>> GOOD AFTERNOON. I THINK WE'LL GET STARTED. I KNOW MANY OF YOU ARE CONFUSED AS TO WHAT YOU'RE DOING HERE ON A THURSDAY BUT THIS IS A WEDNESDAY AFTERNOON LECTURE ON THURSDAY. AND I'M REALLY DELIGHTED ON BEHALF OF THE LAMDA LUNCH INTEREST GROUP TO INTRODUCE TODAY'S SPEAKER JO HANDELSMAN. JO IS A PIONEER IN THE STUDY OF MICROBIAL COMMUNITIES AN INTERACTION OF BACTERIA WITH HOSTS AND I THINK YOU'RE GOING TO HEAR ONE STORY OR SOME OF THAT TODAY. SO JOSEPHED HER UNDERGRADUATE EDUCATION AT CORNELL AND Ph.D. IN MOLECULAR BIOLOGY AT THE UNIVERSITY OF WISCONSIN. I DON'T WANT TO DISCOURAGE ALL THE POST-DOCS IN THE AUDIENCE BUT AFTER A YEAR OF POST-DOC SHE BECAME ASSISTANT PROFESSOR AT WISCONSIN AND STAYED THERE AND ROSE THROUGH THE RAINGS AND EVENTUALLY MOVED TO THE DEPARTMENT OF BACTERIOLOGY WHERE SHE BECAME CHAIR IN 2007. IN 2010 SHE MOVED TO YALE WHERE SHE'S PROFESSOR IN THE DEPARTMENT OF MOLECULAR AND DEVELOPMENTAL BIOLOGY. SO HER WORK ON STUDYING MICROBES IN THE INSECT GUT HAS SERVED AS A MODEL FOR DISU DIS -- STUDYINGS OF THE HUMAN MICROBIOME AND SHOULD BE OF GREAT INTEREST TO A LOT OF THIS COMMUNITY. IN ADDITION TO HER SCIENCE WHICH IS REALLY GROUND BREAKING, SHE'S ALSO BEEN A LEADER IN SCIENCE EDUCATION AND FOR THOSE OF YOU WHO ARE INTERESTED, THERE WILL BE A SESSION, SHE'LL SPEAK TOMORROW MORNING AT NINE IN THE NATIONAL LIBRARY OF MEDICINE VISITORS CENTER ABOUT SCIENCE EDUCATION. THAT'S SPONSORED BY THE AA (INAUDIBLE). SHE'S NOW WITH WISCONSIN AND NOW YALE, SHE'S BEEN HHMI PROFESSOR DEVELOPING NOVEL TEACHING STRATEGIES, CO-ORGANIZED INSTITUTE ON UNDERGRADUATE EDUCATION AND BEEN INTERESTED IN DEVELOPING PROGRAMS FOR TEACHING MENTORING WHEREVER SHE'S BEEN. SO THERE'S -- HEAR MORE ABOUT IT TOMORROW IF YOU WOULD LIKE. SO I WON'T SPEND TOO MUCH TIME ON THE LIST OF MON NORS, THEY REFLECT THESE TWO STRONG THREADS IN HER CAREER, INNOVATIVE SCIENCE AND EXCELLENCE IN TEACHING. I DISCOVERED YOU HAD ONE THAT I HAD NEVER ACTUALLY KNEW EXISTED WHICH IS THE PRESIDENTIAL AWARD FOR EXCELLENCE IN SCIENCE MATHEMATICS AND ENGINEERING MENTORING. WHICH SHE RECEIVED IN 2011. SO THAT'S -- WE LOOK FORWARD TO YOUR TALK, THE TITLE IS UP THERE AND THANK YOU FOR COMING TO VISIT US. >> THANK YOU SUZANNE, THANK YOU ALL FOR THE INVITATION TO BE HERE AND FOR JOINING US THIS AFTERNOON. FOR THOSE OUT IN THE BOONDOCKS, THIS IS LIKE COMING TO MECCA. SO I HAVE BEEN REALLY ENJOYING FINDING OUT WHAT INTRAMURAL RESEARCH AT NIH IS ABOUT. AND ENJOYING THE BEAUTIFUL WEATHER, IT'S MUCH BETTER HERE THAN IN NEWHAVEN