ANTIBIOTICS FOR TWO WEEKS.
WE DID THIS MEASURE OF DRUG
PREFERENCE, CONDITIONED PLACE
PREFERENCE, AN APPARATUS WITH
TWO CHAMBERS, EACH DAY SALINE IN
THE MORNING, OPIOID IN THE
AFTERNOON, CONFINED TO THE OTHER
SIDE, LAST DAY OPEN THE DOORS
AND SEE HOW MUCH TIME THEY SPEND
ON EACH SIDE, INDEX OF DRUG
PREFERENCE.
WHAT WE SAW AND PUBLISHED
EARLIER ON MICROBIOME DEPLETION
LED TO MARKED REDUCTION AND
FORMATION OF CONDITIONED PLACE
PREFERENCE.
HIGHER DOSE, DECREASED PREACH
-- PREFERENCE.
TRIED GOING HIGHER WITH EVEN
LARGER DOSE OF MORPHINE VERSUS
STRONGER PREFERENCE IN CONTROL
ANIMALS HERE, ANIMALS LACKING
MICROBIOME FORMED NO PREFERENCE
FOR THIS DRUG.
THAT WAS KIND OF INTERESTING.
IN COMBINATION WE'VE BEEN
LOOKING AT A NUMBER OF PAPERS
SUGGESTING THAT CHANGES IN THE
MICROBIOME AND METABOLITES CAN
AFFECT TRANSCRIPTIONAL
REGULATION IN THE BRAIN SO IN
THE SAME PAPER WE ALSO PERFORMED
RNA SEQUENCING OF THE VENTRAL
STRIATUM, NUCLEUS ACCUMBENS.
AND WE FOUND ANIMALS WITH THE
MICROBIOME KNOCKED DOWN TREATED
WITH MORPHINE ARE DYSREGULATED
GENE EXPRESSION.
THIS IS A VOLCANO PLOT COMPARING
MORPHINE-PAIRED GROUPS,
DYSREGULATION OF THOUSANDS OF
GENES EVEN COMPARED TO
MORPHINE-TREATED CONTROLS.
THIS IS AFFECTING CELLULAR
PATHWAYS AS DIVERSE AS CHROMATIN
MODIFICATION, EPIGENETIC
REGULATION, CELL ADHESION, GLIAL
PATHWAYS, NEUROGENESIS, WE'VE
BEEN MOVING TOWARD MODELS WITH
HIGHER TRANSLATIONAL RELEVANCE.
HOW DO MICROBIOME MANIPULATIONS
DRIVE DRUG SEEKING IN
TRANSLATIONALLY RELEVANT MODELS?
ARE MOW ELECTRIC ALREADY CHAINS
-- MOLECULAR CHANGES IN THE
BRAIN EXHIBITED AT PROTEIN
LEVEL?
I'LL ADDRESS THAT IN THE LAST
TEN MINUTES HERE.
THE WAY WE'RE MODELING SUBSTANCE
USE DISORDER IN THE RODENT,
HIGHER RELEVANCE, WITH DRUG SELF
ADMINISTRATION, WE TAKE ANIMALS,
IMPLANT WITH CATHETER, TRAIN
THEM TO LEVER PRESS FOR
INFUSIONS OF A DRUG.
THIS ALLOWS US TO LOOK AT HOW
ANIMALS ACQUIRE DRUG-TAKING
BEHAVIORS AND MORE IMPORTANTLY
AND MORE RELEVANT FOR ADDICTION
MODELS LOOK AT DRUG SEEKING WITH
RESPONSES, CHANGING HOW MANY
TIMES ANIMALS HAVE TO PRESS THE
LEVER TO GET INFUSION OF DRUGS.
AND ALSO WE CAN LOOK AT MODELS
OF RELAPSE, TEACH ANIMALS TO
SELF-ADMINISTER, PUT THEM IN
HOME CAGE FOR ABSTINENCE AND
RETURN THEM TO MODELS OF DRUG RE
RE RELAPSE.
FOR BEHAVIORAL EXPERIMENTS,
SIMILAR TO PUBLISHED WORK,
CONTROLLED WATER OR ANTIBIOTICS
FOR TWO WEEKS, TRAIN TO
SELF-ADMINISTER FENTANYL, TO
TRAIN THEM TO LEVER PRESS TO GET
AN INFUSION OF DRUG.
AND WE SPEND TIME BECAUSE WE
WANTED TWO GROUPS TO START ON
EQUAL FOOTING AFTER
SELF-ADMINISTRATION, TO LEARN TO
ADMINISTER EQUALLY.
SO WHEN WE USE THIS INTERMEDIATE
DOSE WE SEE BOTH CONTROL AND
MICROBIOME DEPLETED ANIMALS
LEARN BEHAVIOR EQUALLY, EACH
GETTING ABOUT 30 INFUSIONS PER
SESSION BY THE END, THIS IS A
ROBUST DOSE OF FENTANYL WHERE
THEY ARE RESPONDING NICELY FOR
IT.
WE CAN TAKE THIS ACQUISITION AND
TRANSITION TO SOME MORE COMPLEX
TASKS, INTERROGATE DRUG-SEEKING
BEHAVIOR.
FIRST THING IS INCREASED FIXED
RATIO, WE CAN NOW INCREASE THE
NUMBER OF TIMES THEY HAVE TO
PRESS THE LEVER TO GET DRUGS.
WE SEE WHEN WE INCREASE THAT TO
TWO PRESSES PER INFUSION,
ANIMALS DON'T HAVE MUCH OF A
DIFFERENCE.
WE MAKE THEM WORK HARDER AND SEE
A SEPARATION OF GROUPS.
ANTIBIOTIC ANIMALS HAD TO PRESS
THREE TIMES TO GET INFUSION,
START TO PRESS AT HIGHER LEVELS
TO ACHIEVE INFUSIONS.
AS WE MAKE IT EVEN HARDER, TO
GET INFUSIONS, PRESS FIVE TIMES
TO GET A SINGLE INFUSION,
ANTIBIOTIC ANIMALS ARE WORKING
MUCH HARDER TO GET INFUSIONS OF
OPIOIDS.
SUGGESTING AS WE DO THIS MORE
COME BEHAVIOR WE SEE INCREASE IN
DRUG-SEEKING BEHAVIOR.
WE PERFORMED ANOTHER EXPERIMENT,
PROGRESSIVE RATIO, WITHIN A
SINGLE SESSION WE PROGRESSIVELY
INCREASE HOW MANY TIMES ANIMALS
PRESS THE LEVER TO GET A SINGLE
INFUSION OF DRUG AND LOOK AND
SEE HOW MANY TIMES THEY WILL
WORK OR PRESS TO GET EVEN ONE
INFUSION.
OVER A SESSION CONTROL ANIMALS
WILL PRESS, ON THE LAST TIME,
ABOUT 25 TIMES TO GET SINGLE
INFUSION.
THEY EVENTUALLY GIVE UP BECAUSE
IT'S TOO HARD TO GET IT.
WE SEE OUR MICROBIOME DEPLETED
ANIMALS WORK MUCH HARDER TO GET
SINGLE INFUSIONS, 50 TIMES OR
MORE TO GET THE SINGLE INFUSION.
WE'RE STARTING TO SEE HOW THIS
MICROBIOME MANIPULATION IS
AFFECTING THESE DRUG-SEEKING
BEHAVIORS.
GOING TO ACTIVE ADMINISTRATION
MOVING TO THE RELAPSE MODEL.
MICROBIOME KNOCKDOWN PERIOD,
ANIMALS TRAINED TO
SELF-ADMINISTER EQUALLY.
AFTER THEY LEARN BEHAVIOR THEY
GO BACK TO HOME CAGE FOR TWO
WEEKS, TWO WEEKS OF ABSTINENCE,
PUT THEM BACK IN THE BOX AND DO
REINSTATEMENT TASKS.
WE PUT THEM IN THE BOX, NO DRUG
AVAILABLE BUT LEVERS THEY
PREVIOUSLY PRESSED ARE THERE.
AND SO WE CAN SEE HOW MUCH BEING
REEXPOSED MAKES THEM PRESS THE
LEVER TO TRY TO RECEIVE
INFUSIONS.
SIMILAR PATTERN TO WHAT WE SAW
WITH THE INCREASED RATIO,
PUTTING THEM BACK IN THE BOX
MICROBIOME DEPLETED ANIMALS ARE
PRESSING MUCH MORE FREQUENTLY ON
THE LEVER THAT WAS PREVIOUSLY
ASSOCIATED WITH DRUG EVEN THOUGH
NO DRUG IS BEING DELIVERED.
AFTER THIS SESSION WE TURN ON
ONE OF THE CUE LIGHTS TURNED ON
WHEN THE ANIMALS RECEIVED
INFUSION OF DRUG TO SEE IF THAT
WOULD FURTHER SPUR DRUG SEEKING
BEHAVIORS AND THE ANTIBIOTIC
ANIMALS CONTINUE TO PRESS MORE
FOR THE DRUG ASSOCIATED CUE, NO
DRUG IS COMING.
WE SEE HOW MUCH ANIMALS PRESS
OVER BOTH SESSIONS, TO SEPARATE
EARLY ON IN THE SLOPE OF THE
ANTIBIOTIC ANIMALS CONTINUES TO
BE HIGHER THROUGHOUT THE
SESSION.
EVEN OVER THE COURSE OF THE
HOUR-LONG SESSION WE'RE SEEING
ANTIBIOTIC ANIMALS AS YOU ADD
MORE DRUG ASSOCIATED CUES
CONTINUE TO PRESS AT HIGHER
LEVELS, INTERESTING AND
SOMETHING WE'RE DELVING INTO.
BUT ONE OF WHICH IS TRY AND
FIGURE OUT MOLECULAR
UNDERPINNINGS, SO I MENTIONED
EARLIER THAT WE HAVE DONE
TRANSCRIPTOMIC ANALYSIS, WORKING
WITH THE PROTEOMICS SCORE TO DO
QUANTITATIVE ANALYSIS IN THE
BRAIN SO WE'RE TAKING ANIMALS
FROM EXPERIMENTS, DISSECTING
NUCLEUS ACCUMBENS, VENTRAL
STRIATUM, PERFORMING
QUANTITATIVE PROTEOMIC ANALYSIS
USING MASS SPECTROMETRY, NO
LONGER A NEW METHOD, DATA
INDEPENDENT METHOD FOR MASS
SPECTROMETRY, A WAY TO TRY AND
MAKE THIS LESS BIASED, THE
PEPTIDES ARE GROUPED BY MASS TO
CHARGE RATIO AND INDIVIDUAL MASS
TO CHARGE RATIO, ALLOWS US TO
GET MORE DETECTION OF SOME
PROTEINS EXPRESSED AT LOWER
LEVELS RATHER THAN ONLY THOSE
PEPTIDES SAMPLED AT HIGH LEVELS.
WHEN WE DID THIS WE LOOKED AT A
NUMBER OF GROUPS.
THIS IS JUST A VOLCANO PLOT
LOOKING AT DIFFERENT GROUPS.
FOR THOSE NOT FAMILIAR, THE
X-AXIS IS LOOKING AT LOG FOLD
CHANGE, UP OR DOWN.
AND THEN THE Y-AXIS IS THE
NEGATIVE LOG 10 P-VALUE, LOOKING
AT THIS, KIND OF THE BIGGER THE
ERUPTION, THE MORE PROTEINS ARE
REGULATED.
WE SEE SOME PROTEINS CHANGING IN
THE NUCLEUS ACCUMBENS.
OUR EFFECTS IN WATER FENTANYL
CONTROLS ARE MORE ROBUST, 300
PROTEINS UP OR DOWNREGULATED.
AND WHEN WE LOOK AT INTERACTION,
SIMILAR TO TRANSCRIPTOMIC DATA,
ADD IN FENTANYL SEE REGULATION
OF SIGNIFICANTLY MORE PROTEINS.
HE'S ARE HOT OFF THE PRESS DATA,
DIGGING INTO IT NOW.
I WANTED TO GIVE SOME DETAIL AND
PATHWAY ANALYSIS WE'VE BEEN
DOING.
LOOKING AT MOST SIGNIFICANT
PATHWAYS, YOU CAN SEE IF YOU
LOOK AT GENE ONTOLOGY-TYPE
PATHWAY ANALYSIS, NEGATIVE LOG
10 P-VALUE, ANYTHING BEYOND IS
STATISTICALLY SIGNIFICANT.
WHAT WE SEE HERE, THIS IS WHAT
WE EXPECTED, WE SEE EFFECTS MORE
ROBUST IN ANTIBIOTIC ANIMALS,
SIMILAR PATHWAYS REGULATED.
YOU EXPECT SYNAPTIC PROTEINS
REGULATED IN ANIMALS RECEIVING
FENTANYL BUT SEEING A MORE
SIGNIFICANT ENRICHMENT IN
ANIMALS LACKING MICROBIOME,
SIMILAR WITH METABOLIC
PROCESSES, THINGS WE WOULD
EXPECT THAT THE MICROBIOME AND
CHANGES IN METABOLITES REACHING
THE BRAIN ARE AFFECTING CELLULAR
METABOLISM IN RESPONSE TO DRUGS.
WE DO PATHWAY ANALYSIS, TAKING
INTO ACCOUNT VECTOR OF THE OR
DIRECTION OF CHANGE, THERE ARE A
NUMBER OF PATHWAYS CHANGING IN
OPPOSITE DIRECTIONS BETWEEN THE
GROUPS.
PROTEINS ARE INCREASED MORE SO
IN ANTIBIOTIC ANIMALS, DECREASED
IN WATER FENTANYL, SIMILAR WITH
PKA AND INTRACELLULAR CASCADES.
THIS IS SOMETHING THAT'S REALLY
INTERESTING.
I WANT TO ON THE LAST SLIDE DIG
OUT SYNAPTIC RELATED PROTEINS,
TO LOOK BETWEEN THE GROUPS AND
SEE WHAT KIND OF OVERLAP.
IF YOU LOOK AT PROTEINS FROM
THIS SYNAPSE LOCALIZATION GENE
ONTOLOGY, WE SEE SIGNIFICANT
OVERLAP.
THERE ARE A NUMBER THERE ARE
UNIQUE WITH FDR CORRECTION.
WHEN WE START TO LOOK AT THE
STRING ANALYSIS TO SEE HOW THESE
DIRECT, WE SEE WATER FENTANYL
ANIMALS HAVE SMALLER INTERACTIVE
NETWORK OF SIGNIFICANTLY
REGULATED SYNAPTIC PROTEINS.
ANTIBIOTIC ANIMALS HAVE LARGER
CHANGE.
PRE AND POST SYNAPTIC PROTEINS,
BOTH ARE HAVING EFFECTS ON
EITHER SIDE OF THE SYNAPSE.
TO FINISH WITH WORKING MODEL,
THIS IS MORE EPIGENETICALLY
GEARED BUT STILL WORKS, OUR IDEA
HERE IS WITH NORMAL MICROBIOME
IT'S IMPORTANT HOMEOSTATIC
REGULATOR OF PRETTY MUCH EVERY
SYSTEM THROUGHOUT THE BODY, BY
PRODUCTION OF NORMAL SHORT CHAIN
FATTY ACIDS YOU GET APPROPRIATE
GENE EXPRESSION WHICH HELPS WITH
BEHAVIORAL REGULATION AND IF WE
DEPLETE MICROBIOME WE'RE
DEPLETING HOMEOSTATIC FORCE OF
METABOLITES THAT IT'S PRODUCING,
DYSREGULATED GENE AND PROTEIN
EXPRESSION, ABERRANT BEHAVER AND
SYNAPTIC PLASTICITY.
I'D LIKE TO THANK THOSE IN MY
LAB.
I WOULD LIKE TO -- I'M MOVING IN
THE NEAR FUTURE, WE'RE MOVING TO
WAKE FOREST SCHOOL OF MEDICINE
BEGINNING IN JUNE, WE'RE LOOKING
TO HIRE PEOPLE AT ALL LEVELS,
TECHNICIANS, POSTDOCTORAL
FELLOWS, GRADUATE STUDENTS, IF
ANYONE IS INTERESTED HAVE THEM
GET IN TOUCH WITH ME.
>> WE HAVE A FEW QUESTIONS.
HOW DID YOU DECIDE THE DRUG
AMOUNT?
I DID BEHAVIORAL STUDIES,
STRUGGLE TO LEARN HOW MUCH OF
THE DRUG WE COULD USE TO SHOW
GOOD LOCOMOTION EFFECTS.
>> YEAH, WHEN WE'RE STARTING A
NEW PROJECT WE DO A
DOSE-RESPONSE CURVE, WITH THE
CONDITION PLACED PREFERENCE,
USING PREVIOUSLY PUBLISHED DOSES
DID DOSE RANGE FROM 2 1/2
MILLIGRAMS PER KILOGRAM TO 15,
SEEING WHERE WE SEE THE EFFECTS.
THAT'S WHAT WE DO WITH ANY
BEHAVIORAL PHARMACOLOGY THING
LIKE THIS, START WITH DOSE RANGE
AND GO FROM THERE.
>> THANK YOU FOR THE
PRESENTATION.
HAVE YOU PERFORMED FECAL
MICROBIOME TRANSPLANTATION
EXPERIMENTS TO REVERSE IMPACT OF
DEPLETION?
>> YES, GREAT QUESTION.
WE'RE WORKING ON THE TRANSPLANT,
THAT'S KIND OF SORT OF WORKING
BUT THAT'S EARLIER DAYS.
WE HAVEN'T DONE AS MUCH
PROBIOTICS BUT A NUMBER OF
THINGS WITH METABOLITE
SUPPLEMENTATION, PARTICULARLY
SHORT CHAIN FATTY ACIDS,
REVERSED EFFECTS, WE HAVEN'T
DONE IT IN THE
SELF-ADMINISTRATION YET BUT
THAT'S THE AVENUE WE'VE BEEN
MAKING MORE SO THAN PROBIOTICS
SO FAR.
>> A RELATED QUESTION, SO
PATIENTS, I DO HEMATOLOGY,
SICKLE CELL DISEASE EARLY ON,
THEY ARE GETTING EARLY ON IN
LIFE PENICILLIN, PERTURBING
MICROBIOME, I DON'T KNOW IF
THERE'S ANYONE THAT'S LOOKED
BACK TO SEE, NOT EVERYONE IS ON
PROPHYLAXIS, WHETHER THERE'S
CORRELATION BETWEEN PATIENTS WHO
ARE ACTUALLY ON PROPHYLACTIC
MEASURE IN TERMS OF ORAL
ANTIBIOTICS AND GETTING OPIOIDS
AND WHETHER THAT HAS ANY
INFLUENCE ON THE CHANCE THEY
WILL DEVELOP UNFORTUNATELY
ADDICTIVE BEHAVIORS?
>> GREAT QUESTION.
WE'VE BEEN TRYING TO LOOK IN THE
CLINICAL LITERATURE, HAVEN'T
BEEN SUCCESSFUL SO FAR, I'VE
BEEN HOPING TO GET SOME MORE
STUDIES GOING IN THAT.
I WOULD SAY BECCA, THE
I NSTRUCTOR, HER K AWARD IS
LOOKING AT SUBSEQUENT MODELS OF
OPIOID USE DISORDER AND SEES
LASTING EFFECTS IN THE ANIMAL
MODEL, THERE'S SMOKE IF NOT
FIRE.
>> VERY COOL TALK.
WE'LL MOVE ON, A FEW MORE
QUESTIONS IF YOU COULD ANSWER IN
THE CHAT THAT WOULD BE
FANTASTIC.
>> OF COURSE.
>> NEXT SPEAKER STEPHAN RUHL,
UNIVERSITY OF BUFFALO, STRAIGHT
FROM THE MOUTH, NATURAL
GLYCAN-BINDING TOOLS.
I'M TRYING TO GROW -- GO ON
SCREEN SHARE.
THIS SHOULD WORK.
I WOULD LIKE TO BRING THE
ATTENTION BACK TO THE ORAL
CAVITY FROM THE GUT BECAUSE IT'S
BASICALLY ONE CONTINUUM.
I WOULD LIKE TO EMPHASIZE THAT
THE MICROBIOME AND ORAL CAVITY
IS ONE OF THE MOST RICHEST
MICROBIOME IN MAMMALIAN HOSTS.
IT IS CONNECTED WITH THE GUT
MICROBIOME BECAUSE IT'S ALL ONE
CONTINUUM.
AND IT HAS BEEN KNOWN FOR QUITE
A LONG TIME AT LEAST SINCE THE
'70s AND '80s THAT GLYCAN
MEDIATED INTERACTIONS PLAY A BIG
ROLE IN THE BINDING OF MICROBES
TO EACH OTHER AND ALSO IN THE
BINDING OF MICROBES HE BINDING
OF MICROBES TO HOST SURFACES.
IS
SALIVA PLAYS AN IMPORTANT ROLE.
THEY BIND WITH PROTEIN ADHESINS
TO GLYCANS.
GLYCAN BINDING CAN MEDIATE
BINDING TO NEIGHBORING BACTERIA,
AND BEGINNING THE INITIATION OF
BIOFILMS THROUGH MICROADHESION,
A BUILDUP OF COMMUNITIES AND
EVENTUALLY BIOFILMS.
MICROBES CAN BIND TO EPITHELIAL
SURFACES VIA GLYCAN MEDIATED
DIRECTIONS.
THEY CAN ALSO CAUSE
AGLUTENNATION, BEFORE THE
MICROBES REACH HOST SURFACES,
THEY BECOME AGLUTENNATED WHICH
MAY REPRESENT BACTERIAL
CLEARANCE.
THEY CAN DECORATE THEMSELVES,
DIETARY REQUIREMENTS, ALSO MAY
SHIELD FROM HOST IMMUNE SYSTEM,
KIND OF A BACTERIAL MIMICRY,
COVERING THEMSELVES WITH HOST
MOLECULES.
LASTLY MICROBES ARE IMPORTANT
FOR INTERACTION WITH PHAGOCYTES.
MOSTLY NEUTROPHILS, STEMMING
FROM THE GINGIVAL FLUID.
THE FIRST TIME MY LABORATORY
DISCOVERED THESE GLYCAN-MEDIATED
INTERACTIONS EVER ORAL BACTERIA
WHEN WE LOOKED AT THE BINDING TO
SALIVARY GLYCOPROTEINS, WHAT YOU
SEE ON THE LEFT-HAND SIDE IS A
DOT PLOT, DEFINED GLYCAN
SEQUENCES.
WE HAVE OVERLAID THIS WITH
STRING OF STREPTOCOCCUS GORDONII
1 FLUORESCENTLY LABELED.
THESE BIND TO FETUIN, THAT
CARRIES A LOT OF FATTY ACID, BUT
NOT ISIALO-FETUIN AND BIND ONLY
TO ALPHA2-3 AS SHOWN HERE.
BUT NOT TO ALPHA2-6 LINK
GLYCOPROTEINS.
THEY DO NOT BIND TO THE
UNDERLYING GLYCAN.
SO THIS SHOWS SOME SPECIFICITY,
AND THIS SPECIFICITY BECOMES
MORE INTERESTING.
HERE ARE SECRETIONS OF PAROTID
GLAND SALIVA, SUBLINGUAL,
S UBMANDIBULA, WE SEE A BAND I
HAVES ILLIN SUBMANDIBLE, THIS
REPRESENTS THE SALIVARY MUC7,
THIS DISAPPEARED IF TREATED
PRIOR TO OVERLAY WITH THE
BACTERIA.
BINDING ALSO DOES NOT TAKE PLACE
WITH A MUTANT OF THE
STREPTOCOCCUS THAT LOST ITS
SIALIC BINDING ADHESION.
WITH THIS SPECIFICITY IN MIND,
IT WAS INTERESTING WHICH ARE
THESE ADHESINS ON THE SURFACE OF
BACTERIA THAT ARE RESPONSIBLE
FOR BINDING TO SIALIC ACIDS.
IT TURNED OUT, THIS WAS MOSTLY
THE WORK FROM PAUL SULLAM AT
UCSF, THAT THOSE ARE SYRIN-RICH,
EXTENDING FROM THE CELL WALL AND
CONTAIN A NON-REPEAT BINDING
REGION HERE..
THIS CONTAINS A UNIQUE DOMAIN
THAT BOTH ARE RESPONSIBLE FOR
BINDING TO SIALIC ACID.
YOU HAVE TO IMAGINE BACTERIA
BEING COVERED WITH FUR-LIKE
COVER OF MANY, MANY ADHESINS,
THAT MULTIVALENTLY BIND TO
CLUSTERED GLYCAN RECEPTORS ON
THE BACTERIA SURFACE,
VELCRO-LIKE PROCESS THAT I
COMPARE TO BURS STICKING TO YOUR
SWEATER OR MAYBE IF YOU HAVE A
DOG YOU KNOW HOW DIFFICULT THEY
ARE TO REMOVE, ONCE ATTACHED
THEY ARE DIFFICULT TO REMOVE.
HERE IS AFTER SCANNING ELECTRON
MICROSCOPIC IMAGE OF
STREPTOCOCCUS EARLY COLONIZING
TOOTH SURFACE, SPREAD OUT LIKE A
MONOLAYER, LATER ON PILE UP AND
FORM BIOFILMS.
ONE GLYCAN THAT IS A PREFERRED
RECEPTOR IS SIALIC ACID BECAUSE
IT IS ON MAMMALIAN GLYCANS THE
OUTER MOST, FIRST ENCOUNTERED BY
MICROBES.
SIALIC ACID IN NATURE, OCCURS
IN 50 SUBTYPES, DEPENDING ON THE
SUGAR RING OF SIALIC ACID.
A GREAT STEP FORWARD WAS THE
AVAILABILITY OF ARRAYS, SO OUR
GROUP AND ALSO GROUP OF PAUL
SULLAM FORTUNATE TO BE ABLE TO
COLLABORATE WITH THE LAB THAT
DEVELOPED AN ARRAY CARRYING
DIFFERENT SIALIC ACID SUBTYPES.
WHEN WE EXPOSEED RECOMBINANT
SIALIC ACID BINDING PROTEIN, AND
ALSO WHOLE BACTERIA TO THESE
GLYCAN-BINDING ARRAYS WE FOUND
DIFFERENT SPECTRA OF GLYCANS
BEING BOUND.
SO THERE ARE -- THERE'S
ADHESIN-A THAT BINDS TO GLYCANS,
ALSO HAVE 2, 3 LINKED TO
UNDERLYING GLYCAN BACKBONE.
OTHERS HAVE A MORE DEFINED
SMALLER SPECTRUM.
THERE'S SPECIFICITY.
WE'RE WONDERING NOW WHETHER THIS
IS SPECIFICITY MEDIATES HOST
TROPISM.
AND THERE IS ONE NATURAL ANIMAL
MODEL THAT ONE COULD EXPLORE
NAMELY DIFFERENCE BETWEEN HUMANS
AND GREAT APES.
AND THAT GREAT APES EXPRESS
SHOWN HERE, IN ADDITION.
HUMANS HAVE LOST THIS TYPE OF
GLYCAN.
THIS IS DUE TO AN ENZYMATIC
DEACTIVATION AN ENZYME
CONVERTING.
THE LOSS IN HUMANS HAS BEEN
DESCRIBED.
THIS IS THE WORK OF SEVERAL
YEARS OF MY STUDENT NINA
THAMADILOK, WE ISOLATED
STREPTOCOCCI.
OUR HYPOTHESIS WAS THAT WE MIGHT
FIND BACTERIA THAT BIND IN THE
GREAT APES BECAUSE THEY ARE
STILL ABLE TO PRODUCE THIS,
ABSENT IN HUMANS.
SURPRISINGLY, JUST THE OPPOSITE
WAS THE CASE.
WE FOUND STREPTOCOCCUS BINDING
IN THE HUMORAL CAVITY, ABSENT OR
ALMOST ABSENT IN CHIMPANZEES AND
GORILLAS.
WHEREAS OTHER TYPES OF SIALIC
ACIDS WERE STILL BOUND, IN THE
CHIMPANZEES AND GORILLAS.
THIS IS A WHOLE STORY IN ITSELF.
WE USED A SCREENING ASSAY FOR
THAT, LOOKING AT THE
DIFFERENTIAL HEMAGGLUTINATION OF
HUMAN VERSE CHIMPANZEE, STILL
HAVE ACID ON THE SURFACE, HUMAN
DEVOID OF IT.
WE WERE LOOKING FOR THIS
PHENOTYPE OF HEMAGGLUTINATION,
SHOWN BY A PANCAKE-LIKE
APPEARANCE IN THE MICROTITER
PLATE AND NOT OTHER
ERYTHROSITES.
WE HAD A THOUSAND STRAINS WHERE
WE KNEW SPECIFICITY, A POSTDOC
USED A FEW EXAMPLES FROM THESE
DIFFERENT BINDING SPECIFICITIES,
DIFFERENT PHENOTYPES, WHOLE
GENOME SEQUENCING, WITH THE
UNIVERSITY OF MARYLAND, PICKED A
FEW STRAINS FROM EACH PHYLO
GENETIC CLADE.
THIS SORTS BACTERIA ACCORDING TO
SPECIES AFFILIATION.
IF WE CONCENTRATE ON THE CYCLIC
DOMAIN WE SEE THE TREE, BRANCHES
OF THE TREE ARE DIVIDED TO THE
SIALIC ACID SPECIFICITY.
STRAINS EXCLUSIVELY PRESENT IN
ORAL CAVITY OF HUMANS CLUSTER IN
ONE BRANCH.
WE ALSO FOUND THAT AN IMPORTANT
MOTIF BECOMES MODIFIED,
DEPENDING ON SPECIFICITY SO THAT
FOR THE BINDING DOMAINS FTRT OR
YTRT PLODIFICATION OF THIS
CANONICAL MOTIF.
BEN RECOMBINANT EXPRESSED SOME
OF THIS SILGEC SUBDOMAINS AND WE
USED THEM AS PROBES, TESTED
THEM.
I SHOWED TWO EXAMPLES OF MONEY.
ONE IS A STREPTOCOCCUS ST01 THAT
BLINDS, GLYCOARRAY CONFIRMS,
BINDING TOOL.
OTHER SIALIC ACID SUBSTITUTIONS
ARE BOUND WITH MUCH LESS
AFFINITY.
HERE IS ANOTHER STRAIN.
BR3.
EXCUSE ME.
THIS IS MA6.
THAT HAS A VERY DIFFERENT
BINDING SPECTRUM, BINDS TO A 50
ACETYLATED FORM, SO IT'S A MUCH
MORE NARROW SPECIFICITY OF
SIALIC ACID BINDING SHOWING
BACTERIAL LECTINS HAVE DIFFERENT
AND VERY, VERY SPECIFIC
AFFINITIES.
SO, NEXT STEP WAS TO LOOK AT THE
3D STRUCTURE AND THIS WAS DONE
WITH TINA IVERSON AT VANDERBILT
UNIVERSITY.
AND THREE STRUCTURES WERE
DETERMINED IN COLLABORATION WITH
PAUL SULLAM'S LAB, AND WE ADDED
SY10, CR29 AND ST01,
EXPERIMENTAL STRUCTURES
DETERMINED FOR ST 701 AND SY 10,
CR 29 STRUCTURE IS ALMOST DONE.
THESE STRUCTURES ALL RESEMBLE
EACH OTHER BUT THERE ARE FINE
DIFFERENCES TINA FOUND THAT MAY
ACCOUNT FOR THE DIFFERENCES IN
SPECIFICITY.
WHAT BEN ALSO DID WAS BASED ON
THE INSIGHTS GAINED FROM TINA'S
3D STUDIES WE KNEW WHICH AMINO
ACIDS ARE ESSENTIAL FOR HOLDING
SIALIC ACID MOLECULE IN THE
BINDING POCKET AND WHEN MUTATED
AMINO ACIDS -- HE DID SINGLE
AMINO ACID SUBSTITUTION MUTANTS,
BY SUBSTITUTING ARGININE WITH
GLUTAMIC ACID.
BINDING WAS ABOLISHED.
WE HAD PROBES AND CONTROLS
IDENTICAL JUST AS THE DIFFERENCE
THAT ONE AMINO ACID HAS BEEN
EXCHANGED.
AND THIS IS AN IDEAL TOOL SET
TO EXPLORE, TO USE THESE
GLYCAN-BINDING PROTEINS IN
VARIOUS DIFFERENT SETTINGS.
WE HAVE USED THEM IN FLOW
CYTOMETRY LOOKING AT BINDING TO
HUMAN NEUTROPHILS, THIS IS
BINDING OF HSA, YOU CAN SEE IT
BIND TO NEUTROPHILS AND SIALIC
ACID IS BEING REMOVED, BINDING
IS REDUCED.
THIS IN COLLABORATION WITH NISSI
VARK USED THESE PROBES IN LECTIN
HISTOCHEMISTRY LOOKING AT TISSUE
SLIDES.
AND WE HAVE USED THEM ALSO IN
FAR-WESTERN BLOTS, SERUM
PROTEINS AND ALSO LOOKING AT
HUMAN SALIVA AND MORE RECENTLY
ALSO AT MURINE SALIVA, USING A
KNOCKOUT MOUSE SYSTEM DEFICIENT
IN IN N GLYCOAMINNIC ACID,
SHOWS CLEAR DIFFERENCE, IT BINDS
TO WILDTYPE BUT NOT KNOCKOUT
MOUSE PROTEIN.
MORE RECENTLY MY LAB IS ALSO
TRYING TO USE THESE PROTEINS IN
BIOFILMS, AND WE TRY TO LOOK AT
BACTERIAL CO-ADHESION, DONE BY
CHRISTIAN AHERN IN MY LAB.
GOING BEYOND THIS TOOLBOX, WE
HAVE ALSO DONE IN SILICO
ANALYSIS IN COLLABORATION WITH
UNIVERSITY OF MARYLAND, WHAT YOU
SEE HERE IS A PHYLO GENETIC TREE
OF STREPTOCOCCUS, REPRESENTING
ALL THE SPECIES AND STRAINS THAT
HAVE BEEN SEQUENCED.
THE GRAY STICKING OUT REPRESENTS
SUCH STRAINS THAT CARRY A SERINE
REPEAT PROTEIN.
SEVERAL STREPTOCOCCAL SPECIES
HAVE REPEAT PROTEINS BUT ONLY
THESE HIGHLIGHTED HERE HAVE
PROPANES CONTAINS AS CYCLIC,
HIGHLIGHTED IN RED, THE LONG
STICKS HERE.
THE SURPRISING RESULT WAS THAT
THE REPEAT PROTEINS THAT CARRY
CYCLIC DOMAINS CLUSTER IN ONE
REGION OF THE PHYLO GENETIC TREE
AND ALL SPECIES THAT ARE
POSITIVE HERE ARE INHABITANTS OF
THE ORAL CAVITY.
AT LEAST INHABITANTS OF MUCOSAL
SURFACES.
BUT WHAT WE DID FURTHER WAS WE
TOOK NOW THESE RED HIGHLIGHTED
REPEAT PROTEINS AND ONLY ALIGNED
THEIR CYCLIC AND UNIQUE DOMAINS.
HERE ONLY THE CYCLIC AND UNIQUE
DOMAINS ARE ALIGNED, AND
ALIGNMENT NOW DOES NOT REPRESENT
THE DIFFERENT SPECIES ANYMORE,
DOESN'T CLUSTER SPECIES, IT
CLUSTERS PHENOTYPIC ACTIVITY OF
SIALIC.
AND FOR US IT WAS VERY
INTERESTING THAT THIS THESE
BACTERIA WE FOUND BIND, CLUSTER
IN ONE BRANCH OF THE
PHYLOGENETIC TREE, BELONGING TO
THE STREPTOCOCCUS
[AUDIO DISTORTION]
SO WE PREDICT THAT OTHER BRANCH
OF THE PHYLOGENETIC TREE MIGHT
REPRESENT SPECIFICITIES FOR
OTHER SIALIC ACIDS.
SO, THESE BINDING LECTINS, WE
ENVISION AS HELPFUL IN DIAGNOSIS
OF ORAL DISEASE AND PERHAPS
EVENTUALLY ALSO AS THERAPEUTIC
WAY OF INHIBITING BACTERIAL OR
STREPTOCOCCAL ADHESION TO SIALIC
ACID COUNTER RECEPTORS IN THE
HOST.
WITH THIS THANK YOU FOR
ATTENTION.
THIS IS MY LAB IN TIMES OF THE
PANDEMIC.
HERE ARE THE COLLABORATORS WHO
CONTRIBUTED TO VARIOUS ASPECTS
OF OUR PROJECT.
WE COULDN'T HAVE DONE THIS
WITHOUT THE SUPPORT BY NIH AND
NIDCR.
THANK YOU VERY MUCH.
>> WE HAVE A QUESTION IN THE
CHAT.
IN ADDITION TO ATTACHMENT SITE,
IS THERE KNOWN PREFERENCE?
I GOT TRIPPED UP.
>> SO, THE METABOLIC PREFERENCE,
IT IS LIKELY THAT THEY MIGHT
HAVE METABOLIC PREFERENCE FOR
SIALIC ACIDS AND THAT'S ANOTHER
INTERESTING QUESTION WHICH WE
ARE ABOUT TO INVESTIGATE.
[AUDIO DISTORTION]
IT IS KNOWN THAT SOME
STREPTOCOCCUS CAN PRODUCE
SIALIDASE, SOME CANNOT, THERE'S
ALREADY KNOWLEDGE IN THE
LITERATURE BUT WE HAVE CONDUCTED
IN SILICO SEARCH FOR PRESENCE
AND ABSENCE OF SIALIDASE IN THE
GENUS STREPTOCOCCUS, I DIDN'T
BRING THIS DATA BUT MAYBE NEXT
TIME.
I'M SURE THERE'S SPECIFICITY IN
TERMS OF DIETARY REQUIREMENTS.
>> ANOTHER QUESTION, DO YOU
THINK BINDING MICROBES MIGHT
ORIGINATE FROM RED MEAT DIET OF
HUMANS?
>> AN INTERESTING QUESTION.
OF COURSE WE WERE VERY PUZZLED
BY THE FINDING THAT WE FIND
BACTERIA THAT BIND TO GLYCAN.
THAT HUMANS CAN NORMALLY NOT
PRODUCE.
[AUDIO DISTORTION]
SO THE MOST LOGICAL EXPLANATION
THROUGH DIETARY INTAKE, SO IT
COULD EITHER BE THAT THE ACID
BECOMES INCORPORATED INTO HOST
OWN GLYCANS.
HOWEVER WHEN WE LOOK AT SALIVA,
WE NEVER FOUND ANY TRACES OF
5GC, MORE LIKELY THROUGH THE
INTAKE OF ANIMAL FOOD, THIS
COULD BE MEAT, COULD ALSO BE
DAIRY PRODUCTS, N GLYCOMINI ACID
CAN GET INTO THE ORAL MICROBIOME
AND CERTAIN MIKE ROAMS --
MICROBES CAN EXPRESS ON THE
SURFACE, IT'S VERY POSSIBLE THAT
THOSE ARE THE NATURAL COUNTER
RECEPTORS FOR THOSE BINDING
STREPTOCOCCI WE HEAR IN THE ORAL
CAVITY.
>> THANK YOU FOR YOUR SEMINAR.
VERY INTERESTING.
TWO QUESTIONS.
FIRST IS IS THERE ANYTHING KNOWN
ABOUT PATHOGENETICTY OF
STREPTOCOCCUS BINDING TO SIALIC
ACID AND VARIATIONS BETWEEN
SPECIES COULD THAT BE USED TO
IMPROVE THE TOOL IF SOMETHING
BINDS MORE TIGHTLY FROM ONE
SPECIES HAVE YOU LOOKED AT
BINDING AFFINITY IS THE MAIN
POINT OF MY QUESTION.
>> THANK YOU FOR THIS
INTERESTING QUESTION.
TO ANSWER THE FIRST PART, DOES
THIS DIFFERENCE IN SIALIC ACID
SPECIFICITY PLAY A ROLE IN
PATHOLOGY, SO WE KNOW AND THIS
HAS BEEN SHOWN BY OTHERS THAT
BINDING TO SIALIC PLAYS A ROLE,
PARTICULARLY MEDIATED BY BINDING
TO PLATELETS IN BLOOD THAT THEN
ARE ACCUMULATE ON THE HEART
VALVE AND BACTERIAL VEGETATIONS
CAN FORM THERE.
WHETHER THE DIFFERENCE PLAYS A
DIFFERENCE IN THAT REGARD IS A
VERY INTERESTING QUESTION.
BECAUSE IT HAS BEEN SHOWN IN
SOME STUDIES THROUGH DIETARY
INTAKE, IT CAN BE EXPRESSED ON
THE HEART VALVE.
OUR LAB HAS NOT BEEN ABLE YET TO
DO ANY EXPERIMENTS SUPPORTING
THIS HYPOTHESIS, THIS
DIFFERENCE, THAT ONE OF THESE
BACTERIA MAY BE MORE PATHOGENIC
THAN THE OTHER.
THIS WOULD BE AN INTERESTING
NEXT STEP TO DO.
THE
>> THEY ARE DIFFERENT FOR
DIFFERENT SIALIC ACID BUT WE
HAVEN'T TESTED
[AUDIO DISTORTION]
TO DO THAT WOULD NEED TO GO TOP
SUBSTRATES AND THIS WOULD BE AN
INTERESTING NEXT STEP, TO USE
GLYCAN ARRAY.
WE'RE PLANNING TO DO THAT USING
SALIVA GLYCOPROTEINS, RELEASE
THE GLYCANS AND EXPRESS THEM ON
AN ARRAY.
AND THAT WOULD TELL THE
DIFFERENCE.
THANK YOU.
>> WE'LL MOVE TO THE NEXT
SPEAKER.
DR. MICHAEL FISCHBACH FROM
STANFORD UNIVERSITY.
>> THANK YOU.
THANKS TO THE ORGANIZERS FOR
INVITING ME.
A DIFFERENT TOPIC THAN I
ANTICIPATED, A NEW STORY WE'RE
EXCITED ABOUT, I'M LOOKING
FORWARD TO SHARING.
THIS IS ALL WORK LED BY AN
EXTRAORDINARY
PHYSICIAN-SCIENTIST IN THE LAB,
ERIN CHEN, A BIG ASSIST FROM
YASMINE BELKAID.
THE BROAD OVERARCHING QUESTION
THAT WE WERE TRYING TO ADDRESS
IN SOME PART WAS WHAT THE
IMMUNE -- HOW THE IMMUNE SYSTEM
SEES A COMMENSAL, WHETHER --
WE'RE ALL FAMILIAR WITH HOW THE
IMMUNE SYSTEM HANDLES AN ACTIVE
INFECTION BUT THE QUESTION IS
FOR COLONIZING BARRIER SURFACE,
SKIN AND GUT MAINLY, WHAT IS THE
IMMUNE SYSTEM SEEING OR DOING
THAT IT'S COMPLETELY IGNORING
THEM BECAUSE THEY ARE ON THE
OPPOSITE SIDE OF THE BARRIER OR
IS IT SOMETHING ELSE?
SO THIS BROAD QUESTION IS WHAT
GOT US INTERESTED IN THIS.
THERE'S A BUNCH OF WORK DONE
PRIOR, SUMMARIZING HERE WITH
THREE LESSONS FOR IMMUNE
MODULATION BY THE MICROBIOME,
IMPORTANT BECAUSE THEY SHAPE THE
NARRATIVE AND SPECIFIC FORM OF
THE QUESTION WE WANT TO ADDRESS.
HERE I'M GOING US TO ADAPTIVE
IMMUNE RESPONSE, ON THE T-CELL
RESPONSE THERE'S BEEN A LOT OF
WORK IN THAT AREA MORE THAN ANY
OTHER AREA AND THE FIRST LESSON
OF THE THREE IS THAT WHICH BUG
YOU'RE TALKING ABOUT DETERMINES
WHAT KIND OF T CELL COMES OUT ON
THE OTHER EN.
IF YOU COLONIZE MICE WITH STAPH
EPIDERMIDIS, YOU GET A BURST.
SAME WITH SFB EXCEPT T CELLS ARE
TH 27 AND WITH WITH THIS YOU GET
A BURST ON THE OTHER SIDE BUT
THEY ARE REGULATORY T CELLS, SO
THE NATURE OF THE BUG APPEARS TO
WHAT DETERMINE WHAT KINDS OF T
CELL IS INDUCED.
I SHOULD SAY THIS STORY I'M
GOING TO TELL IS ABOUT STAPH
EPIDEHMIDIS.
