>>WELCOME TO THE
WEDNESDAY AFTERNOON LECTURE
SERIES. I'M GIGI S, A PRINCIPLE
INVESTIGATOR AND IT IS MY GREAT
PLEASURE TO INTRODUCE TODAY'S
SPEAKER ON BEHALF OF THE
INTEREST GROUP. SO DR. S. WAS A
TOP VOTE GETTER FOR LAMBDA LUNCH
FOR SEVERAL YEARS IN A ROW SO WE
ARE SO PLEASED THAT HE WAS ABLE
TO COME THIS WEEK.
HE'S THE INCUMBENT OF THE SYLVIA
CHAIR IN THE DEPARTMENT AT THE
WEISSMAN INSTITUTE IN ISRAEL.
HE RECEIVED A BULK OF THE
EDUCATION AT TEL AVIV UNIVERISTY
WHERE HE EARNED A MASTER'S
DEGREE IN GENETICS AND A PH.D.
IN HUMAN GENETICS. I DIDN'T
KNOW THAT. HE FOLLOWED UP WITH
POST-DOCTORAL FELLOWSHIP AT THE
BERKLEY LAB IN CALIFORNIA AND
RETURNED TO ISRAEL WHERE HE WENT
TO THE WEISSMAN AND HAS BEEN
GOING UP THE RANKS, MOVING UP
THE RANKS AT THE WEISSMAN. SO
HE HAS HAD MANY RECOGNITIONS AND
ACCOLADES INCLUDE THE GERMAN
NATIONAL ACADEMY OF SCIENCES,
THE ANDRE PRIZE FOR SCIENTIFIC
RESEARCH. THE RAH PA IMPORTANT
PRIZE FOR EXCELLENCE AND
ELECTION TO BOTH THE AMERICAN
ACADEMY OF MICROBIOLOGY. SO
RECOGNITION ALL AROUND THE
WORLD. BUT MOST IMPORTANTLY
ONTO HIS RESEARCH WHICH COMBINES
COMPUTATIONAL GENOMICS, SYSTEMS
BIOLOGY, METAGENOMICS, HIGH
THROUGHPUT SEQUENCING AND MODERN
EXPERIMENTAL APPROACHES AND
MICROBIOLOGY AND PHAGE BIOLOGY.
AMONG THE MOST SIGNIFICANT
RESULTS ARE MULTIPLE DISCOVERS
ABOUT THE ARMS RACE BETWEEN
BACTERIA AND PHAGE. SO MANY
PEOPLE HAVE HEARD ABOUT KRIS PER
THAT IS NOT THE WHOLE STORY. AS
YOU WILL SEE HERE. EXCITING
FINDINGS THAT HAVE COME FROM THE
LAB INCLUDE THE DISCOVERY OF AN
ASTONISHING NUMBER OF SYSTEMS,
DEFENSE SYSTEMS BEYOND CRSPR.
THE DISCOVERY THAT PHAGES USE
SMALL MOLECULE COMMUNICATION TO
COORDINATE THEIR INFECTION
DYNAMICS. SMALL RNAS AND SMALL
PROTEINS WHICH ARE CLOSE TO MY
HEART ARE INVOLVED IN THE
PROCESS AND MOST INTRIGUINGLY
THE PARALLELS BETWEEN THESE. I
THINK SOME OF THESE TOPICS ARE
THE FOCUS OF THE TALK TODAY.
LET ME STOP THERE AND THANK YOU
THEM FOR COMING TO VISIT US
IN-PERSON.
>>THANK YOU SO MUCH IT'S A
GREAT PLEASURE AND HONOR FOR ME
TO GIVE A LECTURE IN THIS FORUM
AND I THANK THE LAMBDA FORUM FOR
THE INVITATION AND FOR HOSTING
ME FOR SUCH AN INTERESTING
MEETING. THESE LAST TWO DAYS
HAVE BEEN FASCINATING. I WILL
TALK ABOUT OUR ADVENTURE
STUDYING BACTERIA AND WHAT WE
LEARNED IN THE BAHS PAST FEW
YEARS. BACTERIA ARE VERY RARELY
INFECTED BY PHAGES. PHAGES ARE
VIRUSES, THEY USE SPECIFIC
RECEPTOR ON THE BACTERIUM
SURFACE AND WILL INJECT THEIR
GENETIC MATERIAL. AND WITH THE
PHAGES WE STUDY IN THE LAB,
AFTER BETWEEN HALF AN HOUR TO
TWO HOURS THEY BURST OPEN THE
BACTERIA AND ONE HUNDRED PHAGES
WILL EMERGE FROM IT AND SEARCH
BY DIFFUSION FOR OTHER BACTERIA
TO INFECT AND THESE ARE SUPER
ABUNDANT IN THE ENVIRONMENT.
MORE ON THE OTHER AND IF YOU
WEIGH ALTOGETHER THEY MAY MORE
THAN ENTIRETY OF HUMANITY
ALTOGETHER. BUT YOU CAN ONLY
SEE WHEN YOU LOOK AT THE
MICROSCOPE. SO BACTERIA ARE
INFECTED AND HENCE THEY NEED TO
DEVELOP AND EVOLVE AN IMMUNE
SYSTEM AND THE IMMUNE SYSTEM OF
BACTERIA WE KNOW OF IT FOR MANY
YEARS NOW. WE HAVE TWO, OR AT
LEAST WHAT WE KNEW UP TO FOUR OR
FIVE YEARS AGO THAT WE HAVE TWO
ARMS OF IMMUNITY. ONE IS THE
ADAPTIVE IMMUNE SYSTEM CALLED
KRIS PER CAS THAT PUTS PIECES IN
THE BACTERIAL GENOME AND USES
THESE PIECES TO MITIGATE FUTURE
INFECTION BY THE SAME PHAGE. WE
HAVE RESTRICTION MODIFICATION.
THESE ARE BASICALLY RESTRICTION
ENZYMES THAT CLEAVE THE INCOMING
DNA IN SPECIFIC KNOW TIES.
THERE'S ALWAYS GOING TO BE
ANOTHER PROTEIN THAT YOU MODIFY
THE SAME MOTIFS TO PREVENT
AUTOIMMUNITY. PREVENT SELF
CLEAVAGE BUT A NUMBER OF YEARS
AGO WE STARTED THINKING THAT
THERE MIGHT BE OTHER DEFENSE
MECHANISMS IN BACTERIA THAT WE
ARE NOT AOF YET AND THE REASON
WHY WE THOUGHT THERE MAY BE NOT
SUCH MECHANISMS IS FIRST OF ALL
CRISPER IS ABSENT FROM MOST.
AND YOU ASK HOW DO THEY SURVIVE
PHAGES AND KRIS PER WAS
DISCOVERED SO LATE. DISCOVERED
ONLY 1 -- 90 YEARS AFTER THE
DISCOVERY OF THE PHAGE AND
DURING THIS TIME, FANLS HAVE
BEEN THE MAJOR DRIVING FORCE FOR
STUDYING MOLECULAR BAY YOOLG.
HOW TRANSLATION GOES, GENETIC
CODE WAS ALL DISCOVERED UPON
INFECTION OF PHAGES IN BACTERIA
SINCE E. COLI DOESN'T HAVE A
WORKING KRIS PER. SO KRIS PER
AND E. COLI IS NOT WORKING VERY
WELL SO THAT'S WHY IT WAS NOT
DISCOVERED DURING ALL THESE
DECADES OF RESEARCH. WE THOUGHT
THERE MIGHT BE OTHER MECHANISMS
THAT E. COLI DOESN'T HAVE BUT
OTHER BACTERIA MAY HAVE AND
THESE WE COULD DISCOVER. THE
REASON WE WERE SO INTERESTED TO
FIND NEW IMMUNE SYSTEMS IS FIRST
OF ALL, IF WE WANT TO UNDERSTAND
THE EVOLUTION, THE CONFLICT OF
THESE PHAGES THE DEFENSE SYSTEMS
ARE A MAJOR PART OF THAT
CONFLICT. SO WE NEED TO
UNDERSTAND HOW THEY OVERCOME
THEMSELVES AND AND DEFENSE. FOR
A MORE PRACTICAL POINT OF VIEW
IT SEEMS TO US THAT IN THE PAST
WHEN NEW IMMUNE SYSTEMS HAVE
BEEN DISCOVERED AND STUDIED, IT
FREQUENTLY LED TO REVOLUTIONS IN
BIOLOGY SO THE DISCOVERY OF
RESTRICTION ENZYMES ALLOWED THE
FIRST GENETIC ENGINEERING,
BEFORE THAT PEOPLE DIDN'T KNOW
HOW TO DO IT. DISCOVERY OF KRIS
PER LEADS TO A REVOLUTION IN
GENOME ENGINEERING AND IF YOU
THINK ABOUT THE BACTERIA, THE
DISCOVERY OF RN IAI WHICH IS AN
ANTIVIRAL MACHINE THAT SILENCE
EUCARYOTES AND THE WAY WE WORK
WITH ANTIBODIES IS ALSO
PATHOLOGICALLY, YOU KNOW, VERY
RICH TOOL AND THE REASON THEY
ARE SO BENEFICIAL IS AN IMMUNE
SYSTEM BY DEFINITION IS TO
IDENTIFY A SPECIFIC MOLECULAR.