TODAY SO THANKS FOR ARRANGING THAT. SO I THOUGHT I WOULD TALK TODAY ABOUT THE INSECT GUT AND INTERACTIONS AMONG MICROBES IN THE GUT BOTH BENEFICIAL AND DETRIMENTAL TO INSECT HEALTH. MY LAB IS INTERESTED IN INTERACTIONS WITHIN THE MICROBIAL COMMUNITIES AND FINDING MODEL SYSTEMS TO TAKE APART ELEMENTS OF COMMUNITIES AND FIGURE HOW THEY FUNCTION, HOW THEY RESPOND TO THE ENVIRONMENT AND TO INVADERS AND HOW THEY AFFECT THEIR HOSTS. SO I DECIDED IN ADDITION O THE INSECT PART I WOULD GO FURTHER BACK AND SHOW YOU WHERE SOME OF THE MOLECULES THAT WE'RE INTERESTED IN IN THE INSECT SYSTEM CAME FROM AND A PLANT SYSTEM. SO I'M GOING TO START WITH THE PLANT SYSTEM, PLANT MICROBE INTERACTION AND THEN TALK ABOUT TWO INSECTS INTERACTIONS. BEFORE ID THAT, IT'S WORTH MENTIONING THAT MICROBIAL COMMUNITIES ARE IN THE NEWS A LOT THESE DAYS FOR VERY GOOD REASON. WHAT HAS EXPLODED IN THE KNOWLEDGE ARENA IN MICROBIOLOGY THE LAST 30 YEARS IS THE REALIZATION THERE IS ESSENTIALLY NO FUNCTION ON EARTH THAT IS NOT IN SOME WAY MEDIATED BY THE MICROORGANISMS, PARTICULARLY BACTERIA AND ARCEA BUT SOME EUKARYOTIC ONES AS WELL. MOST PEOPLE I FIND IN THE WORLD DON'T THINK ABOUT THE MICROBES FIRST WHEN THEY GO TO THE EAT OR THINK ABOUT TAKING A SWIM OR WHATEVER YOU MIGHT DO, THE MICROBES SHOULD BE YOUR FIRST FOCUS. SO I JUST MADE A LIST OF SOME OF IMPORTANT ELEMENTS OF THE WORLD AROUND US INFLUENCED BY THE MICROBES. THE FIRST IS OF COURSE ONE OF MY GREATEST INTERESTS, THAT'S THE MAMMALIAN GUT. AND THAT'S ONE OF THE MOST COMPLEX ENVIRONMENTS ON EARTH. ONE OF THE MOST COMPLICATED AND DYNAMIC COMMUNITIES OF MICROORGANISMS. AND WE'RE HOPING THAT OUR SYSTEM IN INSECTS WILL BE A MODEL FOR THAT AND I'LL COME BACK TO THAT LATER. DISEASE SUPPRESS SIF SOILS, THESE ARE SOILS THAT DEVELOP THE ABILITY TO SUPPRESS PLANT DISEASE AND KEEP CROPS HEALTHY. THIS HAPPENS AS A MICROBIAL COMMUNITY, NOT A SINGLE ORGANISM RESPONSIBLE, IT'S A COMPLEX SYSTEM AND DYNAMIC SYSTEM OF SPECIES COMING TOGETHER TO HAVE A NOTICEABLE OBSERVABLE AND IMPORTANT AFFECT FOR CROP PRODUCTION. IT'S ONE THAT'S POORLY UNDERSTOOD. AS YOU MIGHT IMAGINE, IT'S VERY DIFFICULT TO TAKE THESE INTERACTIONS APART. AND SO THAT'S ONE MUCH LIKE THE HUMAN GUT THAT WILL TAKE A LOT OF DISSECTION BECAUSE COMMUNITIES ARE SO SPECIES RICH AND THE RESULTS IN MANY CASES ARE DIFFUSE IN TERMS OF MONITORING HOST HEALTH. THINGS LIKE GREENHOUSE GAS REGULATION, DEGRADATION OF POLLUTANTS, THE FACT THAT OUR WATER SUPPLY DOES NOT HAVE GASOLINE IN IT, ALL THOSE ARE AFFECTED BY AND