IT IS CONSERVED IN NON-HUMAN
PRIMATE, NOT SOMETHING PECULIAR
TO THE MOUSE.
T CELLS THAT RESULT EXPRESS
T-CELL RECEPTOR LOOKING FOR A
PEPTIDE THAT DERIVES, THIS
DEGREE OF SPECIFICITY YOU WOULD
ASSOCIATE WITH RESPONSE TO
INFECTION, ALSO PRETTY
INTERESTING FROM A UTILITY
PERSPECTIVE BECAUSE AS WE ALL
KNOW PEOPLE ARE GOING TO GREAT
LENGTHS TO ENGINEER ANTIGEN
SPECIFIC T CELLS BECAUSE THEY
ARE SO USEFUL.
ANTIGEN-SPECIFIC CD8 T CELLS ARE
HELPFUL IN ONCOLOGY, ALSO THE
AGENT OF KILLING FOR INFECTIOUS
DISEASE, T CELL COMPONENT OF
THAT, AND ANTIGEN-SPECIFIC
REGULATORY T CELLS HAVE BECOME
GREAT IF FOR AUTOIMMUNE DISEASE,
THE DEGREE OF SPECIFICITY HAS
CAUGHT OUR ATTENTION AND
ATTENTION OF OTHER PEOPLE IN THE
FIELD.
THE THIRD LESSON IS PERHAPS THE
MOST PROFOUND, THAT ALL THE THIS
HAPPENS WITHOUT A BREACH IN
BARRIER.
TEXT BOOK IMMUNOLOGY TELLS YOU
IN ORDER TO GET ADAPTIVE IMMUNE
RESPONSE YOU NEED INFECTION OR
VACCINATION, NEEDS TO BE
INFILTRATION OF INNATE IMMUNE
CELLS, SWELLING, A FIVE ALARM
FIRE, WHAT RESULTS FROM THAT ARE
ANTIGEN-SPECIFIC T AND B CELLS,
CANONICAL IMMUNOLOGY.
THIS IS THE BIG PIECE OF
CURIOSITY, THAT WHEN YOU
COLONIZE ANIMALS WITH CERTAIN
BUGS FROM THE MICROBIOME, YOU
GET TO THE SAME ENDPOINT,
ANTIGEN-SPECIFIC T AND B CELLS
AND IMMUNE MEMORY, BUT NO
INFECTION.
THIS IS THE PUZZLE WE'RE TRYING
TO FIGURE OUT.
TWO BIG QUESTIONS ABOUT IMMUNE
MODULATION WE AND OTHERS ARE
INTERESTED IN.
ONE IS HOW DOES IT WORK?
SECOND IS WHY DOES IT HAPPEN?
HOW AND WHY.
WE VERY MUCH STARTED OUT ON THE
HOW, AND BY HITTING A POINT OF
FRUSTRATION BECAME MORE
INTERESTED IN THE WHY.
WHY IS HOST DEVELOPING WHAT
APPEARS TO BE PREEMPTIVE
ADAPTIVE IMMUNE RESPONSE AGAINST
COLONISTS?
WHAT DO THESE CELLS FUNCTIONALLY
CAPABLE OF DOING, WHAT IS THEIR
ROLE IN LIFE?
OUR APPROACH WAS
COUNTERINTUITIVE.
I SHOULD TELL YOU ERIN'S POINT
OF ENVY TO DEVELOP A SYSTEM TO
MANIPULATE STAFF EPIDERMIDIS.
VERY FEW MUTANTS WERE MADE.
ERIN DEVELOPED A WAY TO
MANIPULATE GENOME OF STAPH
EPIDERMIDIS.
THIS REMAINS A PAUSED STORY,
INSTEAD OF TRYING TO FIGURE OUT
MECHANISM, WE'RE TRYING TO
FIGURE OUT WHAT FUNCTIONAL THESE
DO.
WE DID SOMETHING THAT'S
COUNTERINTUITIVE BUT
INTERESTING.
I TOLD YOU IN AN EARLIER SLIDE
THAT THE T CELLS THAT RESULT
FROM STAPH EPIDERMIDIS
COLONIZING A MOUSE HAVE A T-CELL
RECEPTOR, HAVE A PEPTIDE ANTIGEN
THEY ARE LOOKING FOR.
WE DECIDED TO TRICK THIS PROCESS
AND REDIRECT BY EXPRESSING TUMOR
ANTIGEN INSIDE.
WHAT WE HOPED WOULD HAPPEN IS
THAT THROUGH THE SAME PROCESS OF
COLONIZATION AND PRESENTATION
COULD GET CD8 T CELLS SPECIFIC
FOR THE BUG BUT SPECIFIC FOR
NON-NATIVE ANTIGEN, TUMOR
ANTIGEN, WOULD BE LICENSED BY
THE SAME IMMUNE PROCESS THAT
RESPONDS TO COLONIST BUT
SPECIFICITY FOR TUMOR IN MOUSE,
SEND THEM OFF TO SEE WHAT THEY
ARE CAPABLE OF DOING.
THERE WERE TWO KIND OF OPPOSITE
HYPOTHESIS.
ONE WAS THAT THESE CD8 T CELLS
WERE UNLIKE OTHER CD8 T CELLS IN
IMMUNOLOGY, MORE COLONIST
INTERACTIVE AND CALM AND WOULD
REMAIN IN THE TISSUE THAT WAS
COLONIZED, NOT DO MUCH OF
ANYTHING, A PEACE-KEEPING ROLE.
WHEREAS THE OTHER POLAR OPPOSITE
HYPOTHESIS, THEY ARE LIKE CD8 T
CELLS ELSEWHERE IN IMMUNOLOGY,
WILLING TO TRAVEL SYSTEMICALLY
THROUGHOUT THE ANIMAL, LICENSED
TO KILL.
WE WERE TRYING TO DIFFERENTIATE
BETWEEN THESE TWO POSSIBILITIES.
ERIN CONSTRUCTED A SERIES OF
STAPH EPIDERMIDIS MUTANTS, USING
PEPTIDES USED AS AN ANTIGEN IN
IMMUNOLOGY, LOTS OF TOOLS EXIST
TO DETECT.
WE DIDN'T KNOW WHERE TO EXPRESS
THIS TO ELICIT RESPONSE ERIN
TRIED CYTOPLASM OR ATTACHED TO
THE WALL OR SMALL EPITOPE ON THE
WALL AND SO ON INCLUDING
SECRETED CONSTRUCTS.
IT TURNS OUT THROUGH SOME IN
VITRO WORK THAT WE DID, WE FOUND
COMBINATION OF WALL ATTACHED AND
SECRETED EPITOPES WOULD BE
USEFUL.
WE TESTED THIS HYPOTHESIS, IN
RETROSPECT NOT THE RIGHT MODEL,
DIFFICULT TUMOR MODEL, WE TAKE
CONVENTIONALLY COLONIZED BLACK 6
MICE, NOT GERM FREE OR
PRE-TREATED, NOT SCRUBBED OR
ANTIBIOTIC TREATED OR SHAVED,
RIGHT OUT OF THE SHIPPING
CONTAINER FROM TAKONIK, INJECT
TUMOR CELLS B 16 MELANOMA FROM
THE BLACK 6 MICE, YOU CAN INJECT
IT, IMMUNE COMPETENT, NOTHING
HAS CHANGED, YOU CAN STUDY
IMMUNE RESPONSE TO TUMOR IN ITS
FULL GLORY.
B 16 MELANOMA CELLS DON'T
RESPOND WELL TO IMMUNE-DIRECTED
THERAPIES, THE TUMOR KEEPS
IMMUNE CELLS OUT EFFICIENTLY.
THIS TURNS OUT TO HAVE BEEN A
HIGH BAR TO SET.
THE BUG IS THE ONLY THING ADDED,
RUBBED GENTLY ON THE HEAD OF THE
MOUSE, THAT'S THE TREATMENT.
SO HERE WE'RE COMPARING APPLYING
NOTHING TO THE MICE VERSUS
APPLYING THE CONTROL STRAIN OF
STAPH EPIDERMIDIS WITH EMPTY
PLASMID VERSUS A WALL EXPRESSED
EPITOPE AND OTHER SECRETED
OVALBUMUNIN CONSTRUCT, THE
OVALBUMUNIN WITH LIGHTER AND
SMALLER TUMORS, JUMPING OUT AT
US.
THIS IS WHAT WE WERE HOPING TO
SEE BUT SURPRISED BY MAGNITUDE
GIVEN HOW AGGRESSIVE TUMORS ARE
WHEN THEY GROW.
ERIN WAS STRUCK BY THE FACT THE
TUMOR CELLS ARE INJECTED INTO
THE FLANK OF THE MOUSE, HIP,
GENTLY APPLYING THE BUG TO THE
HEAD OF THE MOUSE, IN A WAY
THAT'S ACTION AT A DISTANCE,
SEEING A VERY STRONG EFFECT ON
PRIMARY TUMOR SITE IN THE FLANK
OF THE MOUSE FROM A GENTLE
APPLICATION OF BUG TO THE HEAD
AND SO WE OF COURSE WERE
IMMEDIATELY INTRIGUED.
THERE ARE A COUPLE THINGS I NEED
TO TELL YOU ABOUT THAT ARE
IMPORTANT FOR UNDERSTANDING.
FIRST IS THAT IF SHE DOES
EXACTLY THE SAME THING BUT
APPLIES BUG THAT'S BEEN HEAT
KILLED WE SEE NO RESPONSE.
THIS IS NOT SIMPLY THE IMMUNE
SYSTEM RESPONDING TO WHAT WOULD
AMOUNT TO AN INERT ANTIGEN PLUS
ADJUVANT, THIS REQUIRES THE BUG
TO BE ALIVE.
THAT'S POINT ONE.
SECOND POINT, WE DID THE
EXPERIMENT DIFFERENT USING
METASTATIC TUMOR TO ANSWER THE
QUESTION, I TOLD YOU THAT THIS
SEEMS LIKE ACTION AT A DISTANCE,
COLONIZE HEAD, SEE TUMOR IN THE
FLANK GO DOWN IN SIZE.
PROBLEM WAS THAT MICE GROOM EACH
OTHER AND THEMSELVES SO WE
FIGURED THEY WERE SPREADING THE
BUG ALL OVER THEIR SKIN, MAKING
IT POSSIBLE THERE WAS BUG
GROWING ON THE SURFACE OF THE
SKIN NEAR WHERE THE SUBCUTANEOUS
SITE OF THE TUMOR WAS AND WANTED
TO FIGURE OUT DOES THE BUG NEED
TO BE NEAR THE TUMOR TO ACT.
SO IN ORDER TO ANSWER THAT
QUESTION INSTEAD OF INJECTING
TUMOR CELLS SUBCUTANEOUSLY
INJECTED INTRAVENOUSLY IN THE
TAIL VEIN, THEY DEVELOP RAMPANT
METASTASES MAINLY IN THE LUNG,
FAR FROM THE SITE.
DOES THE BUG NEED TO BE NEAR THE
TUMOR?
IT DOES NOT, VERY STRONG
EFFECT, SO THIS WAS ACTION AT A
DISTANCE.
A PIECE OF DATA I'M NOT SHOWING,
ERIN WAS ABLE TO SEE LONG
INDUCED T CELLS TRAVEL FROM THE
SKIN DRAINING LYMPH NODE TO THE
TUMOR, SEES THEM IN BOTH PLACES,
WE DO THINK OF THE IMMUNE
TRAINING HAPPENS IN THE SKIN AND
T CELLS LEAVE THE SKIN AND
TRAVEL TO THE TUMOR.
THEY GENERALIZES IN IMPORTANT
WAYS.
WE'VE BEEN USING OVALBUMUNIN AS
A MODEL BECAUSE IT'S USEFUL AND
HIGHLY IMMUNOGENIC, WE WONDERED
WHETHER THIS WOULD WORK WITH A
REAL WORLD ANTIGEN, NOT ONE
OPTIMIZED IMMUNOLOGICALLY.
INSTEAD WE USED NATURALLY
OCCURRING MUTATED PEPTIDES FROM
THE TUMOR, NEOANTIGENS, TO SEE
WHETHER THIS WOULD WORK IN
ANYTHING LIKE THE SAME WAY.
IT DOES.
ANTI-TUMOR EFFECT IS COMPARABLY
STRONG IF YOU COLONIZE MICE WITH
STAPH EPIDERMIDIS THAT EXPRESS
NEOANTIGENS INSTEAD OF
OVALBUMUNIN, NOT A PECULIARARITY
CAUSED BY OVALBUMUNIN.
FOR HISTORICAL REASONS, THIS IS
THE WAY WE SET IT UP AT THE
BEGINNING, MOST EXPERIMENTS THAT
SHOWN BEFORE NOW, I'LL CLICK
BACK TO THE PREVIOUS SITE SO YOU
CAN SEE, INVOLVE A TIME LINE
WHERE MICE ARE COLONIZED BY
STAPH EPIDERMIDIS AHEAD OF TIME
AND TUMOR CELLS ARE INJECTED
SUBCUTANEOUSLY OR I.V
B 16 MELANOMA GROWS SO FAST IF
YOU WAIT THEY GET OUT OF
CONTROL.
THE IMMUNE RESPONSE TO A
COLONIST, TO ANYTHING IF YOU'RE
LOOKING FOR ADAPTIVE T-CELL
RESPONSE, TAKES 7 TO 10 DAYS,
THE WINDOW OF TIME IS NARROW,
WITH THIS TUMOR MODEL.
IF FIRST INJECT I.V. MELANOMA
CELLS AND COLONIZE WITH STAFF
YOU GET A STRONG EFFECT.
WE DO THINK THAT IN PRINCIPLE
THIS COULD WORK, IN TREATMENT
MODE, NOT JUST IN PROPHYLAXIS
MODE.
I'LL END BY SHOWING TWO
EXPERIMENTS THAT SPEAK TO
TRANSLATABILITY.
WE NOTICED THE T CELLS INDUCED
BY STAPH EPIDERMIDIS AND END UP
IN TUMOR LOOK PARTIALLY --
EXPRESSING MARKER P1 INDICATING
THAT THEY COULD BE DEREPRESSED
FURTHER BY GIVING MICE
CHECKPOINT INHIBITORS, SO WE
TRIED A COMBINATION, SMALL DOSE,
AND ADDED THE BUG.
THE QUESTION WAS WHETHER THE
COMBINATION OF THE TWO WOULD BE
SYNERGISTIC.
AND THE ANSWER STRIKINGLY, THEY
ARE SYNERGISTIC, THE MICE NOT
ONLY IS THE SIZE OF THE TUMOR
SMALLER BUT THE TUMORS ARE IN
MOST MICE CLEARED ENTIRELY.
A
AFTER TWO WEEKS THE BUG IS GONE,
NOT A GOOD COLONIST.
FROM A SMALL EXPOSURE TO THIS
BUG MICE ARE CLEARING TUMORS
ENTIRELY WHICH LEADS TO A STRONG
GAIN IN SURVIVAL, ALL THE
UNTREATED MICE WILL END UP
DYING, ALL THE TREATED MICE WILL
END UP LIVING.
MOREOVER IF YOU TAKE THE SAME
MICE THAT CLEARED THEIR TUMOR
NOW THEY HAVE LONG SINCE STOPPED
SEEING BUG, THE BUG HAS GONE
AWAY, YOU RECHALLENGE WITH TUMOR
CELLS, TUMOR CELLS DON'T GROW.
THIS TRANSIENT EXPOSURE YIELDED
LASTING IMMUNE MEMORY, AT LEAST
LASTING ON THE TIME SCALE OF
THIS EXPERIMENT.
THIS IS PRETTY EXCITING,
SOMETHING THAT WE THINK SUPPORTS
TRANSLATABILITY.
FINALLY IF YOU COMBINE THE
TREATMENT WITH BUG IN TREATMENT
MODE, AFTER TUMOR CELLS HAVE
BEEN INJECTED, YOU GET A STRONG
RESPONSE.
SO AGAIN THIS IS SOMETHING WE
WERE SURPRISED TO SEE BECAUSE
THE THINGS YOU HAVE TO ADD TO
ANTI-PD-1 AND ANTI-CTLA-4 ARE
AGGRESSIVE.
VERSUS TREATMENT HERE, VERY
MILD, COLONIZING THE SKIN GENTLY
WITH Q-TIP WITH A BUG THAT
DOESN'T CAUSE OTHER SIDE
EFFECTS.
SO THERE'S SOMETHING INTERESTING
GOING ON HERE, JUST THAT
COMBINATION OF TREATMENTS IS
YELLING A STRONG ANTI-TUMOR
RESPONSE.
AND WE'RE REALLY EXCITED BY IT.
WE DON'T KNOW EVERYTHING ABOUT
HOW THIS WORKS, WE KNOW VERY
LITTLE ABOUT THE MECHANISM BUT
OUR MODEL TO DATE WE THINK THAT
IN THE SKIN COLONIZATION IS
YIELDING A RESPONSE, I OF WE
THINK THE COMBINATION OF THESE
TWO IS REQUIRED.
THEY THEN TRAVEL TO THE TUMOR.
I'M NOT SURE WHETHER THROUGH THE
BLOODSTREAM OR LYMPHATICS BUT
END UP THERE AND KILL TUMOR
CELLS.
LET ME COMPARE WHAT WE'VE
LEARNED ABOUT THIS COLONIST
RESPONSE TO THE MORE COMMON
INFECTION PROGRAM.
WE KNEW THE PURPOSE OF THE T
CELL COMPONENT OF ADAPTIVE
IMMUNITY IS TO KILL CELLS.
WE HADN'T REALIZED THESE ARE
ALSO LICENSED TO KILL,
PREVIOUSLY BEEN THOUGHT THEY
PLAYED ROLES THAT WERE MORE
HOMEOSTATIC AND TISSUE REPAIR
AND KEEPING THE PEACE IN THE
COLONIZED TISSUE, SO THIS WAS A
NEW FINDING.
WE LEARNED T CELLS ARE WILLING
TO TRAVEL TO OTHER PARTS OF THE
BODY AND ACT AGGRESSIVELY.
WHAT WE THINK THIS SUGGESTS IS
THAT WHAT THE HOST NEEDS TO DO
IN THE PRESENCE OF A COLONIST IS
SEEK OUT COLONIST PREEMPTIVELY
AND DEVELOP IMMUNE RESPONSE
BEFORE INFECTION HAPPENS.
THIS IS NEW.
THE HOST IS SURVEILLING BARRIER
SURFACES PRESUMABLY IT'S
INEVITABLE YOU'LL GET NICKS AND
CUTS AND SCRAPES AND BECOME
BACTEREMIC, AND SO WHAT THE HOST
IS TRYING TO DO HERE IS TO
PREEMPT ACTIVELY DEVELOP
ADAPTIVE IMMUNITY SO WHEN YOU
BECOME INFECTED, YOU WON'T DIE.
DEATH FROM STAPH EPIDERMIDIS IS
EXCEEDINGLY RARE.
WE THINK THIS IS PREVENTING YOU
FROM DYING.
IF WE TRICK THE HOST INTO
THINKING THE TUMOR IS INFECTED,
THEN THE HOST DIRECTS A VERY
AGGRESSIVE IMMUNE RESPONSE
AGAINST THE TUMOR AND THAT'S
WHAT WE THINK IS HAPPENING HERE.
THANK YOU FOR LISTENING.
I WANT TO THANK OUR FUNDERS AND
FOLKS IN THE LAB.
HAPPY TO TAKE QUESTIONS.
>> I DON'T SEE QUESTIONS IN THE
CHAT.
I HAVE A QUESTION.
THERE'S TWO STAFF VARIATIONS,
ONE TETHERED TO SURFACE, ONE
SECRETING.
IF YOU JUST HAVE SECRETION
VERSION, YOU CAN MIX WITH STAPH
IS IT HAVING STAPH PRESENT ABLE
TO TAKE UP STAPH IN THAT
ENVIRONMENT, IS IS THAT
SUFFICIENT OR DOES IT HAVE TO BE
TETHERED TO SEE IF IT'S CAPABLE
OF ELIMINATING THE MELANOMA
MODEL?
>> WE TRIED TO ADD ANTIGEN NOT
COVALENTLY TETHERED, THAT
DOESN'T WORK.
WE HAVEN'T NAILED WHAT THE
ANTIGEN REQUIREMENTS ARE BUT
THINK YOU NEED MINIMALLY CD8 AND
CD4 EPITOPE FOR B 16 MELANOMA AT
LEAST, MAYBE DIFFERENT FOR
OTHERS BUT IN THIS CASE YOU NEED
BOTH.
SOMETHING PEOPLE HAVE SEEN WITH
CAR T CELLS, I DON'T KNOW THE
MINIMAL ANTIGEN REQUIREMENTS.
>> THERE'S A FEW QUESTIONS IN
THE CHAT.
WHAT'S YOUR BEST GUESS HOW IT
WORKS AT A DISTANCE ACROSS THE
BARRIER?
>> THAT'S A GREAT QUESTION.
WE DON'T KNOW AND WOULD LOVE TO
KNOW.
THE WAY I RESTATE, BEGGING
FORGIVENESS, WHAT IS THE
MECHANISM OF SURVEILLANCE, HOW
DOES THE IMMUNE SYSTEM SEE?
THE ANSWER IS I DON'T KNOW.
SOMEHOW, IT COULD BE THROUGH
DENDRITIC CELL OR ANTIGEN
PRESENTING CELL, REACHING
THROUGH THE EPITHELIUM INTO A
FOLLICLE AND GRABBING CELLULAR
MATERIAL BUT THERE'S A SPECIAL
QUESTION HERE, CD8 T CELLS, HOW
THEY ARE INDUCED.
THOSE USUALLY ONLY RESULT FROM
INFECTION INSIDE THE CYTOPLASM,
LIKE LISTERIA OR A VIRUS, SO IT
IS ATTRACTIVE TO SPECULATE THIS
BUG ENDS UP SOMEHOW STUCK INSIDE
OF A CELL.
YOU DON'T THINK OF YOUR COLONIST
ACTING ON THE OTHER SIDE OF THE
BARRIER WALL ALIVE BUT WE THINK
THAT MAY BE HAPPENING HERE AND
THE BUG MAYBE ENDS UP STALLED IN
ENDOSOME.
I THINK THE NICHE MAY BE CLOSER
TO HOME THAN WE INITIALLY
THOUGHT.
SETH, DO YOU HAVE A QUESTION?
>> GREAT WORK.
SO IMPORTANT.
I THINK YOU'RE WORK -- TWO
QUESTIONS.
HOW MUCH OF A LOCAL -- HOW MUCH
LOCAL INFORMATION AND/OR TISSUE
DAMAGE DO YOU THINK THE TUMOR IS
DOING, LEADING TO RECRUITMENT OF
THE ANTI-TUMOR T CELLS?
TWO, RELATED TO HYPOTHESIS OF
SENSING PREDICTIVE SENSING FOR
FUTURE RESPONSES DO YOU THINK
THAT SIMILAR THINGS ARE
HAPPENING IN THE GUT FOR
ANAEROBES MAY NOT BE HAPPENING
FOR PREDICTIVE RESPONSES TO
POTENTIAL PATHOGENS?
>> GREAT QUESTIONS.
FIRST QUESTION IS REALLY
INTERESTING.
I'VE NEVER BEEN ASKED THAT
BEFORE.
I DON'T KNOW -- IT'S AN
ARTIFICIAL PROCESS WHEN YOU GIVE
A SYNGENEIC TUMOR CELLS, IT'S
DIFFERENT IN HOW THE TUMOR
GROSS, MICROENVIRONMENTS ARE
DISTINCT.
FOR THE INTRAVENOUS MODEL,
METASTASES IN LUNG, I WORRY
LESS, I THINK THE PROCESS OF B
16 CELLS SETTING UP SHOP IN THE
LUNG IS DAMAGING TO THE TISSUE,
IT'S A REALLY INTERESTING
QUESTION AND IT STANDS LIKE WHAT
ROLE DOES THAT PLAY IN IMMUNE
CELL RECRUITMENT?
THE OTHER QUESTION YOU ASKED,
WHICH IS TO WHAT EXTENT WE THINK
THIS IS HAPPENING IN THE GUT, I
THINK IT'S HAPPENING A LOT
THROUGH ANOTHER PROJECT JUST
WRAPPING UP IN THE LAB, I
SUSPECT THERE'S A LOT OF -- A
LOT OF WHAT WE'RE SEEING FROM
HELICOBACTER AND OTHERS IS THE
SAME THING, PREEMPTIVE,
IMMUNITY, HOW THE HOST DEALS
WITH DIVERSITY OF BUGS AND
CHOOSES WHICH TO DEVELOP
ADAPTIVE IMMUNE RESPONSE TO IS
THE BIG QUESTION..
>> TO ADD TO THAT WHAT IS A
COMMENSAL, WHAT IS A PATHOGEN,
AND ARE BUGS ATTACHING WITH
PATHOGENIC FEATURES, HOW MUCH
ARE PROGRAMMING.
>> FANTASTIC QUESTION.
I DON'T THINK THE HOST HAS LIKE
A SEQUENCER, OR LIKE THERE'S NO
VOICE SAYING THIS ONE IS A
PATHOGEN, BETTER WATCH OUT.
>> ATTACHMENT RECOGNITION
SYSTEMS.
>> THAT'S TOTALLY IT.
THE HOST JUST SAYS WHOEVER YOU
ARE, WHATEVER YOU ARE, IF YOU
SHOW UP IN THIS NICHE YOU'RE
CLOSE ENOUGH TO TROUBLE THAT I'M
GOING TO SURVEIL AND DOING THAT.
THAT'S NICHE DEPENDENT.
IF YOU'RE IN THAT PLACE AT THAT
TIME, EVERYTHING IS QUIET, WE'LL
DEVELOP A CALM PROPHYLACTIC
RESPONSE.
IF IT'S NOT QUIET, BREACH IN
BARRIER, IMMUNE SYSTEM
RESPONDING TO A BREACH IN
BARRIER COULD GIVE YOU SOMETHING
LIKE CHRONIC INFLAMMATION.
>> THANK YOU SO MUCH FOR THAT
TALK.
WE'RE BEHIND BUT ON TIME ALMOST
FOR 1:00 TALK.
I'M GOING TO TURN THIS OVER TO
KATHERINE.
THESE WILL BE RECORDED SO IF YOU
NEED TO TAKE A BREAK, AS I DO,
GRAB LUNCH, I ENCOURAGE YOU TO
DO SO.
AND WE CAN ONCE AGAIN PROVIDE
LINKS AND SO FORTH SO EVERYONE
CAN WATCH THE TALK LATER ON.
I'LL TURN THIS OVER TO
CATHERINE.
>> WE'RE ON TIME.
WE PLANNED THAT LUNCH BREAK
PERFECTLY.
FOR THE QUESTIONS ASKING ON THE
ONLINE CHAT PLEASE KEEP THEM
GOING.
PRESENTERS ARE GETTING COPIES OF
THE QUESTIONS AND THEY WILL
RESPOND VIA E-MAIL.
AN OPPORTUNITY TO ENGAGE WITH
THE PRESENTER ONLINE THROUGH
E-MAIL AS WELL WILL FOLLOW.
PLEASE KEEP THE QUESTIONS
COMING.
WE APPRECIATE THE VIRTUAL
QUESTIONS COMING IN AS WELL.
THANK YOU SO MUCH.
NOW I'M GOING TO INTRODUCE OUR
NEXT SPEAKER, RICHARD CUMMINGS,
INTERRELATIONSHIPS OF INTESTINAL
GLYCOME.
HOPEFULLY YOU KNOW HOW TO SHARE
A SCREEN.
PERFECT.
>> THANK YOU FOR THE
OPPORTUNITY.
I'M EXCITED ABOUT THIS.
HEARD FANTASTIC TALKS TODAY.
I WANT TO FOCUS ON AN ISSUE NOT
DEALT WITH YET, THE RELATIONSHIP
BETWEEN HOST GLYCOME, MICROBIAL
GLYCOME, INTESTINAL CELLS,
INNATE AND ADAPTIVE IMMUNE
SYSTEM, OTHER GLYCAN BINDING
PROTEINS EXPRESSED BY US AND THE
MICROBES.
SO, IN THAT REGARD I WANT TO
FIRST ACKNOWLEDGE SOME REALLY
GREAT COLLABORATORS ON THIS
PROJECT, I'LL JUST MENTION THEIR
NAMES HERE.
THESE ARE THE COLLABORATORS I'VE
BEEN WORKING WITH OVER THE YEARS
LOOKING AT THESE ISSUES ABOUT
THESE RELATIONSHIPS.
A MUCIN IS HEAVILY GLYCOSYLATED
AND MAY HAVE A VARIETY OF
GLYCANS, HUNDREDS IF NOT
THOUSANDS OF GLYCANS.
WHEN THERE'S DEFICIENCY IN
GLYCOSYLATION OF PROTEIN, A
MUCIN, IT CAN RESULT IN
TREMENDOUS COLLAPSE OF LARGE
NUMBER OF GLYCANS, LOSS OF KEY
MONO SACCHARIDES, KEY CHANGES,
NOT JUST IN STRUCTURE BUT
STABILITY OF THE PROTEIN.
DEFICIENCIES OF GLYCOSYLATION
ARE ASSOCIATED WITH PATHOLOGY,
WE NEED TO KEEP THAT IN MIND AS
WE GO FORWARD.
I WANT TO FOCUS ON THE OLD
GLYCOSYLATION.
BASICALLY IN THE GOLGI, SINGLE
MONO SACCHARIDE, THERE'S ONLY
ONE ENZYME THAT INITIATES CORE
DERIVED O-GLYCANS, THAT ENZYME
IS REGULATED BY A CHAPERONE,
COSMC.
THE COSMC IS ENCODED ON X
CHROMOSOME, THIS FUNCTION IS AS
A KEY MOLECULAR CHAPERONE FOR
ONE PROTEIN.
THIS IS GOING TO COME UP LATER
AS I TALK ABOUT CHILDREN WITH A
MUTATION IN THIS GENE ENCODING
COSMC, IMPORTANT TO UNDERSTAND
HOW THE GLYCOME IS REGULATED
THROUGH THE SIMPLE INTERACTION
IN THE E.R.
PRODUCTION IS REGULATED BY
PROTEIN COSMC, IF NOT FUNCTION
YAM P SYNTHASE WILL DEGRADE,
GLYCOPROTEINS WILL BE SUFFICIENT
IN TERMINAL STRUCTURES, THE
LARGE TABLE I SHOWED YOU.
THERE'S A COLLAPSE OF THE O
GLYCOME.
I'D LIKE TO FOCUS ON WHAT
HAPPENS WHEN YOU LOSE THE O
GLYCAN, CALL THEM THE EXTENDED
O-GLYCANS.
WE TALKED ABOUT THE PRODUCTION
OF THE TN ANTIGEN IN TUMORS, AND
THAT'S A SPECIAL EPITOPE
EXPRESSED MOST CARCINOMAS.
IT'S NOT A BIOMARKER, IT'S A
DRIVER OF TUMORIGENESIS.
THE REALITY WHEN COSMC AND T
SYNTHASE IS COMPROMISED THERE'S
LOSS OF O-GLYCANS.
THERE ARE CORE 3 AND CORE 4
STRUCTURES WHICH HAVE BEEN
STUDIED AND THEY CAN ALSO BE
EXPRESSED IN THE INTESTINE.
CORE 1, 2, 3, 4 AND OTHERS IN
HUMANS VERSUS MICE, WE HAVE TO
ACKNOWLEDGE UNDERSTANDING OF THE
MOUSE GLYCOME IS PARTIALLY
REFLECTIVE OF THE HUMAN GLYCOME.
COSMC IS ON THE X CHROMOSOME,
THERE ARE MANY IMPORTANT GENES
REGULATING GLYCOSYLATION THAT
HAVE BEEN IDENTIFIED ON THE X
CHROMOSOME.
THAT'S ALSO VERY INTERESTING
AREA OF BIOLOGY, SO KEEP THAT IN
MIND.
THE GENES HAVE BEEN FOUND TO BE
ALTERED IN HEMATOPOIETIC CELLS
OCCURRING IN HEMATOPOIETIC STEM
CELLS MORE SO THAN ALMOST ANY
OTHER STEM CELL POPULATION.
A FEW YEARS AGO A WONDERFUL
REVIEW WAS PUT TOGETHER,
DESCRIBING THE PROTEIN
GLYCOSYLATION OF MUCINS AND
GLYCOPROTEINS THAT REGULATE
HOMEOSTASIS AND GUT MICROBIOTA,
EXPRESS OF COSMC PROTEIN OR LOSS
OF EXPRESS LEADING TO CHANGE IN
MICROBIOME.
WE CREATED A MOUSE WITH A
KNOCKOUT IN THE INTESTINAL
EPITHELIAL CELLS DRIVEN BY THE
BILLEN PROMOTER, EXPRESSES TN
ANTIGEN, MALES EXPRESS
EXTENSIVELY SHOWN IN THE MIDDLE
PANEL HERE.
IN THE COLORECTAL REGION AND
SMALL INTESTINE, THE FEMALES
BECAUSE OF THE X-LINKAGE ARE
MOSAIC, YOU CAN SEE EXPRESSION
VERY INTERESTING IN THE FEMALES.
IF YOU DO A SWISS ROLL YOU CAN
SEE IN WILDTYPE VERSUS KNOCKOUT
MICE, THERE'S TREMENDOUS
THICKENING OF THE CLONIC WALL,
DIFFERENT BETWEEN THE WILDTYPE
AND COSMC KNOCKOUT, SO THAT
SINGLE MUTATION LEADING TO A
LOSS OF THESE O-GLYCANS HAS LED
TO A MASSIVE CHANGE IN THE
MORPHOLOGY OF THE ORGAN ITSELF.
EXPRESSION OF FUCOSE, AN
INTERESTING SUGAR A LOT OF
LABORATORIES ARE STUDYING IN THE
INTESTINAL SUGAR, IF WE TAKE THE
LECTIN THAT STAINS, WE SEE
FUCOSE IS EXPRESSED IN SMALL AND
LARGE INTESTINE BUT IN THE COSMC
KNOCKOUT CELLS IT'S MOSTLY LOST
IN THE COLON, MOSTLY THEREFORE
MOST O-GLYCANS, MOST FUCOSE ON N
GLYCANS IN THE INTESTINE,
DIFFERENTIAL EXPRESSION OF
SIMPLE EPITOPES WITHIN THE
DISTRIBUTION IN THE INTESTINE,
THIS IS NOT WELL UNDERSTOOD,
IT'S POORLY UNDERSTOOD IN REGARD
TO SPATIAL AND TEMPORAL
REGULATION OF GLYCOSYLATION AND
THIS SIMPLE EXPERIMENT
ILLUSTRATES THAT.
YOU CAN SEE MANY CELLS DO NOT
EXPRESS FUCOSE DETECTABLE,
DETECTABLE FUCOSE EPITOPES.
IF YOU ELIMINATE EXTENDED O
GLYCAN YOU CHANGE THE STRUCTURE
OF MICROVILLI, THIS IS IS
FANTASTIC WORK.
THE ANIMALS DEVELOP A LOT OF
INFLAMMATION AS A RESULT.
SO THE GLYCOME IS REGULATED TO
SOME EXTENT BY THE PRODUCTION OF
THE O-GLYCANS.
THE LOSS OF INTEGRITY OF THE
O-GLYCANS AND MUCINS IN THE
COSMC KNOCKOUT CELLS LEADS TO A
LOSS OF BARRIER FUNCTION BETWEEN
THE LUMINAL CONTENTS AND
EPITHELIAL CELL LAYER.
THIS IS NOT A MUTATION OF THE
MUCINS BY THE WAY.
THE MUCINS ARE STILL EXPRESSED.
THIS IS A LOSS OF THE
GLYCOSYLATION OF THE MUCINS.
THEY ARE STILL AROUND BUT NO
LONGER FUNCTIONING IN A BARRIER
WAY.
THIS IS POORLY UNDERSTOOD IN
TERMS OF HOW A PROTEIN CAN BE
THERE, NOT A BARRIER, STILL HAS
SUGARS ON IT.
THIS IS A SPECIFIC LOSS NOT OF
GLYCANS, IT'S A CHANGE IN
STRUCTURE OF GLYCANS AND LOSS OF
EXTENDED GLYCANS LEADING TO
CHANGE IN THE BARRIER FUNCTION
EPITHELIAL CELL LAYER.
LOOK AT THE POPULATION OF THE
COLON BY BACTERIA, CLONIC MUCOSA
CHANGED TREMENDOUSLY, THERE'S
LOSS OF BACTEROIDES.
THIS DID NOT CHANGE HOWEVER IN
THE ILIUM MUCOSA.
THERE WERE SOME CHANGES, THEY
WERE MUCH LESS.
LUMINAL CONTENTS DID NOT CHANGE
AT ALL SIGNIFICANTLY.
THERE ARE DIFFERENTIAL EFFECTS.
THERE'S A CHANGE IN HOST GENES,
IMPORTANT IN MICROBIOTA.
THE CONSEQUENCE OF ALTERING THE
SINGLE GENE, OVERALL THE
REGULATED FUNCTION OF THE
O-GLYCANS IS TO CONTROL THE
RELATIONSHIPS OF INFLAMMATION,
UNFOLDED PROTEIN RESPONSES,
MICROBIOME, TUMORIGENESIS AND SO
FORTH, ACCENTUATED WHEN ONE
CHANGES THE GLYCOME OF THE
INTESTINE.
WORK WITH SETH'S GROUP AND
STEPHEN SEGAL LOOKING AT
GLYCOME OF THE INTESTINE OF THE
MOUSE.
WE'RE NOT GROWING TO GO OVER IT
EXTENSIVELY.
THERE ARE BIG CHANGES IN THE
FUCOSLYATED AND SIALYATED
O-GLYCANS.
THE REPERTOIRE IS SUBJECT TO
CHANGE, TO BE MODIFIED BY THE
INFLAMMATORY REACTION, ALSO MAY
BE SUBJECT TO CHANGE BY THE
MICROBIOME.
THAT WILL BE STORIES THAT ARE
COMING OUT LATER FROM SETH'S
LAB.
GENERALS THAT REGULATE
GLYCOSYLATION HAVE BEEN
IMPLICATED IN INTESTINAL BOWEL
DISEASES, NOT WELL EXPLORED IN
THE BIG PICTURE OF IBD AND
CROHN'S.
WE COULD SAY P53 GENE, GUARDIAN
OF THE GENOME, WE ACCEPT THAT.
IT'S IMPORTANT IN REGULATING
GENOMIC STABILITY, PREVENTING
APOPTOSIS, EXHIBITING TO
ANGIOGENESIS, ET CETERA.
BUT WE CAN THINK OF O AND
N-GLYCANS, GENES THAT REGULATE
STRUCTURES AS GUARDIANS OF THE
CELL BECAUSE THEY ARE
ALTERATIONS LEAD TO TREMENDOUS
PATHOLOGY, CHANGES IN CELL
ADHESIONS SIGNALING
LYMPHANGIOGENESIS.
WE MUST IDENTIFY THE INDIVIDUAL
FUNCTIONS OF GLYCANS OF COURSE
AND INDIVIDUAL FUNCTIONS OF
GENES BUT THINK THE STORY IS
VERY CLEAR.
THE HOST GLYCOME IS IMPORTANT IN
MAINTAINING NORMAL HOMEOSTASIS.
WE'VE IDENTIFIED SEVERAL
CHILDREN WITH A MUTATION IN THE
COSMC GENE.
THIS WAS DONE WITH OUR
TREMENDOUS COLLABORATORS IN
COLOGNE, IDENTIFIED A FAMILY
THAT INCLUDES TWO CHILDREN, TWO
BROTHERS, THAT HAVE A HEMIZYGOUS
GERMLINE MUTATION, THAT CHANGES
THE STRUCTURE OF THE
TRANSMEMBRANE DOMAIN ABOUT THE
COSMC PROTEIN.
THE BOYS PRESENTED WITH
DEVELOPMENTAL DAY, SHORT
STATURE, SEVERE
IMMUNODEFICIENCY, BOTH HAVE
ACUTE KIDNEY FAILURE, TREATABLE
WITH ECULIDUMAB, THIS PATHWAY
CRITICAL FOR IgA NEPHROPATHY,
ASSOCIATED WITH INSUFFICIENT
GLYCOSYLATION OF IgA 1.
ONE HAS BEEN HOSPITALIZED
REPEATED LIE.
MANY PATIENTS WITH CONGENITAL
DISORDERS OF GLYCOSYLATION HAVE
GASTROINTESTINAL COMPLICATIONS
INCLUDING DIARRHEA AND RECURRENT
INFECTION, BY THE WAY.
THIS SHOWS THE PEDIGREE OF THE
FAMILY, BOTH FEMALES AND
GRANDMOTHER AND MOTHER ARE
CARRIERS OF THIS MUTANT BUT THEY
ARE ALSO AFFECTED BECAUSE
THERE'S X SKEWING.
THEY WOULD BE A 50-50 MOSAIC,
IT'S NOT LIKE THAT.
THEY ARE SKEWED TOWARD UTILIZING
MUTATED X CHROMOSOME WHICH FROM
A BIOLOGICAL POINT OF VIEW IS
VERY INTERESTING HOW DOES X
SKEWING HAPPEN AND IS THERE A
PRIVILEGE BY HAVING THE
EXPRESSION OF THE TN ANTIGEN IN
CELLS.
THIS MUTATION RULEDDED IN CHAIN
OF ALANINE RESIDUE AT C 20,
LEADS TO A LOSS OF FUNCTION OF
COSMC, CHANGE IN STABILITY,
LEADS TO DECREASE OF THE TOTAL
PROTEIN AND THE T SYNTHASE IS 2
TO 3% OF NORMAL ACTIVITY.
THAT 2 TO 3% ALLOWS 90% OF O
GLYCANS, THAT'S ON YOU THEY
SURVIVED, MAJORITY ARE NORMAL
BECAUSE OF THE SMALL AMOUNT OF
ACTIVITY.
WE ARE HOPEFUL AND RAJ IS
PURSUING THIS MIGHT BE TREATABLE
BY MONO SACCHARIDE THERAPY.
THE LAST THING I WANT TO TALK
ABOUT IS FLY -- GLYCOME, WE
HAVEN'T ABOUT IMMUNOGLOBULIN, A
MAJOR COMPONENT.
IgA IS PROMINENT IN THE GUT
LUMEN, FUNCTIONS IN HOST
COMMENSAL SYMBIOSIS, DEFICIENT
MICE AND HUMANS ARE SUBJECT TO
GI INFECTION AND IBDs.
WE'VE LOOKED AT PRESENCE OF
IgA 1, IgA 2, RECOGNITION OF
GLYCANS.
WE EXAMINED A LOT OF SPECIFICITY
OF IgA 1, BROADLY SPEAKING
MANY OF THE ANTIBODIES
RECOGNIZED FEATURES OF GLYCANS,
WITH TERMINAL N GLUCOSAMINE, BUT
THEY HAVE MANY DIFFERENCES IN
RECOGNITION OF GLYCANS, ONLY 15
GLYCANS WERE CO-TARGETED.
MANY GLYCANS ARE KNOWN TO BE
ATTACHMENT SITES FOR BACTERIA,
THEY ARE PREDICTED TO BE.
THAT'S INTERESTING BECAUSE THE
IgA MAY ALSO BE INTERACTING
WITH HOST GLYCANS, AND AFFECTING
COLONIZATION AND STABILITY OF
THE MICROBIOME ITSELF.
MOST OF THE BACTERIA WE COULD
IDENTIFY WERE ASSOCIATED WITH
SURFACE-BOUND IgA.
WE CAN DEVELOP MONOCLONAL
ANTIBODY, CELLS PRODUCING
DIFFERENT MONOCLONAL ANTIBODY
IgA 2, ALL WERE IgA 2 BY THE
WAY, THIS SHOWS A PATTERN ON A
MICROARRAY, RECOGNIZING A SET OF
GLYCANS WITH OVERLAP, VERY
INTERESTING BECAUSE MOST
IgGs HAVE SPECIFIC BINDING,
IgAs SEEM TO BE MORE PATTERN
RECOGNITION MOLECULES.
SECRETORY COMPONENT, REQUIRED
FOR THE TRANSFER OF IgA INTO
THE LUMEN, SECRETORY COMPONENT
IS ABLE TO BIND CARBOHYDRATES.