IN SELF INFECTING FOREIGN
ELEMENT AND THEN DO SOMETHING TO
THAT MOLECULE, CLEAVE IT OR
REMOVE AND IT THAT'S EXACTLY
WHAT WE NEED AS MOLECULAR
BIOLOGISTS WE NEED THESE
PRECISION TOOLS TO BIND, CLEAVE
OR REMOVE OR MOVE AWAY OR DO
SOMETHING TO A SPECIFIC
MOLECULE. SO WE HOPE THAT IF WE
FIND NEW DEFENSE SYSTEMS, NEW
IMMUNE SYSTEMS THEY MIGHT OH
INVOLVE INTO BECOMING NEW
MOLECULAR TOOLS BENEFICIAL FOR
HUMANITY. HOW CAN WE FIND NEW
IMMUNE SYSTEMS IN A SYSTEMATIC
MANNER GIVEN THAT ALL THESE
IMMUNE SYSTEMS IN THE PAST WERE
DISCOVERED JUST BY CHANCE?
PEOPLE STUMBLED UPON THEM AND
THEN REALIZED WHAT THEY SEE AND
SAW LATER ON SO WE'VE BEEN
TRYING TO FIND IMMUNE SYSTEMS
THROUGH MULTIPLE DIFFERENT KINDS
OF METHODOLOGIES AND WE FAILED
IN MOST OF THEM. BUT THE THING
THAT EVENTUALLY WORKED FOR US
COMES FROM A VERY PECULIAR
BEHAVIOR OF DEFENSE SYSTEMS IF
WE LOOK AT GENOMES OF BACTERIA.
GENOME COLLIE SPEAKING,
CLUSTERED AS DEFENSE ISLANDS
THESE AREAS ARE RICH IN DEFENSE
SYSTEMS. WE CAN SEE THAT IN THE
GENOME OF THIS MICRO ORGANISM
I'M SHOWING YOU A SMALL PIECE OF
THAT GENOME. YOU SEE THE GENES
THAT CODE FOR CRISPR ARE CODED
NOT FOR FOR THOSE RESTRICTION
ENZYMES, THESE ARE THE DIFFERENT
SUB UNITS AND THEY ARE ENCODED
NOT FAR FROM ANOTHER DEFENSE
GENE THAT IS NOKNOWN. SO WE
THOUGHT THAT IF WE'RE -- IF WE
CAN LOOK FOR GENES OR MORE OFTEN
GENE CASSETTE THAT CURRENTLY
OCCUR AND ARE ENRICHED NEXT TO
THESE DIFFERENT SYSTEMS WE MIGHT
FIND NEW IMMUNE SYSTEMS THAT
WERE NOT KNOWN YET. AND SO A
PH.D. STUDENT FROM MY LAB
ANALYZED GENOMES THAT ARE
OCCURRING NEXT TO CRISPR AND
RESTRICTION ENZYMES AND TO OUR
SURPRISE FOUND MANY CASSETTES
THAT WERE OF UNKNOWN FUNCTION
BACK THEN. SO TO SEE WHETHER
THEY'RE INDEED FUNCTIONING IN
DEFENSE AGAINST PHAGES WE
DEVELOPED THIS EXPERIMENTAL
PLATFORM TO TEST THEM SO WHAT WE
DO WHEN WE SUSPECT THAT WE FOUND
NEW DIFFERENTS DEFENSE SYSTEM WE
LOOK IS THE ALL THE GENOMES IN
THE DEFENSE DATABASE THAT HAVE
THIS OPERA AND WE SELECT THE
GENOMES THAT ARE AS CLOSE AS
POSSIBLE TO OUR LAB ORDER
ORGANISMS AND WE SYNTHESIZE THE
DNA AND EXPOSE THIS DE BACTERIA
TO A SERIES OF PHAGES.
SO SINGLE STRAND DNA, RNA, ET
CETERA. AND WE LOOK FOR CASES
IN WHICH THE SYSTEM WE CLONE.
IF THE BACK TEAR YUM PROTECTED
AGAINST THAT PHAGE.
AND WHEN WE STARTED DOING THESE
EXPERIMENTS, WE WERE HOPING TO
FIND ONE OR TWO NEW DEFENSE
SYSTEMS WHICH WE THOUGHT WOULD
BE SUPER EXCITING BUT WE WERE
SUPER, SUPER SURPRISED TO FIND
NUMEROUS DEFENSE SYSTEMS IN
MICROBIAL GENOMES SO FAR I THINK
MY LAB DESCRIBED MORE THAN 40
DIFFERENT DEFENSE SYSTEMS THAT
ARE ABANDONED IN MICROBES AND
OTHER LABS NOW THAT DID
EXPERIMENTS INSPIRED BY US FOUND
DOZENS OF DEFENSE SYSTEMS. NOW
WE KNOW SINCE 2018 OF, YOU KNOW,
ABOUT 100 DIFFERENT KINDS OF
IMMUNE SYSTEMS OR DEFENSE
SYSTEMS THAT BACTERIA ENCODE IN
NATURE AND PROTECT THEM AGAINST
PHAGES AND SO WHAT YOU SEE HERE,
THIS FIGURE LET ME TRY TO
EXPLAIN WHAT YOU SEE. THIS IS A
FIGURE FROM OUR 2018 PAPER WHERE
WE DESCRIBE 9-10 DIFFERENT
DEFENSE SYSTEMS WHAT YOU SEE IS
A TWO GENE SYSTEM. THIS GENE IS
A DOMAIN OF UNKNOWN FUNCTION AND
THEN WE HAVE A CASE DOMAIN THAT
MANIPULATES DNA OR RNA AND THE
SIZE OF THESE GENES IS ABOUT THE
SIZE OF CLASS NINE SO IT'S A
PRETTY LARGE PROTEIN. WE CLONE
IT. AND WE INFECTED THE
BACTERIUM WITH TEN DIFFERENT
PHAGES AND WHENEVER YOU SEE A
RED COLOR HERE MEANS THAT THE
SYSTEM PROTECTED THE BACTERIUM
AGAINST THE PHAGE AND THIS COLOR
MEANS WE NEEDED TO INFECT THE
BACTERIA BY TEN MILLION TIMES
MORE PHAGES OR ONE THOUSAND
TIMES MORE IN ORDER TO SEE THIS.
AND IN THE PAST FOUR OR FIVE
YEARS, SINCE THE DISCOVERIES OF
THE SYSTEMS WE SHIFTED THE
GRAVITY OF THE LABS WORK FROM
MOSTLY GENETICS TO MUCH MORE
BIOCHEMISTRY IN ORDER TO START
SOLVING THE MECHANISMS OF HOW
THESE SYSTEMS WORK. ONE THING
WE SAW QUITE EARLY ON, WHICH WAS
VERY SURPRISING FOR US, IS THIS
SYSTEM THAT TO THE OTHER SYSTEM.
THIS IS ONE OF THE DEFENSE
SYSTEM WE CHARACTERIZE. AND IT
INTERESTED US BECAUSE IT HAS
PROTEINS WITH TIER DOMAINS AND
TIER DOMAINS ARE ALREADY KNOWN
TO PARTICIPATE IN IMMUNITY BUT
IN OUR OWN IMMUNE SYSTEM. THE
IMMUNE SYSTEM OF HUMANS. IT'S
WITH THE RECEPTORS AND THESE ARE
PIECES OF OUR RECEPTORS THAT
PARTICIPATE IN OUR OWN IMMUNITY.
SO THIS GAVE US THE NOTION THAT
FOR SOME DEFENSE SYSTEMS, THE
REASON ANCIENT EVOLUTIONARY LINK
BETWEEN BACTERIA AND HUMAN AND
MORE ACCURATELY, WE HAVE
EVIDENCE THAT SOME IMMUNE
SYSTEMS IN BACTERIA GAVE RISE,
EVOLUTIONARILY TO COMPETENCE OF
OUR OWN IMMUNE SYSTEMS AND WE
STARTED ASKING OURSELVES WHETHER
THERE MIGHT BE OTHER COMPONENTS
OF OUR INNATE IMMUNE SYSTEM THAT
EVOLVED FROM BACTERIA DEFENSE
MECHANISMS AGAINST PHAGE AND THE
ANSWER IS YES, I'M GOING TO SHOW
YOU A FEW EXAMPLES FOR SUCH
COMPETENCE. SO FIRST OF ALL,
LET'S LOOK AT THIS, IT'S CALLED
THE PATHWAY, IT'S AN IMMUNE
PATHWAY IN ANIMALS THAT ALLOW
ANIMAL CELL TO IDENTIFY THAT
THEY HAVE BEEN INFECTED.
BASICALLY, THIS PATHWAY IS
PRESENT IN MANY CELLS. NOT
NECESSARILY IMMUNE CELLS BUT
MANY CELLS IN OUR BODY AND
COMPRISES A PROTEIN CALLED CGAS
WHICH IS THE CYTOPLASMIC DNA.
WHICH WE KNOW IS NOT SUPPOSED TO
BE IN THE CYTOPLASM TO HAVE DNA.
IT'S ILLEGAL IN THE SAY TO
EXAMPLE. THE DNA IS PERCEIVED
AS VIRAL INFECTION.