KEPT GOING BY MICROORGANISMS. SO I THINK IT'S IMPORTANT TO REMEMBER THE MICROBES ARE THE MEDIATORS AND THE BUFFERS OF JUST ABOUT EVERY KIND OF ENVIRONMENTAL CHANGE WHETHER THAT ENVIRONMENT IS THE WORLD AROUND US, THE AIR OR THE SOIL OR THE OCEAN, OR IS A HOST OR HUMAN THAT HOSTS A COMMUNITY. IT'S ALSO IMPORTANT THAT AFTER ABOUT HUNDRED YEARS OF TRYING TO GET BACTERIA AS PURE AS POSSIBLE, TYPICALLY IN SINGLE CULTURE OR WHAT WE CALL PURE CULTURE, WE FINALLY BEGUN TO REALIZE AS A SCIENTIFIC COMMUNITY THAT THE WORK OF MY CROABS IS -- MICROBES IS DONE IN MIXED SPECIES ENVIRONMENTS AND THEY RARELY DO WORK AS SINGLE SPECIES. SO THE CHALLENGE OF THE LAST DECADE AND MOVING AHEAD ONE OF THE BIG CHALLENGES IN MICROBIOLOGY THE NEXT DECADE WILL BE TAKING MAGNIFICENT KNOWLEDGE WE ACQUIRED IN SECOND-OF 20th CENTURY ABOUT MICROBIAL GENETICS AND PHYSIOLOGY AND FUNCTION IN THE UNICELLULAR AND SINGLE SPECIES ENVIRONMENT AND TRABS TRANSLATING THAT KNOWLEDGE INTO THESE VERY COMPLEX MULTI-SMEE SIS ENVIRONMENTS. SO -- MULTI-SPECIES ENVIRONMENTS. SO INTEREST IN THE GUT COMMUNITY COMES FROM RECENT WORK THAT SHOWS THAT INFLAMMATORY BOWEL DISEASE OF VARIOUS TYPE, OBESITY, COLON CANCER, DEPRESSION AN DIABETES SEEM TO BE ASSOCIATED WITH MICROBIAL COMMUNITIES ASSOCIATED WITH THE HUMAN BODY IN PARTICULAR THE HUMAN GUT. THIS IS AN EXAMPLE IN A NON-HUMAN SYSTEM, A MOUSE SYSTEM, OF THE DRAMATIC EFFECT THAT THE MICROBES CAN HAVE ON THEIR HOST, IN THIS CASE MICROBES FROM A FAT MOUSE GENETICALLY OBESE OR TRANSFERRED TO THIS MOUSE HERE WHO USED TO LOOK JUST LIKE THE ONE NEXT TO IT, AND AFTER A NUMBER OF WEEKS WITH THESE MICROORGANISMS IN HER GUT THIS MOUSE BECAME EXTREMELY OBESE. SO THIS IS A CLEARLY A VERY COMPLEX DISEASE THAT HAS A COMBINATION OF LIFESTYLE ISSUES, GENETIC ISSUES AND A VERY STRONG COMPONENT OF MICROBIAL ISSUES. SO MY LAB FINDS THOSE INTERESTING AND THE MODEL SYSTEMS THAT ARE AVAILABLE FOR PARTICULARLY THE FIRST THREE ARE DEVELOPING RAPIDLY IN MICE AND EVEN HUMANS BUT WE THINK THERE ARE FUNDAMENTAL BASIC RESEARCH CHALLENGES THAT NEED SIMPLER SYSTEMS. SO I WILL TALK FIRST ABOUT THE ANTAGONISM BETWEEN BACILLASERUIS ON PLANTS,S THAT TWO PARTNER INTERACTION ON A HOST, THEN I'LL TALK ABOUT THE COOPERATION, THE POSITIVE INTERACTION BETWEEN BACILLSEUS IN AN INSECT AND THEN END WITH COOPERATION BETWEEN BA CRIRKSSS GENERAL SUS AND THE NORMAL GUT COMMUNITY. SO THE MODEL SYSTEM WE LOOKED FOR HAD TO BE SIMPLE, MULTI-SPECIES TO BE A COMMUNITY BUT NOT MANY MULTI-SPECIES. IT HAD TO BE TWO OR THREE OR A FEW