BEFORE THIS WORK I NEVER THOUGHT
MUCH ABOUT IT BUT IT'S A
PROTEOLYTIC CLEAVAGE PROJECT OF
THE IMMUNOGLOBULIN RECEPTOR
SUGGESTING SECRETORY COMPONENT
HAS ABILITY TO BIND SELECTIVE
GLYCANS.
IT DOES RECOGNIZE A PATTERN.
THIS IS SOMETHING WE WANT TO
PURSUE IN THE FUTURE, WHAT IS
THE OVERALL REPERTOIRE OF IgA
ANTIBODIES IN THE INTESTINE,
WHAT DO THEY RECOGNIZE AND WHAT
IS THEIR PATTERNS OR SPECIFIC
LIGANDS THEY BIND TO.
I WANT TO MENTION SOME BEAUTIFUL
WORK THAT WE'RE GOING TO HEAR
ABOUT LATER FROM KATERINA, WE'VE
BEEN FORTUNATE TO WORK WITH HER
IN JUST LOOKING AT SOME ROLES OF
MUCIN GLYCANS ON THE ROLES OF
GLYCANS RECOGNIZED BY BACTERIA.
WE THOUGHT ABOUT BACTERIA THAT
ADHERE TO MUCINS, BUT KATERINA
HAS BEEN LOOKING BEYOND IN A AT
THE INTERACTIONS OF BACTERIA
WITH OLIGOSACCHARIDES OF MUCINS
AND THEY FUNCTION AS A SIGNALING
WAY.
THIS IS THE ONLY SLIDE I'LL
SHOW, THE HOST GLYCOME IS
BIOACTIVE NOT JUST AS ADHESION
AND BARRIER FUNCTION BUT
REGULATORY FUNCTION.
I'LL END BY SAYING WE HEARD
ABOUT THE CROSSROADS BUT THE
ROADS CROSS.
ALL OF THESE INTERACTIONS ARE
OCCURRING SIMULTANEOUSLY, AND
IT'S HARD TO WRAP YOUR HEAD
AROUND ALL THE INTERACTIONS
GOING ON, I'M JUST TALKING ABOUT
A FEW.
WE'VE HEARD ABOUT OTHER
ADHESIONS AND GLYCAN SENSORS AND
LECTINS THAT ARE INVOLVED.
THAT'S THE STORY.
WE HAVEN'T TALKED ABOUT DIETARY
GLYCANS WHICH WILL BE
INFLUENTIAL IN MANY AFFECTS
ALSO.
I'D LIKE TO ACKNOWLEDGE ALL THE
WONDERFUL PEOPLE IN MY LAB.
THANK YOU FOR LISTENING.
>> BEAUTIFUL WORK AS ALWAYS,
RICK.
THANK YOU FOR REMINDING US THEY
INTERSECT.
I'LL BE MONITORING THE CHAT SO
PLEASE GET THOSE IN.
>> I WAS CURIOUS WHETHER OR NOT
YOU THINK THAT MAYBE LOSS OF THE
CHAPERONE MIGHT ALSO CHANGE WHAT
SOME OF OUR HOST LECTINS
RECOGNIZE, THAT MIGHT BE ALSO
PROBLEMATIC TO THE HOST.
>> I THINK YOU'RE EXACTLY RIGHT.
I WOULD PREDICT IT CHANGES IgA
BINDING, LECTIN BINDING, A LOT
OF THE HOST ADAPTIVE AND PERHAPS
INNATE IMMUNE RECEPTORS THAT
PROBABLY INTERACT WITH ALL THOSE
GLYCANS.
THINK IT'S ESPECIALLY
INTERESTING IN CDGs, BY THE
WAY, IN GENERAL WHERE THE
DIFFERENT N AND O-GLYCANS HAVE
BEEN CHANGED IN EXPRESSION,
ASSOCIATED WITH GUT HOMEOSTASIS,
SO THE CLUES ARE THERE.
I WOULD SAY THE CLUES ARE THERE.
SPECIFICITY IS NOT SO CLEAR BUT
THE CLUES ARE STRONG THAT YOU'RE
ABSOLUTELY RIGHT.
A CHANGE IN HOST GLYCOME WILL
CHANGE THE MICROBIOME ITSELF.
>> I WAS THINKING ABOUT IT
BECAUSE MBL HAS ALL THOSE ROLES
IN COMPLEMENT, YOU MENTIONED
THAT.
THERE ARE A NUMBER OF LECTINS
THAT CAN CONTRIBUTE TO THE
PATHWAY, USEFUL TO LOOK
INCISIVELY AT WHAT MIGHT BE IN
THERE.
>> YOU'RE ABSOLUTELY RIGHT.
THERE'S A WHOLE ASPECT OF
COMPLEMENT INVOLVEMENT IN SOME
OF THE CDGs THAT HAVE NOT BEEN
WELL STUDIED.
THANK YOU.
>> GREAT WORK.
IN THE INTEREST OF TIME WE'LL
MOVE ON TO OUR NEXT SPEAKERS.
WITH ALL THE SPEAKERS, I'M
E-MAILING THE QUESTIONS.
PLEASE ASK A QUESTION.
I'M GOING TO INTRODUCE THE NEXT
SPEAKER, NEXT SPEAKER WILL TALK
ABOUT DISRUPTION OF MICROBIOTA.
>> CAN YOU HEAR ME?
>> YES.
>> THANK YOU FOR THE INVITATION
TO PRESENT MY WORK.
I'M GOING TO TALK ABOUT
INTERACTION TO PROTECT HOST AND
SOMETIMES TO CAUSE DISEASE.
A BROAD VIEW OF MY LAB, HOST AND
MICROBIOTA ARE TALKING TO
PROMOTE HEALTH.
WHAT HAPPENS WHEN BENEFICIAL
INTERACTION GETS DISRUPTED AND
MAY LEAD TO HUMAN DISEASE.
WHAT WE KNOW IS THAT DISRUPTION
OF THE GUT MICROBIOTA HAS BEEN
ASSOCIATED WITH A LOT OF HUMAN
DISEASES FROM NECROTIZING
INTERCOLITIS IN INFANTS, IBS,
IBD, THE LIST GOES ON.
ONE PARTICULAR ASPECT OF
DYSBIOSIS, IMBALANCE, THAT IS
ASSOCIATED WITH DISEASE IS THAT
THIS -- I'M TALKING SPECIFICALLY
FOR THE LARGE BOWEL.
WHAT WE SEE IS THAT MICROBIC
COMMUNITY STARTS TO SEE
EXTENSIONEN ANAEROBIC BACTERIA,
WHAT MY LAB IS INTERESTED IN.
WE'RE INTERESTED IN
PROTEOBACTERIA BECAUSE THEY
BEHAVE DIFFERENTLY.
THEY ARE NOT ABLE TO DIGEST
FIBER THE SAME WAY A LOT OF
MICROBES ARE, SOME ARE
PATHOGENIC.
I'M GOING TO REFER TODAY MOSTLY
TO E. COLI STRAINS.
WHAT IS INTERESTING, THEY HAVE A
DIFFERENT METABOLISM THAN
ANAEROBIC BACTERIA.
THEY CAN RESPIRE OXYGEN, BUT
THEY CAN ALSO PERFORM ANAEROBIC
RESPIRATION, IN THE ABSENCE OF
OXYGEN, ANAEROBIC ENVIRONMENT
CAN USE OTHER IONS TO RESPIRE
AND GROW.
WHAT'S INTERESTING, THEY PREFER
OXYGEN FOR RESPIRATION BUT RIGHT
BELOW THERE'S TWO OTHER
RECEPTORS THEY LIKE, AND WE
KNOW BY WORK FROM OUR GROUP AND
OTHERS THAT NITRATES AND
NITRITES ARE PRESENT IN THE GUT.
OUR GROUP HAS PROPOSED A
MICROBIAL SIGNATURE OF
DYSBIOSIS.
WHAT WE WANTED TO DO FOR MY WORK
AS A POSTDOC BEFORE I STARTED MY
LAB TO USE SOME OF OUR KNOWLEDGE
TO TRY TO UNDERSTAND WHY.
THIS IS PUBLISHED WORK.
WHAT WE FOUND USING A MODEL OF
ANTIBIOTICS IN THE HEALTHY GUT
ON THE LEFT WHAT HAPPENS IS THAT
THE HEALTHY MICROBIOTA PROTECTS
THE HOST, BUTYRATE INCREASES
OXIDATIVE METABOLISM OF THE
INTESTINE EPITHELIUM, THROUGH
ACTIVATION OF NUCLEAR RECEPTOR.
ANTIBIOTICS KILL THESE,
INCREASING INFLAMMATORY CONTENT,
ALSO DECREASES OXIDATIVE
METABOLISM LEADING TO INCREASED
OXYGENATION OF THE GUT, AND
USING THIS OXYGEN TO GROW.
MAYBE
THIS COULD BE A SIGNATURE,
EPITHELIUM NOT FUNCTIONING
PROPERLY.
THERE'S A LOT OF DISEASES WE
COULD HAVE FOCUSED ON BUT
DECIDED TO FOCUS ON ONE SPECIFIC
DISEASE, METABOLIC SYNDROME,
SPECIFICALLY DIET INDUCED
OBESITY.
BEFORE COVID, WE KNOW OBESITY
WAS THE EPIDEMIC OF THE 21ST
CENTURY.
IN THE 2000, 20 TO 30% OF WEST
POPULATION WAS CONSIDERED OBESE.
NOW 2019 UP TO 40%.
PREDICTION IS BY 2030 ABOUT 50
TO 60% OF OUR POPULATION WILL BE
OBESE, AFFECTING THE GUT
MICROBIOTA.
TO MIMIC DIET INDUCED OBESITY
DECIDED TO USE A MOUSE MODEL.
CHRONICALLY FEED MICE 10% DIET,
LOW FAT DIET, MATCHING DIET
EXCEPT FOR THE FAT CONTENT TO
60% FAT.
WE CAN SEE MICE WITH HIGH FAT
DIET HAVE INCREASE THE
ANTIBIOTICOSITY IN THE
MICROBIOTA.
100 FOLD MORE IN THEIR GUT THAN
THE MICE WITH LOW FAT DIET.
I LIKE TO TAKE A MOMENT TO
EXPLAIN THE ASSAY, A MODEL WE
USE TO DETERMINE IF SPECIFIC
NUTRIENTS ARE IMPORTANT FOR
MICROBIAL SURVIVAL.
1:1 RATIO OF WILDTYPE AND MUTANT
STRAIN FOR A PARTICULAR NUTRIENT
AND SEE OUTPUT IN THE FECES.
IF THE NUTRIENT IS ABSENT, WILD
TYPE AND MUTANT STRAIN SHOULD
RECOVER.
IF THE NUTRIENT IS PRESENT,
INDEX ABOVE 1, SO FIRST WE
WANTED TO SEE IF AS WE SEE IN
ANTIBIOTIC MODELS HIGH FAT DIET
E. COLI, USING OXYGEN, WE DID
THIS COMPETITIVE INFECTION USING
E. COLI STRAIN THAT CANNOT
PERFORM AEROBIC RESTORATION.
YOU CAN SEE ABOUT ONLY THE MICE
WITH HIGH FAT DIET E. COLI
EXPANDS BY USING OXYGEN,
AVAILABLE FOR BACTERIA TO USE.
IN THIS CASE WE WANTED TO SEEK
NITRATE BECOMES AVAILABLE TO
HOST RESPONSE, PROMOTING E. COLI
GROWTH DURING HIGH FAT DIET,
WHAT WE SEE IS THAT SIMILAR TO
OXYGEN, ONLY HIGH FAT DIET FED
MICE E. COLI IS USING NITRATES
FOR GROWTH.
THE QUESTION HERE THEN IS HOW IS
THIS BECOMING AVAILABLE.
TO REMIND YOU, ANTIBIOTICS
MODEL, ELECTRON ACCEPTORS BECOME
AVAILABLE, METABOLITES ARE
DEPLETED WITH ANTIBIOTICS SUCH
AS BUTYRATES.
WITH SLIGHT DECREASE, NOT THE
SAME REASON WHY ACCEPTORS BECAME
ABLE, CHRONIC EXPOSURE DAMAGES
THE GUT EPITHELIUM.
IT'S ABOUT -- YOU CAN SEE BOTTOM
PANEL THERE'S A LOSS OF CELLS,
INCREASE THE PROLIFERATION,
SUGGESTING MAYBE THE DIET
ITSELF, IT'S IMPORTANT TO SAY WE
SAW THE SAME CHANGES IN
GERM-FREE MICE SUGGESTING THE
DIET MAY BE AFFECTING EPITHELIAL
FUNCTION.
TO CONFIRM, WE USED CELL LINE
EXPOSING CELLS TO INCREASE THE
LEVELS OF FAT TO MIMIC WHAT WE
SAW IN A HIGH FAT DIET AND
LOOKED FOR MITOCHONDRIAL
FUNCTION.
YOU CAN APPRECIATE HERE IS AS
INCREASED EXPOSURE TO FAT
THERE'S A DECREASE IN CAPACITY
AND DECREASE IN ATP COMING FROM
MITOCHONDRIA, SUGGESTING
MITOCHONDRIA IS NOT FUNCTIONING
PROPERLY NOT BECAUSE CELLS WERE
DYING.
WE ALSO SAW INCREASE LEVEL OF
REACTIVE OXYGEN SPECIES COMING
FROM MITOCHONDRIA, WHEN EXPOSURE
TO PALMITATE, SUGGESTING HIGH
FAT DIET DAMAGING MITOCHONDRIA.
SO WE THINK IT'S HAPPENING IN
HIGH FAT DIET, THAT WHILE IN LOW
FAT HEALTHY DIET, MITOCHONDRIA
FUNCTION PROPERLY, PREVENTING
ELECTRON ACCEPTORS TO BE
AVAILABLE.
HIGH FAT DIET IS DAMAGING, A
CONSEQUENCE IS OXYGEN BECOMES
AVAILABLE AND STARTS TO PRODUCE
NITRATES, ACCEPTORS BECOME
AVAILABLE, TO EXPAND IN THE GUT,
ALSO FEED THE RESIDENT
MICROBIOTA.
SO WHAT, RIGHT?
WHAT IS RELEVANCE OF
ENTEROBACTERIA, WHAT IS THE
CONSEQUENCE?
OUR LAB IS INVESTIGATING BUT ONE
THING I WANT TO PRESENT,
SOMETHING RELATED TO INCREASED
RISK FOR CARDIOVASCULAR DISEASE,
AGAIN A NUMBER ONE KILLER IN OUR
COUNTRY IN A WORLD WITHOUT
COVID.
OUR LAB IN PARTICULAR BECAME
INTERESTED IN CARDIOVASCULAR
DISEASE, THE MICROBIOME CAN
METABOLIZE ENZYMES THAT BREAK
DOWN COMPONENTS, TMA, OXIDIZED
INTO TMAO, METABOLIZED, PLAYING
A ROLE IN METABOLIC SYNDROME AND
HEART DISEASE.
AN INTERESTING STUDY TO SEE WHAT
MEMBERS ARE ABLE TO METABOLIZE
SPECIFICALLY CHOLINE, YOU SEE
LARGE NUMBER OF MICROBES ARE
ENTEROBACTERIA.
WE KNEW HIGH CAPACITY TO
METABOLIZE CHOLINE.
THE WEIGH THEY MA METABOLIZE IS
CREATING MICROCOMPARTMENT THAT
ENABLES BACTERIA TO BREAK DOWN
CHOLINE, INTO ENERGY, AND PMAs
INTO THIS METABOLIC PATHWAY.
WHAT DECIDED TO SEE IN VITRO IF
CONDITIONS THAT MIMIC CONDITIONS
IN THE HIGH FAT DIET, OXYGEN IS
AVAILABLE, NITRATE IS AVAILABLE,
WHETHER E. COLI WOULD BE ABLE TO
METABOLIZE CHOLINE.
WE GAVE OXYGEN AND NITRATE, ONLY
UNDER THOSE CONDITIONS E. COLI
IS BREAKING DOWN CHOLINE AND
GROWING USING CHOLINE, MUTANT
STRAIN CANNOT DO THE SAME THING.
THIS IS INTERESTING TO US, WE
WANTED TO SEE IF THIS IS
RELEVANT IN VIVO, WENT BACK TO
THE MOUSE MODEL AND WE DID HAVE
TO SUPPLEMENT CHOLINE BECAUSE
TURNS OUT MOUSE DIET DOESN'T
HAVE A LOT OF CHOLINE, BACK TO
THE ASSAY.
WE SAW ONLY MICE FED HIGH FAT
DIET, WILDTYPE STRAIN OF E
CO-COULD OUTCOMPETE SUGGESTING
ONLY HIGH FAT DIET E. COLI WAS
USING CHOLINE FOR GROWTH.
IT WAS REALLY BECAUSE THE
BACTERIA WAS RESPIRING USING
CHOLINE, IN REPEATED EXPERIMENTS
USING STRAINS COULD NOT PERFORM
RESPIRATION, WILD TYPE COULD NOT
OUTPERFORM, IF THEY CAN'T
RESPIRE THEY CAN'T USE CHOLINE
IN THE INTESTINAL LUMEN.
TO REMIND UP WHAT WAS
INTERESTING ABOUT THE STORY IS
THAT CHOLINE, METABOLISM OF
CHOLINE LEADS TO PRODUCTION OF
METABOLITES HARMFUL TO THE HOST.
SO WE WANTED TO SEE WHAT HAPPENS
IF WE MEASURE IN THE MOUSE FED
LOW FAT OR HIGH FAT DIET,
DIFFERENT E. COLI.
WE CAN SEE THAT HIGH FAT MED
MICE WITH E. COLI ABLE TO
METABOLIZE CHOLINE IN
MICROBIOTA, INCREASE CIRCULATING
TMAO.
IF WE COLONIZE DO NOT
METABOLIZE, DOES IT GO AWAY, THE
SAME IS TRUE IF WE COLONIZE MICE
WITH STRAIN OF E. COLI THAT
CANNOT RESPIRE CONFIRMING
RESPIRATION IS WHAT IS LEADING
TO INCREASED LEVELS OF TMAO IN
THE BLOOD CIRCULATION.
THE LAST QUESTION HERE, THIS WAS
INTERESTING TO US, BUT IF THIS
INTERACTION BETWEEN INTESTINE
EPITHELIUM AND MICROBIOTA IS
DRIVING PRODUCTION, COULD IT
PREVENT PRODUCTION?
TO DO THAT WE CHOSE TO USE A
DRUG WHICH IS USED, FDA APPROVED
USED FOR IBD TREATMENT.
WE'VE SHOWN 5-ASA TARGETS --
RESTORES METABOLISM OF INTESTINE
EPITHELIUM.
WHAT WE DID HERE, WE TREATED
MICE ARE 5-ASA AND LOOKED INTO
FUNCTION AND SAW THAT HERE CELLS
IN RED ARE CELLS PERFORMING
OXIDATIVE METABOLISM.
WE CAN APPRECIATE HERE THAT IN
UNTREATED MICE, HIGH FAT DIET,
METABOLISM OF INTESTINES GOES
AWAY, CAN RESTORE THIS BY GIVING
THIS DRUG TO THE MICE.
NOT ONLY THAT 5-ASA WAS ABLE TO
INHIBIT GROWTH OF E. COLI IN THE
GUT, BY USING CHOLINE.
THAT ACTUALLY LED TO DECREASED
LEVELS OF BLOOD CIRCULATING TMAO
IF YOU RESTORE FUNCTION IN MICE
WITH HIGH FAT DIET.
SO WHAT I SHOWED YOU HERE TODAY
DURING HIGH FAT DIET WE THINK
THERE'S DYSFUNCTION OF INTESTINE
EPITHELIUM, MAY NOT BE HARMFUL
BUT IF IT CAN METABOLIZE CHOLINE
INTO TMA PATHWAYS TURNS ON AND
CAN CAUSE HARM.
WE'RE SHOWING MICROBIAL
METABOLISM IS CONTEXT DEPENDENT,
NOT JUST MICROBES HAVE GENE OR
CAPACITY TO PERFORM METABOLIC
PATHWAY, THEY ARE GOING TO BE
DOING THIS ALL THE TIME.
THE CONTEXT AROUND THIS MICROBES
HOW IMPORTANT TO TURN ON AND
REALLY EXPLAIN WHAT PARTICULAR
METABOLISM IS HAPPENING IN THE
CONTEXT OF HEALTH AND DISEASE,
AND SHOULD KEEP THAT IN MIND TO
UNDERSTAND WHAT MICROBES ARE
DOING IN THE GUT IN OUR MODELS
OF DISEASE.
SO WITH THAT, I CAN THANK THE
PEOPLE THAT HAVE DONE THE WORK,
MY POSTDOC, AND OUR FUNDING
SOURCES.
>> ANY QUESTIONS FROM ANYONE IN
THE AUDIENCE, ZOOM, OR VIRTUAL?
I BELIEVE YOU GOT A QUESTION
EARLY IN THE CHAT, A LOGISTICAL
QUESTION, WHAT TYPE OF HIGH FAT
DIET WAS USED?
>> YEAH, I GET THAT QUESTION A
LOT.
THIS IS NOT A KETOGENIC DIET.
WE'VE NOT DONE WORK WITH THAT
KIND OF DIET.
WE USE TYPICAL RESEARCH DIET.
WHICH IS FAT COMES FROM LARD
USUALLY.
IT'S NOT A SPECIFIC TYPE OF FAT.
>> DO YOU THINK AGE IS A FACTOR,
OF THE ANIMALS YOU USE, OR NO?
>> YES, WHEN I FIRST STARTED THE
PROJECT I DID A COUPLE PILOT
EXPERIMENTS.
THESE 16, 17 WEEKS OF AGE WHEN
DOING EXPERIMENTS.
I'VE DONE EXPERIMENTS WITH MICE
AT 23, 24 WEEKS, LIKE OLDER,
MOST DIFFERENCES STARTED TO GO
AWAY, IT WAS BECAUSE OUR LOW FAT
MICE STARTED TO BE MORE
SUSCEPTIBLE.
HIGH FAT DIET ACCELERATING
MITOCHONDRIA DYSFUNCTION
ASSOCIATED WITH AGE AND MICE ARE
OLDER YOU LOSE THAT PHENOTYPE
BECAUSE OLDER MICE MAY START TO
HAVE DYSFUNCTION.
>> LIKE AN ADDITIVE THING TOO,
AGE AND COMPOUNDING OF HIGH FAT
DIET.
>> WOULD BE, YES.
>> THANK YOU FOR SHARING YOUR
WORK.
WE'LL MOVE ON IN THE INTEREST OF
TIME.
NEXT SPEAKER IS DR. CHRIS STEIN
SZYMANSKI FROM UNIVERSITY OF
GEORGIA, NEW PARADIGMS FOR
CAMPYLOBACTER JEJUNI IN THE GUT.
PERFECT, YOU'RE ON TOP OF IT.
I APPRECIATE YOU BEING HERE, SO
SUFFICIENT.
LOOKING FORWARD TO YOUR WORK.
>> THANK YOU, CATHERINE AND ALL
THE ORGANIZERS FOR INVITING ME.
IT'S BEEN AN EXCITING DAY.
I'M GOING TO TELL YOU ABOUT MY
FAVORITE GUT MICRO,
CAMPYLOBACTER JEJUNI.
IT'S A LEADING CAUSE OF FOOD
BORNE ILLNESS IN HUMANS.
CAMPYLOBACTER CAUSES DIARRHEA
BUT CAN ALSO CAUSE PARALYSIS AND
THE WAY THE PARALYSIS IS FORMED
IS THROUGH THESE GANGLIOSIDE
MIMICKING STRUCTURES ON THE
SURFACE, IN SOME CASES PEOPLE
WILL CREATE ANTIBODIES AGAINST
THESE GANGLIOSIDE AND
CROSS-REACT WITH OUR OWN
NEURONS, LEADING TO PARALYSIS.
A SHOUT OUT TOP A PROJECT FUNDED
BY THE DoD TO LOOK AFTER
PATIENT SERA ON GLYCAN ARRAYS HE
SYNTHESIZED WITH CAMPYLOBACTER
LOS TYPE SUGARS.
WHAT I'M GOING TO FOCUS ON TODAY
IS ANOTHER CONSEQUENCE OF
CAMPYLOBACTER INFECTION, THAT'S
IN LOW- AND MIDDLE-INCOME
COUNTRIES, CAMPYLOBACTER IS A
HUGE COURSE OF GROWTH STUNTING.
THE REASON FOR THIS IS BECAUSE
THE MICROBE DISPLACES BENEFICIAL
MICROBES EARLY IN LIFE, WHICH IS
VERY IMPORTANT FOR DEVELOPMENT,
NOT JUST OF GUT HEALTH BUT
NORMAL HUMAN DEVELOPMENT BUT
ALSO CAUSES SIGNIFICANT
INFLAMMATION AND THAT ALSO LEADS
TO NUTRIENT MALABSORPTION.
IN THIS STUDY FUNDED BY THE BILL
AND MELINDA GATES FOUNDATION
THEY FOUND CHILDREN UP TO 85% OF
CHILDREN WERE INFECTED WITH
CAMPYLOBACTER BY THE AGE OF 1.
AND SO WE'VE KNOWN FOR A LONG
TIME THAT CAMPYLOBACTER COULD
ADHERE TO FUCOSLYATED
FUCOSLYATED SUGARS, AND WE HEARD
A TALK BY RICK CUMMINGS, HOW
IMPORTANT FUCOSE IS IN THE GUT.
WE DIDN'T KNOW IT COULD
METABOLIZE SHOULD GO ASHES, --
SUGARS, IT WAS PREVIOUSLY
THOUGHT THAT CAMPYLOBACTER WAS
ASACHROLYTIC.
WHAT WE DISCOVERED WAS SOME
CAMPYLOBACTERS DIDN'T ONLY
SURVIVE ON PROTEIN SHAKES BUT
THAT THEY ACTUALLY COULD
METABOLIZE FUCOSE.
AND THESE ARE THE PEOPLE
INSTRUMENTAL IN THE STUDIES IN
MY LAB, LOOKING AT THIS FUCOSE
PATHWAY IN CAMPYLOBACTER WHICH
EXISTS AS A SINGLE OPERON.
STRAINS CAN METABOLIZE,
INDICATED AS GREEN, SHOWING
GROWTH ADVANTAGE IN THE PRESENCE
OF FUCOSE, STRAINS DO WHICH DO
NOT SHOW GROWTH ADVANTAGE IN
PRESENCE OF FUCOSE, DENOTED AS
RED.
TOGETHER WE LOOKED AT ALL THE
SEQUENCED CAMPYLOBACTER GENOMES
AND DISCOVERED A SIGNIFICANT
PROPORTION OF CAMPYLOBACTER
ISOLATES, THESE IN RED HAVE THE
OPERON.
JOLENE LOOKED AT PATHWAY SHOWN
HERE, BROKEN DOWN INTO PYRUVATE,
STUDIED THAT ARABINOS IS
METABOLIZED.
I WANTED TO FOCUS ON FOR THIS
TALK A DEHYDROGENASE.
STUDIES WERE DONE SHOWING
WITHOUT FUCOSE, WILDTYPE
CAMPYLOBACTER FORMS REBUST
BIOFILMS BUT IN PRESENCE WILL
DISPERSE AND SWIM TOWARDS THIS
SUGAR STRUCTURE, WE DEVELOPED
THIS ASSAY TO UNDERSTAND THE
SWIMMING BEHAVIOR, CAMPYLOBACTER
AT THE BOTTOM, A FILTER DISC AT
THE TOP WITH REREPELLANT, GAUGE
WHETHER IT SWAM TO THE TOP,
MEASURING RESPIRATION.
IT CAN SWIM TO FEW DOSE.
RED LINE.
THE FUCOSE CAN'T ENTER THE
CYTOPLASM, IT'S STILL ABLE TO
SWIM TO IT BUT WHEN YOU KNOCK
OUT THE DEHYDROGENASE ENZYME CAN
NO LONGER SWIM TO FUCOSE, WHEN I
PUT IT BACK IT RESTORES SWIMMING
BEHAVIOR.
IF YOU TAKE A STRAIN THAT
DOESN'T HAVE PATHWAY, CAN SWIM
TO FUCOSE, GIVE IT THAT FUCOSE
OPERON, IT CAN SWIM.
IF YOU DIVERSITY JUST
DEHYDROGENASE WITHOUT ENZYMES
IT'S STILL ABLE TO SWIM TO
FUCOSE.
SO WHAT THAT TELLS US IS THAT
FUCOSE DOESN'T NEED TO ENTER THE
CYTOPLASM TO TRIGGER CHEMOTAXIS
AND THIS DEHYDROGENASE PLAYS A
KEY ROLE IN CHEMOTAXI.
IT BINDS AND CONVERTS.
WHAT WE THOUGHT WAS SINCE WHEN
WE CRYSTALLIZED NADP CAN BIND
WITHOUT FUCOSE, AND MAY BE
IMPORTANT IN SENSING WHETHER TO
SWIM OR NOT.
WE DID THIS EXPERIMENT HERE,
SUBJECTED THE BACTERIA TO
OXIDATIVE STRESS, HYDROGEN
PEROXIDE, REMOVED CATALASE
ENZYME, PRIMARY MECHANISM FOR
RELIEVING STRESS TO FORCE THE
MICROBE TO USE THE SECONDARY
PATHWAY THAT WOULD USE NADP.
YOU SEE SWIMMING WHEN HYDROGEN
PEROXIDE IS PRESENT, WHEN
CATALASE IS KNOCKED OUT BUT NO
LONGER SWIMMING TO FUCOSE WHEN
BOTH ARE THERE.
THAT'S TELLING US NEDPH IS THE
SIGNAL, TO CONFIRM USED A GLO
ASSAYS.
WHEN ENZYME IS PRESENT SEE
DROPPED IN THAT LEVEL, WHAT I
SHOULD POINT OUT IS THIS IS A
LOG SCALE HERE.
ONE OF THE THINGS QUITE UNUSUAL
IS CAMPYLOBACTER AND ITS RELATED
PROTEOBACTERIA HELICOBACTER HAVE
A FLIPPED RATIO OF NADP/NADPH TO
E. COLI.
WE SHOWED THESE RATIOS WERE
ACTUALLY TRUE USING NMR.
I'LL SAY PHOEBE IN THE LAND --
LAB HAS DETERMINED WHICH
COMPONENTS ARE INVOLVED IN
SENSING, AND SHE'S NARROWED DOWN
TO THESE THREE PROTEINS.
WHICH ARE INVOLVED IN ENERGY
TAXIS IN CAMPYLOBACTER.
WHAT FUCOSE OR NADP IS SIGNALING
ENERGY STATUS TO THE MICROBE, IT
WILL DETERMINE ITS FLAGELLA
ROTATION BASED ON NADP LEVELS.
WHAT WE THINK IS HAPPENING BASED
ON RESULTS IS THAT NADP PH
LEVELS CHANGE IN THE MICROBE,
AND NADPH WILL COME AND REDUCE F
OF FAD CO-FACTOR OF SET B AND C
WHICH ACTIVATES SET A WHICH THEN
INTERACTS WITH CHEMOTAXIS
PROTEIN WHICH INFLUENCES
FLAGELLA ROTATION.
I TOLD YOU ABOUT THE MONO
SACCHARIDE FUCOSE.
YOU KNOW THAT FUCOSE DOESN'T
EXIST AS A MONO SACCHARIDE
TYPICALLY.
IT EXISTS AS PART OF MORE
COMPLEX OLIGOSACCHARIDES SO
YOU'RE WONDERING HOW DOES
CAMPYLOBACTER GET FUCOSE?
WE THOUGHT THAT QUESTION TOO.
MARIO ALLUDED TO THIS EARLIER
THIS MORNING THAT PATHOGENS
SOMETIMES ARE LAZY AND DON'T
HAVE REPERTOIRE AND WILL
SCAVENGE FROM MICROBES READY IN
OUR GUT LIKE BACTEROIDES,
CAPABLE OF RELEASING FUCOSE.
CAMPYLOBACTER CANNOT RELEASE
FUCOSE BUT USED BACTEROIDES
VULGATUS, WE SEE ENHANCED
GROWTH, THAT'S FUCOSE PATHWAY
DEPENDENT.
WHAT THIS TELLS US IS THAT
CAMPYLOBACTER FUCOSE PATHWAY
BECOMES UPREGULATED WHEN
BACTEROIDES IS SUPPLYING FUCOSE.
WE USED MARIO'S PROTOCOL,
INCUBATED WITH MUCIN, SHOWING WE
COULD GET RELEASE OF FREE FUCOSE
FROM THIS MEDIA.
AND SO WE THEN WONDERED IS
FUCOSE BENEFICIAL TO
CAMPYLOBACTER?
WORK SHOWED WILDTYPE STRAIN
COULD OUTCOMPETE FUCOSE
METABOLIZING DEFICIENT STRAIN IN
THE PIGLET DIARRHEAL MODEL AND
WANTED TO INVESTIGATE IN
BREASTFEEDING AND HUMAN MILK
OLIGOSACCHARIDES.
AND SO WHAT WE WANTED TO FEMALE
FOLLOW UP ON REALLY ELEGANT
PIONEERING WORK THAT DAVID
NEWBERG'S GROUP PUBLISHED,
SHOWING IT BINDS AND THEY ACT AS
DECOYS TO REMOVE CAMPYLOBACTER
TO INTESTINAL TRACT.
AT THAT TIME, WE WEREN'T AWARE
IT COULD METABOLIZE FOE COAST --
FUCOSE.
WE'RE SEEING SO MUCH INFECTION
IN INFANTS, OR ARE THEY ACTING'
DECOYS AND PROTECTING CHILDREN
AND WE'RE SEEING OTHER
CAMPYLOBACTERS THRIVING?
THIS WAS THE QUESTION XIAOMIN G
WANTED TO ANSWER, PIGGYBACKING
OFF GEMS STUDY, ISOLATES FROM
BREAST FEDS AND NON-BREASTFED
INFANTS LESS THAN ONE YEAR OF
AGE, ALSO FECAL SAMPLES AND DNA
TO LOOK AT LEVEL OF
CAMPYLOBACTER IN THESE CHILDREN.
AND WHAT WE FOUND WAS ACTUALLY
QUITE SURPRISING.
NOT ONLY WERE THEY ALL COLONIZED
WITH CAMPYLOBACTER BUT WE SAW
SIGNIFICANTLY HIGHER PROPORTIONS
OF CAMPYLOBACTER IN KIDS THAT
WERE BREASTFED, SO THERE'S
ACTUALLY MORE CAMPYLOBACTER IN
THE BREAST-FED CHILDREN THAT HAD
DIARRHEA THAN CHILDREN THAT HAD
CAMPYLOBACTER DIARRHEA THAT
WEREN'T BREASTFED.
WE WANTED TO FOLLOW UP ON THAT,
DO OLIGO OLIGOSACCHARIDES ACT
AS DECOYS OR PROVIDING NUTRIENTS
FOR SUCCESSFUL COLONIZATION?
AND SO THIS IS SUMMARY OF
LOOKING AT ALL OF THOSE ISOLATES
WE OBTAINED FROM THE GEM STUDY
SO WE FIRST LOOKED FOR PRESENCE
OF FUC PG, CONFIRMED PATHWAY WAS
ACTIVE BY DOING GROWTH ANALYSIS
OF EACH STRAIN AND I SHOULD
POINT OUT FOR EVERY CHILD ONLY
ONE STRAIN WAS ISOLATED.
SO THEN WHAT WE LOOKED AT IS THE
PROPORTION OF FUCOSE
METABOLIZING VERSUS
NON-METABOLIZING CAMPYLOBACTERS.
IF NOT BREASTFED YOU SAW EQUAL
NUMBERS IN THEIR FECAL SAMPLES
BUT IF THE CHILDREN WERE
BREASTFED THEN YOU ACTUALLY SAW
SIGNIFICANTLY LESS
CAMPYLOBACTERS THAT WERE FUCOSE
METABOLIZING, SO THIS DOES
SUPPORT DAVID NEWBERG'S THEORY,
PROBABLY SENSING MONO SACCHARIDE
AND WILL SWIM TOWARD IT AND BE
RELEASED FROM INTESTINAL TRACT
WHEREAS THEN WHAT'S LEFT BEHIND
ARE THE CAMPYLOBACTERS THAT DO
NOT METABOLIZE FUCOSE AND
PROBABLY DO HAVE A COMPETITIVE
ADVANTAGE BECAUSE THEY ARE WITH
ALL OF THESE CARBOHYDRATE
METABOLIZERS IN THE GUT AND THEY
HAVE THIS EXPANDED REPERTOIRE OF
METABOLIZING TCA CYCLIN AND
AMINO ACIDS.
TO INVESTIGATE WHETHER THIS IS
TRUE, PHOEBE WITH JOLENE HAS
DONE RNAseq EXPERIMENTS,
PATHWAY NOT INDUCED IN BREAST
MILK, PATHWAYS ARE BEING
EXPRESSED, AND TO DETERMINE
WHICH ONES ARE THE MOST
IMPORTANT FIB -- PHOEBE DID
EVOLUTION STUDIES, WHEN
CAMPYLOBACTER IS GROWN DIRECTLY
WITH MILK IT IS ACTUALLY TOXIC
TO THE MICROBE.
AND SO YOU CAN SEE HERE WHETHER
IT HAS THE FUCOSE PATHWAY OR NOT
CAMPYLOBACTER WILL NOT BE ABLE
TO GROW IN MILK ON ITS DOWN
WITHOUT BENEFICIAL MICROS IN THE
GUT TO BREAK DOWN CERTAIN
COMPONENTS.
SHE HAD TO EVOLVE STRAINS TO
GROW IN MILK AND GENOME SEQUENCE
FOG SEE WHAT HAS CHANGED IN
CAMPYLOBACTER STRAINS AND WE'VE
ACTUALLY DISCOVERED THAT
ATTENTION SPECIFIC CHANGES IN
TRANSPORTERS THAT ARE INVOLVED
IN NUTRIENT ACQUISITION.
SO THAT'S WHAT WE'RE FOLLOWING
UP ON NOW.
IN CONCLUSION WE'VE DESCRIBED
PATHWAYS, FUCX IS NECESSARY FOR
CHEMOTAXIS.
IT DEPENDS ON COMMENSAL MICROBES
LIKE VULGATUS.
CO-CULTURE STUDIES SHOW ENHANCE
GROWTH WITH MUCIN AND VULGATUS,
AND MASS SPECTROMETRY SHOWED
EVIDENCE OF FUCOSE RELEASE FROM
MUCINS BY BACTEROIDES VULGATUS.
BREASTFED INFANTS IN
LOW-RESOURCE SETTINGS HAVE
HIGHER PROPORTIONS OF
CAMPYLOBACTERS WITHIN FECAL
MICROBIOTA, THIS COULD BE
RELATED TO A SELECTION FOR
STRAINS WITH ENHANCED AMINO ACID
METABOLISM.
FINALLY, I'VE ALSO TOLD YOU
HUMAN MILK IS TOXIC TO
CAMPYLOBACTER BACKUP MICROBE CAN
EVOLVE TO RESIST TOXICITY.
I'D LIKE TO ACKNOWLEDGE A LOT OF
THE PEOPLE THAT HAVE BEEN
INVOLVED IN THESE STUDIES.
I'M NOT MOVING LIKE SOME OTHER
SPEAKERS BUT I'M INVOLVED IN
SEVERAL COMMITTEES, AMBASSADOR
FOR THE AMERICAN SOCIETY OF
MICROBIOLOGY SO I'LL THROW UP
THIS ADVERTISEMENT, AND ALSO
CONTRIBUTOR TO THE ESSENTIALS OF
GLYCOBIOLOGY, I WANTED TO
HIGHLIGHT THIS BOOK AS WELL.
THANK YOU FOR YOUR ATTENTION.
>> THANK YOU FOR A FANTASTIC
TALK AND SERVICE WORK.
REALLY IMPORTANT.
THANK YOU SO MUCH.
WE HAVE A QUESTION, JUST ONE,
I'LL USE ONLINE TO ENCOURAGE
PEOPLE TO COMMUNICATE.
GRACE HENRY SAYS DID YOU LOOK AT
HMO COMPETITION OF THE WOMAN WHO
BREASTFED WHERE MAYBE IF THEY
WERE OR NOT SECRETORS, DID YOU
NOTICE ANY TRENDS?
>> IN OUR STUDIES WITH EVOLUTION
AND WITH RNAseq, WE USED TWO
DIFFERENT DONORS.
AND IN BOTH CASES THEY ARE
SECRETERS BUT THERE WAS ANOTHER
STUDY PUBLISHED NOT LONG AGO
SHOWING THERE ISN'T A
CORRELATION BETWEEN SECRETERS
AND NON-SECRETERS, IF I REMEMBER
CORRECTLY.
>> THANK YOU.
IN THE INTEREST OF TIME WE HAVE
A 20-MINUTE BUFFER TO 5:00 SO
WE'LL TRY GET THIS MEETING
FINISHED BY FIVE.
SWITCHING SPEAKERS, THANK YOU
FOR THE TALK.
NEXT SPEAKER IS AMANDA LEWIS,
FROM UNIVERSITY OF SAN DIEGO IN
CALIFORNIA, TALKING ABOUT
RESIDENT MICROBES WHICH SHAPE
VAGINAL EPITHELIAL GLYCAN
LANDSCAPE.
I HOPE YOU SEE INTERSECTION AS
WORK IS BUILDING ON EACH OTHER.
IF YOU HAVEN'T MET PEOPLE YOU'RE
MEETING IN THIS COMMUNITY AND
ABLE TO USE THE CHAT FEATURE
WITHIN THE ZOOM.
IT'S AVAILABLE FOR YOU TO TALK
TO EACH OTHER.
THANK YOU.
>> GOOD AFTERNOON.
TODAY WE'LL TALK MORE ABOUT THE
VAGINA, IT'S NICE TO NOT BE THE
ONLY PERSON IN THE ROOM TALKING
ABOUT VAGINAS, SO THANKS TO
EMILY BYNDLOSS WHO INTRODUCED
THE TOPIC EARLIER.
I WANTED TO START WITH
ACKNOWLEDGMENTS, KAVITA IS THE
FIRST AUTHOR, AND WARREN LEWIS
IS THE LAST AUTHOR ON THIS.
A LOT OF GOOD WORK.
WE'LL START WITH A BRIEF
INTRODUCTION.
MANY WOMEN HAVE MICROBIOTA
DOMINATED BY LACTOBACILLUS, A
LOT OF OTHER WOMEN HAVE OTHER
ORGANISMS THAT CAN OVERVIEW,
GARDNER VAGINALUS IS ONE.
IT'S A DYSBIOSIS WITH HEALTH
COMPLICATIONS INCLUDING SEXUALLY
TRANSMITTED INFECTIONS, URINARY
TRACT INFECTIONS, ASCENDING
INFECTIONS AT THE UTERUS,
PRE-TERM BIRTH, PELVIC
INFLAMMATORY DISEASE,
INFERTILITY, AMONG OTHER
OUTCOMES.
IT'S RARE FOR ME TO HAVE ONE
SLIDE THAT TALKS ABOUT MOUSE
WORK, I'M GOING TO GIVE A BRIEF
OVERVIEW WHY WE BELIEVE
GARDNERELLA IS POTENTIAL LIP A
CAUSAL FACTOR, FROM OUR WORK NOT
IN HUMAN STUDIES BUT RATHER IN
MICE.
WE KNOW GARDNERELLA ON ITS OWN
WHEN INTRODUCED INTO THE HAVE
THE VAGINA OF MICE CAN TRIGGER
VAGINOSIS.
I WON'T SHARE ALL THAT TODAY.
IN THE BLADDER WHICH WE HAPPENS
WITH HYGIENIC AND SEXUALLY
ACTIVITIES, EXPOSURE, CAUSES
BLADDER APOPTOSIS OF EPITHELIAL
CELLS AND WITH SMALL EXPOSURE
TO KIDNEY CAN CAUSE IL-1
DEPENDENT KIDNEY INJURY, ENABLES
COLONIZATION BY OTHER ORGANISMS,
MOUSE MODELS WHERE WE'RE DOING
SINGLE OR CO-INFECTIONS AND
COMPARING OUTCOMES, IN RECURRENT
UTI MODEL IF WE EXPOSE MICE IN A
ALREADY HAVE E. COLI RESERVOIRS
INSIDE THEIR BLADDER EPITHELIAL
CELLS IT TRIGGERS RECURRENT UTI
WHICH CAN ALSO GO UP INTO THE
KIDNEYS TO CAUSE KIDNEY
INFECTION.