AND THE SENSE FOR THAT IS THE
PROTEIN CALLED CGAS AND THE NAME
CGAS PRODUCES A UNIQUE
SIGNALLING MOLECULE WHICH IS
CYCLIC GMP-AMP. THESE ARE TWO
NUCLEAR TYPE. WHICH ARE BOUND
ONE TO EACH OTHER IN A SICKLY
SIZED FORM. AND THE MOLECULE
ACCUMULATES IN THE CELL AND
BINDS ANOTHER PROTEIN CALLED
STING. AN ACRONYM. AND I
REALLY ENVY THE ACRONYMS. THEY
ARE SUPER COOL AND INFORMATIVE
BECAUSE WHAT IT DOES IS IT
ACTIVATES AN ESCAPE THAT
EXPRESSES INTERFERON AND OTHER
INTERFERON RELATED GENES SO THIS
PATHWAY WAS DESCRIBED ABOUT TEN
YEARS AGO. 2013. IN SEMINAL
PAPERS. BUT AROUND THE SAME
TIME, THE LAB IN HARVARD FOUND
THAT SOME BACTERIA CAN ALSO
PRODUCE THE SAME MOLECULE,
CYC
CYCLIC GMP-AMP L IN QUITE
RECENTLY THE BIOLOGICAL ROLE WAS
COMPLETELY UNKNOWN.
AND THE REASON I'M SHOWING YOU
THIS IS BECAUSE WHEN WE LOOK AT
THE PROTEIN, THE BACTERIAL
PROTEIN, AND WE LOOK AT THE
BACTERIAL SIGNALS WE SEE IT IS
FOUND NEXT TO CRISPR SO IN THIS
IT'S FOUND NEXT TO A CRISPR AND
IN THIS IT'S FOUND NEXT TO A
RESTRICTION ENZYME AND IN
ANOTHER BACTERIA, THE GENE IS
FOUND NEXT TO OTHER DEFENSE
SYSTEMS THAT WE CHARACTERIZE
RECENTLY AND THIS
CHARACTERIZATION WAS DONE BY
DEMINIMUM FROM MY LAB AND
MOREOVER YOU CAN SEE THAT THE
CGAS IS PRESENT. ONE I WILL
TALK ABOUT THIS, GENE, SHORTLY.
AND SO THE REASON -- I MEAN, THE
FACT THAT THIS OPERATING SO
FREQUENTLY BEING PRESENT NEXT TO
CRISPR AND RESTRICTION LED US TO
THINK THAT IT MIGHT HAVE AN
ANTIVIRAL ACTIVITY AND AN
ANTIVIRAL SYSTEM IN THE
BACTERIAL CODE. AS I TOLD YOU,
WHAT WE DO WHEN WE EXPECT IT TO
FUNCTION IN AN ANTIVIRAL SYSTEM
WE TAKE IT FROM ITS ORIGIN AND
CLONED IT TO E. COLI THAT
DOESN'T ENCODE IT. AND THEN WE
INFECT THE E. COLI BY PHAGES AND
THIS IS A TYPICAL EXPERIMENT
DONE IN MY LAB EVERYDAY I GUESS.
WHAT YOU SEE HERE, THE LIGHTER
PARTS ARE LONG OF BACTERIA AND
GROWING ON A PETRI DISH AND WE
PUT THE FEW PHAGES HERE, TEN
TIMES MORE PHAGES, A HUNDRED
TIMES, A THOUSAND TIMES MORE AND
MORE PHAGES IN EACH SERIAL
DILUTION. WHEN ONE PHAGE
TOUCHES ONE BACTERIUM ON THAT
PLATE. ONE HUNDRED WILL EMERGE
AND INFECT CELLS AND WE SEE THIS
AREA OF CLEARANCE WHICH ARE THE
PLAQUES. REGIONS WHERE THE
SINGLE PHAGE HAS BEEN AND KILL
THE BACTERIA. WHAT YOU SEE HERE
IS WHEN THE BACTERIA ENCODES THE
SYSTEM NOW WE NEED TO INFECT THE
BACTERIA WITH MORE PHAGES BUT
MORE PHAGES MEANING THAT THE
SYSTEM PROTECTS THE BACTERIA
AGAINST THE PHAGE. AND THIS
OBSERVATION THAT WAS DONE BY
SARAH IN MY LAB LED US TO, YOU
KNOW, YOU KNOW, STARTED
ADVENTURE WHICH INVOLVED
GENETICS AND BIOCHEMISTRY AND
EVENTUALLY WE REALIZED OUT THE
SYSTEM WORKS OR AT LEAST HOW THE
MAJOR PARTS OF IT WORKS. SO WE
UNDERSTAND NOW THAT THE
BACTERIAL CGAS ENZYME IS
EXPRESSED MORE MATERIALLY.
PHAGE ACTIVATION ACTIVATES THIS
PROTEIN. BUT IT'S NOT THE DNA
OF THE PHAGES THAT ACTIVATES.
IT'S SOMETHING ELSE WITHIN THE
PHAGE INFECTION. THE DNA IS NOT
A GOOD ACTY VA DOOR FOR SENSING
DNA WOULD BE FUTILE. IT'S
SOMETHING ELSE THAT ACTIVATES
THE PROTEIN.
WE EXPECT IT IS PART OF THE
CAPSID OF THE PHAGE AND WHEN THE
PROTEINS ACTIVATED IT PRODUCES
CYCLIC GMP-AMP THAT BIND AND
ACTIVATE IT. AND THIS ACTIVE
PHOSPHOLIPASE, EACH INNER
MEMBRANE OF THE CELL AND KILLS
IT. AND THE CELL DIES BEFORE
THE PHAGE REPLICATION IS
COMPLETE AND I KNOW THAT FOR
SOME PEOPLE IT SOUNDS WEIRD THAT
BACTERIA WOULD KILL THEMSELVES
BEING SINGLE CELLED ORGANISMS.
WHY WOULD BACTERIA KILL
THEMSELVES AS PART OF THEIR
DEFENSIVE MEASURE. SO
BASICALLY, VERY FREQUENTLY IN
NATURE BACTERIA LIVE IN COLONIES
EITHER IN SOLID SURFACES OR IN
LIQUID CULTURE AND THESE
COLONIES ARE NEARLY SAY AYE SEW
GENERALIC MEANING THEY START
FROM ONE BACTERIA THAT
REPLICATED AND THEN WE HAVE A
SMALL COLONY OF BACTERIA.
AND PHAGES DON'T HAVE A WAY TO
BE MOBILE. SO THE INFECTION
USUALLY STARTS BY INFUSION OF A
SINGLE PHAGE IN THAT COLONY AND
THIS PHAGE REPLICATES WITHIN
THAT BACTERIUM AND THEN AFTER
AWHILE, PHAGE PARTICLES EMERGE
FROM THE BACTERIUM AND
EVENTUALLY, THE ENTIRE COLONY
WILL DEMISE, BUT IF THE BACTERIA
HAVE AN ABILITY TO SENSE THAT
THEY HAVE BEEN INFECTED, AND
THEN KILL THEMSELVES THE SINGLE
CELL THAT IS INFECTED WOULD KILL
ITSELF BEFORE THE CYCLE IS
COMPLETE THEN ONLY ONE CELL IS
LOST AND THE COLONY SURVIVES SO
BASICALLY GENETICALLY SPEAK,
EVOLUTION PREFERS THAT. AND
WE -- NOW UNDERSTAND THAT MOST
BACTERIA IN NATURE HAVE AT LEAST
ONE DEFENSE SYSTEM THAT WORKS
THIS WAY. MEANING THAT WE'LL
SENSE THE PHASE INFECTION AND
THEN KILL THE CELL. THERE ARE
MULTIPLE PRINCIPLES THAT GOVERN
DEFENSE -- THAT WORKS THIS WAY.
ONE OF THEM IS THAT THESE
SYSTEMS USUALLY BECOME ACTIVE
RELATIVELY LATE IN THE INFECTION
CYCLE OF THE PHAGE SO THAT
DEFENSE SYSTEMS THAT CAN
OVERCOME THE PHAGE WITHOUT
KILLING THE CELL COULD WORK
FIRST LIKE CRISPR SHOULD BE ABLE
TO WORK FIRST. IF THEY DON'T
WORK ONLY THEN THE OTHER DEFENSE
SYSTEMS THAT KILL THE CELL COME
INTO ACTION. SO WE SEE THAT
VERY FREQUENTLY. SO AROUND THE
SAME TIME WE DISCOVER THESE CGAS
SYSTEM IN BACTERIA. PHILLIP AND
AARON FROM HARVARD MEDICAL
SCHOOL, THEY FOUND THAT THE
BACTERIA CGAS IS JUST ONE
REPRESENTATION OF A LARGER
FAMILY OF PROTEINS THAT PRODUCE
THIS PROTOTYPE. SO WHAT THEY
FOUND IS THAT THIS REPRESENTS
THE FAMILY TREE. THE TREE OF
THIS FAMILY OF ENZYMES, THE
CGAS-LIKE ENZYMES. SOME OF THEM
PRODUCE CYCLIC GMP BUT OTHER
PRODUCE OTHER MOLECULES LIKE
CYCLIC AMP OR CYCLIC UU OR
CYCLIC TRINUCLEOTIDES AND IN MY
LAB LOOKED AT WHETHER THESE
FAMILIES OF PROTEINS FREQUENTLY
OCCUR NEXT TO KNOWN DEFENSE
SYSTEMS SO IN 64% OF THE CASES,
GENES THAT PRODUCE PROTEINS THAT
PRODUCE GMP-AMP, THE GENES WERE
NEXT TO SYSTEMS BUT THIS WAS
ALSO TRUE FOR OTHER PROTEINS
FROM THIS FAMILY. AND THIS LED
US TO REALIZE THAT ALL THESE
PROTEINS PARTICIPATE IN DEFENSE
AGAINST PHAGE AND THAT WHAT WE
ACTUALLY SEE AND THIS IS --
FOUND IN -- BETWEEN 13-15% OF
THOSE IN THE GENENOMAS WE NOW
UNDERSTAND THAT THERE IS A LARGE
DIVERSITY OF ANTIPHAGE
SIGNALLING SYSTEMS IN THESE
GENOMES AND THESE HAVE A
UNIFYING PRINCIPLE OF ACTIVITY.