SPECIES. WE NEEDED A SYSTEM THAT WE COULD USE HIGH THROUGH PUT SCREENING WITH BECAUSE THE BACKBONES OF WHAT WE DO IS LOOKING FOR MUTANTS, BACTERIAL MUTANTS ALTERED IN A PARTICULAR CAPABILITY TO IDENTIFY GENES THAT FUNCTION IN PARTICULAR WAY, EITHER IN A TWO PEE SIS INTERACTION WITH HOST OR CONTEXT OF A COMPLEX COMMUNITY WHICH I THINK IS THE REALLY EXCITING WORK THAT'S STARTED TO EMERGE. WE NEED AD SYSTEM THAT COULD BE MANIPULATED WHERE YOU HAVE INPUTS AND MEASURE OUTPUTS AND IT NEEDED TO BE LARGE ENOUGH THAT WE COULD DO SOME SORTS OF IMAGING AND ALSO TAKE BLOOD SAMPLES. WE KNEW WE WERE MOVING TO INSECT. OR ANIMAL. WE NEEDED A SYSTEM THAT WOULD PROVIDE ALL THOSE THINGS WHICH MEANT THAT IT WAS SIMPLE YET LARGE. SO WE TRIED TO GET A SYSTEM IN DROSOPHILA BUT IT'S VERY HARD TO GET DROSOPHILA BLOOD. SO WE HAVE A MAJOR CHALLENGE WITH THAT. WE HAVE WORKED ON IT ON THE SIDE AND I'LL PRESENT A LITTLE DATA AT THE VERY END ABOUT WHAT'S HAPPENED WITH DROSOPHILA. I THINK IT IS A GOOD MODEL SYSTEM. BUT INSTEAD WE MOVE TO AN INSECT THAT IS MUCH LARGER, CATERPILLAR AND WE GOT LED TO THAT BY IT NOT ONLY BEING A GREAT MODEL SYSTEM BUT AN INTERESTING CIRCUITOUS ROUTE TO TELL YOU ABOUT BEFORE I MOVE TO THE INSECT SYSTEM. SO WHEN I WAS THE UNIVERSITY OF WISCONSIN IN PLANT PATHOLOGY DEPARTMENT, I KNEW ABSOLUTELY NO PLANT PATHOLOGY WHEN I STARTED MY JOB SO I STARTED LEARNING IT. AND I DISCOVERED THIS INTERESTING DISEASE CAUSED BY THE OMICETES, A GROUP OF PROTUS THAT CAUSE DEVASTATING PLANT DISEASES, ONE IS THE CAUSAL AGENT OF THE IRISH POTATO FAMINE AND MANY OTHER DAMAGING EPIDEMICS OF PLANTS. AND THEY CAN INFECT THE HOSTS AS SEIDLEINGS OR AFFECT THEM AS LARGER PLANTS OR AFFECT THE FRUIT AS YOU CAN SEE HERE. SO THIS IS AN EXAMPLE OF PITH YUM, ONE OF THE OMYSETE SERKS WE STUDIED GROWING ON A QUEUE CUCUMBER, THAT'S 24 HOURS OF GROWTH FROM 500 CELLS OF THE OMYCE TO THAT MASSIVE QUANTITY OF BIOMASS TO LIQUIFYING THE CUCUMBER UNDERNEATH SO THIS IS A SERIOUS SET OF DEES FOR FAMPLERS AND THEY'RE LOVELY TO STUDY BECAUSE -- FARMERS BECAUSE THEY PRODUCE GORGEOUS STRUCTURES AND HAVE SOME VERY INTERESTING MOTILITY BEHAVIORS AROUND BOTH OTHER MICROORGANISMS AND PLANT HOSTS. SO THESE ARE THE CRITICAL STRUCTURES OF (INDISCERNIBLE) IN THIS CASE WHICH ARE THE SEE SPORES WHICH CAN SWIM IN AN AQUEOUS TARGET AND FIND A TARGET BY CHEMO TACTIC ATTRACTION AND INFECT THE PLANT. SO NORMALLY WE WORK IN THIS PART OF THE PROJECT WITH ALFALFA SHOWN HERE, PARTLY BECAUSE IT'S A TINY LITTLE PLANT AND THEREFORE YOU CAN GROW