WE'VE SHOWN IN CO-INFECTIONS
WITH PREVOTELLA, ANOTHER
ORGANIZATION ASSOCIATED WITH
BACTERIAL VAGINOSIS WHEN IT'S
TOGETHER IT CAUSES MUCH WORSE
ASCENDING INTRAUTERINE
INFECTIONS.
IN PREGNANCY MODELS WE'VE SHOWN
THAT GROUP B STREPTOCOCCUS IS
MORE LIKELY TO COLONIZE IN WOMEN
WITH BV, FINE DISTINCTION, IN
THE PRESENCE OF GARDNERELLA,
MORE LIKELY TO COLONIZE
VAGINALLY BUT ALSO MORE LIKELY
TO CAUSE ASCENDING INFECTIONS
AND I'M SHOWING ON THE RIGHT A
PICTURE OF GROUP B STREP WITHIN
THE PLACENTAL TISSUE.
WE'VE ALSO SHOWN IT LIBERATES
CARBOHYDRATE RESOURCES THAT
ENABLES GROWTH OF OTHER
ORGANISMS.
NOW WHAT I WANT TO DO IS TALK
ABOUT GLYCOBIOLOGY.
WE BECAME INTERESTED IN THIS
LARGELY BECAUSE SIALIDASE
ACTIVITY HAS BEEN KNOWN FOR
QUITE SOME TIME, DECADES, TO BE
ASSOCIATED WITH BACTERIAL
VAGINOSIS, FOUND IN THE VAGINAL
FLUIDS OF WOMEN WITH BACTERIAL
VAGINOSIS BUT NOT WITHOUT THIS
CONDITION.
SO WE HAVE DONE WORK IN THIS
REGARD AND HAVE BEEN ABLE TO
SHOW THAT GARDNERELLA VAGINALIS
PRODUCED SIALIDASE, ABLE TO
CLEAVE IN CONTEXTS INCLUDING
FROM MUCINS, SECRETEORY IgA,
DEPLETING FROM MUCOSA, IN BOTH
HUMANS AS WELL AS MICE WHEN WE
JUST LOOK AT SORT OF THE WHOLE
VAGINAL SAMPLE.
AND WE'VE BEEN ABLE TO IDENTIFY
THE SIALIDASES THAT ARE
RESPONSIBLE FOR THIS ACTIVITY,
AND SHOW THAT BOTH THE
RECOMBINANT AND SIALIDASES THAT
ARE PRESENT WITHIN CLINICAL
SPECIMENS ARE ABLE TO CLEAVE OFF
SIALIC ACIDS IN VIRTUALLY ALL
CONTEXTS WE'VE TESTED.
GOING FURTHER THAN THAT WE KNOW
THAT GARDNERELLA IS ABLE TO
CATABOLIZE, NOT JUST CLEAVE.
YOU'RE HEARD A THEME THROUGHOUT
A FEW TIMES THAT SOME ORGANISMS
CAN PRODUCE HYDROLASES, SOME CAN
TAKE ADVANTAGE.
THIS IS ALSO HAPPENING IN THE
VAGINA WHERE WE HAVE ADDITIONAL
SPECTRA OF BACTERIA IN THIS
CONTEXT UP HERE ABLE TO EAT THE
SIALIC ACIDS BUT DON'T
THEMSELVES PRODUCE SIALIDASES.
ALL RIGHT.
BUT WHAT I REALLY WANT TO TALK
TO YOU ABOUT TODAY GOES FURTHER
THAN JUSTS SO MOLECULES, IF THEY
ARE ACTING ON THE EPITHELIAL
GLYCOCALYX.
YOU CAN SEE THIS COVERED IN
BACTERIA.
THESE BACTERIA ARE LIVING RIGHT
ON THE SURFACE OF THE EPITHELIAL
CELLS.
WE CAN DO TRADITIONAL STAINING,
NEGATIVE STAINING WITH RED.
AND PETRONIUM OXIDE.
I'VE GOT THAT WRONG.
WE USED LECTINS TO PROBE
EPITHELIAL CELLS AND SHOWED
MAL-II IS ABLE TO BIND VERY WELL
TO NO BV CELLS, AS YOU CAN SEE
WHEN YOU INCREASE LEVEL OF
SIALIDASE ACTIVITY PRESENT IN
THE SAMPLE, WITH ONE EXCEPTION
HERE YOU HAVE FEWER OF THESE
CELLS LIGHTING UP THAT ARE
STAINING FOR 2, 3 LINK SIALIC
ACID, THE FIRST EVIDENCE THEY
MAY BE DEPLETED.
SO TAKING THIS A STEP FURTHER
AND USING THESE PRIMARY HUMAN
VAGINAL EPITHELIAL CELLS
ISOLATED FROM VAGINAL SWABS, WE
DID GLYCAN ANALYSIS WITH A SCORE
AT UCSD AND WERE ABLE TO SHOW A
STARK LOW LEVEL OF SIALYTATED N
AND O.
IF DEPLETED IT MIGHT BE THAT WE
SHOULD BE ABLE TO SEE REVEALING
OF CARBOHYDRATE RESIDUES, USING
PEANUT AGGLUTININ, RECOGNIZING
UNDERLYING GALACTOSE RESIDUE,
SHOWING BY FLOW CYTOMETRY, YOU
SEE LOTS OF STAINING WITH PNA
BUT ONLY WHEN UP ADD EXOGENOUS
SIALIDASE.
YOU SEE YOU CAN'T REVEAL ANY
MORE PNA STAINING, IT'S ALREADY
HIGHLY STAINED AND CAN'T REVEAL
ANY MORE BY ADDING EXOGENOUS
SIALIDASE SUGGESTING THESE ARE
REMOVED AND THIS UNDERLYING
LIGAND IS COMPLETELY UNCAPPED.
AND SO HERE YOU CAN SEE IN OF IF
YOU DO THIS OVER A NUMBER OF
SAMPLES, WE HAD 15 IN EACH
GROUP, YOU CAN SEE A VERY HIGHLY
STATISTICALLY SIGNIFICANT
DIFFERENCE BETWEEN THIS BINDING
OF PNA TO THE EPITHELIAL CELLS
IN BV.
GOING FURTHER TO LOOK AT N AND
O-GLYCANS WE'VE DONE ANALYSIS
WITH GLYCOANALYTIC SCORE AT
UCSD.
I'M SHOWING TO SHOW PROFILES OF
N-GLYCANS RELEASED FROM
EPITHELIAL CELL LYSATES.
WHEN WE SEPARATE YOU CAN SEE
HIGH MANNOSE GLYCANS BUT IF YOU
LOOK IN WOMEN WITHOUT BV YOU'LL
SEE QUITE A LOT OF PEAKS THAT
ARE SIALYTED.
PREPARING FOR MASS SPENDING WE
DID FOLLOW-UP FLOW CYTOMETRY TO
LOOK AT STRUCTURES.
WE CAN SEE IN WOMEN WITHOUT BV,
WE SEE A LOT OF CORE 2
O-GLYCANS, SOME CORE 1 AS WELL.
THE MOST PROMINENT ONE, HIGHEST
SENSETY, CONTAINING POTENTIAL
LEWIS X, IF WE TOOK THOSE
EPITHELIAL CELLS AND TREATED
WITH A COMMERCIAL SIALIDASE,
USED ANTIBODY AGAINST LEWIS X
ABLE TO SHOW THAT WE CAN SHOW
THAT LEWIS X IS THERE BY FLOW
CYTOMETRY.
ALSO POINTING TO THIS GLYCAN
HERE, WHEN WE FRAGMENT AND WERE
ABLE TO SEE SIALYATED ANTIGEN
CONFIRMING THAT WHAT WE WERE
SEEING EARLIER WITH PNA WHEN WE
RELEASE THE SIALIC ACID WE SEE
THAT UNDERLYING ANTIGEN.
IN CONTRAST WHEN YOU LOOK AT
WOMEN WITHOUT -- WITH BV, WE SEE
THERE'S A LOT OF HYPO SIALYATED
STRUCTURES.
SENT OF THE 1878 PEAK YOU SEE
LOW INTENSITY, AND INSTEAD
HIGHER INTENSITY PEAK OF WHAT
WOULD BE UNDERLYING GLYCAN
STRUCTURE.
SO MOVING BACK TO, YOU KNOW,
10,000-FOOT VIEW HERE, WE'RE
GOING BACK TO TRANSMISSION
ELECTRON MICROSCOPY TO LOOK AT
THE SURFACE OF THE CELL AND LOOK
AT THE GLYCOCALIX, YOU CAN OFTEN
SEE A LOT OF BACTERIA HERE.
WE DID MORE THAN JUST TAKE A
COUPLE PICTURES AND CALL THEM
REPRESENTATIVE.
WE TOOK VAGINAL EPITHELIAL CELLS
AND WE DID A SCORING PROCEDURE
IN WHICH WE HAD THREE OBSERVERS,
THE PERSON WHO TOOK ALL OF THE
IMAGES WAS BLINDED TO THE
CLINICAL STATUS OF THOSE
SAMPLES, AND ALL PEOPLE WHO
SCORED THE SAMPLES WERE BLINDED
TO THE STATUS OF THE SAMPLES.
AND SO WHAT WE CAN SEE IS THAT
IN THE CONTEXT OF WOMEN WITHOUT
BV YOU SEE GLYCOCALYX, IN MORE
THAN HALF ALL THREE SAID THERE
WAS ZERO GLYCOCALIX PRESENT.
WE'VE BEEN ABLE TO STUDY IN
VITRO, WE HAVE RECOMBINANT FROM
GARDNERELLA, DECIDED TO TAKE
NORMAL CELLS AND TREAT WITH
SIALIDASE TO DETERMINE WHETHER
WE'RE STILL SEEING THIS WHOLE
PHENOTYPE, WERE SIALIC ACID
REMOVAL IS RESPONSIBLE OR MORE
MIGHT BE GOING ON.
WE CAN SEE THIS SIALIDASE
TREATMENT WITH GARDNERELLA
REDUCED APPEARANCE AND TO
PHENOCOPY WHAT WE'RE SEEING IN
BACTERIAL VAGINOSIS.
KIND OF TAKING THIS BACK TO,
OKAY, WHAT DO WE THINK IS
HAPPENING, WHY IS THIS
IMPORTANT, IF YOU ARE DOING A
WHOLE SCALE REMOVAL THIS COULD
HAVE A LOT OF DOWNSTREAM
EFFECTS, AS I'M SURE THIS
AUDIENCE CAN APPRECIATE.
AND SO SOME OF THE EXPERIMENTS
THAT WE'VE DONE, WE HAVE
DIFFERENT AVENUES TO FOLLOW, BUT
SOME EXPERIMENTS THAT WE'VE DONE
INVOLVE LOOKING AT WHETHER THE
CHANGES TO THE GLYCANS ON THE
CELL SURFACE MAY INFLUENCE
ABILITY OF EITHER GARDNERELLA OR
OTHER BACTERIA TO BIND TO
EPITHELIAL CELLS AND COLONIZE
THE VAGINA, MAYBE HAS SOMETHING
TO DO WITH HOW YOU DEVELOP
COLONIZATION AND OVERGROWTH YOU
SEE IN BV.
THESE ARE NOT WITH EPITHELIAL
CELLS.
THIS HAPPENS A LITTLE BIT
EARLIER IN THE PROCESS OF THE
LAB.
BUT INSTEAD WE'RE USING
HEMAGGLUTINATION TO LOOK AT
BINDING OF GARDNERELLA VAGINALIS
AND EVALUATE IMPACT OF GLYCANS.
FIRST I'M GOING TO TELL YOU THAT
WE WERE ABLE TO OBSERVE THAT
SIALIDASE TREATMENT IMPROVED
HEMAGGLUTINATION TO SHEEP
RBCs, AND THEN WE COULD
ACTUALLY OUTCOMPETE THAT WITH
GALACTOSE OR LACTOSE.
BUT NOT WITH THESE OTHER SUGARS.
WE THINK THIS IS GOOD EVIDENCE
IN RELATION TO THIS MODEL WITH
RBCs THAT GARDNERELLA CAN BIND
TO GALACTOSE RESIDUES UNDERLYING
SIALIC ACID, MAY BE RELEVANT TO
EPITHELIUM.
TO ASK DOES IT REALLY IMPACT
BINDING, I WANTED TO SHARE ONE
MORE EXPERIMENT WITH YOU GUYS.
THIS IS A LITTLE BIT MORE
PRELIMINARY.
WE STILL NEED TO FOLLOW UP.
THIS IS MORE RELEVANT TO HUMAN
CONTEXT USING CELLS ISOLATED
FROM VAGINAL SWABS.
IN THIS CASE WE'RE USING
EPITHELIAL CELLS FROM NORMAL
WOMEN WITHOUT BV.
AND THEN WE HAVE ALSO DEVELOPED
THIS RESOURCE WHETHER WE'VE
BROUGHT VAGINAL SWABS TO THE LAB
UNDER ANAEROBIC CONDITIONS AND,
WELL, CAN FREEZE THEM DOWN,
REMOVED THEM FROM FREEZERS AND
CULTIVATE.
THIS IS FROM A COMPLETELY
DIFFERENT COHORT, THAT DOESN'T
MATTER, BUT THE EXPERIMENT IS
ULTIMATELY TO TAKE THESE PRIMARY
EPITHELIAL CELLS, TREAT THEM
WITH OR WITHOUT SIALIDASE, TAKE
THESE COMMUNITIES THAT WE'RE
ABLE TO GROW IN VITRO, INCUBATE
WITH CELLS, DO FLOW CYTOMETRY TO
EVALUATE BINDING.
WHAT WE CAN SEE WITH A COUPLE
DIFFERENT POOLS FROM THE WOMEN
WITHOUT BACTERIAL VAGINOSIS
WE'RE ABLE TO SEE THAT IN THE
CONTEXT OF SIALIDASE MUCH BETTER
INTERACTION OF THIS COMMUNITY
WITH THE CELLS.
SO THIS IS SORT OF SOME
PRELIMINARY TEASER EVIDENCE THAT
WE THINK SIALIDASE MAY BE
INVOLVED IN HELPING SOME OTHER
BACTERIA ATTACH TO THE HOST AND
THEREFORE COLONIZE AND
POTENTIALLY CAUSE BV.
A TINY BIT FURTHER, THIS IS
OLDER DATA BUT I WANTED TO SHARE
IT BECAUSE IT'S RELEVANT.
IN SOME EARLIER STUDIES WE WERE
LOOKING AT ABILITY OF
GARDNERELLA AND THE GLYCOSYLASES
PRESENT IN THE CONTEXT TO
DEGRADE SUBSTRATE, PROVIDING
IgA, HIGHLY GLYCOSYLATED,
LABELED TO OBSERVE ON THE GEL.
WHEN WE INCUBATE THIS SIGA-FITC
OF WOMEN WITH OR WITHOUT
BACTERIAL VAGINOSIS CAN SEE
ANALOGOUS TO EARLIER STUDIES
WITH IMMUNOFLUORESCENCE, EXCEPT
THIS TIME LOOKING AT SNA, 2, 6
LINKED, TOP IS SECRETORY
COMPONENT, BOTTOM IS HEAVY
CHAIN, LABELING FROM FIT-C, IN
THE CONTEXT OF THE BV STATUS YOU
SEE THAT THERE'S, SORRY, THIS
ONE IS NOT COMING FROM FIT-C,
FROM SNA, EARLIER.
YOU SEE VERY LITTLE STAINING,
YOU DO SEE THAT AS YOU WOULD
EXPECT ON OF THE IgA OF THE
WOMEN, SAMPLES FROM WOMEN
WITHOUT BV BEING TREEDED HERE.
IF YOU TAKE THAT A STEP FURTHER
WE WANT TO KNOW IS THERE
PROCESSIVE DEGLYCOSYLATION.
WE TESTED TO SEE IF WE COULD SEE
REVEALING OF CON-A BINDING
SITES.
IN FACT EXPERIMENTS CAN BE
MESSY, BUT WHEN YOU LOOK HERE,
WHEN THE SUBSTRATE WAS ADDED TO
SAMPLES OF WOMEN WITHOUT
BACTERIAL VAGINOSIS YOU SEE MORE
CLEAN BANDS HERE AND HERE AND
HERE.
THIS IS THE CONTROL WHETHER
WE'RE TAKING COMMERCIAL ENZYMES
AND TREATING SO YOU CAN SEE HOW
IT SMEARS DOWNWARDS, AND IN THE
CONTEXT OF BACTERIAL VAGINOSIS
YOU SEE A SIMILAR PHENOTYPE,
SMEARING DOWNWARDS OF CON-A
BINDING SITES, HERE AND HERE.
THIS IS THE QUANTITATION OF
THOSE BANDS.
AND SO JUST COMING BACK TO THE
BEGINNING I HOPE THAT I HAVE
CONVINCED YOU THAT RESIDENT
MICROBES IN VAGINA SHAPE THE
EPITHELIAL LANDSCAPE AND MAY BE
RELEVANT FOR THE BEEN OF
BACTERIAL VAGINOSIS, AND KAVEETA
IS THE FIRST AUTHOR, SHE'S ON
THE JOB MARKET IF YOU'RE
INTERESTED.
THANKS TO THE COLLABORATORS AND
PATIENTS WHO MADE THIS WORK
POSSIBLE.
I'LL TAKE ANY QUESTIONS.
>> SUPER TALK.
DO WE HAVE QUESTIONS FROM ANYONE
LISTENING ON THE ZOOM?
YOU CAN USE YOUR MICROPHONES OR
I'LL MONITOR THE CHAT.
I'LL LET YOU CAN, LAURA.
>> I WAS CURIOUS, I'LL SOUND
LIKE A BROKEN RECORD ASKING THE
SAME KIND OF QUESTION OVER AND
OVER.
SEEMS LIKE THE LOSS OF SIALIC
ACID COULD OPEN SITES FOR
GALECTINS, WONDERING IF YOU'VE
EVER LOOKED TO SEE IF YOU SEE AN
INCREASE IN GALECTINS, MAYBE
BEING RECRUITED TO THE MUCOUS OR
SORTS OF LAYERS AND BRINGING
THAT UP BECAUSE SEAN MIGHT BE
INTERESTED.
>> YEAH, WE HAVEN'T LOOKED AT
THAT YET BUT I THINK IT'S VERY
INTERESTING AND SINCE WE KNOW WE
HAVE AN EXFOLIATION PHENOTYPE IN
BACTERIAL VAGINOSIS AND IT'S
ALSO SHOWN THAT THOSE CELLS ARE
UNDERGOING APOPTOSIS AS THEY ARE
EXFOLIATING AND COULD BE
REVEALING GALACTOSE EPITOPES
CAUSES GALECTIN 1 OR OTHERS TO
ACT IN THAT CONTEXT.
>> THERE ARE SOME, I DON'T KNOW
WHICH ONES, BUT I THINK IT WOULD
BE INTERESTING TO LOOK AT.
>> COMPLETELY AGREE.
I WON'T COMMENT FOR TIME BUT
THIS IS BEAUTIFUL WORK.
>> THANK YOU FOR A GREAT TALK.
REALLY NICE.
IN THE INTEREST OF TIME WE'LL
MOVE ON BECAUSE WE DO WANT TO
WRAP BY FIVE.
THE FAIL-SAFE SHY OF -- IS I
WON'T GIVE MY TALK.
NEXT IS MOHAMMED ABDEL FROM THE
WISTAR INSTITUTE, FLY COMBIC
MODULATION OF THE GUT
MICROBIOME.
>> MY PRESENTATION WILL FOCUS ON
VIRAL INFECTION.
IN PARTICULAR I WANT TO
HIGHLIGHT IMPORTANCE OF GLYCANS
IN REGULATING RELATIONSHIP
BETWEEN THE MICROBIOME AND
IMMUNITY DURING VIRAL
INFECTIONS.
BECAUSE OF THAT I WANT TO GIVE
TWO EXAMPLES.
FIRST EXAMPLE I WILL FOCUS ON
HOST, IN PARTICULAR GUT
GLYCOSYLATION AND REGULATING
RELATIONSHIP BETWEEN MICROBIOME
AND IMMUNITY AND I WILL USE HIV
AS AN EXAMPLE.
AND IN THE SECOND EXAMPLE I WANT
TO MOVE FROM HOST TO HOST
GLYCANS IN MICROBIOME AND HOW DO
GLYCANS IN BACTERIA AND FUNGUS
IN OUR MICROBIOME IMPACT THE
RELATIONSHIP BETWEEN HOST AND
IMMUNITY IN SARS-COV-2.
STARTING WITH THE FIRST EXAMPLE
IN HIV INFECTION I WANT TO START
BY HIGHLIGHTING WHY THE GUT IS A
MAJOR SITE FOR HIV PATHOGENESIS.
SO HIV CAUSES SYSTEMIC
INFLAMMATION THROUGH CYTOKINE
STORM, SOME CAN INJURY THE GUT,
CAUSING PERMEABILITY, LEAKY GUT,
MEANING IT ALLOWS MICROBES AND
BYPRODUCTS TO TRANSLOCATE FROM
THE GUT TO THE BLOOD BEING
RECOGNIZED BY SYSTEMIC IMMUNE
SYSTEM CAUSING EVEN FURTHER
INFLAMMATION, FURTHER CYTOKINE
RELEASE, WHICH HAPPENS TO BE THE
THING HIV NEEDS TO EVEN INFECT
MORE CELLS.
HIV CAN ALSO DIRECTLY INFECT T
CELLS IN THE GUT TO EVEN FURTHER
FEED THE CYCLE, EVENTUALLY THIS
VICIOUS CYCLE CONTRIBUTES TO THE
DEVELOPMENT OF SEVERAL
INFLAMMATION-ASSOCIATED DISEASES
IN PEOPLE LIVING WITH HIV
INCLUDING NEUROLOGICAL
IMPAIRMENTS, CARDIOVASCULAR
DISEASES, SEVERAL KIND OF
CANCERS.
EVEN IF PEOPLE LIVING WITH HIV
ARE ON SUPPRESSIVE THERAPY,
VIRUS IS SUPPRESSED, THIS CYCLE
NEVER COMPLETELY RECOVERS AND
NEVER COMPLETELY STOPS,
CONTINUES CAUSING CHRONIC
INFLAMMATION IN PEOPLE WITH HIV
EVEN AFTER LONG-TERM THEY
LONG-TERM -- THERAPY.
THERE'S A LOT OF INTEREST IN
FINDING A WAY OR WAYS TO BREAK
THIS CYCLE AND PREVENT THIS
INFLAMMATION AND PREVENT THESE
COMORBIDITIES.
NOW AS MENTIONED EARLIER TODAY
IN MULTIPLE TALKS, IN THE LAST
COUPLE YEARS IT'S BECOME CLEAR
GUT GLYCANS PLAY A ROLE IN
REGULATING GUT AND MICROBIOME.
THERE'S SEVERAL BACTERIA THAT
CAN RELEASE HIGH LEVEL OF GLYCAN
DEGRADING ENZYMES BECAUSE
BACTERIA IS TRYING TO USE THIS
SUGARS AS FOOD SOURCE.
GUT MICROBIOME CAN IMPACT THE
GLYCOME.
THE GUT GLYCOME IS FOOD
SOURCEING SITE.
WHEN BACTERIA CONSUMES SUGAR
IMPACTION VIRULENCE.
THIS IS EXPRESSED BY EPITHELIAL
CELLS IN THE GUT, WHEN CONSUMED
CAN REPRESS VIRULENT GENES.
AN INDIVIDUAL WHO DOES NOT HAVE
ABILITY TO SUPPRESS MORE PRONE
FOR GUT INFLAMMATION IN
INFLAMMATORY BOWEL DISEASES.
WE WANT TO ASK THE QUESTION DOES
HIV INFECTION EVEN AFTER LONG
TERM SUPPRESSIVE THERAPY
RELATED?
WE ISOLATED CELL MEMBRANE
PROTEINS FROM EPITHELIAL CELLS,
APPLIED TO LECTIN ARRAY, AN
ARRAY WITH IMMOBILIZED LECTINS,
HIGHER BINDING TO INDICATE
HIGHER EXPRESSION OF SUGAR.
IN THIS HEAT MAP AN EXAMPLE FROM
THE ILIUM, SIMILAR FROM THE
COLON.
RED IS HIGHER EXHIBITION, BLUE
IS LOWER EXPRESSION.
GUT GLYCOME SHOWING SEVERAL
DIFFERENCES COMPARED TO GUT
GLYCOME FROM CONTROL.
NOTABLY, A LOWER LEVEL OF THIS
PROTECTIVE FUCOSE AS INDICATED
BY LOWER BINDING TO SEVERAL
LECTINS IS SPECIFIC FOR ALPHA.
WE CAN CONFIRM USING
IMMUNOHISTOCHEMISTRY, HERE AN
EXAMPLE OF SLIDES FROM SAME
DONORS.
LEFT HAVING THE LOWEST SIGNAL
FROM LECTIN ARRAY, ONE ON THE
RIGHT SHOWING HIGHEST SIGNAL BY
LECTIN ARRAY, AND ALSO YOU CAN
SEE FROM LEFT TO RIGHT A HIGHER
LEVEL BY IMMUNOHISTOCHEMISTRY
USING THIS LECTIN.
WE CAN SEE NICE BURST OF
CORRELATION MEASURED BY
IMMUNOHISTOCHEMISTRY.
WE THINK HIV INFECTION EVEN
AFTER LONG-TERM THEY WERE
ASSOCIATED WITH FUCOSLYATED
GLYCANS.
NEXT WE WANT TO EXAMINE
CORRELATION BETWEEN LEVEL OF
THESE BRANCHES, FUCOSLYATED
GLYCANS, INFLAMMATION, FEATURES
OF THE MICROBIOME.
IN THIS PLOT, RED IS INDICATING
SIGNIFICANT BURST OF
CORRELATION.
I WANT TO SHOW CORRELATION
BETWEEN MICROBIOTA AND
INFLAMMATION.
THIS MARKER IS USED AS MARKER,
THE MORE DIVERSE THE MICROBIOTA,
THE MORE HEALTHY THE GUT.
AS EXPECTED, THERE'S A NEGATIVE
CORRELATION BETWEEN IT, AND
PRO-INFLAMMATORY MARKERS,
EXPRESSED HIV INFECTION.
WHEN WE LOOK AT THE MICROBIOME
WE FOUND IT IS THE LEVEL OF THIS
PARTICULAR BACTERIAL SPECIES
MEASURED IN ILIUM, RIGHT COLON,
SIGMOID COLON, CORRELATION IN
THE GUT DURING HIV INFECTION,
QUITE INTERESTING BECAUSE AS I
MENTIONED EARLIER, BACTERIA
VULGARIS, HIGHER LEVEL OF THIS
BACK TEAR YEA -- BACTERIA,
CONSISTENT WITH
ANTI-INFLAMMATORY PROPERTIES OF
THIS BRANCH OF FUCOSE WITHOUT
FOUND ITS LEVEL EXHIBIT A BURST
OF INFLAMMATION, AND CORRELATION
WITH IL-10, ANTI-INFLAMMATORY
MARKERS.
OBVIOUSLY THE LOWER LEVEL OF GUT
FUCOSYLATION WE SEE IN HIV
INFECTION IS ASSOCIATED WITH
LOWER MICROBIAL DIVERSITY.
WE WANTED TO EXPLORE POTENTIAL
MECHANISM WHY LOWER FUCOSE
CORRELATES.
WE DID RNA SEQUENCING AND
FOCUSED ON THE GENES, EXHIBITING
BURST OF CORRELATION WITH
INFLAMMATION, OR WITH LEVEL OF
HIV IN THE GUT.
FUCOSE IS ANTI-INFLAMMATORY, BY
DEFAULT NEGATIVE CORRELATION
WITH FUCOSE.
WHEN WE DID ANALYSIS WE FOUND
LOWER LEVEL OF FUCOSE DURING HIV
INFECTION ASSOCIATED WITH
ACTIVATION OF THESE SIGNALING
PATHWAYS, A SIGNALING PATHWAY
KNOWN TO ACTIVATE THE GUT
INFLAMMASOME, WE THINK MAYBE THE
RELATIONSHIP BETWEEN LOWER
FUCOSE AND HIGHER INFLAMMATION
DURING HIV INFECTION MAY BE
RELATED TO THE ACTIVATION OF THE
GUT INFLAMMASOME MEDIATOR.
WE DID THIS ANIMAL STUDY USING
HUMANIZED MICE OF HIV INFECTION,
IT CANNOT INFECT REGULAR MICE,
INFECTED WITH HIV, WE TREATED
THEM OR NOT WITH GLYCOMIC
MODULATOR INCLUDING L FUCOSE AND
AS YOU CAN SEE THE MICE, TREATED
MICE IN RED, SHOWED LOWER LEVEL
OF IMMUNE ACTIVATION, IN THE
BLOOD AND GUT COMPARED TO
UNTREATED MICE IN GRAY BARS.
SOME DATA HAS BEEN PUBLISHED,
YOU CAN READ MORE.
ON THE HOST GLYCANS, IMPORTANCE
IN VIRAL INFECTION, LOWER LEVEL
OF ANTI-INFLAMMATORY FUCOSLYATED
GLYCAN ASSOCIATED WITH HIGHER
ABUNDANCE, IN PARTICULAR
BACTERIA VULGARDIS, HIGHER LEVEL
OF INFLAMMATION AND THESE
FINDINGS WERE CORRELATED, OR
ASSOCIATED WITH ACTIVATION OF
INFLAMMASOME TO SIGNALING
MESSENGER LEVEL.
THEY WERE ONE OF THE FEW
EVIDENCE THAT EXIST PREVIOUSLY
UNAPPRECIATED FACTOR IN
VIROLOGY.
I SHOWED AN EXAMPLE OF HIV BUT
THIS COULD BE TRUE FOR SEVERAL
VIRAL INFECTIONS WHERE GUT
MICROBIOME AND GUT ARE
IMPLICATED.
FOCUSING IN THE GUT
GLYCOSYLATION I'LL MOVE TO
SECOND EXAMPLE, THE OTHER
MEMBER, THE MICROBIOME, THE
FUNGUS AND BACTERIA IN THE GUT
HEAVILY COVERED WITH GLYCANS AND
SHOWING AN EXAMPLE HOW GLYCANS,
BACTERIA AND FUNGUS MAY PLAY
IMPORTANT ROLE REGULATING
RELATIONSHIP BETWEEN HOST AND
IMMUNITY BUT NOW DURING
SARS-COV-2 INFECTION.
BEFORE THE SARS-COV-2 PANDEMIC
THERE WERE SEVERAL EVIDENCE OF
THIS EMERGING CONCEPT, GUT-LUNG
AXIS, IN THE LUNG INFECTION
OTHER DAMAGE THAT IT CAN CAUSE
SYSTEMIC INFLATION, RELEASE OF
HIGH LEVEL OF CYTOKINES, CAN
INDIRECTLY ALSO LEAD TO HIGHER
INTESTINAL PERMEABILITY AND HIGH
LEVEL OF MICROBIAL
TRANSLOCATION, THIS CAN CAUSE
FUEL THIS SYSTEMIC INFLAMMATION
OF CYTOKINE IN A MATTER THAT CAN
WORSEN ORIGINAL LUNG INFECTION
OR DAMAGE.
IN SARS-COV-2 WE KNOW IT'S ALSO
SARS-COV-2 CAN DIRECTLY INFECT
INTERIOR SITE IN THE GUT TO
MAYBE FEEDING THE CYCLE.
WE ASKED THE QUESTION IS THERE
LINK BETWEEN LUNG INFLAMMATION
AND GUT IN COVID-19?
THAT WAS NOT THE FOCUS OF MIGHT
HAVE EXAMPLE TODAY.
WE EXAMINED THE PLASMA SAMPLE OF
SARS-COV-2 NEGATIVE AND POSITIVE
INDIVIDUAL WITH DIFFERENT
DISEASE SEVERITY, MILD,
MODERATE, SEVERE.
AS YOU CAN SEE INDEED WE FOUND
HIGHER LEVEL OF MARKERS AND
DRIVER OF TIGHT JUNCTION
PERMEABILITY IN PLASMA OF
INDIVIDUALS WITH SEVERE
COVID-19 COMPARED TO CONTROL,
ALLOWING MICROBES AND BY-PRODUCT
TO TRANSLATE TO THE BLOOD USING
MARKERS TO MEASURE, OR MARKER OF
FUNGAL TRANSLOCATION, A GLYCAN
COVERING SURFACE OF FUNGUS.
IN BOTH CASE THERE'S EVIDENCE OF
BACTERIA AND FUNGUS CAN
STIMULATE IMMUNE SYSTEM, IN THIS
EXAMPLE YOU CAN SEE STRONG BURST
OF CORRELATION.
THERE WAS ROBUST BODY OF
LITERATURE USING STOOL SAMPLE
SHOWING COVID-19 IS ASSOCIATED
WITH STATE OF GET MICROBIAL
DYSBIOSIS, IN A MANNER
ASSOCIATED WITH HIGH
INFLAMMATION AND HIGH -- WORSE
DISEASE SEVERITY.
MY EXAMPLE TODAY WILL FOCUS ON
WHAT HAPPENED AFTER RECOVERY
FROM ACUTE COVID-19.
AND WE NOW KNOW THERE'S AT LEAST
10 TO 30% OF PEOPLE WHO RECOVER
FROM ACUTE COVID-19 CAN
EXPERIENCE WIDE VARIETY OF
SYMPTOMS, EITHER PERSISTENCE,
COVID-ATTRIBUTED SYMPTOMS FOR
THREE TO FOUR MONTHS FOLLOWING
ACUTE COVID-19, LONG COVID,
POST-ACUTE SEQUELAE OF COVID-19.
THIS PHENOMENON IS VERY
IMPORTANT, CAN IMPACT LIFE OF
MILLIONS OF PEOPLE RECOVERED AND
RECOVERING FROM COVID-19.
THERE'S A LOT OF INTEREST IN
UNDERSTANDING MECHANISM DRIVING
THIS, ASSOCIATED WITH SUSTAINED
LEVEL OF INFLAMMATION,
MULTI-FACTORIAL IN NATURE BUT IN
OUR CASE WANTED TO EXAMINE DOES
MICROBIAL TRANSLOCATION
BACTERIAL OR FUNGAL RESIST AFTER
CONVALESCENS.
WE ASKED SAMPLES FROM UCSF
COHORT, FROM INDIVIDUALS THREE
TO FOUR MONTHS AFTER RECOVERY IN
ACUTE COVID-19.
THAT WERE EITHER STILL SUFFERING
FROM SOME SYMPTOMS, PASC, OR
NOT.
WE LOOKED AT MARKERS.
YOU CAN SEE IN BLUE INDIVIDUALS
STILL SUFFERING FROM PASC HAVING
HIGHER LEVEL OF MARKER OF TIGHT
JUNCTION PERMEABILITY, THAT
COULD BE IN THE GUT OR LUNG.
AND INDEED WHEN WE LOOK AT
MARKER OF FUNGAL TRANSLOCATION,
THIS IS POLYSACCHARIDE, SUGAR,
WE FIND HIGH LEVEL OF FUNGAL,
YOU MIGHT NOTE WE DRAW A LINE AT
40, A CLINICAL CUT OFF,
DIFFERENTIALING INDIVIDUAL WHO
WILL HAVE FUNGAL TRANSLOCATION
IN A MANNER LEADING TO CLINICAL
OUTCOME AND WORSE INFLAMMATION,
INDEED YOU CAN SEE INDIVIDUAL
WITH NO PASC, THERE ARE SEVERAL
INDIVIDUALS WITH PASC WITH
HIGHER THAN 40-MICROGRAM PER M
MIL CUTOFF OF BETA GROUP CAN.
EVEN 18 SYMPTOMS IN SOME
INDIVIDUALS, ALSO CORRELATING
NEGATIVELY WITH QUALITY OF LIFE
THAT IS REPORTED BY THOSE
INDIVIDUALS OR PATIENTS.
WHEN WE DIVIDE INDIVIDUALS WHO
ARE SUFFERING FROM PASC,
INCLUDING G.I.-RELATED SYMPTOMS,
YOU CAN SEE INDIVIDUALS WHO HAVE
PASC WITH G.I.-RELATED SYMPTOMS
HAVE HIGH LEVEL OF BETA GLUCAN
COMPARED TO INDIVIDUALS WITHOUT
G.I.-RELATED SYMPTOMS OR NO
PASC, HIGHLIGHTING POTENTIAL
ROLE OF GUT IN THIS PHENOMENA.
WE SEE TREND TOWARD HIGH LEVEL
TRANSLOCATION
WHY FUNCTIONAL TRANSLOCATION
WILL BE ELEVATED, AND WE KNOW
THESE POLYSACCHARIDES BETA
GLUCAN CAN DRIVE ON SURFACE OF
MYELOID CELLS, THE BINDING CAN
ACTIVATE SYK SIGNALING LINKED TO
NF-kappaB ACTIVATION, LEADING
TO MYELOID INFLAMMASOME.
ONE COULD HIGH POTENTIAL --
HYPOTHESIZE SOME LEVEL EXHIBITS,
MAYBE BETA GLUCAN CONTRIBUTES TO
INFLAMMATION.
THE LEVEL OF BETA GLUCAN IN
PLASMA IN THE Y-AXIS CORRELATES.
THIS IS JUST CORRELATION AND WE
WANT TO EXAMINE IF THE LEVEL OF
BETA GLUCAN IN PLASMA OF
INDIVIDUALS CAN INDEED LEAD TO
NF-kappaB MEDIATED
INFLAMMATION.
IN ORDER TO EXAMINE THAT WE USED
THIS MODEL, A CELL LINE EXPRESS,
RECOGNIZED BY BETA GLUCAN,
LEADING TO SYK SIGNALING AND
NF-kappaB ACTIVATION WHICH YOU
CAN MEASURE IN THE CULTURE AND
USE THIS INHIBITOR FOR SYK
SIGNALING.
I WANT TO POINT YOUR ATTENTION
TO THIS SIDE OF THE GRAPH, CAN
YOU SEE CLEARLY THE PLASMA FROM
INDIVIDUALS CAN LEAD TO HIGHER
NF-kappaB SIGNALING COMPARED
TO THE PLASMA FROM SARS-COV-2
NEGATIVE INDIVIDUALS USING THIS
INHIBITOR TO MITIGATE
ACTIVATION.
WE ALSO FOUND POSITIVE
CORRELATION BETWEEN LEVEL OF
BETA GLUCAN WE CAN EXAMINE IN
PLASMA AND DEGREE OF NF-kappaB
SIGNALING CAN ELICIT IN THIS
CELL MODEL.
THE LEVEL IN POST ACUTE SEQUELAE
ARE CAPABLE OF INDUCING SOME
MANNER LINKED TO ACTIVATION
OF-AND-A-HALF SIGNALING PATHWAY.
WE THINK IT'S PARTIALLY WHAT
HAPPENED, MIGHT BE DISRUPTION IN
TIGHT JUNCTION, THIS
POLYSACCHARIDE CAN BIND TO ITS
SUGAR BINDING PROTEIN DECTIN 1
LEADING TO ACTIVATION INHIBITED
USING THIS SMALL MOLECULE
INHIBITOR FOR SYK.
THE THIRD BAR, THIS COULD BE A
TOPIC OF SEPARATE PRESENTATION
AND WOULD LIKE TO GO TO MY
ACKNOWLEDGMENT SLIDE.
I WOULD LIKE TO THANK MY
COLLABORATORS.
I WILL TAKE QUESTIONS OR FEEL
FREE TO E-MAIL ME.
THANK YOU.
>> FANTASTIC.
DO WE HAVE QUESTIONS FROM PEOPLE
IN THE ROOM OR PEOPLE ONLINE?
WHAT ABOUT USING NF-kappaB
INHIBITORS RIGHT NOW, COULD YOU
MULTIPURPOSE THEM?
>> I'M NOT SURE HOW TOLERABLE
THAT WILL BE,-AND-A-HALF IS
OBVIOUSLY IMPORTANT PATHWAY FOR
IMMUNITY.
I WILL BE AFRAID IT WILL CAUSE
SYSTEMIC IMMUNE SEPARATION ALSO
NOT VERY ATTRACT ATTRACTIVE,
IT'S STILL SYK SIGNALING IS
IMPORTANT, MAYBE LESS BROAD,
SOMETHING SPECIFIC FOR BETA
GLUCAN WILL BE EVEN MUCH BETTER
OR SOMETHING LIKE TO INCREASE
THE TIGHTNESS OF THE TIGHT
JUNCTION.
FOR EXAMPLE, ACTUAL LIP THERE
ARE SMALL MOLECULE INHIBITORS
THAT CAN PREVENT, MAYBE EVEN
GOING TO THE SOURCE OF THE
PROBLEM, MAYBE MORE SPECIFIC AND
SAFER.
>> TIMELY.
PEOPLE ARE SUFFERING.
THANK YOU FOR A FANTASTIC TALK.
THANK YOU.
I WILL KEEP YOU POSTED.
IF PEOPLE POSE QUESTIONS I'LL
MAKE SURE I GET THOSE E-MAILED
OUT.
KEEP QUESTIONS COMING ONLINE.
SEAN WILL INTRODUCE THE NEXT
SPEAKER.
WE'LL TAKE A SWITCHING BREAK.
>> WE'LL NEXT HAVE THE
OPPORTUNITY TO HEAR FROM SETH
RAKOFF-NAHOUM, GLYCAN-METABOLITE
EMERGENT PROPERTIES IN THE GUT
MICROBIOME.
>> THANK YOU FOR THE INVITATION
TO SPEAK TODAY.
I'M GOING TO SET MY TIMER.
AND TALK TO YOU ABOUT A STORY
THAT WAS RECENTLY PUBLISHED IN
THE LAB.
BUT I WANT TO START WITH
REALLY -- I'LL PUT MY POINTER
UP.
HOW IMPORTANT WE THINK THIS IS,
ACROSS ORGAN AND TISSUE AND
PHYSIOLOGICAL SYSTEMS AND
PATHOPHYSIOLOGICAL SYSTEMS.
WE CAN ALL AGREE CHALLENGES THAT
HAVE EMERGED FROM OUR
ASSOCIATIONS BETWEEN DIFFERENT
MICRO BIOMES AND THESE HEALTH
AND DISEASE SPACE ARE TRYING TO
UNDERSTAND MORE ABOUT THE
MICROBIOME ITSELF, WHAT ARE THE
MICROBES DOING, HOW DO THEY
COLONIZE AND SURVIVE?
AND REALLY FROM THIS, ACT ON THE
MICROBIOME FOR HEALTH, AND TO
REALLY REALIZE THE
THERE ARE, OF COURSE, A NUMBER
OF NEEDS THAT ARE REQUIRED TO
MEET THESE GOALS, AND I WOULD
SAY THAT THEY FALL INTO FOUR
NON-MUTUALLY EXCLUSIVE
CATEGORIES, WHICH IS WE NEED TO
BECOME ENVIRONMENTAL MICRO
BIOLOGISTS FOR EACH OF THESE
MICROBES THAT WE'VE NEVER HEARD
OF.
MOLECULAR BIOLOGISTS FOR THESE
MICROBES, FOR REDUCTION OF
STUDIES, USING CLASSIC BACTERIAL
GENETIC APPROACHES, AND THEN
ECOLOGISTS AND EVOLUTIONARY
BIOLOGISTS TO UNDERSTAND HOW
THESE MICROBES ARE LIVING THERE
AND WHAT TYPES OF RELATIONSHIPS
THEY HAVE WITH EACH OTHER AND
WITH THE HOST.
AND SO FROM AN ECOLOGICAL
PERSPECTIVE, WE CAN ASK THE
QUESTION OF WHAT CONTROLS THE
MICROBIOME'S FORM AND ITS
FUNCTION AND REALLY BREAK THIS
UP INTO THREE NON-MUTUALLY
EXCLUSIVE CONCEPTUAL CATEGORIES.
SO THESE CAN BE ABIOTIC, DIET,
WHICH PROVIDES NUTRIENTS AS
DEPICTED IN A GREEN POSITIVE
ARROW, OR A NEGATIVE FACTOR SUCH
AS ANTIBIOTICS, WHICH KILLS
MICROBES IN THE GUT.
THERE'S, OF COURSE, A PLETHORA
OF ECOLOGICAL -- THOSE SPAN
COMPETITION TO EXPLOITATION AND
POSITIVE AND NEGATIVE
DIRECTIONAL INTERACTIONS BETWEEN
IN PAIR WISE INTERACTIONS
BETWEEN MICROBES.