THERE'S ALWAYS A PROTEIN FROM
THE CGAS FAMILY THAT RECOGNIZES
PHAGE INFECTION AND PRODUCES A
SIGNALLING MOLECULE. THE
SIGNALLING MOLECULE COULD BE
CYCLIC GMP-AMP THAT CAN BE
ANOTHER KIND OF SIGNALLING
MOLECULE AND BINDS AN INFECTOR
THAT KILLS THE CELL THAT COULD
BE A PHOSPHOLIPASE BUT COULD
ALSO BE A DNA AND THE NUCLEASE
THAT DEGRADES THE BACTERIUM AND
OF THE PHAGE TO KILL BOTH OF
THEM DURING INFECTION. AND
COULD ALSO BE THE PROTEINS THAT
DEPOLARIZE THE MEMBRANE AND WE
NOW HAVE PAPER COMING OUT
SHOWING THAT SOME EFFECTORS
DEGRADE THE ATP POOL FROM THE
CELL DURING INFECTION TO ABORT
THE REPLICATION OF THE PHAGE AND
WE CALL THIS FAMILY OF
SIGNALLING SYSTEM, WE CALL THEM
IN THE UNIFYING NAME, CALLED
CBAS WHICH IS THE ACRONYM OF THE
NUCLEAR BASE ANTIPHAGING
SYSTEMS. SO AGAIN TO PUT -- TO
PUT THE PRINCIPLE OF CBASS INTO
WITHIN -- BLUE COLLARED FIG
INSTEAD OF THE RED COLORS.
THE UNIFYING PRINCIPLE IS
THERE'S AN ENZYME THAT
RECOGNIZES PHAGE. PRODUCES A
SIGNALLING MOLECULE THAT BINDS
AN EFFECTOR THAT KILLS THE
CELLS. WE IDENTIFIED CBASS
WHICH IS FOUND WITHIN 13-15% OF
GENOMES BUT LATER ON WE FOUND
THAT THERE ARE ADDITIONAL
FAMILIES OF IMMUNE SYSTEMS THAT
WORK ACCORDING TO THE SAME
PRINCIPLE. SO ONE OF THEM WE
CALL PIXAR AND THIS IS A STUDY
DONE WITH PHILLIP FROM HIS LAB
AND PH.D. STUDENT FROM MY LAB
AND WE FOUND THIS FAMILY PRODUCE
CYCLIC CMP OR SO MONO NUCLEOTIDE
THAT ACTIVATE AN EFFECTOR
PROTEIN AND THEN WE FOUND
ANOTHER FAMILY OF IMMUNE SYSTEMS
THAT WORK IN THE SAME PRINCIPLE.
THIS IS A TWO AREA SYSTEM IN
WHICH TWO MAIN PROTEINS PRODUCE
CYCLIC ATP RIBOSE ISOMER WHICH
WE WILL NOT TALK MUCH ABOUT
HERE. SO ALTOGETHER, MORE THAN
20% OF ALL BACTERIA IN NATURE OR
AT LEAST ALL THE BACTERIA FOR
WHICH WE HAVE THE GENOME
SEQUENCES HAVE AT LEAST ONE OF
THESE DEFENSE SYSTEMS. VERY
ABUNDANT DEFENSE SYSTEMS IN
MICROBIAL GENES. HOW CAN PHAGES
OVERCOME THAT? CAN PHAGES
DEFEND BACTERIA AND APPARENTLY
THEY DON'T STUDYINGS FROM OUR
COLLEAGUE, AS YOU CAN SEE WE
HAVE A VERY DEEP DEEP
UNDERSTANDING WITH PHILLIP. THE
LAB HAS DISCOVERED ENZYMES THAT
SOME PHAGES BRING THAT CLEAVE
THE SIGNALLING MOLECULE AND
INACTIVATE THEM ESCAPING DEFENSE
BY PHAGES AND A SIMILAR KIND OF
CLEAVING ENZYMES WERE FOUND ALSO
IN VIRUSES THAT INFECT US. SOME
WILL HAVE AN ENZYME THAT CLEAVES
THE MOLECULE OF HUMANS.
SOMETHING THAT WAS ALSO
DISCOVERED BY PHILLIP AND IN A
MORE RECENT STUDY TOGETHER WITH
PHILLIP LED BY SOMEONE FROM MY
LAB AND ALAN FROM PHILLIP'S LAB
WE FOUND THAT OTHER ANTIDEFENSE
PROTEINS, PHAGES L BIND THE
SIGNALLING MOLECULE VERY TIGHTLY
AND SEQUESTER IT AND THEREBY,
AGAIN, NOT ALLOWING THE
SIGNALLING MOLECULE TO BIND THE
EFFECTOR PROTEIN AND SAVING THE
PHAGE FROM THE DEFENSE SYSTEM.
ALL RIGHT SO LET'S GO BACK TO
THE CELL AUTONOMOUS IMMUNE
SYSTEM THAT OUR OWN BODY HAS.
CELLS IN OUR BODY HAVE THE
ABILITY TO SENSE THAT THEY HAVE
BEEN INFECTED AND I'M NOT
TALKING ABOUT IMMUNE CELLS OR T
CELLS OR B CELLS BUT ANY KIND OF
CELL IN OUR BODY HAS A CELL
AUTONOMOUS INNATE IMMUNE SYSTEM
THAT ALLOW THE CELL TO IDENTIFY
THAT IT HAS BEEN INFECTED AND
THEN THROUGH SOME KIND OF
SIGNALLING CASCADE TO ACTIVATE
FIRST OF ALL SECRETION OF
INTERFERON AND SECOND OF ALL THE
PRODUCTION OF EFFECTORS WITHIN
THE CELL THAT ALLOW THE CELL TO
FIGHT AGAINST THE INFECTION OR
AT LEAST THE CELL TRIES TO FIGHT
AGAINST THE INFECTION BEFORE IT
SPREADS. AS I TOLD YOU, SOME OF
THE SENSORS ARE CONSERVED
BETWEEN BACTERIA AND HUMAN AND
THIS IS NOT THE ONLY THING
CONSERVED IN THE INNATE IMMUNE
SYSTEM OF HUMANS. AM GOING TO
SHOW YOU ANOTHER STORY OF A
PROTEIN CALLED VIE WRITTEN WHICH
IS PRODUCED BY INFECTED CELLS
AFTER THEY SENSE THEY HAVE BEEN
INFECTED AND HAS ANTIVIRAL
CAPACITY. SO IT WAS KNOWN FOR
MANY YEARS I THINK MAYBE TWO OR
THREE DECADES THAT THE VIPER RIN
PROTEIN. IT'S ANOTHER CALL
ACRONYM VIRUS INHIBITOR PROTEIN
SOMETHING SOMETHING. NICE
ACRONYM. AND IT WAS KNOWN THAT
IT PROTECTS CELLS FROM
INFECTION, IF YOU OVER EXPRESS
IT CELLS ARE LESS INCLINED TO BE
INFECTED BY SEVERAL DIFFERENT
FAMILIES OF VIRUS BUT ONLY ABOUT
FIVE YEARS AGO THE MECHANISM OF
ACTION WAS DISCOVERED BY A GR
GROUP -- A CLEVER GROUP IN MOUNT
SINAI I THINK AND WHAT THEY
FOUND IS THAT THE VIPER RIN IS
AN ENZYME THAT TAKES THE
NUCLEOTIDE AND MODIFIES IT BY
REMOVING THE THREE PRIME OH AND
INSTALLING A DOUBLE BOND INSTEAD
AND NOW INSTEAD OF A REGULAR
NUCLEOTIDE, THIS MODIFIED
NUCLEOTIDE IS A POISON FOR THE
VIRUS REPLICATION AND THE REASON
IT FORMS A POISON WHICH S WHEN
THE VIRUS TAKES THE NUCLEOTIDE
TO POLYMERIZE ITS NASCENT CHAIN
YOU TAKE THE STRAND AND PUT IT
AGAINST THE TEMPLATE STRAND.
YOU REMOVE THE TWO PHOSPHATES
AND THE LAST PHOSPHATES YOU
ATTACH IT TO THE PREVIOUS
NUCLEOTIDE SO IF THE VIRUS HAS
TAKEN THE MODIFIED NUCLEOTIDE
THERE'S NOW NONE. AND THE VIRUS
CANNOT CONTINUE TO REPLICATE ITS
GENOME AND THIS NUCLEOTIDE IS
NON TOIKSIC TO HUMAN CELLS MOST
PROBABLY BECAUSE THE IT DOESN'T
NEED TO WORK AS FAST AS THE
POLYMERASE OF THE VIRUS AND IT
CAN BE MODIFIED. THE REASON I'M
TELLING YOU THIS IS BECAUSE WE
FOUND THIS PROTEIN IN BACTERIA.
THIS WAS FOUND BY A POSTDOC IN
MY LAB. SHE HAS HER OWN LAB, A
VERY SUCCESSFUL LAB AT AN
INSTITUTE NOW AND WHAT SHE FOUND
IS THAT THERE ARE LOADS OF THE
VIPER RIN. THESE ARE FOUND.
AND THESE ARE VERY, VERY
FREQUENTLY FOUND NEXT TO CRISPR
AND ENZYMES. WE THOUGHT AS YOU
KNOW NOW, THIS TELLS US THAT
THIS PROTEIN HAS A FUNCTION, HAS
AN ANTIVIRAL FUNCTION IN
BACTERIA AS WELL SO, AGAIN, WE
TOOK A BUNCH OF THESE PROTEINS,
WE CLONED THEM IN BACTERIA. IN
THIS CASE I'M SHOWING YOU T 7.
ONE, TEN, THESE ARE HUGE
PLAQUES. T 7 HAS A SHORT CYCLE.
THAT'S WHY IT FORMS A HUGE
PLAQUE AFTER OVERNIGHT.
AND WHAT YOU SEE IS THAT WHEN WE
EXPRESS -- THE VIPER RIN WITHIN
E. COLI CELLS WE SEE MUCH FEWER
PLAQUES. THIS TOOK US TO
ADVENTURE THAT INVOLVES A LOT OF
CHEMISTRY. AND EVENTUALLY WHAT
AUDE FOUND WAS THIS PRODUCED A
FAMILY OF MOLECULES. SOME OF
THEM ARE THE SAME LIKE THE HUMAN
MOLECULE WHILE OTHERS ARE BASED
ON A DIFFERENT NUCLEOTNUCLEOTID
CASES THE OH FROM THE 3 PRIME
END IS DEHYDRATED AND A DOUBLE
BOND BECOMES -- IS INSTALLED AND
POISONS THE PHAGE REPLICATION.
SO WHEN WE TAKE ALL THE
BACTERIAL ENZYMES AND PUT THEM
ON THE TREE WHAT WE SEE IS THAT
EWE SEE MULTIPLE CLAIFS THAT ARE
FORMED. SOME OF THEM HAVE MORE
TENDENCY TO PRODUCE THE OTHER
NUCLEOTIDES AND WHEN WE ADD TO
THIS TREE, THE SEQUENCES OF ALL
THE YOU KARYOTIC. WE SEE ALL OF
THEM CLUSTER ON A SINGLE CLAYED
THAT EMERGES FROM THE CLAYED
THAT PRODUCES THE CTP AND THAT
IS MOSTLY UNREACHED. SO WE
THINK WE HAVE PRETTY STRONG
REASONS TO BELIEVE NOW THAT
THERE HAS BEEN A SINGLE
ACQUISITION EVENT IN THE VERY
EVOLUTION OF EUKARYOTES WHERE
THE EARLY EUCARYOTE ACQUIRED ONE
VIPER RIN FROM BACTERIA AND
STARTED ON THIS ACQUISITION AND
LED TO WHAT WE SEE TODAY IN
HUMANS AND OTHER EUKARYOTES.
ONE THING THAT REALLY -- AGAIN,
THERE ARE MANY SURPRISES IN THIS
STORY. THAT WAS ANOTHER
SURPRISE THAT BACTERIA CAN
PRODUCE ANTIVIRAL MOLECULES.
WE'RE USED TO THINKING ABOUT
BACTERIA HAS A SOURCE MORE
MOLECULES BUT THEY MOUSSE MORE
MOLECULES TO PROTECT THEMSELVES
FROM PHAGES. THESE OF YOU WHO
UNDERSTAND HOW ANTIVIRALS WORK,
REALIZE THAT IN MANY CASES,
ANTIVIRALS THAT WE USE AGAINST
VIRUSES THAT INFECT US ARE ALSO
BASED ON MODIFIED NUCLEOTIDES.
THIS MOLECULE IN THE MIDDLE IS
PART OF THE THREE DRUG COCKTAIL
THAT PEOPLE TAKE AGAINST HIV.
AND THIS IS A NUCLEOTIDE THAT
HAS INSTEAD OF THE THREE PRIME
OH HAS THREE NITROGENS. THIS IS
THE DRUG THAT PEOPLE TAKE
AGAINST HERPES VIRUSES THAT YOU
PUT ON YOUR LIPS, THIS OINTMENT
WHEN YOU HAVE HERPES, THIS IS A
MODIFY NUCLEOTIDE THAT BLOCKS
THE BOTTOM PART OF THE VIRALS.
THIS WORKS THE SAME WAY. IT
TAKES IT IN AND SO WE THINK NOW
REALIZING THAT BACTERIA PRODUCE
AGAINST PHAGES WE WONDER WHETHER
THESE MOLECULES CAN WORK AGAINST
VIRUSES THAT INFECT US AND WE
ARE TRYING TO SEE WHETHER THEY
CAN BE NEW DRUGS IN THE FUTURE.
HERE'S ANOTHER DEFENSE SYSTEM
THAT IS VERY, VERY -- STUDIED
ONE DAY IN HUMANS. SO IN ANIMAL
CELLS INCLUDING IN OUR OWN CELLS
THERE IS A SYSTEM THAT, OR AN
APPARATUS CALLED THE INFLAM MA
SOME THAT RECOGNIZES INFECTION
AND ONCE THE INFECTION IS
RECOGNIZED. PROTEINS CALLED CAS
PACES BECOME ACTIVATED.
AND THIS CAS PACE IS BY DOING
SOME CLEAVAGE EVENT ON A PROTEIN
CALLED GAS DER MIN, ONCE IT'S
CLEAVED BECOMES ACTIVITY AND
MAKES A GIANT PORE IN THE CELL
MEMBRANE AND BASICALLY THE CELL
LEAKS OUT AND DIES AT THE SAME
TIME THAT THE CELL DIES IT
SECRETES INTERNAL TO THE SITE OF
THE INFECTION AND THAT'S WHY
THIS IS CALLED PYRO TO SIS
BECAUSE IT'S DEATH THAT CAUSES
INFECTION. IN MY LAB, WAS
DISCOVERED HOMOLOGS OF THE HUMAN
GAS DER MIN AND FOUND THAT IN
ALL CASES, THIS HOMO LOGS ARE
FOUND NEXT TO A PROTEASE EITHER
FROM THE CAS PACE FAMILY OR
ANOTHER KIND OF PROTEASE SO WE
SUSPECTED AND ALSO THESE ARE
FOUND NEXT TO CRISPR SO WE
SUSPECTED THIS IS ANOTHER
DEFENSE SYSTEM IN BACTERIA. AND
TOGETHER WITH OTHERS, THEY
SHOWED THAT INDEED DURING
INFECTION, THIS IS FROM MY LAB,
DURING INFECTION THE CASPASES
ARE ACTIVATED. THE CASPASE-LIKE
PROTEINS ARE SENSING THE PHAGE
AND WE VERIFY THE CLEAVAGE AND
THEY MAKE GIANT HOLES IN THE
MEMBRANE THAT KILL THE CELL. A
SYSTEM THAT KILLS THE CELL
BEFORE THE PHAGE IS READY.
BEFORE THE PHAGE PARTICLES ARE
RIPE WITHIN THE INFECTED CELL
LET ME KNOW YOU ANOTHER PECULIAR
MODE OF DEFENSE. THIS IS FOUND
IN MY LAB BY A PH.D. STUDENT.
SOMEONE WHO HAS BEEN VERY
SUCCESSFUL IN HER DISCOVERS.
THERE ARE FAMILIES OF ENZYMES
THAT RECOGNIZE PHAGE INFECTION,
MOST PROBABLY THEY RECOGNIZE THE
PHASE AS INHIBITED FROM THE
TRANSCRIPTION AND ONCE THIS IS
SENSED THESE ENZYMES TAKE ONE OF
THE NUCLEOTIDES IN THE CELL
WHICH ARE THE BUILDING BLOCKS
FOR DNA REPLICATION AND
BASICALLY DEGRADES THAT
NUCLEOTIDE. SO SHE FOUND TWO
FAMILIES OF ENZYME ONE IS THAT
THE DPASE AND ANOTHER FAMILY OF
ENZYMES IS THE AM MIN NICES
WHICH REMOVED THE NH FROM THE
CTP. BASICALLY THESE ENZYMES
HIDE ONE OF THE OXY NUCLEOTIDES
AWAY FROM THE PHAGE AND THEY TRY
TO REPLICATE THE GENOME AND
WITHOUT THE NUCLEOTIDE IT GETS
STUCK AND IS NOT REPLICATED.