AND THEN OF COURSE THERE'S THE
ROLE OF THE HOST WHICH HAS BEEN
TALKED ABOUT IN A NUMBER OF
DIFFERENT WAYS TODAY, BOTH AT
THE ANTIBODY LEVEL AND HOST
SECRETED LECTINS THAT PLAY ROLES
IN EITHER NEGATIVELY REGULATING
WHICH IS THE -- WHEN WE THINK
ABOUT ANTIMICROBIAL PEPTIDES BUT
ALSO POTENTIALLY FEEDING OR
KEEPING CERTAIN MEMBERS OF THE
MICROBIOME, SUCH AS IN POSITIVE
SELECTION.
HOWEVER, IF WE THINK ABOUT THE
MICROBIOME AND TRYING TO
UNDERSTAND WHAT ARE THE FACTORS
ALONG THESE THREE CATEGORIES
THAT WOULD IMPACT ANY INDIVIDUAL
MEMBER OF THE MICROBIOME,
WHETHER IT'S A BUG YOU WANT TO
COLONIZE THE GUT OR YOU DON'T
WANT TO COLONIZE THE GUT, WE CAN
THINK ABOUT HOW DIET IMPACTS THE
FITNESS OF AND FOR FUNCTION OF
EACH INDIVIDUAL MEMBER, BUT OF
COURSE DIET IS MADE OUT OF
THOUSANDS OF MACRO AND MICRO
MOLECULES.
SIMILARLY, MY KROAPS MICROBES THEMSELVES
MAKE A PLETHORA OF METABOLITES,
PROTEINS, SMALL MOLECULES, AND
THEN, OF COURSE, THERE'S THE
HOST WHICH HAS 19,000 GENES IN
THE IMMUNE SYSTEM AND A NUMBER
OF GLYCANS AND LECTINS THAT ARE
LIKE THE IMPACTING MEMBERS OF
THE MICROBIOME.
SO WHEN WE THINK ABOUT THIS
FUNDAMENTAL NICHE OF ALL THE
POTENTIAL FACTORS THAT CAN BE
ACTING ON ANY GIVEN MICROBE, IT
CAN HURT YOUR HEAD.
AND OF COURSE EACH OF THESE
FACTORS COULD IMPACT THE
ECOLOGICAL INTERACTIONS BETWEEN
WHICH MEMBERS AND WE DON'T KNOW
WHICH MEMBERS HAVE THOSE PAIR
WISE INTERACTIONS, WE DON'T KNOW
WHETHER THEY'RE PASSTIVE AND
NEGATIVE, AND OF POSITIVE AND
NEGATIVE AND WE DON'T UNDERSTAND
THE MECHANISTIC RELATIONSHIP AT
THE GENETIC OR MOLECULAR LEVELS.
SO THAT'S THE GOAL OF MY LAB,
AND OF COURSE MANY OF YOU THAT
ARE OUT THERE.
AND I'LL JUST USE 30 SECONDS TO
KIND OF TALK ABOUT REALLY -- OUR
LAB REALLY IS AT THE CROSSROADS
OF THE MICROBIOME AND
GLYCOSCIENCE, AND THAT ACTUALLY
IS SOME PROJECTS IN THE LAB
WHICH ARE JUST LEARNING OF HOW
BIZARRE INDIVIDUAL MICROBES IN
OUR GUT CAN USE GLUCOSE.
AND HOW CERTAIN BISACCHARIDES
CAN ACT ASKEWS FOR THE
MICROBIOME AND HOW THEY HAVE
COMPLETELY DIFFERENT SENSING
SYSTEMS FOR SIMPLE SUGARS SUCH
AS MALTOSE.
WE HAVE PROJECTS IN THE LAB
LOOKING AT THE DARK MATTER OF
DIET SUCH AS THE PLANT
PHYTOCHEMICALS AND HOW THOSE ARE
METABOLIZED AND IMPACT THE HOST.
WE, OF COURSE, ARE DEEP WITHIN
THE FEMALE GENITAL TRACT
MICROBIOME, IN PARTICULAR, HOW
HOST AND ENVIRONMENTAL GLYCANS
IMPACT FTT MICROBIOME
COMPOSITION AND FUNCTION.
AND WORK THAT RICK ALLUDED TO
EARLIER AS WE'RE TRYING TO
DEFINE A GLYCAN ATLAS IN THE
MOUSE AND HUMAN INTESTINE ACROSS
HEALTH AND DISEASE.
TODAY I'M GOING TO TELL YOU A
STORY WE'VE BEEN, WOULDING ON
PRETTY MUCH WORKING ON,
THE SHORT CHAIN FATTY ACIDS THAT
WERE INTRODUCED EARLIER, AND
THERE ARE ACTUALLY UNEXPECTED
RELATIONSHIPS BETWEEN GLYCANS.
AND SO BEFORE I INTRODUCE OUR
WORK, I'M GOING TO TELL YOU A
LITTLE BIT -- MARIO INTRODUCED
IT AND OTHER PEOPLE HAVE A
LITTLE BIT.
SO ABOUT HALF OUR MICROBIOME
NUMERICALLY ACTUALLY FALL INTO
THIS ONE ORDER OF BACTERIA
CALLED THE BACTEROIDALES, ABOUT
UP TO 15 DIFFERENT SPECIES
WITHIN THIS GENUS.
OF COURSE THAT DIFFERS FROM
WHERE YOU ARE IN THE WORLD, AND
WHAT'S REALLY EMERGED IS THAT
DIFFERENT SPECIES AND STRAINS
HAVE VERY IMPORTANT PROPERTIES
IN HUMAN HEALTH AND DISEASE
RANGING FROM WHETHER CERTAIN
STRAINS HAVE CERTAIN TOX I INS TOXINS THA T
MIGHT BE INVOLVED IN THE
DEVELOPMENT OF COLON CANCER OR
CERTAIN SPECIES AND STRAINS THAT
HAVE CERTAIN ANTIGENS THAT MIGHT
BE INVOLVED IN AUTOIMMUNITY AND
ANTITUMOR RESPONSES OR PRO TUMOR
RESPONSES.
AND SO THERE'S A GREAT
IMPORTANCE AND UNDERSTANDING THE
ECOLOGY OF -- AT THE STRAIN
LEVEL AND SPECIES LEVEL OF THE
HUMAN GUT BACTEROIDETES.
MARIO ALSO INTRODUCED HOW THEY
COMPETE IN THE GUT, THROUGH AN
EVOLUTIONARY ARMS RACE, THEY
KEEP ADDING AND ADDING MORE
POLYSACCHARIDE UTILIZATION TO
USE DIETARY AND HOST GLYCANS AS
THEIR ENERGY SOURCES.
MARIO TALKED A LITTLE ABOUT
THESE LOCI WHICH ALLOWS A GIVEN
STRAIN OR SPECIES OF THE SPECIES TO CHOP THEM
UP AND USE THEM AS ENERGY.
PREVIOUS WORK IN MY LAB OR MY
POSTDOCTORAL FELLOWSHIP WAS
LOOKING OW THEY USE DIFFERENT
SUGARS AND THEY'RE INVOLVED AS
PUBLIC GOODS TO FEED OTHER
BACTERIA, AND THERE'S THIS
LINEARITY ACTUALLY IN DIET AND
DIETARY SUGARS, HOW THOSE CHANGE
THE COMPOSITION OF THE
MICROBIOME, OF COURSE
COMPOSITION OF THE MICROBIOME
IMPACTS THE HOST, BUT A REALLY
IMPORTANT ASPECT OF THIS DIETARY
GUT MICROBIOME HOST ACCESS IS
THE FERMENTATION PRODUCTS OF
POLYSACCHARIDE AND SUGAR
UTILIZATION BY MEMBERS OF THE
GUT MICROBIOME, IN PARTICULAR,
THE FERMENTATION PRODUCTS KNOWN
AS THE SHORT CHAIN FATTY ACIDS.
THESE ARE IN MILIMOLAR
CONCENTRATIONS WITHIN THE GUT.
AND THESE PLAY A NUMBER OF
IMPORTANT ROLES THROUGH MULTIPLE
MECHANISMS IN HOST HEALTH.
BUT WHAT'S REALLY NOT WELL
APPRECIATED IS FOR THESE
MOLECULES THAT ARE MILIMOLAR
CONCENTRATIONS WITHIN THE GUT
IS, ARE THEY PLAYING ANY ROLES
ON THE MICROBES THEMSELVES.
AND SO THERE ARE -- THEY ARE
USED IN ASSIMILATION AS ELECTRON
DONORS FOR ENERGY AND THAT'S
BEEN WELL APPRECIATED SINCE THE
1960s, BUT WE KIND OF TOOK A
LITTLE BIT OF A DIFFERENT
APPROACH AND REALLY ASK THE
QUESTION, DO THESE IMPACT THE
FITNESS OF MICROBES THAT REALLY
AREN'T KNOWN TO DO -- TO
ASSIMILATE SHORT CHAIN FATTY
ACIDS AS ENERGY SOURCES.
AND SO WE TOOK A PANEL OF BACT
EROIDES AND WITH MINIMAL MEDIA,
WE HAPPEN TO GROW OUR BUGS ON
GLUCOSE, AND WHAT I'M ABOUT TO
SHOW YOU IS WE THREW DIFFERENT
SHORT CHAIN FATTY ACIDS ON THESE
MEMBERS.
ACROSS ONE TO THREE CARBON
INCLUDING A FOUR-CARBON SHORT
CHAIN FATTY ACID ORGANIC ACID
SUCCINATE, IF YOU LOOK DOWN EACH
STRAIN, YOU DON'T SEE MAJOR
DIFFERENCES IN THE GROWTH OF THE
BACTE ROIDES SO THEY DON'T SEEM
TO MIND THESE SHORT CHAIN FATTY
ACIDS TOO MUCH.
BUT FOR ONE, BUTERATE, WE
OBSERVED SOME BACTEROIDES
MEMBERS DON'T SEEM TO CARE BUT
OTHERS, DIDN'T GROW AT ALL AND
THIS WAS REALLY THE START OF THE
PROJECT.
WE REALLY EXPANDED THE BASKET
BACTEROIDES THAT WE LOOKED AT,
AND WHAT WE FOUND IS THIS
ANTIMICROBIAL EFFECT WAS
ACTUALLY STRAIN-DEPENDENT.
I'LL GIVE YOU AN EXAMPLE HERE,
YOU'LL BE SEEING THESE, BUT THIS
IS BASE BASICALLY A TIME SERIES AT
DAY ONE, DAY TWO, DAY THREE, DAY
FOUR, AND ON THE TOP ROW IS
WITHOUT BUTYRATE.
THIS OTHER STRAIN IS HIGHLY
RESISTANT.
YOU CAN SEE THE TWO STRAINS OF
THE SPAIN SPECIES, A RESISTANT
STRAIN ON THE RIGHT AND A HIGHLY
SENSITIVE STRAIN ON THE LEFT.
WE MOSTLY STUDIED SUGAR
UTILIZATION.
WE KNOW THEY'RE SEEING ALL THESE
DIFFERENT SUGARS IN THE GUT, AND
THAT'S HOW THEY COMPETE TO
COLONIZE THE GUT.
SO WE WANTED TO KNOW WHETHER
THIS BUTYRATE INHIBITION ON THE
BACTEROIDES WAS ROBUST ACROSS
DIFFERENT SUGARS.
SO WE WENT TO THE CANDY STORE IN
THE LAB AND THREW A NUMBER OF
DIFFERENT SUGARS.
WHAT I'VE JUST SHOWN YOU WAS IN
GLUCOSE, THEN WE START TO SHOW
OTHER MONOSACCHARIDES SUCH AS
FRUCTOSE.
WHAT YOU SEE IS THIS KEY MATCH,
IF YOU SEE
MAP, IF YOU SEE A RED BOX,
THEY'RE HIGHLY SENSITIVE.
IF YOU LOOK ACROSS FOR THE GIVEN
DIFFERENT ISOLATES OF THE HUMAN
GUT BACTEROIDES, YOU DON'T SEE
THAT MANY DIFFERENCES, IF
THEY'RE SENSITIVE IN GLUCOSE,
THEY'RE SENSITIVE IN MANOSE.
WE KEPT ON ADDING MORE SUGARS
AND WHAT WE STARTED TO SEE IS
DEPENDING ON WHAT SUGAR A GIVEN
STRAIN OF THE BACTEROIDES WAS
USING AS CARBON SOURCE, IT WAS
EITHER RESISTANT OR SENSITIVE TO
BUTYRATE.
AND SO WHAT YOU CAN SEE HERE IS
THESE COLORS ARE CHANGING AS YOU
ADD -- IF YOU GO -- THIS IS ONE
OF THE FEW TIMES WHERE I GET TO
TALK ABOUT THESE SUGAR, THE
PEOPLE THAT CARE ABOUT THESE
SUGARS, AND WHAT YOU CAN SEE
HERE IN CERTAIN SUGARS, AND I'LL
JUST JUMP TO THE EXAMPLES HERE
IS, IF YOU GROW THIS BACTEROIDES
STRAIN IN GLUCOSE, IT'S HIGHLY
RESISTANT TO BUTYRATE.
BUT IF YOU GROW IT IN GLICNAC OR
GALNAC, IT'S DEAD.
AND THIS WAS REALLY BIZARRE TO
US.
WE THEN MOVED ON TO ASK, WELL,
IN THE GUT, IN THE DISTAL COLON
OR THE COLON, I SHOULD SAY, IS
THE BACTEROIDES, THEY COMPETE TO
USE DIVERSE DIETARY AND HOST
GLYCANS.
THAT'S HOW THEY REALLY COMPETE
AND SURVIVE IN THERE.
SO HERE WE TOOK A PANEL, AND A
LOT OF THESE DIETARY GLYCANS
YOU'LL SEE HERE, THESE ARE
PRESENT IN PLANTS BUT THEY'RE
ALSO CANDIDATES AS PREBITE IK OH PREBIOTIC S.
SO HERE'S A PANEL AND YOU CAN
SEE HERE, THESE ARE ALL BACTERIA
THAT USE THESE SHOE IMARS SUGARS THAT WE
PROVIDE TO THEM.
BUT IF YOU PUT IN BUTYRATE, YOU
CAN'T PREDICT WHICH STRAINS IN
WHICH GLYCANS WILL BECOME
SENSITIVE, ARE BASICALLY
INHIBITED BY BUTYRATE.
SO THIS IS A REALLY CLEAN
EXAMPLE OF THAT.
SO HERE'S ONE PARTICULAR GENERAL
LIST.
THIS IS BO CLO3.
THIS IS LOTS OF DIETARY GLYCANS.
IT USES PECK TIN, GLUCOMANNAN.
DEPENDING ON WHAT TIE TRI FIBER
IT'S USING, IT'S EITHER HIGHLY
RESISTANT TO BUTYRATE OR HIGHLY
SENSITIVE TO BUTYRATE.
AND I DON'T NORMALLY SHOW THIS
DATA BUT SINCE THIS IS A
GLYCOSCIENCE AUDIENCE, IF YOU
LOOK AT LEVAN, IT'S A POLYMER OF
FRUCTOSE, IF YOU GROW IT IN
FRUCTOSE.
IF YOU GO TO -- YOU GROW IT IN
PULLULAN -- IF YOU GROW IT IN
CHONDRIOTIN SULFATE -- IT'S
SENSITIVE IN.
SIMILARLY, GLUCOMANNAN IS HIGHLY
RESISTANT TO GLUCOSE OR MANNOSE
OR GLUCOSE AND MANNOSE.
THIS ACTUALLY -- AT THE BOND OF
A DIE SACCHARIDE, IN SUCROSE,
ALPHA-1 BETA TWO LINKAGE, IT'S
RESISTANT ALSO, BUT -- IT'S
SENSITIVE TO BUTYRATE AND THAT'S
TRUE ALSO FOR HOMO DIMERS OF
GLUCOSE, WHERE IT'S RESISTANT IN
GLUCOSE AND MALTOSE.
SO SUGARS ACT GREATER THAN THE
SUM OF THE PARTS IN CONTROLLING
ANTIMICROBIAL -- AT STRAIN
LEVEL.
SO WE'VE IDENTIFIED SPECIFIC
MICROBIAL METABOLITES, THAT THE
SPECIFIC MICROBIAL METABOLITE
BUTYRATE IS ANTIMICROBIAL TO THE
BACTEROIDALES, THAT BUTYRATE
ANTIMICROBIAL ACTIVITY IS
STRAIN-SPECIFIC AMONG THE
BACTEROIDES.
AND SUGARS ACT AS FITNESS
SWITCHES BUTYRATE, MORE AS A
DIMMER WITH EXTREMES ON AND OFF
AND IN BETWEENS, WITH INDIVIDUAL
SUGARS RENDERING BUTYRATE
VULNERABILITY.
AND THAT'S WHERE WE SEE THAT
STRAIN LEVEL FITNESS OF THE
BACTEROIDES SEEMS TO BE
UNPREDICTABLE IN THE
INTERACTIONS OF BUTYRATE WITH
INDIVIDUAL SUGARS.
AND SO I KNOW THERE'S A TIME
DELAY IN THIS PRESENTATION, I
THINK, AND THIS IS WHERE WE
START TO ASK WHAT THE FUKE IS
GOING ON HERE.
WE THEN, THROUGH OUR GENETIC
TOOLKIT ON THE BACTEROIDALES
ASKED THIS QUESTION.
WE STARTED WITH A STRAIN WHICH
IS HIGHLY RYE SES TANT TO
BUTYRATE.
WE BASICALLY BLASTED THE GENOME
WITH TN INSERTION TO LOOK FOR
SENSITIVE MUTANTS.
WE HIT THIS GENE WITH BT 3942.
WHAT WE FOUND WHEN WE PURIFIED
IT IS IT CLEAVES BUTYRAL COA AND
EVEN SOME MEDIUM CHAIN LINK
FATTY ACIDS, AND SO THIS
DEMONSTRATED TO US THAT AN
ACYL-COA THIOESTER HYDROLASE --
WHEN WE DID TNC CURE, WE HIT
THIS GEEP CALLED BU -- BVU1163.
THAT ACTUALLY IS AN ACYL COATE
TRANSFERASE.
IT SEEMS TO BE SPECIFIC FOR
TRANSFERRING BUTYRAL COA WITH
ACETYL COA, SO WE FOUND THAT A
SHORT ACYL-COA NEED YAIDZ
SENSITIVITY TO BUTYRATE IN
BACTEROIDES VULGATIS.
SO WHAT'S THE MECHANISM FOR THIS
SPECIES LEVEL PHENOTYPIC
VARIATION.
WHAT WE TOTALLY DIFFERENT
EXPECT, THESE ARE GENES IN THE
CORE GENOME, IS THAT THE
RESISTANT STRAIN AND -- IN THE
SENSITIVE STRAIN ARE ONLY 51%
CONSERVED AT THE AMINO ACID
LEVEL, AND WHEN WE CLONE THEM,
THEY HAVE TOTALLY DIFFERENT
FUNCTIONS, SO BASICALLY IN THE
RESISTANT STRAIN AS IT CONFERS
RESISTANCE TO BUTYRATE, BUT IT'S
HOMOLOGUE IN THE SENSITIVE
STRAIN DOES NOT.
AND WHEN WE DID SOME ENZYME
ASSAYS, WE FOUND THAT THEY HAD
VERY DIFFERENT KINETICS IN THESE
TWO DIFFERENT SPECIES OF THIS
FOR BUTTE ROLL SEEN IN BLACK. BUTYRAL SEEN IN BLACK.
WHAT ABOUT THIS ACYLCOATE
TRANSFERASE.
IT WAS 80% CONSERVED AT THE
AMINO ACID LEVEL, AND IT HAS THE
SAME FUNCTION, WHICH IS YOU CAN
CLONE BOTH OF THEM FROM THE
RESISTANT STRAIN OR THE
SENSITIVE STRAIN IN CONVERSE
SENSITIVITY, BUT WHAT WE FOUND
WAS THAT THIS CONSERVED CORE
GENOME GENE WAS DIFFERENTIALLY
EXPRESSED IN THE GROWTH PHASE,
SO THE RESISTANT STRAIN DOWN
REGULATES THIS GENE, AND THE
SENSITIVE STRAIN UPREGULATES THE
STRAIN JUST WHEN IT'S GROWN.
AND PROTEIN LEVELS ARE
COMMENSURATE.
SO OKAY, THESE TWO DIFFERENT
SPECIES ARE REGULATING THESE
CORE GENOME GENES AND THAT'S
MEDIATING THIS METABOLIC DEFENSE
TO BUTYRATE SO WE CLONED OUT THE
PROMOTERS AND MADE THESE
CONSTRUCTS WITH SWAPPING OUT THE
PROMOTERS FOR THE DIFFERENT
CONSERVED GENES.
AND WHAT WE FOUND WAS THAT THE
PROMOTERS ARE RESPONSIBLE FOR
THIS HIGH LEVEL EXPRESSION OR
THIS LOW LEVEL EXPRESSION, AND
THEN WHEN WE SWAP -- SO THIS IS
THE SENSITIVE STRAIN VERSUS THE
RESISTANT STRAIN HAS HIGH
EXPRESSION OR LOW EXPRESSION,
WHICH IS MINUS 200 NUCLEOTIDES
UPSTREAM AND THAT ACTUALLY
CONFERS SENSITIVITY OF
RESISTANCE.
WE THEN ASKED WHAT'S IN THAT
MINUS 200, SO THAT'S -- THERE'S
CANONICAL MINUS 7 AND MINUS
33 BINDING SITES, AND THERE WAS
BASICALLY THESE TWO TYPES OF
POLYMORPHISMS TO THE TWO SPECIES
OF THE BACTEROIDES.
SO WE MADE A NUMBER OF
CONSTRUCTS WHICH WE MU MUTATED
THESE DOUBLE TYPE POLYMORPHISMS
AND WE CAN CON VIRMT THE LOW
EXPRESSION FROM THESE SNPS INTO
HIGH EXPRESSION, AND WE CAN TAKE
THE OTHER BACKBONE, MAKE THOSE
SAME SNPs, CHANGE THE SNPs,
CONVERT HIGH EXPRESSION TO A LOW
EXPRESSION, CONVERT A SENSITIVE
ORGANISM TO A RESISTANT ORGANISM
JUST BY THESE SINGLE NUCLEOTIDE
POLYMORPHISMS IN THE BINDING
SITES OF THIS CORE GENOME GENE.
SO THIS PREDICTED TO US, AND
HERE'S OUR MODEL BASICALLY, IF
YOU'RE EXPRESSING HIGH AMOUNTS
OF THIS GENE IN PRESENCE OF HIGH
BUTYRATE, YOU'RE DEAD.
AND WE WANTED TO UNDERSTAND WHAT
THE HUMAN SIGNIFICANCE WAS, AND
WHAT WE NEEDED FOR THAT WAS A
MULTI-OMICS COHORT WHERE WE HAVE
METAGENOMES LOOK AT GENE
ABUNDANCE, SUCH AS THIS GENE AS
A PROXY FOR BACTEROIDES
ABUNDANCE IN THE ECOSYSTEM, META
TRANSCRIPT SO WE CAN LOOK AT THE
TRANSCRIPT LEVELS AS PART OF OUR
MODEL, AND THEN OF COURSE
MEASURE THE METABOLITES AND THE
AMOUNT OF METABOLITES, IN
PARTICULAR, BUTYRATE.
AND WHEN WE CORRELATED ALL THESE
THINGS TOGETHER IN HUMAN FECES,
WE FOUND -- WE ACTUALLY PROVED
OUR PREDICTION, WHICH IS THAT IF
YOU EXPRESS HIGH AMOUNTS OF THIS
ACYL COA TRANSFERASE, ONLY IN
HIGH BUTYRATE ECOSYSTEMS, YOUR
FITNESS IS DECREASED FOR THE
BACTEROIDS.
SO WHAT WE FOUND USING BOTTOM-UP
HYPOTHESIS AND MECHANISM IN A
HUMAN COMPLEX OMICS DATASET WAS
TO ASSIGN UNKNOWN FUNCTION TO
THESE NOVEL GENOME GENES TO THIS
NOVEL INTERACTION AND FOR
FITNESS AND CONTEXT DEPENDENT
MANNER THAT'S DEPENDENT ON
TRANSCRIPT LEVELS.
AND SO BASICALLY GOING IN VITRO,
FINDING EMERGENT PROPERTY FOR
COMBINATION OF METABOLITES AND
GLYCANS, ASSIGNING CORE GENOME
REGULATION GOING TO BIG HUMAN
DATA.
I'M GOING TO SKIP OUR SUMMARY
SLIDES, WHICH IS BASICALLY THAT
EVERY DIFFERENT STRAIN OF THE
BACTEROIDES DEPENDING ON WHAT
SUGAR IT USES AND IT COULD USE
LOTS OF DIFFERENT SUGARS, THAT
ONE MICROBIAL METABOLITE
BASICALLY TURNS IT HAPPY OR
SAID.
LIVE OR DEAD.
AND WE'VE DEFINED THE MECHANISMS
HERE, WHAT OUR WORKING IN MODEL
IS THAT BUTTE RIGHT IS
DIFFERENTIALLY METABOLIZED AND
EACH STRAIN OR SPECIES HAVE
THESE IDIOSYNCRATIC METABOLIC
STATE THAT LEAD TO THE BUTYRATE
VULNERABILITY.
SO WE'D LIKE TO FICK OUT WHAT
THE FIGURE OUT WHAT
THE LEVELS ARE AT THE GENETIC
REGULATORY METABOLIC LEVELS AND
THAT'S WORK WE WOULD LIKE TO
PURSUE IN OUR LAB.
AND REALLY TO UNDERSTAND THE
ECOLOGICAL IMPACT IN USING
BUTYRATE.
SO BUTYRATE COMES FROM --
MICHAEL FISH BACK BOX FISHBACH, WITH HIS
KINDNESS, WE'D LIKE TO -- NOW
THAT WE CAN DEFINE BY ITS GENES,
ITS PARTS AND TRANSCRIPT, WE'D
LIKE TO RE-CREATE IT IN MICE,
AND TRY TO TOGGLE GENETICALLY
AND BY GLYCANS STRAIN LEVEL
COMPOSITION POTENTIALLY IN
COMPETITION WITH KLOSS TRID YA
IN ENCLOSE STRID
YA IN THE HUMAN GUT.
WE'D LIKE TO TURN BACK TO
MULTI-YOE MIBG DATASETS WHERE
WE'VE BEEN ABLE TO LOOK AT
COMPOSITIONS OF MICROBIOME, TAKE
THESE EMERGENT PROPERTIES AT THE
LEVEL OF GENE ASSIGNING
FUNCTION, TRANSCRIPTS ASSIGNING
FUNCTION AND THEN METABOLITES AS
TUTORS AND REALLY OPENING THIS
UP TO GLYCOMICS IN COLLABORATION
WITH THE WORK THAT WE'RE DOING
WITH RICK, WHICH IS TO SEE HOW
FINE-TUNED WE CAN LEARN ABOUT
STRAIN LEVEL COMPOSITION IN
THESE EMERGING PROPERTIES FOR
DIETARY GLYCANS AND HEALTH
GLYCANS.
SO WITH THAT, YOU REALLY THANK
EVERYONE FOR THEIR TIME.
I REALIZE I SPOKE VERY QUICKLY.
HOPEFULLY THAT WAS FOLLOWABLE.
AND I'D LIKE TO THANK THE NIH IN
PARTICULAR FOR THEIR SUPPORT, MY
KO8 JUST ENDED FROM NIAID, AND
THANKING FOR OTHER SUPPORT FROM
THE NIH AND OF COURSE PRIVATE
FUNDERS IN THE LAB, OF OUR
LABORATORY.
THANKS.
>> AWESOME.
SUPER.
YOU HAVE TO BRING YOUR LOVE OF
SUGARS AND CARBOHYDRATES OUT OF
THIS COMMUNITY AND EVERYWHERE
YOU SPEAK AND DO NOT HIDE THOSE.
I THINK IT WOULD BE REALLY
IMPORTANT FOR YOU TO DO THAT.
BUT IT WAS AMAZING, AMAZING,
AMAZING.
NEVER HIDE THAT.
IF YOU HAVE QUESTION, YOU'RE
LEADING THIS, SEAN.
GO AHEAD.
>> DOES ANYONE HAVE ANY
QUESTIONS?
>> EVERYBODY UNDERSTOOD
EVERYTHING I WAS SAYING.
>> THERE IS A QUESTION IN THE
CHAT FROM MARK.
HE SAYS IF BUTYRATE, A KETONE IS
RESISTANT TO HEALTH, IS THAT --
TO DIETARY KETOSIS?
>> YEAH, SO, YOU KNOW, THIS IS
INTERESTING.
SO HYDROXYBUTYRATE AS A KETONE
IN KETOSIS, DEFINITELY, AS FAR
AS BUTYRATE PER SE, -- I'M GOING
TO GO OFFLINE ON THAT ONE
BECAUSE THAT'S A FREE
ASSOCIATION WITH A COUPLE OF
THEM THAT I'M NOT EXACTLY SURE,
IF I CAN FIND THAT CHAT.
>> WE WILL EMAIL TO YOU.
>> THAT'S A GREAT QUESTION BUT
IT'S GOT LIKE FOUR DIMENSIONS
THAT I'M GOING TO SPARE THE TIME
ON.
>> THAT'S OKAY.
THAT'S GREAT.
EVAN, I SEE YOUR HAND IS UP IN
THE ZOOM ROOM.
MAYBE WE CAN DO THAT VIA CHAT
WITH SETH BECAUSE WE'RE REALLY
PRESSED FOR TIME AND SEAN AND I
ARE ALREADY -- WE'VE BEEN
SCHEMING HOW WE'RE GOING TO MAKE
OUR TALKS 3 MINUTES.
SO GREAT JOB.
THANK YOU.
>> ALL RIGHT.
WE'RE GOING TO SHIFT GEARS TO
DR. KATHARINA RIBBECK, PARTNERS
IN SLIME, HOW MUCOUS REGULATES
MICROBIAL VIEWER EU VIRULENCE.
>> THANK YOU SO MUCH, CATHERINE
AND SEAN FOR INCLUDING ME IN
THIS REALLY OUTSTANDING
CONFERENCE.
I'VE HAD A VERY GOOD TIME, I'VE
LEARNED SO MUCH.
WE'VE TALKED A LOT ABOUT THE
MICROBIOME, AND I WANT TO SHIFT
BACK TO THE HOME OF THE
MICROBIOME FOR A MOMENT TO THE
MUCOUS BARRIER.
THE GOAL OF MY LAB IS TO
UNDERSTAND HOW IT INTERACTS WITH
MICROBES AND THEN LEVERAGE THESE
CONCEPTS AND MOTIF FS WE CAN TO
MANAGE PROBLEMATIC PATHOGENS.
SO LET'S GO BACK A FEW STEPS.
MUCOUS, YOU MAY RECALL, IS A
HYDROGEL THAT IS BUILT FROM
POLYMERS THAT ENTANGLE, THEY'RE
HIGHLY HYDRATED, AT MUCUS COATS
ENORMOUS SURFACE AREA IN THE
BODY, 2,000 SQUARE FEET IN THE
INTESTINE ALONE, BEST KNOWN FOR
ITS ABILITY TO PRESERVE MOISTURE
IN UNDERLYING EPITHELIA, IT ALSO
PRESERVES YOUR STOMACH FROM
DIGESTING ITSELF.
BUT WHAT I WANT TO FOCUS ON
TODAY IS THE ROLE OF MUCUS AS AN
ECOLOGICAL NICHE THAT
ACCOMMODATES MICROBES, TRILLIONS
OF MICROBES THAT LIVE INSIDE THE
MUCUS IN YOUR GAS TOE GASTROINTESTINAL
TRACT.
MANY ARE BENIGN BUT THERE ARE
ALSO A NUMBER OF PATHOGENS THAT
CAN CAUSE SERIOUS INFECTION.
MUCUS IS REALLY UNSUNG HEROS FOR
MAINTAINING PROBLEMATIC
PATHOGENS AND IT DOES SO WITH
THE HELP OF POLYMERS, THEY LOOK
LIKE TINY BOTTLE BRUSHES, AND
THEY ARE -- THIS IS WHY I'M
SPEAKING HERE TODAY, THEY
PROTRUDE DENSE AREAS OF GLYCANS
INTO THE LUMEN OF MUCUS, AND
THEY INTERACT WITH MICROBES
THEMSELVES BUT ALSO WITH
COMPONENTS FROM THE IMMUNE
SYSTEM.
WHEN MUCINS AREN'T PRODUCED
CORRECTLY OR NOT IN HIGH ENOUGH
AMOUNTS, THEN THE HOST CAN
BECOME INFLAMED, COMPROMISED,
AND MORE SERIOUS DISEASES CAN
EMERGE, CYSTIC FIBROSIS, WE'VE
HEARD A NUMBER OF CASES ALREADY.
SO I'M REALLY EXCITED ABOUT
MUCUS BECAUSE I'M CONVINCED THAT
UNDERSTANDING HOW IT MANAGES
PATHOGENS CAN HELP US FIND NEW
STRATEGIES TO CONTROL PATHOGENS
OURSELVES.
SO THIS IS REALLY THE GOAL OF
THE LAB.
IF YOU LOOK THROUGH TEXTBOOK
MODELS, HOW MUCUS EXACTLY
MEDIATE BARRIER FUNCTION, YOU'LL
FIND TWO PREVAILING MODELS.
ONE IS MUCOUS AS A PHYSICAL
BARRIER THAT KEEPS MICROBES AT A
DISTANCE FROM UNDERLYING
EPITHELIUM, YOU'LL FIND THAT IN
CERTAIN PARTS OF THE GUT, FOR
EXAMPLE.
THE OTHER MECHANISM THAT YOU'LL
FIND IN TEXTBOOKS IS ONE WHERE
MU
MUCIN ES DIRECTLY BIND AND
THEREFORE -- WHEN MUCOUS GETS
REMOVED FROM THE SURFACE OF THE
BODY.
THESE MODELS HAVE THEIR PLACE.
THEY ARE CRITICAL TO MUCUS
FUNCTION.
HOWEVER, THEY DO NOT CAPTURE HOW
MUCUS CAN ACCOMMODATE POTENTIAL
PATHOGENS.
AND THIS IS WHAT I WANT TO ZOOM
IN ON NOW AND TAKE APART WITH
YOU.
I WILL TELL BUT THREE ABOUT THREE
HIGHLIGHTS WE'VE DISCOVERED OVER
THE PAST COUPLE OF YEARS.
THE FIRST IS WHAT WE'VE LEARNED
IS THAT MUCIN POLYMERS, THE POLT
MERES THEMSELVES ARE REALLY GOOD
AT ATTENUATING THE VIRULENCE OF
OPPORTUNISTIC PATHOGENS.
SO WHEN WE FIRST STARTED
THINKING ABOUT HOW TO UNDERSTAND
HOW MUCOUS REGULATES IN
PARTICULAR PATHOGENS, WE WERE
LOOKING FOR A PATHOGEN THAT CAN
RESIDE IN HEALTHY MUCOSAL
BARRIERS WITHOUT CAUSING
PROBLEMS BUT THEN ALSO HAS THE
ABILITY TO CAUSE INFECTIONS,
PSEUDOMONAS IS ONE OF THEM
PROBABLY BEST UNDERSTOOD FOR ITS
BIOFILMS OR DENSE AGGREGATES
INSIDE CYSTIC FIBROSIS MUCUS.
NOW OTHER GOAL WAS TO
INTERROGATE TO MUCUS OUR
INTERACTION WITH THE MICROBES
STARTING WITH THE MU CIN
POLYMERS AND BUILD UP
SUCCESSIVELY AT OTHER FACTORS,
BUT FIRST BUILDING A THREE
DIMENSIONAL NETWORK THAT REALLY
WAS MEANT TO BE COMPLEMENTARY TO
ALREADY ONGOING IMPORTANT
RESEARCH THAT USES TWO
DIMENSIONAL COATINGS OF MUCIN
POLYMERS.
SO THIS IS WHAT WE'LL DO, AND
OUR DAILY BREAD IS NOT GLAMOROUS
BUT IT'S NEEDED, WE PURIFY
MUCINS AND RECONFIGURE THEM INTO
3D NETWORKS AND EMBED MICROBES
INTO THESE NETWORKS, THE
INDUSTRIAL/COMMERCIALLY
AVAILABLE MUCINS DO NOT HAVE
MANY OF THE PROPERTIES,
MECHANICAL AND ALSO BIOCHEMICAL,
THAT YOU'LL NEED FOR THIS TYPE
OF STUDY.
SO FIRST EXPERIMENT TO LOOK
AT -- WHEN YOU JUST LOOK AT --
THAT IS UNCOATED BY MUCIN
THEY'LL BIND READILY TO THE
SURFACE.
IF THE SURFACE IS, HOWEVER,
LINED WITH MUCIN, YOU CAN DO THE
SAME EXPERIMENT WITH MUCUS,
YOU'LL SEE THAT THEY HAVE A MUCH
HARDER TIME ASSOCIATING TO THE
UNDERLYING SURFACE.
MU CINS ARE ACTUALLY REALLY GOOD
AT BINDING TO THE SURFACE, BUT
ALSO EPITHELIAL CELLS, LIVING
EPITHELIAL CELLS.
SO THIS IS EXCITING BECAUSE THAT
MEANS THERE IS THIS
PROTECTIVE -- THAT'S NOT SO
SURPRISING AFTER ALL MUCUS IS
THIS THREE DIMENSIONAL NETWORK,
IT MIGHT GET STUCK IN THE VOLUME
AND NEVER PENETRATE THROUGH.
IF THAT IS THE CASE, ONE SHOULD
BE ABLE TO SEE THAT IF ONE
SWITCHES TO THE VOLUME OF THE
CENTER OF THE GEL.
IF YOU LOOK AT WHAT MICROBES DO
WHILE THEY'RE IN MUCUS, YOU'LL
FIND THAT THEY SWIM.
THIS IS JUST IN BUFFER FOR NOW.
YOU'LL FIND THAT THEY SWIM
OUTSIDE OF MUCUS PRETTY RAPIDLY
IN A PARTICULAR SWIMMING
PATTERN.
IF YOU NOW SURROUND THEM WITH
MUCINS, NORMALLY WHEN I'M IN A
LIVE AUDIENCE, I LIKE TO ASK
STUDENTS WHAT THEY EXPECT WILL
HAPPEN.
MOST OF THE TIME, THAT WAS MY
PREDICTION AS WELL, YOU'LL HEAR
MICROBES WILL PROBABLY GET TRAP
AND ARRESTED BY THE MUCOUS
BARRIER.
AFTER ALL, WE THINK OF IT AS
SOME SORT OF STICK ALL, TRAP ALL
FILTER.
BUT THAT IS NOT THE CASE, MUCINS
DO NOT TRAP MICROBES.
THIS IS ONLY ONE.
A SERIES OF MICROBES WE LOOKED
AT FOR THIS PURPOSE, IN FACT,
THE MICROBES SWIM JUST FINE IN
CERTAIN CASES THE TOP SPEED IS
HIGHER EVEN.
AND IF YOU LOOK AT OUTSIDE OF MU
KU AND IF YOU LOOK AT LONGER
TIME POINTS, YOU'LL FIND REALLY
IN THE ABSENCE OF MUCUS, THEY
STACK UP MORE AND MORE CELLS,
BUILD THICK BIOFILMS, THEY DON'T
DO THAT IN THE PRESENCE OF
MUCINS, THAT OF COURSE HAS ALL
SORTS OF CONSEQUENCES FOR THEIR
ABILITY TO INTERACT WITH EACH
OTHER TO RESPOND TO AND SIGNAL
WITH THE ENVIRONMENT, AND WE
THINK, BY THE WAY, ADHESI IN.
S WILL BE ESSENTIAL FOR THE
ABILITY TO DISPERSE THEM.
AND WE CAN TALK ABOUT THAT
LATER.
SO THIS IS INTERESTING, THIS IS
MATERIAL THAT HAS THE ABILITY TO
PREVENT MICROBES ALONE BY
SEPARATING THEM.
THE QUESTION WAS, IS THIS
SOMETHING THAT -- FOR
PSEUDOMONAS OR IS THIS A GENERAL
FEATURE OF MU MUCINS THAT -- FOR
THAT WE TURN TO CANDIDA ALBICANS
FOR A MOMENT WHICH CONVENIENTLY
HAS TWO MORPHOLOGICALLY DISTINCT
FORMS.
ONE, THE YEAST FORM IS DEBIEN
SUPPOSEDLY BENIGN,
WHEN YOU LOOK AT YEASTS THAT YOU
GROW INSIDE MEDIUM, IN THE
ABSENCE OF HOST FACTORS, THEY'LL
SWITCH THE HYPHAE FORM, IF YOU
SURROUND YEAST CELLS WITH MUCIN
POLYMERS, WHAT THEY'LL DO IS
PROLIFERATE, BUT WHAT THEY WON'T
DO IS SWITCH INTO THE
FILAMENTOUS FORM, WHAT THAT
MEANS IS THEY'RE LOCKED IN A
STATE THAT IS HOST COMPATIBLE,
AND THE ABILITY OF MUCINS TO
PREVENT THE TRANSITION TO THE
HYPHAE FORM IS SO ACUTE AND
STRONG THAT YOU CAN TAKE ALREADY
EXISTING FILAMENTS, IF YOU JUST
LET THEM DO THEIR THING, THEY'LL
CONTINUE TO PRODUCE FILAMENTS.
BUT IF YOU EXPOSE THESE ALREADY,
THESE FILAMENTS TO MUCINS,
YOU'LL SEE THAT THE NEXT
GENERATION HAS REVERSED BACK TO
YEAST CELLS.
AND WE ARE OFTEN ASKED, IS THIS
SOMETHING THAT POLYMERS
GENERALLY DO OR IS THIS SPECIFIC
TO THE MUCIN BIOCHEMISTRY?
AND IT IS SPECIFIC TO THE MUCIN
BIOCHEMISTRY BECAUSE ANY POLYMER
THAT WE'VE TESTED SO FAR THAT
CAN REMOTELY CONSTITUTE MUCIN
PROPERTIES DOES NOT HAVE THIS
EFFECT.
SO THIS REALLY MEANS THAT MUCINS
PRESENT SOMETHING THAT CAN
REGULATE TAP INTO THE PHYSIOLOGY
OF THE MICROBE THAT PREVENTS IT
FROM TRANSITIONING INTO THAT
FILAMENTOUS STATE.
AND SO HOW DOES THAT WORK?
AND THIS IS THE SECOND
OBSERVATION WE'VE MADE.
MUCINS MANAGE PATHOGENS NOT BY
KILLING THEM, BUT BY DISARMING
THEM.
AND I'LL TELL YOU WHAT THIS
MEANS EXACTLY.
WHEN YOU NOW EXPOS THE SUED EXPOSE TO DIF FERENT
SURFACES IN THE BODY, WHAT
YOU'LL SEE IS THAT MANY GENES
ARE DOWN DIAGNOSE DOWNREGULATED AND
UPREGULATED, BUT IF YOU
EXPECT -- YOU'LL FIND MANY OF
THOSE ARE ASSOCIATED WITH
VIRULENCE BEHAVIOR, INCLUDING
BIOINFORMATION, QUORUM SENSING
TOXINS, CERTAIN TYPES OF
SECRETION SYSTEMS ARE ALL
DOWNREGULATED IN THE PRESENCE OF
MUCINS.
AND WE HAVE VALIDATED THAT IT
PUBLISHED WORK THAT INDEED THE
MICROBES ARE PASSIVATED IN THE
PRESENCE OF MUCINS FROM
PHENOTYPIC ACIDS ALL THE THE WAY
FROM CELL CULTURE TO ANIMAL
MODELS.
NOW THIS IS EXCITING BECAUSE
MUCINS, THOSE COMPLEX POLYMERS
SEEM TO BE VERY POTENT IN
MANAGING MICROBES IN THAT THEY
PASSIVATE THEM.
THEY DON'T SEEM TO KILL THEM.
BUT WHAT EXACTLY CAN WE DO WITH
THEM?
THEY'RE TOO COMPLEX AS IS, SO
WHAT WE DECIDED TO DO A COUPLE
YEARS AGO WAS TAKE THEM APART,
TAKE THEM APART, TAKE THEIR DAWN
TINGLY COMPLEX BIOCHEMISTRY AS
FAR AS WE CAN, REMOVE THE
GLYCANS FROM THE BACKBONE OF THE
MUCIN POLYMERS, AND THEN
INTERROGATE IN GLYCAN POOLS FOR
THEIR FUNCTIONALITIES.
THE BIG QUESTION, OF COURSE,
WAS, DO THESE GLYCANS BY
THEMSELVES HAVE THE ABILITY TO
REGULATE, DOWNREGULATE,
SUPPRESS, DISARM THE MICROBES IN
THE SAME WAY AS MUCINS DO.
I SHOW YOU THE EXPERIMENT TO
HIGHLIGHT THE COMPLEXITY OF
GLYCANS THAT MUCINS DISPLAY,
YOU'LL HAVE HUNDREDS OF
STRUCTURES, DIFFERENT DISTINCT
STRUCTURES THAT ARE EXPOSED,
DISPLAYED BY MUCIN, SO THEY'RE
REALLY PLATFORMS OF SMALL
MOLECULES THAT INTERACT WITH THE
ENVIRONMENT.
SO IF YOU TAKE THIS POOL HERE OF
SOLUBLE GLYCANS IN THE ABSENCE
OF THE BACKBONE, MUCIN BACKBONE,
YOU'LL FIND THAT THEY'RE REALLY
POTENT IN SUPPRESSING VIRULENCE
TRAIT.
THE SAME GENE EXPRESSION
CASCADES THAT ARE RELATED TO
VIRULENCE AND ENABLE THE MICROBE
TO INFECT, AND THAT, AGAIN, YOU
CAN SEE AT BOTH THE GENE
EXPRESSION LEVEL BUT THEN
VALIDATE PHENOTYPICALLY BINDING
ASSAYS TO SURFACES, FOR EXAMPLE,
WHERE MUCIN GLYCANS, IN THE
ABSENCE EVEN OF THE GEL
STRUCTURE ARE REALLY EFFECTIVE
AT PRESENTING SURFACE
ASSOCIATION, AND ONE IMPORTANT
CONTROL IF YOU SEPARATE THE
MUCIN GLYCAN STRUCTURES INTO
THEIR COMPOSITES, MONO
SACCHARIDES, YOU LOSE THAT
EFFECT.
THEY DO NOT ACHIEVE THE SAME
KIND OF ASSAY, JUST A DIFFERENT
QUANTIFICATION, MUCIN GLYCANS
REALLY ARE EFFECTIVE AT
PREVENTING GROUP FORMATION
MONOSACCHARIDES DO NOT DO THIS,
AND NUMEROUS GENES RELATED TO
VIEWER EU LENS
VIRULENCE ARE DOWNREGULATED.
THE QUESTION HERE AGAIN BACK TO
CANDIDA BRIEFLY IS, HOW SPECIFIC
IS THIS VIEWER EU LENS
ATTENUATION EFFECT OF THESE
GLYCAN POOLS?
CANDIDA, I REMEMBER THAT MUCIN
POLYMERS ARE REALLY EFFECTIVE AT
PREVENTING THAT TRANSITION TO
FILAMENTS, AND SO ARE THE
GLYCANS.
THE GLYCANS, NO MATTER FROM
WHICH SURFACE HUMAN OR PIG IN
THIS CASE, SALIVA,
GASTROINTESTINAL AND INTESTINE
STOMACH AND INTESTINE, WE
CHOSE -- THEY WERE ALL REALLY
POTENT AT SUPPRESSING THAT
TRANSITION TO THE FILAMENTOUS
FORM WITHOUT KILLING THE
MICROBES.
AND THAT IS REALLY AN IMPORTANT
POINT OF THEIR FUNCTION.
AND THE PICTURE THAT IS EE EMERGING
NOW, I CAN ONLY TELL YOU THESE
TWO STORIES, I DON'T HAVE MORE
TIME BUT I'M HAPPY TO CONNECT
LATER, THE PICTURE IS EMERGING
THAT WE'VE SEEN GLYCANS HAVE THE
REGULATORY POTENTIAL TO SUPPRESS
ALL SORTS OF MICROBES, PASSIVATE
ALL SORTS OF PROBLEMATIC
PATHOGENS.
YOU'LL SEE A COUPLE OF STORIES
COMING OUT IN THE NEXT COUPLE OF
WEEKS AND MONTHS, BUT THE LIST
IS GROWING, AND IN FACT WE
HAVEN'T SEEN A PATHOGEN,
OPPORTUNISTIC PATHOGEN SO FAR
THAT HAS NOT RESPONDED TO MU MUCIN
GLYCANS IN THE WAY CERTAIN GENES
ARE DOWNREGULATED.
NOW THIS IS EXCITING BECAUSE
THIS MEANS THAT WE MIGHT HAVE
ACTUALLY TOOLS TO APPLY FOR
THERAPEUTIC PURPOSES.
THE PROBLEM IS, WE'RE STILL
WORKING WITH VERY COMPLEX GLYCAN
POOLS, SO THE QUESTION NOW
BECAME, HOW CAN WE REDUCE THIS
COMPLEXITY TO SINGLE STRUCTURES,
AND IF YOU LOOK ACROSS THE
BOARD, YOU SEE THAT DIFFERENT
MUCIN POOLS ARE AT PLAY, AND
EXPOSED, BUT YOU ALSO HAVE
CERTAIN GLYCANS THAT OVERLAP
THAT ARE CONSISTENT BETWEEN THE
DIFFERENT SUSH SURFACES, AND THOSE
BECAME OUR FOCUS BECAUSE WE SAW
THESE VIRULENCE ATTENUATION
EFFECTS TOWARDS CANDIDA
SPECIFICALLY, SO WE SAID WHY
DON'T WE LOOK AT THE SIX MOST
COMMON STRUCTURES IN THIS
OVERLAPPING POOL THAT ARE
EXPOSED AND PRESENT ON ALL
SURFACES AND SYNTHESIZE THEM SO
WE HAVE REALLY SINGLE
STRUCTURES.
LONG STORY SHORT, THESE ARE THE
MOST COMMON SIX STRUCTURES
YOU'LL FIND ON SURFACES AND ONE
OF THEM, THE CORE STRUCTURE, IS
REALLY EFFECTIVE.
SO YOU CAN TAKE THE SINGLE
GLYCAN, PRESENT THE TRANSITION
TO FILL MEN TAKES TO THE HYPHAE
FORM BY SINGLE STRUCTURE AND
SOME MODIFICATIONS ARE
TOLERATED, OTHERS LIKE -- ACID
ARE SIALIC
ACID, A NOT.
ARE NOT.
THAT NOW BECOMES LEVERAGABLE,
YOU SHOULD SEE THE STORY VERY
SHORTLY, IN THE NEXT TWO OR SO
WEEKS, IF YOU'RE INTERESTED.
SO TO SUMMARIZE, MUCINS ARE
THERAPEUTIC GOLD MINES.
THEY ARE REALLY RESERVOIRS OF
SMALL MOLECULES THAT ARE
BIOACTIVE, HAVE A BROAD RANGE OF
VIRULENCE ATTENUATING BEHAVIOR,
AND WE HAVEN'T EVEN STARTED TO
LOOK AT COMEN -- THE NEXT TIME YOU
BLOW YOUR NOSE, I WANT YOU TO
REMEMBER MUCINS ARE VERY GOOD AT
MANAGING PATHOGENS, I DID NOT
HAVE TIME TO TALK ABOUT THIS,
BUT AS YOU CAN GUESS ALSO FROM
SETH'S WORK, THIS NOT ONLY
PREVENTS INFECTION OUTGROWTH OF
CERTAIN MICROBES SPECIFICALLY,
BUT GENERALLY DRIVES DIVERSITY
BECAUSE WE HAVE DECREASED
INTERSPECIES COMPOSITION.
I WANT TO THANK A BIG TEAM OVER
THE YEARS, STUDENTS WHO HAVE
REALLY CHARTED NEW TERRITORY AND
REALLY WERE CREATIVE IN THE
TECHNIQUES THEY DEVELOPED AND
THE CONCEPTS THEY HAVE TESTED
AND PROVED AND DISPROVED OVER
AND OVER AGAIN UNTIL THE DATA
REALLY HELD.
AND OUR COLLABORATORS, OUR
EXPERTS IN GLYCAN SCIENCES AND
MICROBIOLOGY, A NUMBER OF THEM
ARE HERE, AND OF COURSE WITHOUT
FUNDING, WE WOULD NOT HAVE DONE
MUCH OF THIS WORK AT ALL.
AND I THANK YOU FOR YOUR
ATTENTION.
>> THANK YOU SO MUCH, THAT WAS A
BEAUTIFUL TALK.
ONCE AGAIN, I ENCOURAGE EVERYONE
TO SUBMIT THEIR QUESTIONS INTO
THE CHAT.
I'M NOT SURE IF YOU HAVE
QUESTIONS IN GENERAL BUT JUST
RIGHT OFF THE BAT JUST KIND OF A
VERY GENERAL QUESTION REGARDING
MECHANISM, I KNOW THIS IS NOT
SIMPLE STUFF THAT YOU'RE
STUDYING BUT EVEN WITH THE LAST
PART OF THE STUDY, JUST SOME OF
THE IDEAS YOU HAVE REGARDING HOW
THAT VERY SIMPLE DIE SACCHARIDE
IS ACTUALLY REGULATING SOME OF
THESE MICROBES.
>> SO WE HAVE DIFFERENT MICROBES
HAVE DIFFERENT MECHANISMS FOR
PSEUDOMONAS, WE WERE ABLE TO
IDENTIFY TWO COMPONENT SYSTEMS
THAT DIRECTLY BINDS TO GLYCANS.
FOR CANDIDA, THAT ES A LITTLE
MORE DIFFICULT.
WE HAVE THE PATHWAYS THAT LEAD
TO THE REPRESSION, WE DO NOT
HAVE THE RECEPTOR YET.
BUT THOSE WILL BE THE KINDS OF
STUDIES THAT NEED TO COME OUT,
AND I'M ANTICIPATING THAT
NUMEROUS -- WILL PLAY NUMEROUS
ROLES AND.
>> GREAT TALK.
BEAUTIFUL.
SO I'M WONDERING, IN CYSTIC
FIBROSIS PATIENTS, WHAT HAPPENS
TO THE MUCINS OR MAYBE IT'S NOT
TO THE MUCINS BUT THAT ALLOWS
THE PSEUDOMONAS TO BEGIN TO
PRODUCE BIOFILM AND BECOME
PROBLEMATIC OR INFECTIVE, WHAT
CAUSES THE IMBALANCE?
>> CYSTIC FIBROCIRCLES JUST LIKE
ON OTHER SURFACES AS WELL, WE
KNOW ONE FACTOR THAT CHANGES IS
GLYCANS INDEED CHANGE, AND THERE
HAS JUST A STUDY BEEN PUBLISHED,
I THINK IT'S PUBLISHED ALREADY,
NOT FROM US, UNC CHAPEL HILL ON
GLYCAN -- HOW THEY CHANGE,
CONSEQUENCE IS NOT CLEAR.
FOR US, THE MOST SIGNIFICANT
FACTOR THAT TRIGGERS THAT CAN
OVERRIDE MUCIN FUNCTION IS
ACTUALLY DNA.
SO EXTRACELLULAR DNA AS IT'S
EXPELLED BY MICROBES, AFTER A
CERTAIN CONCENTRATION, IT
COMPLETELY OVERRIDES MUCIN
EFFECTS.
MU SIGN IT'S REALLY OTHER
POLYSACCHARIDES OR DEGRADATION,
LOSS OF MUCINS THAT ARE
PRESUMABLY MOST PROMINENT.
>> THANK YOU.
>> SO WE'RE GOING TO SHIFT GEARS
A LITTLE BIT.
STEVE TOWNSEND ASKED TO GO EARLY
AND ACTUALLY WAS SCHEDULED TO GO
AT 3:40 SO TO HELP HIM ACTUALLY
BE ABLE TO GIVE HIS TALK AT THE
TIME HE WAS SLOTTED FOR
ORIGINALLY, WE'RE GOING TO HAVE
HIM GO NEXT.
STEVE IS FROM VANDERBILT, AND
HE'S GOING TO TALK ABOUT HUMAN
MILK OLIGOSACCHARIDES AS
MICROBIOME MODULATORS.
>> I APPRECIATE THAT, SEAN, AND
ALL OF THE ORGANIZERS AND
EVERYONE FOR PUTTING ON A GREAT
SYMPOSIUM AND ACCOMMODATING ME
SO I COULD MAKE TO PICK UP MY
ANGRY TODDLER ON TIME.
ALTHOUGH IF SHE SPENT THE
WEEKEND THERE, I PROBABLY
WOULDN'T BE DEVASTATED.
BUT WE'RE HERE TO TALK ABOUT
HUMAN MILK OLIGOSACCHARIDES IN
MODULATING THE MICROBIOME.
IN ADDITION TO THAT, WE'RE GOING
TO TALK ABOUT SOME OF OUR MORE
RECENT STUDIES AND TARGET
IDENTIFICATION.
THESE ARE IN COLLABORATION WITH
MIGRATE FRIEND AND COLLEAGUE AND
COLLABORATOR, DR. FANG YAN FROM
VANDERBILT UNIVERSITY MEDICAL
CENTER.
THE WAY WE CAN START TO TALK IS
GIVE A QUICK INTRODUCTION TO OUR
GROUP.
SO FOR THE PAST EIGHT YEARS AT
VEAN DER
VANDERBILT, WE'RE INTERESTED IN
GLYCOSCIENCE.
THE BIGGEST PROGRAM THAT WE'VE
WORKED ON SO FAR IS OUR HUMAN
MILK OLIGOSACCHARIDE PROGRAM.
AND YOU CAN SEE FROM THE
DISPLAY, WE BECAME INTERESTED IN
THESE COMPOUNDS KNOWN AS HMOs
BECAUSE THEY'RE NOT PRESENT IN
COW'S MILK IN LARGE QUANTITY.
WE THUS WANTED TO FIGURE OUT WHY
THESE COMPOUNDS ARE SPECIAL,
WHAT TYPES OF HEALTH BENEFITS
MIGHT THEY CONFIRM TO FORMULA
THAT FORMULA-FED BABIES WOULDN'T
HAVE ACCESS TO.
OFTENTIMES WE GET THE QUESTION
IS HUMAN MILK REALLY THAT
SPECIAL IN ITS MACROMOLECULAR
CONTENT, SPECIFICALLY
CARBOHYDRATES, AND THE ANSWER IS
CERTAINLY YES.
HUMANS PRODUCE ABOUT 200 UNIQUE
OLIGOSACCHARIDES TO THE TUNE OF
ABOUT 8 TO 20 GRAMS PER LITER.
SO THIS IS A SIGNIFICANT AMOUNT
OF -- MOLECULES AND WE DON'T
REALLY SEE THIS CYBER PATTERN IN
ANY OTHER ANIMAL, SPECIFICALLY
LOOKING AT BOVINE.
NOW, IF WE WERE TO ASK ANYONE IN
THE AUDIENCE TODAY WHAT ARE THE
PURPOSE OF CARBOHYDRATES, MOST
WOULD GIVE A METABOLIC ANSWER OR
SAY THAT THESE ARE THE SOURCES
OF TARGETS.
WELL, IT TURNS OUT IN ONE OF THE
WONDERS OF HUMAN EVOLUTION, A
WOMAN WILL SPEND ABOUT TREE 50
TO 500-CALORIES PER DAY
SYNTHESIZING MULTIPLE GRAMS OF
OLIGOSACCHARIDES, YET THE CHILD
WON'T GET ANY TYPE OF NUTRITION
FROM THEM.
ONLY ABOUT 5% ACTUALLY REESM THE
CIRCULATION AND THE REST GO OUT
THE OTHER DIRECTION.
SO WITH THAT BEING SAID, WHAT
ARE THEY GENERALLY FOR?
WELL, WE KNOW THAT THEY ACT ON
MICROORGANISMS IN THE 80s AND
90s, IT WAS TEASED APART THAT
THESE COMPOUNDS ARE PREBITE BIOTICS,
WE KNOW THEY'RE ANTIADHESIVE
ANTIMICROBIALS, SO IN THIS
MECHANISM, YOU COULD THINK ABOUT
THEM SERVING AS RECEPTOR DECOYS
SO THEY BIND PATHOGENS TO
PREVENT THEM FROM BINDING FROM
YOU.
OUR GROUP AND OTHERS HAVE DONE A
GOOD DEAL OF WORK BOTH FROM A
PHENOTYPIC AND MECHANISTIC
STANDPOINT, OF CHARACTERIZING
THEIR BEHAVIOR, AND -- AND
EVERYTHING WE DESCRIBED OF
COURSE IS ACTING ON
MICROORGANISMS, BUT WHAT ABOUT
THAT 5% THAT REACH SYSTEMIC
CIRCULATION?
WE KNOW THAT THESE COMPOUNDS ARE
ABLE TO MODULATE THE IMMUNE
SYSTEM.
THEY ACT ON THE BRAIN SO THEY'RE
RESPONSIBLE IN PART FOR SYSTEM
AND BRAIN DEVELOPMENT.
WE ALSO KNOW THESE COMPOUNDS
MODULATE THE GROWTH AND
DEVELOPMENT OF THE EPITHELIUM
AND HAVE -- MUCOSAL LAYER EARLY
ON IN LIFE.
SO IT WAS WITH THAT PRINCIPAL IN
MIND THAT WE REALLY TOOK AN
INTEREST TO FIGURE OUT HOW CAN
WE REALLY THINK ABOUT APPLYING
THESE COMPOUNDS AS THERAPEUTICS.
I WANT TO BRING YOUR ATTENTION
TO THE EPIDERMAL GROWTH FACTOR
RECEPTOR AND ALSO THE CONCEPT OF
MUCOSITIS.
THIS REALLY REFERS TO MUCOSAL
DAMAGE THAT REALLY IS A COMMON
SYMPTOM OF A LOT OF ILLNESSES,
SUCH AS IRRITABLE BOWEL
DISEASES.
IN THIS CASE, WE'RE TALKING
ABOUT CROEN DISEASE, ULCERATIVE
COLITIS.
ONE CAN ALSO THINK ABOUT THIS
BEING A SIDE EFFECT OF CELIAC
DISEASE -- CHEMOTHERAPY
RADIATION.
SO IN TOTALITY, ABOUT 10% OF ALL
AMERICANS ARE GOING TO DEAL WITH
SOME TYPE OF MUCOSAL TRAUMA OVER
THE COURSE OF THEIR LIFE, IT'S
TYPICALLY QIENT PAINFUL,
REQUIRES PAIN RELIEF.
MY MOM IS A 10 YEAR BREAST
CANCER SURVIVOR, SHE TALKED
ABOUT HER MUCOSITIS AND HOW IT
WAS SO DAMAGING BUT THEY
ACTUALLY HAD TO GIVE HER A
DIFFERENT CHEMOTHERAPY.
SO WHEN WE THINK ABOUT
PREVENTING THIS TYPE OF ILLNESS,
WHETHER IT'S FOR CROHN'S OR
CANCER TREATMENT, ONE OF THE
FRONTIER TREATMENTS FOR HEALING
MUCOSITIS IS THE IDEA OF
TARGETING THE EPIDERMAL --
RECEPTORS BECAUSE IF YOU DO
THAT, YOU CAN THINK ABOUT
INDUCING DISEASE REMISSION JUST
BY DIRECTLY TARGETING MUCOSAL
HEALING.
SO AT THIS POINT IN TIME --
PUTTING TOGETHER THIS PROGRAM
THAT WE DEVELOPING THE
HYPOTHESIS THAT HUMAN MILK
OLIGOSACCHARIDES MIGHT ACT ON
THE HUMAN GROWTH FACTOR
RECEPTOR -- HELP THAT MUCOSAL
LAYER, IN A REALLY ROBUST
MANNER.
IF WE CAN ACTIVATE THIS WITH
HMOs, WE CAN THINK ABOUT
PREVENTING MUCOSITIS.
SO THE QUESTION BECOMES WHAT
COMPOUNDS DO WE WANT TO USE.
BECAUSE WE HAVE A LOT OF
CHOICES.
THERE ARE 200 COMPOUNDS IN HUMAN
MILK.
FROM A MECHANISTIC STANDPOINT,
WE DIDN'T WANT TO -- ISOLATED
DIRECTLY FROM MILK BECAUSE THEN
WE CAN'T -- SO JUST TO GET TO
THE POINT, WE DECIDED TO USE --
FUCOSYLATED -- MOST COMMON IN
MILK FOR ALL PEOPLE BUT IT'S NOT
UBIQUITOUS.
80% OF THE POPULATION WILL BE
DEFINED AS SECRETERS, THUS CAN
GENERATE SUGARS THAT HAVE THAT
GEICO SYTIC BOND AND FOR THOSE
PEOPLE, THE MAJORITY OF SUGAR IN
MILK WILL BE 2 PRIME FL.
INTERESTINGLY FOR THOSE WHO ARE
NON-SECRETER, THEY DON'T HAVE
THIS -- MACHINERY, WILL BE 3FL
OR L NFP2, THE MOST COMMON SUGAR
IN MILK FOR NON-SECRETERS ARE
LNT.
I LIKE TO SAY FOR ALL THE
TRAINEES IN THE AUDIENCE, IF I
CAN GIVE YOU A SALES PITCH OR
TRY TO INSPIRE YOU, I WOULD SAY
GO OFF INTO THE WORLD OF
SYNTHETIC BIOLOGY AND
CHEMOENZYMATIC SYNTHESIS TO TRY
TO MAKE THESE SUGARS.
OVER THE PAST COUPLE OF YEARS,
WE HAD SOME VERY GENEROUS
FRIENDS IN THE FIELD, BOTH
ACADEMIA AND INDUSTRY, WHO HAVE
HELPED GET US -- BUT ULTIMATELY
THESE ARE ALWAYS SMALL
QUANTITIES.
SO IF I WANT TO DO SOME ANIMAL
STUDIES LIKE I'M GOING TO SHOW
YOU TODAY, WE NEED 100 GRAMS OF
COMPOUND.
SO THE SECOND I SAY WE NEED
100 GRAMS OR 10 GRAMS, PEOPLE NO
LONGER ARE SMILING AND THE SMILE
TURNS UPSIDE-DOWN.
SO JUST TO GET TO THE POINT,
WE'RE USING SYNTHETIC MATERIAL
FOR ALL OF OUR MOUSE STUDIES.
USING CHEMICAL SYNTHESIS, YOU
CAN STILL GET 2 PRIME FL AT 50
CENTS A GRAM.
FOR NLT, LNT, I CAN HAVE AN UNDERGRAD
WAIT MAKE A KILO -- REALLY STILL
NOT COST-EFFECTIVE USING MOST
MODERN STRATEGIES.
SO GOING FORWARD RIGHT AGAIN,
WE'RE THINKING ABOUT THIS
ADDITIONAL HYPOTHESIS THAT HUMAN
MILK OLIGOSACCHARIDES, IN THIS
CASE WE'RE GOING TO BE WORKING
WITH 2 PRIME FL AND ACTIVATE
GROWTH FACTOR RECEPTOR, HERE'S A
WESTERN BLOT THAT SHOWS YOU AT
LEAST PRELIMINARILY THAT THIS IS
THE CASE, MANY DOWNSTREAM
TARGETS LIKE ERK1 AND ERK2 IN
MOUSE EPITHELIAL CELLS.
AT THIS POINT IN THE GAME, WHICH
HAVE A LOT OF OPERATION,
MECHANISTIC DRIVEN THEIR PEW
TICK, AND WE WANT TO SEE WHAT WE
CAN ACTUALLY DO WITH IT.
AT THIS POINT, I'M GOING TO
INTRODUCE YOU SO THE MODEL OF
COLITIS.
THIS IS AN ANIMAL MODEL THAT'S
USED OFTEN BECAUSE IT SHARES A
LOT OF HOMOLOGIES WITH HUMAN
CROHN'S DISEASE.
SO THE WAY THIS EXPERIMENT WORKS
IS WE'RE GOING TO GIVE IT FOR
ABOUT FOUR WEEKS, INDUCED
INFLAMMATION USING TNBS.
FOUR DAYS, THEN DO AN AUTOPSY IN
ORDER TO FIGURE OUT WHAT THE
OUTCOME OF THE EXPERIMENT WAS.
SO IN THIS CASE IT'S PRETTY
COOL, WE ARE ABLE TO SEE THAT IF
WE DOSE THE TOXIN ALONGSIDE
FUCOSYLLACTOSE -- IF WE GO AHEAD
AND LOOK AT A COUPLE OF PICTURES
IT TURNS OUT THE EPITHELIAL
LAYER, SPECIFICALLY THE
JUNCTIONS AND THE VILLI, THEY
ALL REALLY MIRROR THAT OF A
CONTROL, SHOWING WE CAN MITIGATE
DAMAGE BY THIS REALLY NICE MODEL
OF COLITIS.
NOW TO HIT THE POINT HOME A
LITTLE BIT FURTHER, AT THIS
POINT IN THE GAME WE'RE GOING
BACK INTO THE EXPERIMENT AND
WE'RE COMPARING A STANDARDS WILD
TYPE MOUSE TO THAT OF ONE THAT
HAS THE KNOCKOUT FOR EGFR, THIS
AGAIN IS IN THEIR EPITHELIAL
CELLS.
WHAT'S PRETTY COOL IS WE CAN
SHOW THE 2 PRIME FL STIMULATION
OF THIS RECEPTOR IS RESPONSIBLE
FOR PREVENTING COLITIS IN THIS
MODEL, AND SORT OF REALLY LIKE A
NICE COOL PRELIMINARY FINDING AS
WE GO FORWARD.
OKAY.
SO WE KNOW WE HAVE ACTIVATION OF
EGFR IS AT PLAY FOR THE HEALING
THAT WE ARE EXPERIENCING.
WE ALSO WANTED TO TURN OUR
ATTENTION TO THE MICROBIOME, AND
WHAT EFFECTS THAT MIGHT HAVE ON
PREVENTING MUCOSITIS.
SO WE WENT IN TO ANALYZE THE
FLORA OF THE MOUSE IN THE
CONTEXT OF THE WILD TYPE VERSES
ONE -- AND THE DRINKING WATER OF
THE MICE.
THIS GOES ON FOR ABOUT FOUR
WEEKS, AND THEN WE ARE DOING
SEQUENCING OF THEIR FECAL
MICROBIOME.
SO OF NOTE WE FOUND THAT 2 PRIME
FL JEPLY HAS GENERALLY HAS THE ABILITY TO
INCREASE THE COUNTS OF
MICROORGANISMS ASSOCIATED WITH
ANTIINFLAMMATION BUT ALSO BY
DECREASING THE SPECIES THAT ARE
PRO INFLAMMATORY, SO A PRETTY
COOL FINDING ONCE AGAIN.
NOW AT THIS STAGE OF THE GAME,
KEEP IN MIND WE KNOW FROM
PRECEDENT IN BOTH OUR MOST
RECENT DATA THAT HUMAN MILK
OLIGOSACCHARIDES ACT ON SPECIFIC
PROTEIN TARGETS, WE KNOW THEY
HAVE A PROTEIN IN THE -- THIS
TIME FROM THE STANDPOINT OF
CHEMOTHERAPY.
SO WHAT I'M SHOWING YOU HERE AND
WHAT WILL BE REALLY ANALYZED FOR
THE REMAINING 8 MINUTES OR SO IS
OUR DATA THAT COMES OUT OF THIS
MODEL OF CHEMOTHERAPY AND
RADIATION.
SO 5FU IS A REALLY PHENOMENAL
MODEL BECAUSE IT REALLY MIMICS
AND SHOWS A LOT OF HOMOLOGY WITH
PATIENTS THAT ARE RECEIVING HIGH
DOSE CHEMOTHERAPY OR RADIATION.
IT CAUSES SIGNIFICANT DAMAGE,
AND WE FELT IF WE COULD REALLY
MAKE A DIFFERENCE HERE IN
PREVENTING DAMAGE FROM
CHEMOTHERAPY OR FROM HEALING
CHEMOTHERAPY, THIS WOULD BE
REALLY COOL FOR PATIENTS AS WE
TRY TO TRANSLATE MAYBE INTO THE
CLINIC.
SO IN THIS CASE RIGHT OFF THE
BAT, WE WENT IN TO EXAMINE
WHETHER 2 PRIME FL COULD INDUCE
APOPTOSIS, THIS IS A MECHANISM
HOWL MANY CHEMOTHERAPIES KILL
TRANSFORM CELLS.
INTERESTINGLY IT DOES PREVENT
CHEMOTHERAPY FROM PREVENTING THE
GROWTH OF HEALTHY MOUTH EP THREE
YELL CELLS, BUT INTERESTINGLY,
IT DOES NOT PROVIDE ANY
PROTECTION FOR TRANSFORMED
CANCER CELLS SUCH AS AGS OR
AT29.
SO THAT WAS ANOTHER COOL
FINDING, WE COULD USE 2 PRIME FL
TO PROTECT NON-HEALTHY TRANSPORT
CELLS BUT IT DOESN'T PROVIDE THE
SAME BENEFIT TO THOSE THAT ARE
ALREADY CANCERS.
MOVING FORWARD WE INITIATED A
STUDY WHERE WE WANTED TO
EVALUATE INFLAMMATION IN THE
SMALL INTESTINE OF A MOUSE
COHORT WITH VARYING LENGTH OF
EXPOSURE AND VARYING LENGTH OF
TREATMENT.
WHAT WE HEARD, REGARDLESS OF HOW
LONG THE MICE ARE ON
CHEMOTHERAPY, TYPICALLY
DECREASES INFLAMMATION
SIGNIFICANTLY BUT THAT
COTREATMENT DOESN'T WORK IF THE
HOUSE HAS ALREADY BEEN GIVEN
CHEMO BEFORE IT WAS GIVEN
2 PRIME FL.
NOW COMPARED WITH 5 URACIL
TREATMENT ALONE, IT TURNS OUT
THE 2 PRIME FL PRE-TREATMENT --
DECREASE BODY WEIGHT AND
IMPROVE -- SCORES.
SO IF YOU THINK ABOUT
CHEMOTHERAPY, IT'S GOING TO
CAUSE SIGNIFICANT ULCERATION AND
INFLAMMATION IN ANY SUBJECT, BUT
THESE ABNORMALITIES ARE ALWAYS
DECREASED WHEN WE PRE-TREAT WITH
2 PRIME FL.
NOW, LASTLY BEFORE RETURNING
BACK TO THE MICROBIOTA, IT'S
REALLY IMPORTANT TO NOTE, I
GUESS, FOR MANY OF THE TRAINEES
IN THE AUDIENCE, YOU'VE HEARD A
LOT OF THE MUCUS SURFACES TOED.
IF YOU THINK ABOUT IT, THE
MUCOSAL SURFACE IS REALLY
IMPORTANT IN PROTECTING --
BACTERIAL INFECTION, VIRAL
ASSAULT, EERCH MECHANICAL DAMAGE
FROM JUST A NORMAL FUNCTION OF
THE DP G.I. TRACT.
SO CENTRAL TO THIS WHOLE ARE
GOBLIN CELLS, THESE ARE MUCIN
PRODUCING.
THEY GO PEL THE CELL, KOWNDZ ARE
DECREASED AND THESE CELLS ARE
REALLY JUST ATTACKED AND
ERADICATED.
SO WHAT WAS COOL ABOUT THIS
PORTION OF THE EXPERIMENT THAT
I'M SHOWING YOU IS WOULD HE
FOUND OUT THE 2 PRIME FL -- WE
CAN KEEP A NICE MAINTENANCE OF
THIS POPULATION THROUGHOUT THE
DURATION OF TREATMENT WITH THE
CHEMOTHERAPY.
ALL RIGHT.
SO AS WE RETURN BACK TO THE
MICROBIOME WHEN WE CLOSE THIS
OUT, THIS WAS A PRETTY COOL
FINDING AND I REALLY LIKE THE
IDEA OF USING THESE COMPOUNDS TO
HIT SPECIFIC PROTEIN TARGETS,
BUT ALSO MODULATING THE PLY CROW
BIOME TO GET ADDED BENEFIT.
IN THIS CASE WHAT WE OBSERVED IS
THAT 2 PRIME FL DECREASES
5 FLUOROURACIL AND INDUCES
ULCERATION OF THE MICROBIOTA IN
OUR MOUSE MODEL.
SO THIS CAN BE CONSIDERED
IMPORTANT BECAUSE IT'S BEEN
REPORTED THAT CHEMOTHERAPY IS
OFTEN ASSOCIATED WITH DYSBIOSIS
OF PATIENTS, SO WE WANTED TO
EVALUATE WHAT HAPPENS TO THE
OVERALL FECAL BACTERIAL
COMPOSITION, SUCH AS BETA
DIVERSITY SHOWN IN THE MIDDLE OR
OUR SHANNON AND SIMPSON INDICES
WHICH IS A REPRESENTATION OF
ALPHA DIVERSITY WHEN WE TREAT
THESE ANIMALS WITH 2 PRIME FL
VERSUS JUST CHEMOTHERAPEUTIC.
SO WE GENERALLY OBSERVE, AGAIN,
THAT 2 PRIME FL PRE-TREATMENT
AND ACTUALLY CONCURRENT
TREATMENT BOTH ELICIT A PARTIAL
SHIFT OF THE COMPOSITION OF THE
GUT MICROBIOTA AND ANIMALS THAT
WERE EXPOSED TO CHEMOTHERAPY
BACK TO NORMAL, BACK TO THE WILD
TYPE.
AND AS YOU MIGHT EXPECT,
DIFFERENT SPECIES OF BACTERIA
THAT ARE KNOWN TO BE
ANTI-INFLAMMATORY OR SLIGHTLY
UPREGULATED AND THOSE THAT ARE
PRO INFLAMMATORY ARE GENERALLY
DOWNREGULATED.
SO WITH THAT, WE'VE HIT THE
16-MINUTE MARK.
THE ONLY THING LEFT TO DO IS
THANK ALL OF THE FANTASTIC
TRAINEES WHO WORKED ON THIS
SPECIFIC PROJECT.
IN THIS CASE THERE ARE THREE
FROM THE YAN LAB AND FROM MY
GROUP -- THEY'VE DONE PHENOMENAL
WORK IN THESE STUDIES THAT ARE
REALLY TRANSLATIONAL, POWERFUL.
COULDN'T HAVE DONE IT WITHOUT
THEM.
ALSO WANT TO THANK NIH AUTHOR
FAIR SUPPORT, AND TAKE ANY
QUESTIONS THAT YOU MIGHT HAVE.
>> THANK YOU.
AND I LIKE YOUR STATEMENT,
SUPPORT THE NIH, THAT'S GOOD.
YOU HAD YOUR HAND UP.
>> WHAT'S UP, STEVE.
I ALWAYS WANTED TO MEET YOU SO
IT'S NICE TO MEET YOU IN PERSON.
>> SAME HERE, AND I LIKE YOUR
HAT.
>> IT'S A GOLDEN STATE WARRIORS
HAT.
IT WAS JUST FROM TRAVELING.
ANYWAY, SORRY.
SO OKAY.
SO I RECALL A PAPER A WHILE AGO
WHICH WAS ABOUT MAYBE LIKE 3SL
IN COLITIS.
AND REALLY -- I THINK THEY
PROPOSED THAT IT ACTUALLY WAS
ACTIVATING TOLL-LIKE RECEPTORS
DIRECTLY IN A PRO-ACTIVATION
MODE AND WORSE, COLITIS.
WHICH LEADS TO KIND OF LIKE THIS
QUESTION I HAVE WHICH IS, OF THE
DIFFERENT SYLATED OR FEW COST
LATED HUMAN MILK
OLIGOSACCHARIDES, DO YOU THINK
THERE ARE FUNCTIONAL
DIFFERENCES, EVEN EVOLUTIONARY,
BETWEEN THESE TWO BECAUSE IT
SEEMS LIKE YOU HAVE A FEW COST
LATED ONE THAT'S PROTECTIVE AND
YOU HAVE ONES THAT ARE
PATHOGENIC IN SOME WAY.
KIND FTD RHYME OR REASON OF
DIFFERENT HUMAN MILK
OLIGOSACCHARIDES.
MAYBE NOT SO MUCH AT THE
SUGARRIZATION LEVEL OF THE
MICROBIOME OR -- IT'S
OPEN-ENDED.
WHY WOULD ONE OF THEM BE PRO
COLITIC AND ONE ANTI-COLITED?
>> THAT'S A REASONABLE QUESTION.
SOL THE WHY, I DON'T KNOW
BECAUSE I HAVEN'T DOVE INTO THE
EVOLUTIONARY THOUGHT ABOUT IT.
SAME THING ABOUT WHY DOES DNA
HAVE FEWER KNOW CYST INSTEAD OF
PYRO KNOW CYST WHEN PYRO KNOW
CYST ARE THERMALLY MORE STABLE.
WHEN IT COMES TO FEW COST LATED
SUGARS, IF WE EXAMINE MILK,
SOMEHOW GARS DOMINATE OVER
SIALYLATED SUGARS.
IT ABOUT 85 TO 15, WHEREAS IN
COW'S MILK, THEY'RE MY SYLATED.
IF WE BOUNCE THIS BACK TO
COLITIS, EVEN THOUGH THERE'S NOT
A TON OF SUGARS IN THERE FROM
THOSE THAT ARE FORMULA-FED FROM
A MECHANISTIC PERSPECTIVE, I
THINK THAT'S TBD, BUT THERE'S
REASONS WHY HUMANS HAVE -- --
[INAUDIBLE] GLYCOSYLATED WHERE A
LOT OF FOUR CHAMBERED STOMACH
ANIMALS HAVE SYLATED COMPOUNDS
AND VERY LITTLE FUCOS.
I JUST KNOW THE WHY.
>> DO YOU THINK THERE MIGHT BE
SOME HOST RECOGNITION SUCH AS
PERHAPS SIGNALING -- FOR SOME
MODIFICATION OF FUCOSE?
I DON'T KNOW OF TOO MANY, BUT
NOT SO MUCH FUCOSES, ALTHOUGH
MAYBE THIS IS WHERE THEY SAY NO,
THERE'S A LOT OF FUCOSYLATION
THAT LEAD TO SOME SINGLING
LECTINS.
>> SO I DON'T KNOW THAT, AND I
HAVEN'T READ A TON OF LITERATURE
OR CAN RECALL A TON OF
LITERATURE THAT'S TEASING ANY OF
THIS APART, WHICH I THINK FOR
OUR TRAINEES WHO ARE WATCHING,
GO AHEAD AND GET YOURSELF TENURE
WITH ONE OF THESE TOPICS.
YOU
>> MAYBE TBR THE MICROBICIDE,
BUT I DON'T KNOW MANY FROM OUR
SIDE.
>> -- THE MODIFICATION OF
LACTOSE BUT I DON'T THINK IT
BEEN LOOKED AT.
RICK DID SOME BEAUTIFUL STUDY,
BUT I THINK THERE'S A LOT BE
DONE BEYOND THE SPECIFICITY,
WHICH IS AN IMPORTANT ONE TO ASK
AND NOT AN EASY ONE TO ANSWER,
WHAT DOES THAT MEAN WITH RESPECT
TO WHAT THEY'RE DOING HALF THE
TIME ANYWAY FRANKLY.
BECAUSE THEY'RE EVERYWHERE AND
THEY BIND EVERYTHING.
AS MUCH AS I LOVE THEM.
BUT YEAH, THREE QUESTIONS.
GREAT QUESTIONS.
I DON'T WANT TO SHIFT GEARS, I
WISH WE COULD HANG OUT ALL NIGHT
ON ZOOM, BUT I DO WANT TO TRY TO
END BY 5:00 BECAUSE A FEW PEOPLE
NEED TO GO.
ONE OF THE PEOPLE THAT NEED TO
CATCH -- WE HAVE A FEW DAYCARE
APPOINTMENTS.
I WISH MY KIDS WERE IN DAYCARE
STILL.
BUT THEY'RE NOT.
SO -- WAIT UNTIL YOU HAVE
TEENAGERS AND YOU'LL UNDERSTAND
WHAT I'M TALKING ABOUT.
SO NADINE SAMARA IS GOING TO
TALK NEXT FROM NIDCR, "DI DIVERSE
SUBSTRATE SPECIFICITIES OF
MUCIN-TYPE
O-GLYCOSYLTRANSFERASES.
>> SO LET ME MOVE THIS ZOOM
CAMERA SO IT NOT BLOCKING MY
SLIDES.
SO FIRST OF ALL, THANK YOU SO
MUCH FOR THIS IP I INVITATION.
THIS HAS BEEN A GREAT SYMPOSIUM.
AND I WILL TRY TO BE QUICK AND
GET EVERYBODY HOME FOR THE
WEEKEND, SO MY NAME IS NADINE
SAMARA.
I'M AT THE NATIONAL INSTITUTE OF
DENTAL AND CRANIOFACIAL RESEARCH
AT THE NIH.
TODAY I'LL BE TELLING YOU ABOUT
THE DIVERSE SUBSTRATE
SPECIFICITIES OF MUCIN-TYPE O
GLYCOSYL TRANSFERASES.
MY TALK IS GOING TO BE A LITTLE
MORE ME CAN MECHANISTIC, JUST A WARNING
TO EVERYBODY WHO'S STILL
WATCHING.
BUT JUST BEFORE I GET INTO THE
RESEARCH, I JUST WANT TO
INTRODUCE MYSELF AND MY GROUP.
SO I ESTABLISHED THE STRUCTURAL
BIOCHEMISTRY UNIT AT THE DENTAL
INSTITUTE IN AUGUST 2019.
WHICH IS GREAT TIMING.
BUT ANYWAY, SO OUR STUDIES OF
GLYCOSYL TRANSFERASES AND WE
WERE ALSO INTERESTED IN THE O
GLYCANS AND THEIR ROLES AT THE
HOST MICROBE INTERFACE, SO WE
SPROJS ON BOTH SIDES, ON THE
HOST SIDE, AND THOSE ARE -- YOU
KNOW, I THINK YOU'VE ALREADY
HEARD ABOUT THE MUCIN
GLYCOSYLATION SO I'LL BE TALKING
ABOUT THAT TODAY.
MY LAB IS SHOWILY SHIFTING
TOWARDS THE MICROBICIDE, AND WE
HAVE SOME PRELIMINARY PROJECTS
TO TRY AND UNDERSTAND
EXOPOLYSACCHARIDE BIOSYNTHESIS
AND THE ROLE NOT JUST IN THE
MATRIX BUT ALSO IN
PERIODONTITIS, SO WE'RE USING A
VARIETY OF DIFFERENT METHODS,
INCLUDING MICROBIOLOGY,
COMPARATIVE GENOMICS AND
HOPEFULLY STARTING TO USE SOME
IMEU LOGICAL METHODS AS WELL TO
TRY TO UNDERSTAND THE ROLE OF
THOSE EXOPOLYSACCHARIDES IN
DISEASE AS WELL.
SO FOR TODAY, I'M GOING TO BE
TALKING ABOUT MUCIN TYPE OF
GLYCOSYLATION, AND I THINK
KATRINA DID A GREAT JOB
INTRODUCING MUCINS, BUT JUST AN
OVERVIEW, SO THIS IS A POST
TRANSLATIONAL MODIFICATION.
IT OCCURS ON MANY SUBSTRATES,
ALMOST 80% OF PROTEINS THAT GO
THROUGH THE SECRETORY PATHWAY
ARE MUSE MUCYLATED.
SOME OF THEM ARE LOCATED ON THE
CELL SURFACE, OTHERS ARE
SECRETED INTO THE MUCOUS LAYER.
AND SO BOTH MEMBRANE TETHERED --
PROTECT AGAINST PATHOGENS,
PROTECT THE UNDERLYING LAYERS
FROM PATHOGEN AND VISION, AND
SECRETED MUCINS HAVE SIMILAR
RULES AND PROTECTION AGAINST
INFECTION, AS WELL AS MODULATING
ATTRACTIONS OF THE MICROBIOME
AND PLOA PROTECTING OTHER LAYERS FROM
PHYSICAL AND CHEMICAL DAMAGE.
SO THESE ROLES ARE REALLY
IMPORTANT AS WE'VE HEARD TODAY
FROM OTHER TALKS, AND IN
MAINTAINING HOMEOSTATIC STATE
AND IMMUNE RESPONSE.
SO THE THE ENZYMES THAT WE STUDY ARE
THE -- BASICALLY I'D LIKE TO
THINK THAT THER RESPONSIBLE FOR
ESTABLISHING THE FOUNDATION FOR
THIS VERY IMPORTANT
MODIFICATION.