THE REASON I'M SHOWING YOU THE
CONTEXT OF THIS IS BECAUSE, WE
WERE VERY PROUD OF OURSELVES FOR
DISCOVERING A NEW MODE, A NEW
STRATEGY TO PROTECT AGAINST
INFECTION BUT THEN WE REALIZED
THAT A LONG TIME AGO IT WAS
ALREADY DISCOVERED IN THE HUMAN
IMMUNE SYSTEM AND THIS IS
MANIFESTED BY A PROTEIN CALLED H
21. IT IS AN ANTI-HIV FACTOR
THAT BLOCKS INFECTION BY
DEGRADING THE OXY NTPS INTO THE
THREE PHOSPHATES AND SUGAR AND
BASE MOIETY BUT IT DOES THAT FOR
ALL PROTOTYPES. SO WE NEVER
REALIZED THAT MULTIPLE
COMPONENTS OF OUR INNATE IMMUNE
SYSTEMS HAVE PARALLELS IN THE
BACTERIAL IMMUNE SYSTEM AND IN
SOME CASES WE HAVE VERY SOLID
PROOF THAT THE HUMAN COMPONENT
HAS EVOLVED FROM THE BACTERIAL
COMPONENT. YOU KNOW,
GENETICALLY BASED PROOF. SO
WHAT WE THINK HAPPENED IS THAT,
I MEAN, WHAT CAN EXPLAIN THAT IS
THAT PHAGES, AND THAT'S VERY
WELL ACCEPTED. THAT PHAGES HAVE
BEEN IN THE WORLD WELL BEFORE
EUKARYOTES EVEN WERE HERE. SO
PHAGES WERE INFECTING BACTERIA
VERY LONG TIME AGO, VERY EARLY
ON AFTER THE BACTERIA WAS
INVENTED AND DEFENSE SYSTEMS
HAVE INVOLVED IN BACTERIA BEFORE
EUKARYOTES CAME TO BE AND THESE
DEFENSE SYSTEMS HAVE BEEN IN
BACTERIA FOR HUNDRED OF YEARS
AND AT SOME POINT THE FIRST
EUKARYOTE CAME TO BE MOSTLY BY
DIFFUSION AND THESE THIS
BACTERIUM USED TO HAVE DEFENSE
SYSTEMS IN THEM AND THESE
DEFENSE SYSTEMS WERE THE BASIS
OF THE IMMUNE SYSTEM THAT WE
KNOW TODAY SO THIS TEACHES US
ABOUT THE EVOLUTION OF OUR OWN
IMMUNE SYSTEM BUT CAN ALSO BE
BENEFICIAL FOR UNDERSTANDING
IMMUNE SYSTEMS OF HUMANS. AND
WHAT DO I MEAN? WE ARE GOING TO
FIND NOW DEFENSE SYSTEMS THAT WE
KNOW EXIST IN HUMAN BUT WE DON'T
KNOW HOW THEY WORK AND WE NOW
FIND THEM IN BACTERIA. SO THIS
IS ONE INTERESTING IMMUNE
SYSTEM. IT'S CALLED I 15. IT'S
A YOU WICK WE TIN LIKE PROTEIN
THAT IS PRODUCED BY INTERFERON.
IT'S DEDUCED TOGETHER WITH
UBIQUITIN CHARGING ENZYMES THAT
ARE SPECIFIC AND PROTECTS
OURSELVES AGAINST SOME VIRUSES.
AND HAS SOME ROLES IN COVID
PROTECTION FROM COVID INFECTION.
BUT WE DON'T KNOW HOW IT WORKS.
PEOPLE IN IMMUNOLOGY FEEL THEY
DON'T KNOW HOW IT WORKS. NOW WE
REALIZE THAT SUCH A SYSTEM
EXISTS IN BACTERIA. THERE IS AN
INTERFERON IN PROTEINS AND THE
UBIQUITIN DOMAIN AND WE SHOW IT
PROTECTS AGAINST PHAGES. NOW
WE'RE TRYING TO FIGURE OUT WHY
AND WE HOPE IF WE SOLVE THE
MECHANISM IN BACTERIA THIS CAN
TRANSLATE INTO UNDERSTANDING HOW
THE HUMAN MECHANISMS WORK AS
WELL. WE HAVE A BUNCH OF IMMUNE
MECHANISMS THAT ARE FOUND BOTH
IN HUMANS AND IN BACTERIA BUT WE
DON'T KNOW YET HIGH THEY WORK IN
HUMAN. WE HOPE TO SOLVE THIS
RIDDLE. NOT ALL OF THE DEFENSE
SYSTEMS HAVE HOMO LOGS IN
HUMANS.
FOR EXAMPLE, WE FOUND THIS
PROTEIN CALLED RADAR. THIS IS A
SYSTEM THAT WAS FOUND. WHICH
COMPRISES TWO VERY BIG GENES.
ONE OF THEM AS A DOMAIN, THE
OTHER HAS AN ATPASE DOMAIN. AND
TOGETHER IN THE LAB, THEY SHOWED
THAT THESE PROTEINS FORM A GIANT
COMPLEX IN THE CELL. THIS GIANT
COMPLEX IS BUILT FROM TWELVE
MONOMERS OF THIS. THEY FORM
LIKE THIS KIND OF A SOCCER BALL.
AND THIS PROTEIN FORMS THE HEP
TAMIR AND EACH HEP TAMIR SITS ON
ONE FACE OF THE SOCCER -- THE
DECK KA HER TO FORM A GIANT
COMPLEX.
THAT IS FOUR TIMES AS HEAVY AS A
RIBOSOME SO A VERY BIG COMPLEX
AND WHAT WE SHOWED IS THAT THIS
COMPLEX CONVERTS MASSIVELY ATP
TO ADEKNOW SIGN DURING INFECTION
WE DON'T KNOW HOW IT PROTECTS
AGAINST PHAGE.
BUT THIS CONVERSION, MAYBE IT'S
PROTECTION AGAINST PHAGE, WE'RE
STILL STUDYING THAT. THE
COMPLEX IS SOMETHING LIKE -- WE
HAVEN'T SEEN ANYTHING LIKE THAT.
IT'S SUCH A GIANT COMPLEX IN
ORDER TO MEDIATE THIS ACTIVITY.
ANOTHER THING THAT IS VERY
INTERESTING TO US WE FOUND IS
SOMETHING CALLED LET TRONS.
THIS IS A VERY PECULIAR ENTITY
IN BACTERIA. IT'S COMPRISING
RNA AND THERE ARE REVERSE
TRANSCRIPTASE THAT CONVERT INTO
CDNA LEAVING IT BOUND. SO THIS
IS RNA AND DNA THAT WAS KNOWN IN
BACTERIA FOR MANY YEARS IT'S
FOUND IN MORE THAN 10% OF ALL
BACTERIA AND THE FUNCTION OF
THIS WAS NOT KNOWN. WE FOUND
THAT IT PARTICIPATES IN DEFENSE
AGAINST PHAGES AND WE FOUND THAT
ONE OF THESE -- IT'S CALLED A
LET TRON. ONE OF THESE LET TRON
IS A GUARDIAN OF ANOTHER DEFENSE
SYSTEM. SO THERE IS A DEFENSE
SYSTEM OR THERE IS A COMPLEX
CALLED RECK BCD IN THE CELL THAT
ALSO CLEAVES LINEAR DNA.
YOU KNOW WHEN THE PHAGE EJECTS
THIS FROM THE CELL IT EATS THE
DNA VERY RAPIDLY AND FOR THIS
REASON MANY PHAGES ARE ALSO
BRINGING PROTEINS TO INHIBITORY
RECK BCD. WE FOUND THESE GUARD
THE REC BCD AND WHEN IT COMES
INTO ACTION AND TOGETHER KILLS
ANOTHER CELL. IT'S A GUARDIAN
OF ANOTHER DEFENSE SYSTEM AND IF
THIS OTHER DEFENSE SYSTEM
BECOMES INHIBITED BY PHAGE, THIS
GUARDIAN KILLS THE CELL. WE
DON'T YET UNDERSTAND WHY WE NEED
THIS A CHI MARA BETWEEN DNA AND
RNA. THERE ARE MANY OPEN
QUESTIONS. THIS HAPPENS FAST,
THESE DISCOVERS AND WE STILL
DON'T UNDERSTAND MUCH. FOR
EXAMPLE, FOR MANY SYSTEMS WE
KNOW WHAT HAPPENS AFTER THE
PHAGE. IN SOME CASES THEY DO
SOMETHING ELSE. BUT WE DON'T
KNOW WHAT IS BEING RECOGNIZED.
HOW THEY RECOGNIZE PHAGE
INFECTION. NOW WE HAVE SOMEONE
FROM MY LAB, SHE RECENTLY DID A
VERY LARGE SCREENING EXPERIMENT.
WHERE SHE TOOK ALL THE DEFENSE
SYSTEMS THAT WE HAVE. ABOUT 50
DEFENSE SYSTEMS THAT WE HAVE IN
THE LAB AND EACH DEFENSE SYSTEM
SHE INFECTED THE CELL THAT
ENCODES THE SYSTEM WITH A PHAGE
THAT IS NORMALLY BLOCKED BY THE
SYSTEM AND SHE SEARCHED FOR
PHAGE MUTANTS THAT CAN OVERCOME
THE DEFENSE SYSTEM THEN
SEQUENCED THE PHASE GENOME TO
SEE WHAT GOT MUTATED AND IN MANY
CASES IT'S THE GENE THAT GETS
RECOGNIZED BY THE DEFENSE
REALIZED AND SHE REALIZED THEY
FREQUENTLY IDENTIFY EITHER THE
COMPONENTS OF THE PHAGE. THESE
ARE THE PROTEIN THAT FORM THE
REPLICATION MACHINERY. THIS IS
A VERY RELATIVELY CONSERVATIVE.
OTHER SYSTEMS RECOGNIZE
STRUCTURAL COMPETENCE OF THE
PHAGE. WOULD BE, YOU KNOW, THE
PHAGE TWO PROTEIN OR THE CAPSID
OR SOMETHING ELSE THIS THE
STRUCTURE. AND SO OTHER SYSTEMS
RECOGNIZE THE PHAGES TAKING OVER
THE CELL. YOU KNOW, SOME OF
THEM RECOGNIZE THAT ANOTHER
DEFENSE SYSTEM GETS INHIBITED
AND SOME OTHERS RECOGNIZED THAT
TRANSCRIPTION BECOMES INHIBITED.
THIS BECOMES UNIFYING PRINCIPLE
IN HOW DEFENSE SYSTEMS ARE
RECOGNIZED, INFECTION BY PHAGE.