IF YOU DON'T HAVE THIS INITIAL
FOUNDATION, YOU CAN'T LET THE
PROPER GLYCAN, I GUESS -- I
DON'T KNOW, CAN'T FIND THE RIGHT
WORD, FRIDAY AFTERNOON.
BUT SO THESE ARE GOLJI TYPE
PROTEINS AND CONTAIN TWO DOMAINS
VERY IMPORTANT FOR THE FUNCTION.
FIRST YOU HAVE THE CATALYTIC
DOMAIN SHOWN IN GREEN, THIS IS
TETHERED TO THE LECTIN DOMAIN.
SO WE HEARD EARLIER ABOUT LECTIN
FROM LAURA, SO THESE FAMILY OF
ENZYMES ARE RARE AGAINST THE
GLYCOSYL TRANS PHRASE FAMILIES
IN THAT THEY CONTINUE TO -- THIS
LECTIN DOMAIN, AROUND 10 AMINO
ACID -- I'LL TELL YOU MORE ABOUT
IT LATER BUT IT'S IMPORTANT FOR
SUBSTRATE BINDING AND
RECOGNITION.
SO THE ACTIVITY, THE CHEMISTRY
HAPPENS IN THE CATALYTIC DOMAIN,
SO WHAT YOU HAVE ARE TWO
SUBSTRATES, YOU HAVE THE DONOR
SUBSTRATE WHICH IS A U VP -- YOU
CAN HAVE THE -- AND A MANGANESE
DEPENDENT MANNER, THE TRANSFER
OF GALNAC.
THEY EXTEND THESE CHAINS TO FORM
THESE DENSE, DENSE -- MOST OF
THE TIME, THERE'S A CAVEAT HERE
AND I'LL MENTION IT IN A SECOND,
MOST OF THE TIME YOU HAVE DENSE
OLD GLYCANS ON THESE --
ESPECIALLY THESE MUCIN
SUBSTRATE.
SO HERE'S THE BIG QUESTION, YOU
HAVE NUMEROUS DIVERSE
SUBSTRATES, SO MANY DIFFERENT
TYPES OF SUBSTRATES, THEY DON'T
SHARE CONSENSUS SEQUENCE.
MANY OF THESE SUBSTRATES LIKE
MUCINS ARE DENSELY GLYCOSYLATED
BUT THERE ARE -- A LOT OF TIMES
IT DISREGULATES THE PROTEOLYTIC
CLEAVAGE OF A SUBSTRATE.
SO IN A SITUATION LIKE THIS, HOW
DOES NATURE ACCOMPLISH THIS TASK
OF CORRECT CORRECTLY OR EFFICIENTLY
GLYCOSYLATING THESE SUBSTRATES
SO THEY'RE FUNCTIONAL OR THAT WE
CAN UNDERGO FURTHER GLIE
GLYCOSYLATION -- SO WHAT NATURE
HAS DONE IS MADE 20 DIFFERENT
VERSIONS OF THIS ENZYME AND AT
LEAST IN HUMANS, THERE ARE 20,
AND THESE ENZYMES SHARE A LOT OF
SIMILAR FUNCTIONS AND WE KNOW
THIS BECAUSE YOU HAVE A MOUSE
KNOCKOUT, IT'S NOT NECESSARILY
LETHAL, SO THERE'S SOME OVERLAP.
AS A SIDE NOTE, WE'VE BEEN
THINKING ABOUT SUBSTRATE
SPECIFICITY, I RECENTLY HAD A
CONVERSATION WITH JESSICA KRAMER
WHO STUDIES MEU SINS AS MUCINS AS WELL.
IT MADE ME THINK ABOUT
CONFIRMATION, AND SO YOU HAVE A
LOT OF PROLINES AND I WAS JUST
THINKING ABOUT IT.
ON THE OVERALL CONFIRMATION OF
THESE MUCINS.
AS YOU ADD MORE SUGARS, THAT'S
ALSO GOING TO INFLUENCE THE
OVERALL CONFIRMATION.
AND ONE THING I WAS THINKING
ABOUT TODAY IS THAT IF YOU THINK
ABOUT SOME OF THESE TRANSFERASE,
THEY'RE ACTUALLY PRETTY
CONSERVED AMONGST LIKE, LET'S
SAY, T1 IS SIMILAR IN HUMANS AND
MICE BUT A LOT ARE NOT -- MAYBE
WHAT'S NOT IMPORTANT SL THE --
BUT THE CONFIRMATION AND HOW
THAT CONFIRMATION FITS INTO THE
AK SIEVE SITE OR THE REGION OF
THE ENZYME THAT'S POSH FOR IMPORTANT FOR
ACTIVITY.
BUT THAT'S A SIDE NOTE JU TO GET
BACK TO THE POINT OF THIS TALK.
A LOT OF THESE ENZYMES ALSO HAVE
SPECIFIC FUNCTIONS AND
SUBSTRATES, FOR EXAMPLE, T1 IS
INVOLVED IN HEART DEVELOPMENT,
ALTHOUGH WE DON'T KNOW WHAT
SUBSTRATE IT IS.
-- WHICH REGULATES THE --
CLEANAGE OF THAT PROTEIN.
TIER 3 IS SPECIFICALLY
ASSOCIATED WITH THE PATIENT
MUTATIONS IN THIS ENZYME AS WELL
AS IN THE SUBSTRATE -- ONE OF
ITS SUBSTRATES, FGF23.
T12 WHICH IS ONE OF THE ENZYMES
WE'RE INTERESTED IN IS
ASSOCIATED WITH COLON CANCER,
AND THAT'S BEEN SHOWN IN SEVERAL
DIFFERENT PATIENT COHORTS.
AGAIN IN THIS CASE WITH SORT OF
THE MISSING FACTOR HERE IS THAT
WE DON'T KNOW WHAT THE SUBSTRATE
OR SUBSTRATES ARE, SO WE THINK
PERHAPS MAYBE THIS IS TOTAL HAND
WAVING, INFLUENCING THE MY
COBIOTA OR SOMETHING IN THE
IMMUNE SYSTEM BUT WE DON'T
REALLY KNOW, SO THAT'S STILL
KIND OF A BIG QUESTION.
BUT THOSE MUTATIONS ACTUALLY
DECREASE THE ACTIVITY OF T12 AS
I'LL SHOW LATER.
DOWN AT T11 IS INVOLVED IN
REGULATING KIDNEY FUNCTION
THROUGH THE GLYCOSYLATION OF THE
KIDNEY PROTEIN.
THEY SHOW GAL KNACK
D2 ACTIVATES -- THROUGH GLIE
COST LIN OF THE RECEPTOR.
SO THERE'S SO MUCH GOING ON
HERE, SO MUCH DIVERSITY, BURR
THERE'S ALSO MANY LAYERS OF --
YOU HAVE LAYER AT EXPRESSION
LEVEL ACROSS DIFFERENT CELLS
TYPES SO THEY'RE NOT EQUALLY
EXPRESSED THROUGH CELLS.
AND YOU ALSO HAVE DIFFERENTIAL
EXPRESSION THROUGH DEVELOPMENT.
AND WHAT WE'RE INTERESTED IN AS
STRUCTURAL BIOLOGISTS AND ENS
I'MOLOGISTS IS THE RULES, THE
DIVERSE RULES FOR HOW THEY
RECOGNIZE SUBSTRATES AND HOW
THEY CATALYZED THE REACTION.
SO SOME OF THESE DIVERSE RULES
CAN BE SORT OF PUT INTO BOXES
BUT THEY'RE VERY GENERAL BOXES,
SO A LOT OF THE ENZYMES CAN
RECOGNIZE SOME OF THE
UNGLYCOSYLATED PROTEIN OR TEP
TIED AND MODIFY THAT.
AND SO AGAIN, I SAID THERE'S NO
CONSENSUS SEQUENCE BUT I'LL COME
BACK TO SOME CERTAIN
CHARACTERISTICS THAT CAN BE
RECOGNIZED BY THESE ENZYMES.
A LOT OF THE ENZYMES CAN
RECOGNIZE PRIOR GALNACs EITHER
VIA THE LECTIN DOMAIN, WHICH IS
NOT SURPRISING, THESE CONTAIN
HASH KIED RATES, AND OFTEN TOIMS
THEY GO QUITE UNIQUE TO THE
ENZYMES THAT HAPPEN.
AND IN MOST CASES, THE ENZYMES
IS A COMBINATION OF DIFFERENT
BINDING MODES, IT'S NOT JUST ONE
OR THE OTHER.
A LOT IS USUALLY GOING ON IN
TERMS OF HOW THESE ENZYMES
FUNCTION.
SO BASED ON ALL WE KNOW AND ALL
THE LAYERS OF REGULATION WE JUST
MENTIONED, THE CURRENT MODEL --
FIRST YOU HAVE THIS
UNGLYCOSYLATED SUBSTRATE GOING
THROUGH THE GOLGI AND THE SUBSET
OF THESE ENZYMES ARE CALLED THE
EARLY GALNAC TEASE.
ANOTHER SUBSET MIGHT ABLE TO
RM -- ON THE SUBSTRATE AND ADD
MORE, AND THEN YOU HAVE -- AT
FURTHER MODIFY THE SUBSTRATE TO
OBTAIN THIS DENSELY GLYCOSYLATED
PROTEIN.
SO WHAT WE'RE TRYING TO
UNDERSTAND OR THE QUESTIONS WE
ASK IF OUR LAB FOR THIS SET OF
PROJECTS IS WHAT ARE THE PLEAK
ALREADY BASIS OF WHAT ARE
THE MOLECULAR BASIS OF THE
RULES.
WE'RE MOSTLY INTERESTED IN
REALLY LIKE LOOKING AT THE
CRYSTAL STRUCTURES AND DOING
KINETICS TO UNDERSTAND WHAT
THOSE RULES MEAN.
AND SO I DID MENTION THAT THERE
IS NO CONSENSUS SEQUENCE BUT
WHAT I WANT TO SAY IN TERMS OF
JUST THIS POLYPEPTIDE BINDING IS
AS I MENTIONED, MUCIN REPETS,
MOST OF THESE ENZYMES, 17 OUT OF
20 OF THEM, WE THINK -- SO THE
ONLY ONES THAT DON'T FROM WHAT I
KNOW ARE T10, T7 ABANDONS 7 AND T17.
RECOGNIZE A PROLINE, C TERMINAL
TO THE ACCEPTOR SITE.
AND THIS PAROLING POCKET, HERE
I'M SHOWING THIS PAROLING POCKET
FROM T12, HYDROPHOBIC RESIDUES,
SO MOST ENZYMES HAVE THIS
POCKET, SO IF YOU SEE A PROLINE,
FOR EXAMPLE, YOU CAN PREDICT
THAT IS LIKELY TO GET GLIE
GLYCOSYLATE BID MOST ENZYMES
OTHER THAN T10, T17.
I MENTIONED -- HAS BEEN QOIT -- I MENTIONE D COLON
CAB SE SO WHAT I'M SHOWING HERE
IS FROM A PAPER FROM 2009 WHERE
THEY SHOW THAT IF YOU HAVE THESE
DEACTIVATING MUTATIONS, THAT
PEOPLE WITH COLON CANCER, THESE
ARE DIFFERENT CASES, I THINK
EACH ONE IS A SINGLE PATIENT.
THEY HAVE MUTATIONS IN T12 THAT
ARE DIE ACTIVATING. DEACTIVATING.
THE OTHER THING THAT'S REALLY
INTERESTING ABOUT D12, SHOWING SHOWING
PRIOR GALNAC BINDING
PREFERENCES.
I MENTIONED EARLIER MOST OF THE
ENZYMES REALLY PREFER PROLINE AT
THE T12 POSITION, BUT -- LIKES
GALNAC AT THE T12 POSITION.
SO T12 DOES HAVE A -- US A
ALTERNATIVELY CAN -- LET'S SAY
THEIR STUDY SHOWED THAT GALNAC
AT THAT POSITION ENHANCES THE
ACTIVITY OF T12.
AT THE SAME TIME, T12 HAS A
GALN AC BINDING POCKET IN ITS
LECTIN DOMAIN SO IT SEEMS TO
HAVE THESE 2GALN AC BINDING
POCKETS WORKING IN CONCERT,
SUGGESTING IT WORKS LATER IN THE
PROCESS OF GLYCOSYLATION.
SO TO UNDERSTAND THE BASIS OF
THIS ENHANCEMENT, WE SOLVED THE
CRYSTAL STRUCTURE OF GAL KNACK
T12 WITH THIS PEPTIDE.
THE GALNACs ARE SHOAB SHOWN IN
YELLOW, THE PEPTIDES ARE SHOWN
IN MAGENTA.
HERE'S THE 3D -- AND TO SORT OF
UNDERSTAND THE BASIS OF THIS
UNIQUE PLUS-THREE PREFERENCE I'M
GOING TO ZOOM INTO THIS GALNAC
BINDING SITE REAL QUICK SO WHAT
YOU SEE IS MAINLY BACKBONE
INTERACTIONS BETWEEN THE ENZYME
SIDE CHAINS WITH THE EXCEPTION
OF N270.
SO BASICALLY THIS BIND BEING
POCKET IS UNIQUE, BUT ON TOP OF
THAT, THIS N270 OVER HERE IS NOT
CONSERVED IN ANY OF THE
ISOENZYMES IN THE HUMAN FAMILY.
SO IT'S ACTUALLY JUST THIS ONE
RES DUAL THAT BASICALLY DICTATES
THIS PREFERENCE FOR GALNAC AT
THE PLUS 3 POSITION.
ON THE OTHER HAND, THIS GALNAC
BINDING SITE IN THE LECTIN
DOMAIN IS HIGHLY CONSERVED, AND
I'LL TALK ABOUT THAT IN THE NEXT
SLOID BUT I WANTED TO FIRST
INTRODUCE THE LECTIN DOMAIN MORE
SPECIFICALLY, SO THIS LECTIN
DOMAIN CONTAINS THREE REPEATS
WITH SUGAR BINDING POTENTIAL,
AND WHAT WE MEAN BY POTENTIAL IS
THAT YOU HAVE TO HAVE THE RIGHT
RESIDUES TO ACTUALLY INTERACT
WITH THE SUGAR AND THOSE
RESIDUES ARE SHOWN HERE UNDER
STAR -- THAT ALL INTERACT WITH
GALNAC.
SO THIS REPEAT POCKET OR THIS
BINDING POCKET IS CONSERVED, IF
YOU LOOK AT MOST OF THE
STRUCTURES, AT LEAST THE -- CAN
YOU BASICALLY SUPER IMPOSE THOSE
BINDING POCKETS.
AND SO THE POTENTIAL, THE WORD
POTENTIAL COMES FROM THE FACT
THAT IF YOU LOOK AT THE THREE
LECTIN DOMAINS AND YOU LOOK AT
THEM IN ALIGNMENT, YOU CAN SEE,
IN FACT, NOT -- IT'S NOT ALWAYS
THE CASE THAT THESE THREE
RESIDUES OR ONE OF THEM IS
ALWAYS MISSING SOMETHING, MAKES
IT SEEM AS IF THAT BINDING
POTENTIAL FOR TBAL GALNAC IS NOT
NECESSARILY BRENT.
A LOT OF PRESENT.
THAT'S NOT ALWAYS THE CASE, I'LL
SHOW YOU ONE IN A FEW MINUTES
THAT HAS TWO ACTIVE REPEATS.
AND SO WHAT IS INTERESTING ABOUT
THE LECTIN DOMAIN, YES, THE BIND
BEING POCKET IS CONSERVED BUT
THE ORIENTATION OF THE LECTIN
DOMAIN SEEMS TO REALLY INFLUENCE
HOW THESE ENZYMES FUNCTION.
SO HERE WHAT I'M SHOWING ARE TWO
CRYSTAL STRUCTURE, ONE OF GALNAC
T12 AND ONE OF T2.
SO IF YOU SUPER IMPOSE THE
CATALYTIC DOMAIN OF T2 AND T12,
MAYBE BY LOOKING AT THIS ALPHA
HERE AND ALPHA HERE, IT'S NOT
VERY OBVIOUS, I'M JUST LOOKING
AT THE SUPER POSITION, BUT THE
ALPHA REPEATS AND ARE ON
OPPOSITE SIDES COMPARED TO THE
CATALYTIC DOMAIN.
AND SO I JUST WANT TO SAY THAT
THE STRUCTURE OF T4 WAS ALSO
SOLVED PRIOR TO THE STRUCTURE OF
T12 AND IT'S THE CLOSEST
HOMOLOGUE TO T12.
SO T4 AND T12 ARE DOING
BASICALLY THE SAME THING SO
WHAT'S REALLY INTERESTING HERE
IS THAT THE PEPTIDE BINDS FROM
THE END DIRECTION OF THE LECTIN
DOMAIN AND THEN THIS C TERMINAL
OF THIS PEPTIDE IS IN THE
CATALYTIC DOMAIN, WHEREAS IN T
IT, IT STARTS IN THE C-TERMINUS
AND ENDS AT THE END TERMINUS IN
THE CATALYTIC DOMAIN.
AND WHAT DOES THIS MEAN?
SO THIS BASICALLY PROVIDES
EVIDENCE FOR WHAT WAS OBSERVED
FROM BIOCHEMICAL STUDIES OF
DIRECTIONALITY, SO SUBSET OF THE
ENZYMES COMBINE PEPTIDE OR
PROTEIN -- WELL IN THIS CASE
JUST PEPTIDES, AT THE C-TERMINUS
IN THE END TERMINAL DIRECTION,
GLYCOSYLATE IN THE C TERMINAL
DIRECTION.
SO I MENTIONED EARLIER -- WAS
ALSO SOLVED AND THE PEOPLE WHO
DID THIS WORK IN SPAIN -- SHOWED
THAT IF YOU TAKE THE LINGER FROM
T2 AND THEN YOU PUT IT INTO T4,
YOU CAN ACTUALLY SWITCH THE
DIRECTIONALITY OF T4.
SO WHAT THEY PROPOSE WAS THE
LINGER WHICH DICTATES THE LECTIN
DOMAIN POSITION WHICH INFLUENCES
DIRECTIONALITY OF THE ENZYME.
SO THIS IS INTERESTING AND I
THINK IT REALLY DOES APPLY TO
THAT CASE, BUT ONE THING THAT I
WAS THINKING ABOUT FOR A WHILE
IS I DID THE SAME SORT OF
THOUGHT EXPERIMENT WITH GALNAC
T1 AND T2 AND THESE ARE SORT OF
THE HOUSEKEEPING ENZYMES THAT
CAN GLYCOSYLATE ANYTHING.
THEY'RE THE MOST ROBUST AND SO
FARWELL STUDIED OF ALL THE
ISOENZYMES IN THIS FAMILY, AND
IF YOU DO SORT OF THE SAME
EXPERIMENT HERE, YOU COULD SEE
THAT T1s ALPHA AND -- ARE ALSO
NOT ON THE SAME SIDE OF THE
CATALYTIC DOMAIN.
BUT THEY SEEM TO HAVE THE SAME
DIRECTIONALITY BASED ON STUDY
FROM TOM'S GROUP AT CASE
WESTERN.
SO WHY IS THAT?
SO I WAS THINKING ABOUT THIS
RIGHT BEFORE THE PANDEMIC HIT
AND THE POSTBAC IN THE LAB IS
LOOKING FOR A PROJECT AND SO WE
WERE JUST KIND OF STARING AT
THIS CRYSTAL STRUCTURE, THAT
CONTAINS GALNAC AT THE
C-TERMINUS SO THIS IS BOUND IN
THE ALPHA SUBUNIT STARTING FROM
THE C TERMINAL END, AND THEN I
WAS ALSO STARING AT THIS CRYSTAL
STRUCTURE OF T1 THAT WAS SOLVED
MANY YEARS AGO IN LARRY TABAK'S
LAB SO THIS IS LIKE THE FIRST
CRYSTAL STRUCTURE THAT DOESN'T
CONTAIN ANY SUBSTRATES BUT JUST
BY STARING AT THESE TWO PROTEIN,
I NOTICED THE ALPHA AND BETA
REPEAT WERE BASICALLY IN THE
SAME POSITION IN SPACE COMPARED
TO THE CATALYTIC DOMAIN, AND SO
WHAT I PROPOSED WAS THAT IT'S
POSSIBLE THAT MUC5AC13 WAS BOUND
TO T1 USING THE BETA AND NOT THE
ALPHA REPEAT.
SO YOU'RE GOING TO ASK ME, YOU
SAID ONLY ONE LECTIN REPEAT IS
USUALLY ACTIVE BUT IN FACT,
THERE ARE JUST A FEW ENZYMES
WHERE TWO LECTIN REPEATS ARE
ACTIVE AND IN THIS CASE, T1 IS
ONE OF THOSE ENZYMES WHERE BOTH
ALPHA AND BETA WERE SHOWN TO
HAVE ACTIVE LECTIN REPEATS BY A
GROUP A WHILE BACK AND I'LL CITE
THEM IN THE NEXT SLIDE.
BUT IF YOU CRYSTALLIZE T1 WITH
MUC5 -- THAT KIND OF EXPLAINS
WHY THE TWO ENZYMES HAVE THE
SAME DIRECTIONALITY.
IT'S NOT REALLY BECAUSE OF
THE -- POSITIONED THE SAME WAY
IN SPACE.
AND SO WE JUST -- WE'RE
CURRENTLY TRYING TO ASSESS WHAT
THIS MEANS, SO THE GROUP I
MENTIONED THAT FIRST DISCOVERED
THIS THAT BOTH REPEATS IN T1 ARE
ACTIVE, FROM 2002, BUT WE'RE
SORT OF FOLLOWING UP DOING
FURTHER STUDIES ON THIS, AND
HERE'S -- NOW WE'RE REALLY
TRYING TO UNDERSTAND FIRST OF
ALL WHY DOES T1 EVEN HAVE TO
REPEAT, WHY DOES IT NEED TO HAVE
TWO, WHEN IT'S REALLY ACTIVE
AGAINST A LOT OF SUBSTRATES THAT
DON'T EVEN NEED TO BE
GLYCOSYLATED TO FURTHER -- TO
ADD AN INITIAL GALNAC?
SO THAT'S REALLY NOW THE
QUESTION AND THE BASIS OF THE
STUDY WE'RE TRYING TO WRAP UP.
BUT IF YOU LOOK AT THE SECOND --
AGAINST THESE DIFFERENT
PEPTIDES, SO THESE PEPTIDES
EITHER CONTAIN TWO, SO THE BLUE
ONE CONTAINS TWO ACCEPTOR SITES,
AND THE RED AND THE GREEN ONES
CONTAIN EITHER AN ACCEPTOR SITE
AT THE END TERMINUS OR AN
ACCEPTOR SITE AT THE C-TERMINUS,
AND IF YOU LOOK AT JUST THIS
SPECIFICITY OF THIS ENZYME
AGAINST ALL THESE DIFFERENT
LIGHT SUBSTRATES, WHAT WE'LL
NOTICE IS IN THE WILD TYPE,
GALNAC IS ACTUALLY ENHANCING THE
ACTIVITY OF THIS ENZYME, WHETHER
IT'S THIS SUBSTRATE OR THAT
SUBSTRATE.
AND IF YOU MAKE A MUTATION -- TO
KNOCK OUT THE BINDING YOU CAN
REALLY REDUCE THE SPECIFICITY
TOWARDS THIS SUBSTRATE AND WITH
NO INFLUENCE ON THE SPECIFICITY
OF THIS SUBSTRATE.
AND THEN IF YOU MAKE A MUTATION
IN THE BETA, YOU CAN REDUCE THE
SPECIFICITY TOWARDS THE BETA
SUBSTRATE, AND SOMEHOW REDUCE
THE ALPHA SUBSTRATE BUT THE
ERROR BAR HERE IS HIGH AND
THERE'S A TWOFOLD DIFFERENCE SO
I DON'T WANT TO OVERTHINK THIS.
AND THEN IF YOU REMOVE BOTH BETA
AND ALPHA REPEAT BINDING YOU
REDUCE THE SPECIFICITY TOWARDS
BOTH GLYCOSUBSTRATES AND IT'S
ESSENTIALLY AS IF THERE'S NO
LECTIN DOMAIN OVER THERE.
SO THESE LECTIN REPEATS ARE
CERTAINLY ENHANCING THE ACTIVITY
OR SPECIFICITY OF GALNAC
T1 TOWARDS GLYCOSYLATED
SUBSTRATES.
SO I JUST WANT TO BRING YOU THE
LAST SLIDE, I HOPE I DIDN'T GO
TOO LONG AND I APOLOGIZE IF I
DID AND I HOPE CATH LIN CATHERINE STOP ME
IF I AM.
I SAID EARLIER I KIND OF PUT
THESE ENZYMES IN DIFFERENT
BOXES, EARLY, INTERMEDIATE AND
LATE, BUT IT'S WAY MORE
COMPLICATED THAN THAT, RIGHT,
BECAUSE I SAID THAT GALNAC
T1 CAN DO -- GLYCOSYLATE
SOMETHING THAT'S NEVER BEEN
TOUCHED AND IT SEEMS TO BE
ENHANCED BY EITHER ONE OR TWO OR
SO WE'VE BEEN USING THESE SHORT
PEPTIDES BUT IF YOU ENVISION A
LONGER PEPTIDE WITH TWO GALNACS
ON IT, YOU CAN ENVISION THAT
GALNAC T1 MIGHT BE JUST
GLYCOSYLATING BETWEEN TWO
GALNACs THAT HAVE BEEN ADDED
BY SOMETHING ELSE, MAYBE -- BUT
THE POINT IS LIKE -- CONTEXT
MATTERS AND IT'S REALLY
IMPORTANT LIKE WHAT THE
SUBSTRATE IS, WHAT THE CELL IS,
WHAT HAPPENED BEFORE OR AFTER.
SO IT'S NOT AS SIMPLE AS THESE
ENZYMES JUST FITTING INTO
CERTAIN CATEGORIES.
IT REALLY -- YOU KNOW, IT'S
REALLY MUCH MORE COMPLICATED
THAN THAT AS WE'RE SEEING WITH
THE T1 EXAMPLE.
SO THESE ARE THE CONCLUSIONS.
I HOPE TODAY THAT I SHOWED YOU
THAT SELECTIVITY IS REALLY
COATED WITHIN VERY SPECIFIC
RESIDUES OR REGIONS, AND EACH
FAMILY MEMBER SEEMS TO FOLLOW
ITS OWN RULES, AND OF COURSE
FROM MORE BIOMEDICAL
PERSPECTIVE, KNOWING THAT EACH
ONE IS SPECIFIC IN TERMS OF ITS
FUNCTION, CAN WE DESIGN SPECIFIC
INHIBITORS OR ATTENUATORS, AND
USE THIS INFORMATION TO
CORRECTLY PREDICT BUY LOGICAL
SUBSTRATES BECAUSE AS I
MENTIONED A LOT OF TIMES WE
DON'T REALLY KNOW WHAT THE
SUBSTRATES OR SITES ARE OF
GLYCOSYLATION.
I WANT TO FINISH MY TALK BY
SAYING OUR LAB IS GROWING, IT'S
PRETTY AWESOME, I LOVE HOW MUCH
IS BEING DONE AND HOW EXCITED
EVERYBODY IS.
WE COLLABORATE A LOT WITH KELLY
10 HAGEN AND LARRY TABAK,
THEY'RE BOTH AMAZING MENTORS.
A LOT OF THE PEOPLE THAT DID THE
WORK I SHOWED TODAY ARE IN GREEN
AND THOSE CONTINUING TO DO WORK
HIGHLIGHTED IN GREEN AS WELL.
WE COLLABORATED WITH TOM GERKEN.
WE'RE CONTINUING TO COLLABORATE
WITH BEN SCHUMANN AT THE CRICK
ON T12 TRYING TO FIGURE OUT WHAT
THESE SUBSTRATES MIGHT BE.
WITH THAT I WANT TO SAY THANK
YOU FOR YOUR ATTENTION, I KNOW
IT'S THE END OF THE DAY.
HOPEFULLY EVERYONE HAS TIME TO
TELL THEIR STORIES.
THANK YOU VERY MUCH.
>> REALLY GREAT, NADINE.
THANK YOU.
WE HAVE TIME FOR ONE QUICK
QUESTION, JOHN?
WHAT DO YOU THINK?
>> SURE.
SOMEONE HAS A QUESTION, DO THESE
LECTIN DOMAINS ALSO BIND GEL OR
ARE GAL OR
ARE THEY VERY GALNAC --
>> THAT'S A GREAT QUESTION.
SO I CAN SAY THAT LIKE FOR THE
T1 STORY, WE'RE DOING A LOT OF
COMPETITION ASSAYS.
JUST TO MAKE SURE -- WE'RE
DOING -- MY POSTBAC WAS DOING
COMPETITION ASSAYS WITH GALNAC
TO MAKE SURE THAT WE'RE ACTUALLY
REMOVING GALNAC BINDING, AND SHE
DID TRY DIFFERENT SUGARS, AND I
DON'T REMEMBER THE DATA, BUT
THERE WERE SOME OTHER SUGARS
THAT SEEMED TO BE COMPETING WITH
THE PEPTIDES.
SO I CAN GET BACK TO YOU LATER,
LAURA.
I JUST HAVE TO KIND OF GO BACK
AND LOOK AT THAT TO SEE.
I THINK MANNOSE WAS ONE THAT WAS
COMPETEING WITH THE GLYCOSYLATED
PEPTIDE BUT I'M NOT 100% SURE.
SORRY.
>> I JUST THOUGHT MAYBE THAT N
GLYCANS MIGHT HELP RECRUIT THE
ENZYME, AND THAT MIGHT BE AN
INTERESTING ROLE.
>> YEAH.
YEAH, THAT'S A REALLY GOOD
POINT.
SO ACTUALLY IF YOU LOOK AT THE
CRYSTAL STRUCTURE OF T1, IT ALSO
HAS N GLYCANS ON IT SELF, AND
SOMETIMES I THINK ABOUT THE WAY
THEY'RE PACKING IN THE CRYSTAL
STRUCTURE AND I'M WONDERING IF
THAT CAN GIVE YOU ANY INSIGHT
INTO NOT JUST, YOU KNOW, N
GLYCANS ON THE SUBSTRATE BUT
ALSO N GLYCANS ON THE PROTEINS
THEMSELVES AND HOW THEY COULD BE
MODULATING THE ENZYME FUNCTION.
BECAUSE IN SOME CASE, I CAN
REMOVE THE N GLYCANS OF THE
PROTEINS FINE AND THE ACTIVITY
IS NOT TOTALLY GONE, BUT IN
OTHER CASES IF I REMOVE THE
N-GLYCAN, IT KILLS THE ENZYME.
I DON'T KNOW IF IT'S BECAUSE
IT'S JUST DESTABILIZINGITY
OVERALL STABILITY BUT IT'S
SOMETHING I THINK ABOUT TOO, SO
THAT'S A REALLY GREAT POINT.
>> THANK YOU.
>> THANKS, NADINE, FOR A GREAT
TALK.
OUR NEXT SPEAKER IS SEAN
STOWELL, AND HE'S GOING TO TALK
TO US ABOUT DISCOVERING
DETERMINANTS OF NATURALLY
OCCURRING ANTIBODY FORMATION.
SO THANKS, SEAN.
>> ALL RIGHT.
SO I REALLY APPRECIATE THE
OPPORTUNITY TO WORK WITH
CATHERINE ON THE SYMPOSIUM.
I REALLY WANT TO GIVE HER TIME,
IT'S BEEN WONDERFUL TO LISTEN TO
EVERYONE'S SCIENCE AND I'M JUST
GOING TO -- A OPPORTUNITY TO
TAKE A BREAK FROM EVERYTHING
ELSE AND LISTEN TO A LOT OF
GREAT TALKS ON AREAS RELATED TO
THE MICROBIOME AND GLYCOBIOLOGY
IN GENERAL.
SO ONE OF THE THINGS THAT OUR
LAB IS INTERESTED IN IS ANTIBODY
REPERTOIRES AND HOW THEY
DEVELOP.
MOST OF US ARE VERY FAMILIAR
WITH OBVIOUSLY ACQUIRED
DEVELOPMENT OR CHANGES IN
ANTIBODY REPERTOIRE AS A RESULT
OF INFECTION OR VACCINATION, OF
COURSE WITH COVID IT'S BEEN A
TOM IK
TOPIC DU OP JOUR.
SOMETHING THAT RECEIVES A LITTLE
LESS ATTENTION IS ANTIBODIES.
THESE ARE M SO OF SOME OF OF THE FIRST
ANTIBODIES DISCOVERED, THEY ARE
REALLY IMPORTANT IN TRANSPLANT,
TRANSFUSION TOLERANCE, AND ALSO
HAVE BEEN SHOWN TO REGULATE
AUTOIMMUNITY.
THE FUNDAMENTAL QUESTIONS ABOUT
HOW THESE ANTIBODIES FORM AND
HOW THEY SHAPE THE REPERTOIRE
EACH OF US HAVE INDIVIDUALLY,
EXTREMELY DISTINCT AND HAVE VERY
LITTLE OVERLAP AS YOU CAN SEE
HERE IN THE CENTER OF THIS
SCHEMATIC.
THE FIRST NATURALLY OCCURRING
ANTIBODIES THAT WERE DESCRIBED
IN MANY WAYS TO BE THE MOST
COMMONLY KNOWN, THOSE ARE
ANTIBODIES AGAINST A AND B BLOOD
GROUP ANTIGENS.
-- THOSE ARE IMPORTANT
ANTIBODIES IN TERMS OF IMMUNE
DIFFICULTIES WITH RESPECT TO
TRANSPLANTATION AND TRANSFUSION
THAT CAN CAUSE IMMUNE REJECTION
AND COMPLICATIONS, THEY'RE VERY
SEVERE, AND WERE ACTUALLY THE
ANTIGENS TARGETED BY THESE
ANTIBODIES -- LIKE CARL
LANSTEINER IN 1900, OVER 20
YEARS AGO, WHEN SHE GOT THE
NOBEL PRIZE IN 1930, SO THERE
HAVE BEEN A LOT OF DIFFERENT
IDEAS ABOUT HOW NATURALLY
OCCURRING ANTIBODIES DEVELOP.
THE EARLIEST STUDIES ACTUALLY
BEFORE ANTIBODIES WERE REALLY
EVEN DEFINED SUGGESTED MAYBE
THERE WAS A HAPLOTYPE, OBVIOUSLY
BEFORE SCIENTIFIC DISCOVERIES AS
A RESULT IN THE UNDERSTANDING
OF -- GERM-FREE MICE SUGGESTED
THAT MICROBIAL EXPOSURE HAD NO
INFLUENCE REALLY ON IGM LEVELS
OR REPERTOIRE AND THESE STUDIES
WERE REALLY IMPORTANT IN SHAPING
CURRENT UNDERSTANDING REQUIRING
POTENTIAL INFLUENCE OR LACK
THEREOF THE MICROBIOTA ON THE
DEVELOPMENT OF ANTIBODIES.
THESE STUDIES ARE CORROBORATED
BY A STUDY BEAUTIFULLY DONE A
FEW YEARS BACK LOOKING AT BOTH
IGM AND MOSTLY IGA IN TERMS OF
THE ACTUAL REPERTOIRE OF B CELL
RECEPTORS PRESENT IN MICE.
THEY FOUND THAT THE ANTIBODIES
PRESENT EITHER IN GERM-FREE OR
CONVENTIONALLY HOUSED MICE WERE
REALLY NOT TOO DIFFERENT, THEY
WERE QUITE POLYREACTIVE IN
NATURE, SUGGESTING THAT ONCE
AGAIN THEET ANTIBODIES NOT ONLY
FORMED BUT THEY -- THESE RESULTS
SUGGESTED ONCE AGAIN THAT THE
MICROBIOTA ARE YOU THINK UNLIKELY INVOLVE D
IN SHAPING -- ONE OF THE
CHALLENGES IN STUDYING
CLINICALLY RELEVANT NATURALLY
OCCURRING ANTIBODIES THE
ANTIGENS ARE NOT POLYMORPHIC IN
MOST OF THE MODELS WE STUDIED IN
PARTICULAR MICE.
THEY DO NOT MAKE ANTIBODIES
UNTIL LATE IN LIFE, SO FOUR OR
FIVE MONTHS, PEOPLE REPORT THESE
ANTIBODIES, THEY DON'T DEVELOP
ON A TIME SCALE THAT DEVELOPS
NORMALLY.
AS ALL OF YOU KNOW, A AND B
ANTIGENS ARE GLYCANS AND THEY'RE
SYNTHESIZED DUE TO THE --
SYNTHESIS.
DUE TO THE -- WE WANTED TO KNOW
IF WE TOOK MICE AND KNOCKED OUT
THIS ENZYME WHEN WE CAN MAKE AN
O AND B PAIR TO STUDY THE
FORMATION OF SPONTANEOUS
FORMATION IN NATURALLY OCCURRING
ANTIBODIES AGAINST THE B
ANTIGEN.
THIS IS IN PART IMPORTANT ONCE
AGAIN BECAUSE OF THE BARRIERS OF
NATURALLY OCCURRING -- CAUSE IN
TRANSFUSION AND TRANSPLANTATION
CLINICALLY INVOLVED IN
TRANSFUSION MEDICINE, SO THIS IS
SOMETHING WE TEST EVERY DAY AND
PROBABLY THE MOST COMMON
IMMUNOLOGICAL TEST DONE IN THE
WORLD BECAUSE ANYONE WHO GETS
ANY TYPE OF BLOOD TRANSFUSION
GETS AN ABO TYPING WHICH
INCLUDES BOTH THE ANTIGEN AND
THE ANTIBODY.
SO THE FIRST THING YOU WANT TO
DO IS MAKE SURE THESE O MICE OR
DETERMINE WHETHER THESE O MICE
MAKE NATURALLY OCCURRING
ANTIBODIES.
O MICE FORM ANTI-B ANTIBODIES.
NOT ONLY IGM BUT ALSO IGG.
SO THESE ARE ANTIBODIES THAT
FORMED AS THE MICE WERE HANGING
OUT DOWNSTAIRS WITHOUT ANY KNOWN
STIMULUS.
THE NEXT THING WE WANT TO DO IS
DETERMINE WHERE THESE ANTIBODIES
ARE CAPABLE OF CAUSING A
REACTION.
WE ACTUALLY PERFORM ANTIBODIES
AGAINST A VARIETY OF -- THAT ARE
POLYMORPHIC THAT ACTUALLY ARE
NOT CLINICALLY RELEVANT TO
INDUCE -- IF WE'RE GOING TO
MODEL WHAT WE CARE ABOUT
CLINICALLY FOR THESE ANTIBODIES
ARE CAPABLE OF CAUSING -- OF THE
HEMOLYSIS WE'RE TRYING TO AVOID.
SO TO DO THAT WE LABELED UP THE
RED CELLS AND TRANSFUSED FIRST B
POSITIVE RED BLOOD CELLS, NO
CLEARANCE OBSERVED WHICH IS WHAT
YOU'D EXPECT WITH A FULLY
COMPATIBLE TRANSFUSION.
WHEN WE TRANSFUSED THAT SAME
BLOOD INTO OB BLOOD WE SAW
VARYING LEVELS OF CLEARANCE.
CLEARED THE BLOOD COMPLETELY AND
SOME EXPERIENCED LITTLE
CLEARANCE AT ALL.
THIS VARIABILITY IS NOT EASY TO
STUDY WHEN WE'RE LOOKING AT
REDUCTION OF SYSTEMS BUT IT'S
ACTUALLY EXACTLY WHAT HAPPENS
CLINICALLY.
WHEN PEOPLE GET ABO PANEL BLOOD
WHICH DOESN'T HAPPY -- SOME
PEOPLE CAN GET A WHOLE UNIT OF
BLOOD AND EXPERIENCE NO CLINICAL
SEQUELAE.
LEL IT'S NOT A CORRELATION STRONG
ENOUGH TO PREDICT AND RESULT IN
ANY TYPE OF CLINICAL
DECISION-MAKING BASED ON THIS.
BUT IT IS SOMETHING THAT WE SEE
IN SOME WAYS CLINICALLY AS WELL.
SO THE QUESTION WE HAD WAS,
WHERE DOES THIS VARIABILITY IN
ANTIBLOOD -- B ANTIBODY COME
FROM IN THESE MICE BECAUSE THESE
MICE ARE A MODEL AND ONCE AGAIN
A REDUCTION -- ACTUALLY CAN
CAUSE A HEMOLYTIC TRANSFUSION
REACTION.
WE DIDN'T WANT TO USE -- THE
IMMUNE SYSTEM IS OBVIOUSLY VERY
DIFFERENT IN THOSE MICE AND
ALTHOUGH THERE'S A LOT OF
IMPORTANT STUDIES THAT CAN BE
DONE THAT WAY, WE REALLY WANTED
TO UNDERSTAND WHAT THIS
DIFFERENCE LOOKS LIKE UNDER
CONVENTIONAL CONDITIONS AT LEAST
INITIALLY IN MOMS THAT HAD
DIFFERENT LEVELS OF ANTI -- SO
WE TOOK MOMS AND DID HAVE SOME
IN STERILE CONTAINERS AFTER
THEY'VE BEEN CONVENTIONALLY
HOUSED AND ONLY DID THAT SIMPLY
TO REDUCE IN OUR MIND OVER
MULTIPLE GENERATIONS.
AND EVALUATE ANTIB ANTIBODIES
OVER TIME.
WE DID THE SAME THING WITH MOMS
HIGHER ANTIBODIES, WE TOOK THEIR
PUPS AND CROSS FOSTERED WITH
MOMS THAT HAD THE HIGHEST
ANTIBODY LEVELS OVER SUBSEQUENT
GENERAL RAIGS TO SEE IF THIS
ANTIBODY WAS A TRAIT WE COULD
FOLLOW OVER TIME AND IN SELECT
FORM.
USING THIS APPROACH WHAT WE
FOUND WAS THAT MOMS THAT HAD LOW
ANTIBODY LEVELS PRODUCED PUPS
THAT HAD LOW ANTIBODY LEVELS AND
OVER SUBSEQUENT GENERATIONS WERE
ABLE TO SELECT A KOL KNEEL THAT
VIRTUALLY PRODUCED VERY LITTLE
IF ANY -- USING TRADITIONAL
ASSAYS EMPLOYED IN THE BLOOD
BANK, BUT WHEN WE TOOK MOMS THEY
HAD AS CONTROL ANTIBODY LEVELS
THAT WERE HIGHER AND ONCE AGAIN
EVALUATED THEIR ANTIBODY LEVELS
OVER TIME OVER SUBSEQUENT
GENERATIONS FOUND THEY HAD
SUSTAINED ANTIBODY LEVELS.
SO THESE RESULTS SUGGEST THAT
THERE MAY BE AN ENVIRONMENTAL
FACTOR THAT INFLUENCES
ANTIBODIES AND SO WE DID WHAT
EVERYONE DOES, WE DID A SEQUENCE
ANALYSIS OF THE MYOBIOME AND
FOUND THERE WAS VARIABILITY, DID
NOT CORRELATE WITH THESE
ANTIBODIES.
SO NEXT WE WANT TO MAKE SURE
THAT THESE PUPS AND THE MOMS
THAT WE WERE FOLLOWING COULD
ACTUALLY MAKE ANTIBODIES OR
DETERMINE IN CONTRAST WE
SELECTED FOR -- A STRAIN OF MICE
THAT FOR WHATEVER REASON
COULDN'T MAKE ANTIBODIES OVER
TIME.
WE TOOK MICE THAT HAD BEEN
FOSTERED OR ACTUALLY OFFSPRING
OF A MOM THAT DEPARTMENT HAVE DIDN'T HAVE
ANTIBODIES AND CROSS FOSTERED
INTO A MOM THAT HAD ANTIBODY,
TOOK LITTER MATES AND CROSS
FOSTERED A MOM THAT DIDN'T HAVE
ANTIBODIES.
WHAT WE FOUND IS WHEN WE DID THE
LATTER, CROSS FOSTERING PUPS ON
TO MOMS THAT DIDN'T HAVE --
RESULTED IN OFFSPRING THAT ALSO
AFTER FOSTERING WERE UNABLE TO
PRODUCE ANTIB ANTIBODIES BUT
WHEN WE TOOK LITTER MATES THAT
HAD ANTIBODIES, NOW THESE PUPS
WERE ABLE TO PRODUCE BOTH IG PL.
ANTI-B ANTIBODIES AND ALSO IGG.
THAT ANTIBODIES COULD BE INDUCED
AND THIS WAS LIKELY AN
ENVIRONMENTAL OUTCOME OF THE
TYPE OF EXPOSURE EVENTS THAT ARE
OCCURRING.
THE FOLLOWING SLIDES GO WITHOUT
SAYING WITH RESPECT TO THIS
AUDIENCE, OBVIOUSLY THE TYPICAL
STRUCTURE WE LOOKED AT IN TERMS
OF SCHEMATIC MEMORY IS
RELATIVELY SIMPLE -- YOU CAN'T
SEE ANYTHING, IT'S VERY SIMILAR,
IT JUST MAKES SENSE FOR ME IN
MANY WAYS OF A SMALL TOWN IN THE
MIDWEST WHERE YOU CAN SEE A LOT
OF DIFFERENT COMPONENTS OF THIS
TOWN IN TERMS OF SCHOOLS AND
CHURCHES AND STREETS, AND A FEW
TREES HERE.
BUT THIS IS NOT AT ALL AS WE
KNOW THE SURFACE OF A CELL LOOKS
LIKE, THAT'S COVERED IN THE
GLYCOHELIX MUCH MORE SIMILAR TO
URBAN ATLANTA, WHICH IS WHERE I
DID MY TRAINING IN THE CUMMINGS
LAB, THERE'S A WHOLE CITY HERE
BETWEEN EMORY WHICH IS WHERE
THIS PICTURE WAS TAKEN IN
DOWNTOWN ATLANTA BUT THAT'S
REALLY DIFFICULT TO PERCEIVE
BECAUSE OF THIS RILEY RYE REALLY URBAN
FOREST THAT COVERS THE CITY.
THE QUESTION IS IS THERE A --
RED CELL THAT MAY BE RESPONSIVE
FOR STIMULATING THESE
ANTIBODIES.
SO THE ANSWER TO THIS QUESTION,
WE USE A REALLY OLD BLOOD BANK
TRICK IN THE 30s, WE TOOK
SERUM THAT HAD ANTI-B ANTIBODIES
PRESENT, WE SIMPLY WASHED AWAY
THE ANTIBODIES THAT DID NOT
BOUND, ASKED FOR THESE
ANTIBODIES, REACTED WITH -- SO
USING THIS APPROACH WE WERE ABLE
TO DETERMINE THERE WAS A SMALL
POPULATION OF MICROBES THAT WE
COULD DETECT THAT APPEARED TO
REACT WITH THE ANTIBODY AND WHEN
WE SORTED THEM OUT, TO
INDIVIDUAL POPULATIONS AND THEN
WE CULTURED THOSE POPULATIONS.
WE WERE ABLE TO IDENTIFY ONE
STRAIN IN PARTICULAR THAT WHEN
WE TOOK THAT SAME ANTIBODY
PREPARATION, INK INCUBATED WITH THIS
INDIVIDUAL COLONY THAT HAD
SIGNIFICANT REACTIVITY, THIS WAS
NOT SOMETHING COMMON AMONG CUBS
BECAUSE WE LOOKED AT ANOTHER
STRAIN THAT WAS ISOLATED THAT
HAD REALLY A VERY LITTLE IF ANY
REACTIVITY.
SO THE NEXT QUESTION WE WANT TO
ASK WAS WHETHER THERE WAS
SPECIFICITY TO THIS INTERACTION
BETWEEN THE ANTIBODY AND THE
ACTUAL ANTIGEN.
SO TO DO THIS, WE ACTUALLY
ISOLATED THE POLYSACCHARIDE FROM
THESE MICROBES WE'VE BEEN DOING
THIS WITH A LOT OF OTHER
MICROBES BUT I WANT TO
SPECIFICALLY FOCUS ON THE
MICROBES WE ANALYZED WITH THIS
PARTICULAR -- THIS EXPERIMENT WE
WERE DOING.
AS I MENTIONED PREVIOUSLY, MANY
ANTIBODIES WERE THOUGHT TO BE
POLYSPECIFIC, INCLUDING A LOT OF
SO CALLED AUTO ANTIGENS THAT
WERE USED IN THE PREVIOUS STUDY
THAT ARE THOUGHT TO BE LIKE
EVIDENCE OF OR SUGGESTIVE OF
POLYMER ACTIVITY IN GENERAL.
HOWEVER WHEN WE TOOK THE
ANTIBODIES -- THE POSITIVE RED
CELL WE USED TO BIND THESE
MICROBES, YOU HAVE THIS B
ANTIGEN AND THE ONE STRAIN OF
LPS THAT WE ISOLATE FROM THAT
MICROBE.
SO THESE RESULTS SUGGEST AT
LEAST SOME NATURALLY OCCURRING
ANTIPO DE -- MAY INDEED BE
INDUCED BY INDIVIDUAL STRAINS OF
MICROBES THAT ARE DECORATED WITH
UNIQUE GLYCAN SIGNATURE.
AND OF COURSE THERE'S ONCE AGAIN
NO CROSS REACTIVITY WITH ALL
THESE OTHER ANTIGENS THAT HAVE
BEEN DEMONSTRATED FOR OTHER
ANTIBODIES WHICH CERTAINLY
OCCURS.
NEXT WE WANTED TO KNOW WHETHER
THEY WERE ACTUALLY CAPABLE OF
INTRODUCING ANTIBODY FORMATION
SO TO DO THAT WE ACTUALLY
EXPOSED THE MICE TO MICROBES AND
WE FOUND ANTIBODIES COULD BE
DETECTED IF THE O MICE TREATED
OR EXPOSED TO THE BLOOD GROUP
POSITIVE MICROBES.
WE DID NOT SEE MICE FORM IF WE
GAVE THEM THE NEGATIVE MICROBES.
THIS IS NOT A RESULT OF
DIFFERENCES IN COLONIZATION
BECAUSE THEY'RE VERY SIMILAR
COLONIZATION RATES BETWEEN THE
TWO.
FINALLY WANT TO DETERMINE
WHETHER THERE WAS AN ABILITY NOT
ONLY OF THESE MICROBES TO INDUCE
ANTIBODIES BUT WHETHER THESE
ANTIBODIES ARE ABLE TO CAUSE --
WE DID THE EXACT SAME
EXPERIMENT, WE EXPOSED O MICE TO
THE POSITIVE OR NEGATIVE MICROBE
AND TRANSFUSED THESE MICE.
THE O RECIPIENTS THAT GOT THE
POSITIVE MICROBES WERE ABLE TO
INDUCE A HEMOTRANSFUSION
REACTION BUT IF WE TOOK THEM AND
GAVE THEM NEGATIVE MICROBES,
THEY WEREN'T ABLE TO -- SO THESE
RESULTS SUGGEST MICROBES EXIST,
THEY'RE ABLE TO DECORATE
THEMSELVES IN SUGAR STRUCTURES
THAT LOOK VERY SIMILAR TO RED
CELLS AND THAT EXPOSURE TO THESE
MICROBES CAN STIMULATE ANTIBLOOD
GROUP ANTIBODIES THAT ARE
CAPABLE OF CAUSING POTENTIAL
HEMOTRANSFUSION REACTION
TRANSPLANT REJECTION AND MAYBE
RELEVANT OR OTHER SETTINGS WRR
EXPOSURE TO STUFF LIKE ANTIGENS
COULD IB FLEUNS INFLUENCE THE REPERTOIRE
OF ANTIBODY.
WITH THAT, I'LL THANK CONNIE
ARTHUR, SHE HAS HER OWN LAB NOW
AND SHE'S ACTUALLY DOWN THE HALL
FROM ME.
RICK CUMMINGS WHO WE'VE BEEN
DOING THESE STUDIES WITH AND
SETH WHO WE'VE BEEN
COLLABORATING WITH ON THIS
PROJECT AS WELL, AND WITH THAT,
I WILL TAKE ANY QUESTIONS THAT
YOU HAVE.
SORRY I WENT THROUGH THAT REALLY
QUICKLY BUT I'M TRYING TO GET US
BACK ON TIME.
>> FANTASTIC TALK, JOHN.
I'M MONITORING THE CHAT NOW.
I NO YOU'RE ALSO A PRACTICING
CLINICIAN WHICH I THINK IS
AMAZING SO IF YOU PUT THAT HAT
BACK ON IT AND THINK ABOUT HOW
YOU CAN USE WHAT YOU FOUND NOW
IN THE CLINIC, WHAT DO YOU THINK
ABOUT -- HOW WOULD YOU DO THAT?
WHAT WOULD YOU DO?
>> APPRECIATE THE QUESTION.
I THINK THE MOST RELEVANT
SETTING ACTUALLY IS PROBABLY NOT
MANIPULATION OF THE
MICROBIOTA -- PATIENTS WITH
SICKLE CELL DISEASE OR
THALASSEMIA OR IN THE SETTING OF
HEART TRANSPLANT, SO IF YOU HAD
A LITTLE KIDDO WITH A CONGENITAL
HEART DISEASE, IT IS THE NUMBER
ONE BARRIER TO TRANSPLANTATION.
WE THINK OF HLA BECAUSE THAT'S
THE THING THAT IT'S REALLY HARD
TO MATCH FOR BECAUSE THERE'S SO
MANY DIFFERENT POLYMORPHISMS BUT
THE FIRST QUESTION IS ALWAYS
ABOUT ID YOE COMPATIBILITY.
YOU CAN TRANSPLANT ACROSS
BARRIER, THERE'S A TOLERANCE
PROGRAM THAT'S LISTED, SO WE'RE
HOPEFUL THAT IF WE CAN
UNDERSTAND MECHANISTICALLY HOW
THESE ANTIBODIES FORM AND HOW
THE MICROBIOTA SHAPES THESE
ANTIBODIES, WE COULD FAVORABLY
MANIPULATE AND SELECT PATIENTS
IN NEED OF TRANSPLANTATION OR
TRANSFUSION THERAPY SO
IMMUNOLOGICAL BARRIERS ARE NOT
AS PRESENT, THERE'S MORE
OPPORTUNITY -- FOR THEM.
SO WE'LL SEE.
YOU KNOW HOW SCIENCE GOES.
>> NO, IT COOL.
THAT'S REALLY NEAT.
THANKS.
LAURA, I SAW YOU TURNING YOUR
CAMERA ON BUT YOU'RE MUTED.
>> I DIDN'T KNOW IF WE HAD TIME.
CAN I JUST ASK REALLY QUICKLY, I
WONDER IF YOU COULD FLIP THIS
AROUND BECAUSE CLEB CAN COLONIZE
MANY PANCREATIC TUMORS, AND I
WAS WONDERING IF YOU THINK IT
MIGHT BE POSSIBLE TO FLIP THIS,
TO USE THIS KIND OF THING TO
YOUR ADVANTAGE.
>> I COULDN'T AGREE WITH YOU
MORE.
THAT'S ONE OF THE THINGS I'VE
BEEN INTERESTED IN THE GET-GO
IS, BEING ABLE TO SHAPE ANTIBODY
REPERTOIRES, TUMOR ASSOCIATED
CARBOHYDRATE -- I THINK REALLY
IF WE COULD NOT ONLY SHAPE IT
WITHIN THE MICROBIOME OF THE GUT
BUT ACTUALLY WITHIN THE TUMOR, I
THINK EVERYONE IS AWARE THESE
MICROBES ARE PRESENT IN TUMORS
AT A LEVEL I THINK I DIDN'T
APPRECIATE 10 YEARS AGO, THERE
MAY BE A REAL OPPORTUNITY TO
LEVERAGE THIS IN A POSITIVE WAY
TO SHAPE ANTIBODY REP REPERTOIRES
FOR THOSE THINGS WE WANT THEM TO
BE DOING, TYPICAL STRUCTURES
THAT ARE NOT PRESENT IN NORMAL
TISSUE, SO ABSOLUTELY, I THINK
IT'S AN EXCELLENT IDEA.
I THINK THE CHALLENGE IN TERMS
OF VACCINATION IN THIS KIND OF
STUFF IS THAT REALLY FROM THE
IMMUNE PERSPECTIVE, WE DON'T
UNDERSTAND THE RULES.
DOES THIS RESPONSE PRODUCE -- IS
IT CONTINUAL STIMULATION, ALL
THESE QUESTIONS ABOUT IMMUNOLOGY
I THINK ARE PRETTY -- I DON'T
MEAN TO OFFEND ANYONE, I THINK
THEY'RE PRETTY OPEN-ENDED AND
UNKNOWN, SO THIS IS AN
OPPORTUNITY TO REALLY START TO
FERRET THIS OUT.
AT LEAST THAT'S OUR HOPE.
THANK YOU.
>> SUPER NICE STUFF, JOHN.
>> THANK YOU SO MUCH.
ALL RIGHT.
SO IT IS MY ABSOLUTE PLEASURE TO
BE ABLE TO INTRODUCE OUR FINAL
SPEAKER.
WE SAVED THE BEST FOR LAST
OBVIOUSLY.
AND SO FOR EVERYONE KIND OF
HANGING ON HERE UNTIL THE VERY
END, CATHERINE GRIMES IS AT THE
UNIVERSITY OF DELAWARE, AND
SHE'S BEEN AN ABSOLUTE PLEASURE
TO WORK WITH, HAS BEEN THE
PERSON THAT REALLY HAS HELD THE
GLYCOMICS -- OR THE GLYCOTOOL
MEETINGS TOGETHER FOR MANY YEARS
AND PATIENTLY NUDGES US WHEN WE
FAIL TO RESPOND TO EMAILS IN A
VERY KIND AND UNDERSTANDING WAY
AND IS REALLY THE BIG DRIVER
BEHIND THIS MEETING THAT WE'VE
ALL BEEN ABLE TO PARTICIPATE IN
TODAY, SO JUST A BIG SHOUT OUT
TO CATHERINE, ALL OF HER WORK
NOT ONLY FOR THIS MEETING BUT
JUST FOR THE FILM.
>> THANKS, SEAN.
IT'S BEEN REALLY FUN ORGANIZING
THIS WITH YOU TOO, AND I FEEL
LIKE THIS WAS AN AMAZING
SYMPOSIUM AND EVERY TALK, I
THOUGHT THEY WERE SUPER FUN,
AMAZING TALKS.
SO SUPER JOB ORGANIZING IT.
I ALSO WANT TO THANK PAM FOR
INVITING WHOEVER WE WANTED, SHE
WAS LIKE GOOD, GO AHEAD AND TRY
AND INVITE THEM.
IT WAS REALLY AMAZING.
I WILL TRY TO CAP IT OUT IN A
WAY THAT YOU GUYS CAN APPRECIATE
AT THIS LATE HOUR.
I KNOW IT WILL BE HOPEFULLY
SIMPLE IN THAT SENSE.
SO IT BEEN REALLY FUN BEING PART
OF THAT GLYCOSCIENCE -- I'LL
TALK TO YOU ABOUT THE WORK MY
GROUP HAS DONE AS PART OF THAT
AS WELL.
SO FIRST OFF JUST IN CASE WE DO
WRUN OUT
RUN OUT OF TIME, I WANT TO
ACKNOWLEDGE MY GROUP, I HAVE A
FANTASTIC GROUP OF INDIVIDUALS,
ALL OF YOU HAVE AMAZING TEAMS,
I'M LUCKY TO HAVE AN AMAZING
TEAM AS WELL IN FUNDING AND A
GROUP OF COLLABORATORS SO I'M
GRATEFUL FOR ALL OF THAT AND
THEY ARE THE FORCE BEHIND THE
WORK I'M GOING TO TELL YOU
ABOUT, WHICH IS ALL IN THESE
BACTERIAL PEPTIDOGLYCANS WHICH
SURROUND ALL THE BACTERIA THAT
WE HEARD ABOUT TODAY, THEY MAKE
AN AIZ MAZING TARGET FOR
ANTIBIOTICS, TARGETS THE
DESTRUCTION OF THESE BUGS BUT
THEY ALSO MAKE AMAZING IMMUNE
RECOGNITION CALLING CARDS SO
YOUR BODY IS NOT UNAWARE OF THE
FACT THAT INDEPENDENT OF THE
PRESENCE OF AN ANTIBIOTIC, THEY
CAN USE THESE FRAGMENTS THEY'RE
IN TO GENERATE A PROPER IMMUNE
RESPONSE AND GENERATE AN INNATE
IMMUNE REACTION.
SO THAT'S THE TYPE OF THING THAT
MY GROUP AND I ARE REALLY
INTERESTED IN LOOKING AT, IS THE
INNATE IMMUNE RESPONSE, SO WE
GOT YOUR BODY'S FIRST --
REPRESENTING THE INNATE IMMUNE
SYSTEM.
YOU CAN IMAGINE, THIS MASH PHAGE
LOOKS FOR THINGS DIFFERENT THAN
ITSELF, SUCH AS THESE FLA JE
FLAGELLIN -- AND NOBEL PRIZE
WINNING WORK HELPED IDENTIFY A
SERIES OF INNATE IMMUNE
RECEPTORS THAT RESIZE ON
MEMBRANES AND THESE THAT RESIDE
IN THE CYTOPLASM THAT WORK IN
CONCERT TO GENERATE AN
INFLAMMATORY RESPONSE AND THEN
ALSO GO ON TO ACTIVATE THE
ADAPTIVE IMMUNE RESPONSE.
SO THIS ALL LOOKS NEAT AND CLEAN
BUT AS A CHEMIST AND AS WE SAW
TODAY IN , BIOLOGY IS NOTHING THAT'S
NEAT AND CLEAN.
SO AS WE CONSIDER THE
MICROBIOME, THIS GETS MESSY, SO
WE KNOW WE'RE CARRYING AROUND
LOADS AND LOADS OF BACTERIA.
WE TALKED ABOUT THEM ALL DAY.
SO WE LIKE THIS CARTOON, YOU
HAVE 2 TO 5 POUNDS OF BACTERIA
ON YOU, WE LIKE TO THINK OF IT
MORE COMPLICATED, IF YOU GO BACK
TO THE ENVELOPE OF CALCULATION,
YOU MIGHT BE CARRYING ABOUT 1 TO
3 POUNDS OF THESE THROW IN THE
3 TO 5 POUNDS, YOUR BODY IS
CHARGES WITH A TREMENDOUS TASK
TO RECOGNIZE THE PATHOGEN -- TO
MAKE SURE THAT IT TURNS ON THE
INFLAMMATORY RESPONSE CORRECTLY
AND WE KNOW IF IT DOESN'T, A
VARIETY OF DISEASES CAN ENSUE.
AND SO MY GROUP AND I ARE REALLY
VERY INTERESTED IN THINKING
ABOUT THE BACTERIAL PEP
PEPTIDOGLYCAN AND THEIR
RECEPTOR, SO MY GROUP AND I HAVE
DONE A LOT OF BIOCHEMISTRY TO
KIND OF HELP DELINEATE HOW
NOD-LIKE RECEPTORS BIND TO, BUT
HOW DO WE SENSE AND REA SPOND TO
THESE BACTERIAL WALL FRAGMENTS
AT THE MOLECULAR LEVEL.
IF I'M GOING TO TALK TO YOU
ABOUT MOLECULES, I SHOULD SHOW
YOU SOME MOLECULES.
SO IT'S A REPEATING UNIT OF TWO
SUGARS NOW.
THIS OTHER SUGAR THAT LINKS
TOGETHER GIVING THE JACKETTITY
STRENGTH AND RIGIDITY.
IT LONG BEEN KNOWN THAT VERY
SMALL SYNTHETIC FRAGMENT SUCH AS
THE ONE SHOWN HERE, SO NOW JUST
ONE SUGAR AND TWO AMINO ACIDS,
ARE ABLE TO GENERATE AN IMMUNE
RESPONSE IN HUMANS.
IT'S USED IN ADJUVANTS, FOR
EXAMPLE.
THERE IS SOME SPES SPECIFICITY
INVOLVED.
YOU LOSE THE ABILITY TO HAVE THE
SIM LA TRI ACTIVITY.
SO MY GROUP AND I ARE REALLY
INTERESTED IN THINKING ABOUT HOW
IS THIS POLYMER PROCESSED TO
PRODUCE THESE IMMUNOSTIMULATORY
FRAGMENTS SO THE RED IS
HIGHLIGHTED IN RED SO WON KER
WHAT ENZYMES WONDER
WHAT ENZYMES ARE RESPONSIBLE --
UNIQUE SET OF CELL WALL
BACTERIAL CELL WALL FRAGMENTS
WHICH WOULD BE YET ANOTHER WAY
FROM ALL THE DIFFERENT THINGS WE
HEARD ABOUT TODAY TO KIND OF
DELINEATE BETWEEN THE DIFFERENT
TYPES OF MICROBES THAT ARE
LIVING IN OR ON YOUR BODY.
SO OUR HYPOTHESIS THAT DIFFERENT
BACTERIA PRODUCE A VARIETY OF
FRAGMENTS, THERE'S MORE THAN ONE
WARRANTED TO STUDY AND THINKING
ABOUT IMPORTANT BIOLOGICAL
CONSEQUENCES.
SO WE THINK THERE'S SOME
DIVERSITY INVOLVED NOT ONLY IN
THE WAY THAT THE PEPTIDOGLYCANS
LINK TO OTHER SHEATHS, SO GRAM
POSITIVE FOR EXAMPLE WILL TAKE
ADVANTAGE OF THESE BRIDGE,
GRAM-NEGATIVES AND SOME GRAM
POSITIVES TAKE ADVANTAGE OF
THESE ACIDS WHICH RL THIS
UNUSUAL DIE CARBOYXLATED AMINO
ACID, AND THEN IN ADDITION, SOME
USE ORNATHENE.
ACETYLATION PATTERNS ARE REALLY
IMPORTANT, BECOME RESISTANT --
IMMUNE RECOGNITION.
AND MYCOBACTERIUM HAVE -- WHICH
WILL ADD A HYDROXYL GROUP TO
THAT ACETATE RENDERING IT
RESISTANCE TO SOME OF THESE
INNATE IMMUNE MECHANISMS.
WILL GO TO GREAT LINGT TO
REMODEL ITS CELL WALL TO BECOME
RESISTANT TO THE ANTIBIOTIC
VANCOMYCIN.
SO WE REALLY WANT TO EXPAND THE
DIVERSITY THAT'S AVAILABLE.
WHEN SETH WAS TALKING HE SAID HE
WANTS TO GO TO THE CANDY SHOP
AND BUY SOME THINGS, SO IF YOU
WANT TO BUY SOME STUFF THE ONE
COMPOUND YOU CAN BUY IS --
PEPTIDE THAT'S BEEN READILY
AVAILABLE AND RIGOROUSLY STUDIED
BY IMMUNOLOGISTS FOR A VERY LONG
TIME.
SO WE WONDERED, COULD WE EXPAND
THE TYPES OF FRAGMENTS THAT ARE
AVAILABLE, AND THE ONE WE
THOUGHT ABOUT WAS MAKING THESE
SORT OF N GLYCOALLELE --
DEVELOPED THE SYNTHESIS OF THIS
AND JUST LIKE WHEN STEVE
TOWNSEND WAS TALKING, WE DO
SYNTHTHIS IN OUR GROUP AND IT
REALLY IMPORTANT TO THINK WE CAN
CHEMICALLY ACCESS THESE
COMPOUNDS.
SO I WON'T WALK YOU THROUGH IT
FOR THE SAKE OF TIME, BUT JIM
DEVELOPED A REALLY NICE STRATEGY
THAT MAKES THIS 2 AMINO -- WHICH
WILL NOW ALLOW US TO ACETYLATE
OR ALKYLATE A VARIETY OF THINGS
ON THESE COMPOUNDS TO BEGIN TO
STUDY THEM.
IN ADDITION, WE THOUGHT HOW
ABOUT TAKING AND UNDERSTANDING
WHAT ARE SOME OF THE ONES THAT
COULD BE LIVING IN YOUR GUT, SO
WE SET UP THESE REACTORS IN OUR
LAB WHERE WE GREW UP
LACTOBACILLUS -- AND THEN WE
TREATED IT WITH LISOSIME AND
USED MASS SPECTROMETRY TO
UNDERSTAND WHAT ARE THE NAY
JUROR FRAGMENTS BEING PRODUCED,
AND WHAT WE SEE ARE THESE SORT
OF DIE SK RIDE TYPE FRAGMENT,
TRY PEPTIDE, DID THIS WORK IN MY
GROUP.
SO CLAIRE WENT ON -- YOU CAN'T
BUY THIS GMTT TYPE FRAGMENT SO
CLAIRE WENT ON TO DEVELOP THE
SYNTHESIS OF IT, AND I WILL
BREEZE THROUGH THIS IN RAPID
FASHION, AND THIS IS PUBLISHED,
SO SHE CAN MAKE THIS AND SHE CAN
RAPIDLY ADD ON A VARIETY OF
PEPTIDES, AND THESE ARE ECK QIS
EXQUISITELY PURE.
SO WE THINK ABOUT OTHER
BACTERIA, WE DID THIS IN E OWE E. COLI
MODEL SYSTEM.
THIS IS WHERE WORKING WITH THE
COMMON FUND HAS BEEN AN AMAZING
GIFT TO OUR LAB, SO IN WORKING
WITH THE COMMON FUND, WE'VE BEEN
ABLE TO SHARROW PROBE LARGELY
WITH THE HELP OF MY PARTNERS IN
CRIME ON THIS U01 AWARD THAT WE
WERE GIVEN BY THE COMMON FUND TO
SHARE, PROBE AND VERIFY PROTOCOL
WITH A VARIETY OF DIFFERENT
GROUPS.
SO THIS HAS BEEN REALLY FUN TO
DO AND IT REALLY NICE WHEN WE
GET LABELING BACK AND THAT WE'RE
ACTUALLY ABLE TO SEE LABELING IN
A VARIETY OF DIFFERENT TYPES OF
BACTERIA.
THIS HAS BEEN A LOT OF FUN TO
SEE THESE LABELS OF THESE
DIFFERENT ORGANISMS.
AS WE'RE MAKING AND DISTRIBUTING
MORE AND MORE -- WE WISH WE HAD
A VERY EFFICIENT WAY TO MAKE IT,
SO OUR FIRST GENERATION OF
SYNTHTHIS UTILIZES -- WE TRY NOT
TO DO IT ON TOO HIGH OF A SCALE
BECAUSE WE DON'T WANT OUR LAB
MEMBERS TO LOSE HANDS, SO ASHLEY
BROWN HAS STARTED, ONE OF THE
REGIONS CAN BE A CONTACT -- ASH
LEA BROWN HAS DEVELOPED A NEW
SYNTHESIS WHICH IS SCALABLE SO
NOW WE CAN DO THIS ON 15-GRAM
SCALE TO PRODUCE THIS DERIVATIVE
THIS 2 AMINO THAT WE CAN
DECORATE WITH OUR BIOOR THOG NAT
PROBE SO IT'S A REALLY NICE WAY.
ASH LE HOOS LOOKED AT OPTIMIZING
THIS ALONG WITH STEVEN HIGHLAND
AND GLYCOBIOLOGY AND MAJOR WAY
YOU DO THIS IS EITHER -- OR CAP
WITH THE METHYL ESTER YOUR PROBE
THEAND YOU CAN GAIN MORE
EFFICIENT ENTRY INTO THE
CYTOPLASM, HOWEVER, WE FIND
BACTERIA DO NOT HAVE THE
ESTERASES TO REMOVE THESE
ACETATES FROM THE SUGAR SO IF WE
TRY TO LABEL IT WITH AN
ACETYLATED FRAGMENT, WE CAN NO
LONG
LONGER LABEL BUT WE CAN GET AWAY
WITH THIS METHYL ESTER CAPE.
WONDERING CAN WE DO THIS IN
REALTIME USING COPPER FREE CLICK
CHEMISTRY OR SPEC, WE CAN
PRELABEL OUR BACTERIA WITH SPEC
AND INCUBATE THESE BACTERIA WITH
MIKE PHAGE
MICROPHAGE AND LOOK UNDER A MIKE
SCEEP TO SEE WHAT WE CAN SEE,
HOW THEY'RE INTERACTING WITH THE
MACROPHAGE, WE'RE BEGINNING TO
VISUALIZE THE BREAKDOWN PRODUCTS
WITHIN THESE CELLS, AND NOW
WE'RE DOING ENDO SOMOL STAINING
TO WATCH THE BRAKE BREAK DOWN OF
THE BACTERIA WITHIN THE MACRO
PHAGE.
AS WE THINK ABOUT THIS, WE CAN
SEE SOME MACROPHAGES LOTS OF
DIFFERENT TYPES OF BACTERIA, YOU
CAN SEE THESE LYSING INSIDE, AND
WE WANT TO START TO SEE THE
BACTERIA BREAK DOWN WITHIN THE
MACROPHAGES BUT THEN WE ALSO
WONDER WHAT IS THE FATE OF THESE
FRAGMENTS.
SO IS ONE OF THE I THINK INNER
INTERESTING WAYS WHERE THESE
FRAGMENTS COULD BE GOING IS THE
BRAIN.
WOULD HE THINK WE THINK A LOT ABOUT THINGS THAT
CAN PASS INTO THE BRAIN AND WE
THINK THESE SUGARS MIGHT BE
IMMUNOPRIVILEGES THAT THEY OUGHT
TO BE ABLE TO PASS THE BLOOD
BRAIN BARRIER.
WE KNOW THE INNATE IMMUNE
RECEPTORS -- ARE PRESENT IN THE
BRAIN, SO WE WONDERED, COULD
BACTERIA COLONIZING IN THE GUT
CROSS THE BLOOD BRAIN BARRIER
AND CAUSE A LOCAL EFFECT.
WE THOUGHT ABOUT DOING THIS IN
COLLABORATION WITH JACKIE
SCHWARTZ AT THE UNIVERSITY OF
DELAWARE, WHO'S A BRAIN
SCIENTIST AND STUDIES MICRO --
THE IMMUNE CELLS IN THE BRAIN
AND THEY EXPRESS NOD2 SO ASHLEY
HAS MADE THIS THAT CONTAINS A
BIO ORTHOGONAL ANGLE.
WE INJECTED IT INTO MICE AND
THEN DID 10 COURSES WHERE WE
WERE GOING TO TRACK IN DIFFERENT
ORGANS TO SEE WHAT KIND OF
IMMUNE RESPONSES WE'RE ABLE TO
SEE.
WE SEE IT CAN INDUCE
INFLAMMATION IN THE BRAIN, THE
HYPOTHALAMUS, WE IS SEE BOTH
IL-1B AND I WILL 16 ROBUST
EXPRESSION AFTER FOUR HOURS
IMPLYING THE SYNTHETIC FRAGMENT
IS INDEED REACHING THE BRAIN.
WE CAN EVEN VISUALIZE IT INSIDE
A MICRO GILLIA THAT WE CULTURE
FROM THOSE TREATED RATS AND WE
CAN SEE THE WORK FROM NICOLE
HAAS SHOWING THE MICROGLIA IN IN
GREEN AND RED, WE CAN SEE THE --
PEPTIDE INSIDE THE CELL.
IN ADDITION, ASHLEY HAS WORKED
OUT A REALLY COOL WORKFLOW IN
WHICH WE CAN NOW HARVEST THE
BRAIN, AND WE CAN LOOK INSIDE
WITH THE MICROSCOPE AFTER SHE
LYSES AND WE CAN USE SIM AND WE
CAN START LOOKING AT SPECIFIC
FRAGMENTS INSIDE THE BRAIN, AND
WE CAN SEE THESE FRAGMENTS IN
OUR MASS SPEC.
AND IN A REALLY NICE
COMPLEMENTARY APPROACH THAT WAS
JUST PUBLISHED IN SCIENCE IN
2015, OR THIS PAST -- ON
APRIL 15TH, WE CAN SEE ANOTHER
GROUP USED RADIOACTIVITY IN
WHICH THEY CAN TRACK NOW THESE
FRAGMENTS UP IN THE BRAIN AS
WELL, SO WE THINK THIS IS GOING
TO BE A COMPLEMENTARY WAY TO
START THINKING ABOUT KNOWING THE
MOLECULES MAKING THEIR WAY UP
INTO THE BRAIN.
SO WE'VE DONE A LOT OF WORK TO
BE ABLE TO MAKE SORT OF DESIGNER
BACTERIA WHERE WE CAN REMODEL
THEM WITH A VARIETY OF DIFFERENT
PROBES, WE'RE USING A VARIETY OF
DIFFERENT APPROACHES AND WE'RE
REALLY EXCITED ABOUT THINKING OF
WHERE AND HOW THESE DIFFERENT
FRAGMENTS CAN TURN ON DIFFERENT
TRANSCRIPTIONAL PROGRAMS NOT
ONLY IN THE GUT NOW BUT
POTENTIALLY IF THE BRAIN.
SO I'D LIKE TO THANK MY LAB ONE
MORE TIME.
YOU FOR YOUR TIME AND ATTENTION
FOR HANGING ON SO LONG.
I'M SORRY ABOUT THE INTERRUPTION
FOR MY DAUGHTER, SHE IS
OBVIOUSLY HOME FROM SCHOOL.
AND I WILL -- AGAIN I WANT TO
THANK SEAN FOR DOING THIS WITH
ME, FOR ORGANIZING THIS
SYMPOSIUM.
IT'S BEEN REALLY FUN, AND PAM
FOR THE OPPORTUNITY TO DO THAT.
AND IF YOU FIND YOURSELF THIRSTY
FOR MORE AND WANTING MORE
GLYCOSCIENCE, YOU'RE IN LUCK
BECAUSE YOUR OPPORTUNITY WILL
PRESENT TO YOURSELF NEXT MONDAY,
TUESDAY, WEDNESDAY WHERE WE'LL
HAVE OUR GLYCOSCIENCE COMMON
FUND MEETING THAT'S OPEN TO THE
PUBLIC, AND WE VIDEOCAST IS TO
THE LINK IS THERE, AND WE WOULD
LOVE TO SEE YOU ON THERE AS
WELL.
SO THANK YOU SO MUCH FOR HANGING
OUT WITH US ALL DAY, AND
HOPEFULLY THIS WAS A SWEET END
TO YOUR DAY.
SO THANK YOU.
>> THANK YOU SO MUCH, CATHERINE.
THAT WAS AMAZING.
I THOUGHT YOUR ABILITY TO HANDLE
YOUR DAUGHTER IN A SEAMLESS WAY
WAS QUITE IMPRESSIVE.
>> THAT'S OKAY.
THAT WAS LIKE TRAINING OVER THE
YEARS.
>> REALLY IMPRESSIVE.
LAURA HAS A QUESTION.
>> OH, I THINK I'M GOING TO
DEFER FIRST TO SETH, BECAUSE HE
HAD HIS HAND UP BEFORE ME.
>> I WASN'T PAYING ATTENTION TO
WHO HAD THEIR HAND UP FIRST.
I THINK YOU'RE MUTED, BUDDY.
I LOVE THE QUESTION, I CAN TELL
IT'S GOING TO BE GREAT.
>> MY FIRST ONE IS A COMMENT
WHICH IS, LIKE, OH, MY GOODNESS,
I WAS GIVING A TALK AND I HAD A
BLOODY CHILD COME UP TO ME LIKE,
SO YEAH.
>> BUT SHE WASN'T BLOODY.
SHE WAS PROBABLY JUST DIRTY
BECAUSE SHE PROBABLY ATE A LOT
OF CHOCOLATE OR SOMETHING.
I DON'T THINK SHE WAS BLOODY.
>> THAT WAS GREAT.
I HAD TO DO LIKE FIRST AID
DURING A TALK ONCE.
>> SO YOU HAD TO DO THAT.
OKAY.
GLYCOLIPIDS, THERE'S MAYBE
FUNGAL GLYCANS.
THEY'RE PRETTY DIVERSE
CHEMICALLY.
I DID NOT -- WE'RE TALKING ABOUT
PEPTIDE -- A -- BACKBONE
BASICALLY BUT WITH THESE
MODIFICATIONS THAT YOU'RE
SHOWING.
>> RIGHT.
>> WHICH I THINK IS AWESOME, BUT
MY QUESTION IS, WE HAVEN'T EVEN
UNDERSTOOD HOW TLRS BIND TO
THESE CHEMICALLY DIVERSE -- AND
SOME PEOPLE HAVE THEORIES ABOUT
CD14, MD2, CORECEPTORS.
DO YOU THINK THERE'S ACCESSORY
MOLECULES THAT ARE LEADING TO
THE SPECIFICITY OR AT LEAST THE
SPECIFIC PROGRAMS THAT YOU'RE
SEEING?
>> O OH DEFINITELY.
THAT'S WHAT WE WANT TO START TO
TEASE OUT NOW WITH THE DIFFERENT
FRAGMENTS WE HAVE AT THE PROBES
WE'RE MAKING AND BRINGING
ONLINE.
WEE DONE THE CLEAN SPAIRT BUT I
THINK LAURA HAS DONE THE COOL
EXPERIMENT WHEN SHE STARTS
MIXINGS ALL OF HER BACTERIA
TOGETHER AND STARTS TO SEE
SPECIFICITIES FOR THE LECTIN.
WE CAN BE VERY CONTROLLED NOW
BECAUSE WE'VE MADE THEM WHAT
THEY ARE, I DON'T KNOW IF THAT
MAKES SENSE.
>> IT DOES.
CAN I FOLLOW UP WITH THAT WHICH
IS AN IMMUNOLOGICAL THEORY WHICH
BASED ON THE THEORY OF INNATE
CONTROL OF ADAPTIVE IMMUNITY,
AND BASICALLY HOW DIFFERENT PRR,
DEPENDING ON WHETHER THEY'RE
INTRACELLULAR OR EXTRACELLULAR
OR FUNGAL OR VIRAL OR BACTERIAL
LEAD TO DIFFERENT IMMUNE
MODULES.
DO YOU -- SO THE MODIFICATIONS
YOU SHOWED WERE IN LACTOBACILLI,
NOT INTRACELLULAR, WE THINK.
IN MICOBACTERIA, IN STAPH.
CAN YOU COME UP WITH A RHYME OR
REASON AS TO IF THIS IS REALLY A
HOST ADAPTATION IN ANY WAY FOR
DIFFERENT IMMUNE MODULES BASED
ON THE MODIFICATIONS THAT YOU
MIGHT BE SEEING IN PEP
PEPTIDOGLYCAN, AND CAN THE
HOST -- THE WAY IT WORKS IS THAT
THE HOST BASICALLY SAYS, OH,
IT'S THAT TYPE OF INFECTION,
WE'RE GOING TO DO THAT TYPE OF
IMMUNE RESPONSE.
DO YOU THINK THAT SYSTEM WOULD
BE WORKING FOR NOD2, MAYBE NOD 1 PEPTIDOGL YCAN MODIFICATIONS IN
ANY WAY?
BECAUSE I CAN'T SEE IT, YOU
KNOW?
MEANING, LIKE --
>> I THINK MORE LIKE YOU WERE
ALLUDING TO BEFORE, THERE'S
CORECEPTORS AND THERE'S OTHER --
I DON'T JUST THINK IT'S A NOD2,
NOD 1 TYPE OF PROBLEM.
SO I THINK --
>> CAN IT TELL THE TYPE OF --
LIKE --
>> I DON'T THINK IT'S OPERATING
BY ITSELF.
AND I FEEL LIKE WE'VE BEEN DOING
REALLY GOOD DURING THIS
SYMPOSIUM, RIGHT, LIKE THIS IS A
SYSTEM THAT I'M INTERESTED IN
BUT IT'S OBVIOUSLY OPERATING IN
THIS VERY COMPLEX AREA, BUT WE
HAVE TO UNDERSTAND, JUST LIKE
WHEN NADINE WAS TALKING ABOUT
THE BIOPHYSICAL PROPERTIES, WE
HAVE TO UNDERSTAND THAT TOO, AND
THEN THE CONTEXT OF THIS LARGER
FIELD AND THAT'S WHY I THINK
THIS FIELD IS AWESOME, BECAUSE
WE ALL HAVE TO WORK TOGETHER TO
UNDERSTAND DIFFERENT ASPECTS OF
THOSE COMPONENTS.
DOES THAT MAKE SENSE?
>> OH, TOTALLY MAKES SENSE.
>> I THINK MY QUESTION IS
ACTUALLY REALLY RELATED TO WHAT
SETH WAS ASKING AND I'LL BE
PRETTY FAST.
I JUST WAS THINKING YOU HAVE
SUCH A GREAT TOOL NOW, AND I
KNOW THIS ISN'T EXACTLY WHAT
YOU'RE INTERESTED IN, BUT THE
DIFFERENCE IN UPTAKE BY THE
MACROPHAGES OF THE DIFFERENT
BACTERIA IS REALLY COOL, AND I
THINK YOU COULD GET AT SOME OF
THE HOST PART AND DOWNSTREAM
SIGNALING BY DOING LIKE SINGLE
CELL RNA SEQ ON THESE, BECAUSE
YOU HAVE A GREAT TOOL TO SORT
NOW DIFFERENT --
>> WITH THE BACTERIA INSIDE OF
IT, WE CAN SORT AND LOOK AND DO
SINGLE CELL WITH THE LABELING
TECHNOLOGY PART OF IT, YES.
>> I THINK THAT COULD BE A
REALLY GOOD USE OF YOUR COOL
TOOLS.
>> THIS IS COOL.
I'M REALLY GLAD THESE ARE ALL
RECORDED SO THAT I'LL REMEMBER
ALL THIS STUFF TOO.
THIS IS AWESOME.
THIS IS GOOD.
>> AND YOU GUYS ARE AMAZING,
SEAN AND CATHERINE, AND THEN
I'LL STOP TALKING.
>> I THINK THIS IS AN AMAZING
COMMUNITY.
WE JUST BRING OUT THE BEST IN
EACH OTHER AND IT'S REALLY BEEN
AWESOME AND IT'S FUN GOING
FORWARD.
MARIO, I SAW YOU TURN YOUR
CAMERA ON.
I DON'T KNOW IF THAT MEANS
YOU'RE HAPPY THIS IS ALL OVER.
>> SEE YOU FOR A LITTLE BIT AT
THE END, I THOUGHT IT WAS NICE
TO -- I JUST WANTED TO BE PART
OF IT.
>> GREAT.
NO, I THINK THIS IS A REALLY
AMAZING DAY.
AND I AM GLAD IT IS RECORDED
BECAUSE I THINK IT WILL BE GOOD
TO GO BACK AND WATCH SOME OF
THESE THINGS AGAIN, AND THIS WAS
REALLY FUN AND I THINK THIS IS A
REALLY NEAT COMMUNITY AND SO
HOPEFULLY WE GET TO HANG OUT
MORE AT NIH TBLIE COSCIENCE DAY
AT THE TIME THE COMMON FUND
DOES, ALL OF THOSE THINGS ARE
HOW WE CAN STAY CLOSE TOGETHER,
BUT I FEEL LIKE WE'VE IDENTIFIED
AN AREA WHERE GLYCOBIOLOGY IS
EXTREMELY IMPORTANT, RIGHT?
SO LET'S KEEP WORKING ON IT
TOGETHER.
>> AGREE.
THAT WOULD BE WONDERFUL TO GET
OUT AND GET DINNER TOGETHER.
BUT HOPEFULLY WE CAN DO THIS IN
PERSON.
LOGISTICALLY IT'S DIFFERENT BUT
THIS IS A GREAT OPPORTUNITY.
LIKE CATHERINE SAID, I THINK
IT'S A -- COMMUNITY AND
OPPORTUNITY TO BRING A LOT OF
COMMONLY DISPARATE DISCIPLINES
TOGETHER IN A FUN WAY.
I LOVE THE CHEMISTRY, THE MICRO
AND THE BIOCHEMISTRY AND JUST
IMMUNOLOGY ALL IN ONE PLACE,
WHICH IS NOT COMMON, AT LEAST IN
MY EXPERIENCE.
SO IT'S BEEN A LOT OF FUN.
IT'S BEEN GREAT.
SURE APPRECIATE EVERYBODY GIVING
SUCH WONDERFUL TALKS.
IT'S REALLY BEEN FUN.
>> NO, THIS IS GREAT.
THANK YOU ALL FOR HANGING ON SO
LONG, TOO.
AND MANY OF YOU, WE WILL BE
TOGETHER DOWN IN BETHESDA
MONDAY, TUESDAY AND WEDNESDAY,
SO THAT WILL BE NICE TO SEE MANY
OF YOU IN PERSON TOO.
SO THANK YOU ALL.
THANK YOU.
TAKE CARE.
BYE.
>> THANKS, EVERYONE.