ANOTHER OPEN QUESTION IS, WHAT
ARE THOSE DEFENSE ODDS? SO I
TOLD YOU THAT WE IDENTIFIED
MULTIPLE DEFENSE SYSTEMS BASED
ON THE PRESENCE FREQUENTLY NEXT
TO DEFENSE SYSTEMS. AND THIS
DEFENSE ISLAND WAS VERY FRUITFUL
FOR US WITH THE DISCOVERY OF NEW
SYSTEMS BUT THE QUESTION OF WHAT
ARE THE DEFENSE ISLANDS BOTHERED
US FOR A NUMBER OF YEARS AND A
PH STUDENT IN MY LAB FOUND THAT
THE DEFENSE SYSTEMS ARE CARRIED
USUALLY BY MOBILE GENETIC
ELEMENTS. THIS ISN'T STUDYING
E. COLI. SHE SHOWED THAT
DIFFERENT STRAINS OF E. COLI
HAVE DIFFERENT STRAINS THAT ARE
CARRIED ON DEDICATED MOBILE
GENOMIC ELEMENTS. THIS IS THE
LAB STRAIN THAT LACKS MOST OF
THE DEFENSE SYSTEMS THAT WE
FOUND. AND THESE ARE DIFFERENT
STRAINS OF E. COLI. YOU SEE IN
EACH OF THESE STRAINS AN ISL
ISLAND -- A MOBILE ISLAND HAS
INTEGRATED IN THE SAME PLACE.
AND SO, IF YOU LOOK AT, WE
LOOKED AT ABOUT 1,000 E. COLI
GENOMES. WE SEE OUR ABILITY IN
THE DEFENSE SYSTEMS IN THESE
GENOMES. MOST OF THE SYSTEMS
ARE CARRIED BY MOBILE GENETIC
ELEMENTS SO HERE, THIS IS THE
ELEMENT THAT BRINGS MANY KINDS
OF DEFENSE SYSTEMS INTO THE SAME
POSITION IN THE E. COLI GENOME
BUT IN EACH STRAIN IT'S A
DIFFERENT SET OF DEFENSE SYSTEMS
FORMING THESE DEFENSE ISLANDS
AND THIS PANNED OUT INTO
SOMETHING THAT WE DIDN'T EXPECT.
IN TERMS OF THE DISTRIBUTION OF
DEFENSE SYSTEMS IN MICROBIAL
GENOMES. WE SEE THAT THEY ARE
VERY SPORADICALLY PRESENT IN
MICROBES. WHAT YOU SEE HERE,
EACH ROW HERE IS A STRAIN OF E.
COLI. AND EACH COLUMN IS THE
DEFENSE SYSTEM. SO THIS IS A
RESTRICTION ENZYME TYPE ONE,
TWO, THREE, FOUR. OTHER DEFENSE
SYSTEMS THAT WE DISCOVERED,
CRISPR, WHENEVER YOU SEE THE
COLORED BOX MEANS THIS STRAIN
HAS THE SYSTEM. AND YOU SEE
THAT THE SYSTEMS SPORADICALLY
OCCUR IN STRAINS. THE FIRST
STRAIN HAS THIS SYSTEM AND THE
THIRD STRAIN, YOU KNOW, HAS A
TYPE FOUR RESTRICTION ENZYME.
IT HAS A SYSTEM, ET CETERA. AND
SO THESE SYSTEMS ARE
SPORADICALLY OCCURRING AND WE
CALL THAT THE PAN IMMUNE SYSTEM
OF BACTERIA. WE THINK THAT A
SINGLE BACTERIUM CANNOT ENCODE
ALL THE DEFENSE SYSTEMS AT ONCE
BECAUSE THE TIME IT EMERGES AS
AUTOIMMUNITY OCCURS TO TOO MUCH
TOXICITY BUT ON THE OTHER HAND
THE OTHER BACTERIA THAT ENCODE
COULD CONTRIBUTE THAT THROUGH
HORIZONTAL GENE TRANSFER AND
THESE SYSTEMS COULD BE ACQUIRED
BY NATURE IF THEY'RE NEEDED.
AND SO I WANT TO END, YOU KNOW
BY SHOWING THIS POSTER WE WERE
ASKED TO PUT WHAT WE KNOW ON
DEFENSE SYSTEMS OF BACTERIA ON A
SINGLE POSTER. OBVIOUSLY IT'S
TOO SMALL FOR YOU TO SEE. WE
HAVE RESTRICTION ENZYMES HERE.
WHAT FASCINATES ME ABOUT THE
POSTER, WE PUBLISHED IT LAST
YEAR AND NOW MANY ADDITIONAL
SYSTEMS AND MECHANISMS WERE
DISCOVERED SINCE THEN. BUT WHAT
REALLY FASCINATES ME IS THAT
MANY OF THE MECHANISMS THAT ARE
DESCRIBED HERE ARE ONLY THE
PRODUCT OF STUDIES OF OUR LAB
AND MANY OTHER LABS AND THE
VERY, YOU KNOW, VERY RECENTLY.
MUCH OF THE IMMUNE SYSTEMS WE
JUST DIDN'T KNOW AND NOW IT'S
LIKE, YOU KNOW, LIFTING, OR
DIGGING A GIANT EXCAVATION SITE
AND REALIZING THERE HAS BEEN A
CITY BELOW AND MOVER WHAT YOU
SEE IN THE TINY FONT ARE THE
LIST OF NAMES. OF DEFENSE
SYSTEMS THAT WE KNOW PROTECT
AGAINST PHAGE BUT WE DON'T KNOW
YET HOW. VERY LARGE, YOU KNOW,
VERY LARGE GROUNDS FOR HAVING,
YOU KNOW, MAKING FUN BIOLOGY AND
FUTURE DISCOVERIES. AND I WANT
TO END BY THANKING MY LAB WHICH
ARE VERY TALENTED GROUP OF
PEOPLE. VERY GLAD TO HELP WITH
THEM TO WORK WITH THEM. AND
ALSO OUR DEAR COLLABORATORS
PHILLIP AND MANY MEMBERS OF HIS
LAB. WE'VE BEEN HAVING A
WONDERFUL COLLABORATION OVER A
VERY SHORT TIME PERIOD AND MOST
OF THIS COLLABORATION WAS DONE
UNDER COVID SO WE HAVEN'T MET
UNTIL RECENTLY. WE PUBLISHED
PAPERS TOGETHER AND ONLY MET
ONCE. BUT ON ZOOM. AND THANK
YOU VERY MUCH MORE LISTENING.
>>(APPLAUSE).
>>OF COURSE ANYONE HERE IS FREE
TO USE THE MICS AND AS YOU ARE
MAKING YOUR WAY OVER THERE I DO
HAVE A COUPLE QUESTIONS FROM THE
ONLINE. THIS ONE COMES RATHER
EARLY IN THE TALK. DOES
EXPRESSION OF VIRAL CAPSID
PROTEINS LEAD TO CELL DEATH VIA
THE CGAS SYSTEM?
>>THAT'S A GREAT QUESTION. SO
IN SOME CASES, IN SOME DEFENSE
SYSTEMS, EXPRESSION OF THE
STRUCTURAL COMPONENTS OF THE
PHAGE IS DIRECTLY LEADING TO
TOXICITY, LEADS TO CELL DEATH IN
THE CASE OF CGAS WE COULDN'T
FIND -- IN ANOTHER CALLED PYCSAR
WHERE WE FOUND IT IN THE CAPSID
THERE WAS NO TOXICITY. THE
DEFENSE IS GONE. SO WE THINK
THAT EITHER THE LARGER CONTEXT
OF THE PHAGE THAT'S BEING
ASSEMBLED WITHIN THE MATERIAL IS
THE TRIGGER FOR THE CGAS
ACTIVATION OR YOU NEED MULTIPLE
SIGNALS, YOU KNOW, BOTH THE
CAPSID AND SOMETHING ELSE THAT
WE DON'T KNOW OF YET SO WE DON'T
SEE ACTIVATION JUST BY
EXPRESSING THE CAPSID FOR THE
SPECIFIC CASE.
>>YOU MAY HAVE TOUCHED ON THIS
RIGHT AT THE END AND I MAY HAVE
JUST MISSED IT. DO YOU FIND THE
BACTERIA STEALING PIECES OF THE
FABL
PHAGE AS MAKING A BOARD. OR
MAKING SOME UNITS THAT DON'T
WORK OR OTHER SORTS OF THINGS?
>>WE DON'T FIND EVIDENCE THAT
BACTERIA, THAT THE DEFENSE
SYSTEMS THAT ARE WITHIN THIS
DEFENSE ISLANDS, WE DIDN'T YET
FIND EVIDENCE THAT THEY COMPRISE
ANCIENT PIECES OF PHAGES. IT
DOESN'T MEAN THAT THIS IS NOT A
STRATEGY OF BACTERIA. IT COULD
BE THAT SOME BACTERIA PROTECTS
BY THIS BUT MAYBE THESE PROTEINS
ARE NOT TRAVELING ON DEFENSE
ISLANDS LIKE THESE SYSTEMS.
>>IT WOULD BE A FOLLOW UP WITH
A DOUBLE QUESTION ABOUT THE
ISLANDS. NO QUESTION IS AN
ISLAND I GUESS. ARE DEFENSE
ISLANDS TRANSCRIPTIONALLY
ORGANIZED WITH THEIR RESPONSE TO
PHAGE AND DO DEFENSE ISLANDS
DEFEND AGAINST COMPETITOR
BIOFILMS, PROTEIN AGGREGATES.
>>RIGHT. SO BASED ON THE
ORGANIZATION OF THE GENES IN
DEFENSE ISLANDS WE DON'T THINK
THAT THERE IS ORGANIZATION BY
GENE EXPRESSION. WE SEE THE
DEFENSE SYSTEMS WILL DISCOVER
OUR EXPRESS. AND FIND THOSE
PROTEINS BEFORE IT INFECTS. THE
REASON WE THINK THIS IS
ESSENTIAL IS BECAUSE SOME PHAGES
ARE QUICKLY KILLING THE CELLS.
SOME PHAGES WILL DEGRADE THE
INFECTED CELL AFTER FOUR
MINUTES. THIS IS SOMETHING IT
DOES. AND SO THIS IS NOT ENOUGH
TIME TO ACTIVATE EXPRESSION AND
THEN DEFENSE. SO USUALLY THE
ACTIVATION OF THE SYSTEMS IS
DONE IN THE PROTEIN LEVEL.
THERE ARE EVIDENCE THAT YOU CAN
ELEVATE THE EXPRESSION OF SOME
DEFENSE SYSTEMS USING EXTERNAL
SIGNALS. MOST OF THEM ARE
INVOLVED IN THE DENSITY OF THE
CULTURE. AND SOME OF THEM ARE
NOT DONE YET. SO THAT'S THE
ANSWER FOR THE FIRST QUESTION.
THE SECOND QUESTION, DO DEFENSE
ISLAND --
>>COMPETITORS LIKE PROTEIN
AGGREG
AGGREGATES.
>>NOT THAT I KNOW OF. WE'VE
BEEN STUDYING THE DEFENSE
SYSTEMS IN AN ISOLATED MANNER.
BASICALLY WHAT WE'VE BEEN DOING
IS TAKE A DEFENSE ISLAND AND
STORE IT IN ECO LIE. WE'VE
TAKEN ONE SYSTEM AND STUDY WHAT
IT DOES.
WE DON'T KNOW IF THESE ISLANDS
DO OTHER STUFF.
>>I NOTICED EARLIER IN YOUR
TALK WHEN YOU WERE GIVING US
SPECIFIC DEFENSE MECHANISMS THAT
THE NAMES WERE ACRONYMS BASED ON
WHAT THEY DO. BUT AT THE END
YOU GAVE US A LIST OF FIFTY
NAMES THAT SOUNDED A LOT MORE
IMAGINATIVE. WHERE ARE THOSE
NAMES COMING FROM?
>>THANK YOU FOR THIS QUESTION.
YEAH, WE STARTED QUOTING THE
DEFENSE SYSTEMS AS THE FIRST
SYSTEM WE EVER DISCOVERED WAS
CALLED BRICKS. THE SECOND WAS
DISARM, WHICH WAS WITH
MODIFICATION. AND THEN WE RAN
OUT OF ACRONYMS SO WE STARTED
CALLING THEM OF PROTECTIVE GODS
FROM DIFFERENT MYTHOLOGIES. WE
HAVE THE LITHUANIA GOD. WE HAVE
A CELTIC GOD OF THE FOREST. WE
HAVE AN EGYPTIAN GODDESS.
THAT'S THE NAMES.
>>ON A MORE SERIOUS NOTE, SINCE
A LOT OF THE MECHANISMS KILL
BACTERIA, IS THERE AN OBVIOUS
WAY AND I'M SURE YOU'VE THOUGHT
ABOUT THIS, TO USE THOSE
MECHANISMS, TO ACTIVATE THEM AS
ANTIBIOTICS?
>>THIS IS A GREAT QUESTION.
I'M NOT SURE HOW USEFUL WOULD
THIS BE BECAUSE THE SYSTEMS ARE
SO SPORADICALLY PRESENT IN
BACTERIA. SO, YOU KNOW, THE
CBASS SYSTEM WHICH IS PRESENT IN
ABOUT 15% OF THE GENOMES, YOU
CAN IMAGINE THAT IF YOU GIVE
THEM THE SIGNAL IN HEIGHT OF
CONCENTRATION THAT THE PSYCHLY
GMP-AMP, SOME OF THEM WILL FORCE
THE CELL TO DIE. SOME OF THEM
USED PSYCHLY UMP, TRINUCLEOTIDE
SO YOU'D BE ABLE TO ONLY KILL A
SMALL NUMBER OF THEM AND SINCE
IT'S NOT THAT YOU WILL EVER BE
ABLE TO KILL ALL THE E. COLIS.
BECAUSE THE M SYSTEMS ARE
SPORADICALLY PRESENT.
SO YOU'D BE ABLE TO KILL JUST
SPECIFIC STRANDS OF THE
BACTERIUM AND WILL NOT BE
PROFITABLE FOR A DRUG COMPANY TO
BE SO SPECIFIC I IMAGINE.
>>YES. THERE ARE COLLEAGUE
FROM YOURS AND PROBABLY
SWITZERLAND FOR THE WEDNESDAY
EVENING LECTURE SERIES WE HAVE
THERE. LUKE. GREAT TALK, WHY
ARE RESTRICTION ENZYMES SO
PREVALENT COMPARED TO OTHER
INNATE DEFENSE SYSTEMS?
>>WELL E RESTRICTION ENZYMES
GIVE A VERY GOOD ABILITY TO
PROTECT AGAINST THE PHAGE
WITHOUT KILLING ITSELF.
THAT'S A GOOD REASON TO KEEP
THEM THERE. SOME PHAGES BRING
ANTIRESTRICTION. THAT IS
SOMETHING THAT IS COMMON IN
PHAGES. PROTEINS THAT PROHIBIT
RESTRICTION ENZYMES IN CASES
WHERE YOUR RESTRICTION ENZYMES
ARE PROHIBITED I GUESS THE CELL
WOULD WANT TO ACTIVATE THE
SYSTEM THAT LEADS TO DEATH.
>>AND WHAT, FIFTY YEARS AGO ON
THE SUBJECT, WHEN THE PHAGE
INFECTS E. COLI, THE VERY FIRST
THING THAT HAPPENS, AT THIS I
DON'T KNOW, BUT IMMEDIATELY
INACTIVATES THE POLYMERASE TR
TRANSCRIPTION OF THE E. COLI.
BECAUSE IT HAS THE SIGMA FACTOR
AND ONLY THIS, AS WAS EARLIER
STATED, ON THE POLYMERASE. IT
CAN TRANSCRIBE THE T4 DNA AND
THAT IS HOW THE WHOLE PROCESS
PROCEEDS DOWN THE ROAD. SO THAT
IS I THINK LONG BACK DISCOVERED.
VERY FIRST THING, THE
MODIFICATION. VERY IMPORTANT
SIGMA FACTOR WHICH IS INVOLVED
IN THE INITIATION OF THE
TRANSCRIPTION OF RNA AND DNA.
>>YEAH. YEAH.
>>DO YOU HAVE ANOTHER QUESTION?
>>NO.
>>SO IT'LL BE ONE LAST QUESTION
ONLINE. WE'RE JUST PAST THE
HOUR. GIVEN HEMOLOGY BETWEEN
BACTERIAL AND EUCARYOTE
IMMUNITY, IS THERE ANY EVIDENCE
FOR HEMOLOGY BETWEEN ANTIDEFENSE
GENES OF PHAGES AND VIRUSES
ATTACKING EUKARYOTIC CELLS?
>>IT IS A GREAT QUESTION. WE
HAVEN'T FOUND -- SO FAR WE
DIDN'T DISCOVER TOO MANY PHAGE
PROTEINS THAT INHIBIT DEFENSES.
MAYBE FOUR OR FIVE SUCH FAMILIES
AND WE HAVEN'T FOUND HEMOLOGIES
TO PROTEINS AND VIRUSES THAT
INFECT HUMANS. SOMETIMES THERE
IS ANALOGY. SO AS I TOLD YOU
THERE ARE ENZYMES THAT PHAGES
BRING THAT CLEAVE THE SIGNALLING
MOLECULE TO THE DEFENSE SYSTEM
AND THERE ARE ENZYMES THAT
CLEAVE THE SAME MOLECULE IN
HUMAN BUT THESE ARE NOT THE SAME
FAMILY SO THEY EVOLVED
SEPARATELY IN THOSE LINEAGES.
THERE'S NO STRUCTURAL HEMOLOGY
THERE. I IMAGINE THERE MIGHT
BE. WE HAVEN'T FOUND THEM YET.
>>ONE MORE IF YOU DON'T MIND.
>>LAST ONE.
>>I PROMISE. DO YOU SEE ANY
ROLE FOR MODIFIED NUCLEOTIDES
MADE IN BACTERIA IN A MICROBIOME
CONTEXT BEING TAKEN UP BY THE
SURROUNDING EUKARYOTIC CELLS?
ARE THESE ABSOLUTE CONVERSIONS
OF THE ENTIRE POOL INSIDE THE
CELL?
>>WE HAVEN'T LOOKED INTO THAT.
GOOD QUESTION, BUT WE HAVEN'T
EXPLORED IT.
>>OKAY. MY LIPS ARE SEALED.
>>ON THAT NOTE, ON BEHALF OF
LAMBDA LUNCH I WANT TO THANK YOU
FOR SUCH A FASCINATING TALK AND
THE AUDIENCE FOR SUCH A GREAT
DISCUSSION. I WANT TO INVITE
EVERYBODY HERE, LIVE AND PRESENT
IN THE AUDIENCE TO RECEPTION.
WITH COOKIES AND COFFEE,
SPONSORED BY LAMBDA LUNCH.
THEY'RE REAL COOKIES AND REAL
COFFEE. PLEASE JOIN US OUT
THERE TO CONTINUE THE
DISCUSSION. SO THANK YOU VERY
MUCH.
>>THANK YOU VERY MUCH. THANK
YOU.
>>(APPLAUSE).