IT IN TEST TUBES WHICH MET REQUIREMENTS FOR A MODEL SYSTEM AND PARTLY BECAUSE IT'S VERY IMPORTANT TO THE AGRICULTURE OF WISCONSIN AND PARTLY BECAUSE IT'S SUSCEPTIBLE TO A FEW OF THE DISEASES. SO WE LOOKED FOR WAYS OF MANAGING THESE DISEASES, ONE OF THE HISTORICAL FACT IS THE OMYCETES WERE TYPED AS FUN EYE FOR 150 YEARS AND MANY HEARD A FUNGUS CAUSED THE POTATO FAMINE AND MYLODGENY INDICATED THEY ARE NOT FUNGI, THEY'RE PROTUS AND VERY DISTANT FROM THE FUNGI THAT. EXPLAINED WHY NONE OF THE FUNGICIDES USED TO CONTROL MAJORITY OF DISEASES ON PLANTS ARE THAT EFFECTIVE ON OMYCETES SO WE THOUGHT THERE WERE ORGANISMS OUT THERE ADAPTED TO THE ROOT SYSTEM THAT THE OMYCE THETE INFECTS AND ALSO INHIBIT THE ACTIVITY OF THE OMYCETE. SO THE IDEA WAS THERE ARE BACTERIA ON THE SURFACE OF A ROOT THAT ACT AS A PHALANX AND OUR PREDICTION WAS THAT WE COULD FIND ON HEALTHY ROOTS MEMBERS OF THAT PHALANX THAT IS DISEASE SUPPRESSIVE. SO THERE IS AN EXAMPLE OF OUR PHALANX AND WE LOOKED AROUND LOOKING AT ALFALFA PLANTS THAT WERE HEALTHY, NO EVIDENCE OF INFECTION. ISOLATED, CULTURABLE BACTERIA SHOWN HERE, AND BEGAN TO TEST THOSE BACTERIA THAT CAME FROM HEALTHY ALFALFA ROOTS FOR ABILITY TO SUPPRESS DISEASE. SO THIS IS THE ALFALFA SCREENING SYSTEM, YOU CAN'T SCREEN THOUSANDS OF ORGANISMS AND MUTANTS AND WE HAVE USED IT FOR THAT. VERY RAPID SCREEN AND EASY TO TO MANIPULATE THE INPUTS AN OUTPUTS. THIS IS AN EXAMPLE OF THE DAMAGE THAT THE OMYCETES TO ALFALFA AND THIS IS SURVIVAL AFTER BEING TREATED WITH ONE OF THE BACTERIA WE FOUND IN THIS COLLECTION. SO IN THE FIRST SCREEN WE DID ABOUT A THOUSAND ORGANISMS AND ONLY FOUND ONE THAT WAS EXTREMELY DISEASE SUPPRESSIVE BUT IT WAS EXTREMELY DISEASE SUPPRESSIVE. IT WAS EFFECTIVE. SO WE KNEW THIS WAS AN UNUSUAL ORGANISM SINCE IT WAS EFFECTIVE AND EVERYTHING ELSE WE TESTED OVER 900 ISOLATES WERE EFFECTIVE. IT STATE YOUR TUSHED OUT TO BE A STRAIN OF BACILLA SERIOUS, BECAUSE IT'S IN THE E A GROUP WITH TWO OTHER SPECIES OF BACTERIA WITH DRAMATIC EFFECTS ON HOSTS. ONE OF THOSE IS THE ANTHRAX BACTERIA BACILLA ANTHRAXAS CLOSE TO BACILLA SERIOUS AN DIFFERS LARGELY IN THE?[: PLASMA IT CARRIES THAT MAKES IT VIRULENT AGAINST MAMMALS TESTIMONY OTHER ONE IS THE BACILLAS (INDISCERNIBLE) THAT IS A PATHOGEN OF INSECTS WITH A DRAMATIC EFFECT ON SURVIVAL AS WELL. SO IT WAS INTERESTED WE FOUND A GROUP OF DISEASE SUPPRESSIVE BACILLI IN THE SAME GROUP AS THERE ARE TWO VIGOROUS DISEASE CAUSING AGENTS. SO THIS IS AN EXAMPLE WHEN WE CODED A SEED WITH BACILLA SERIOUS. WE SEE VERY GOOD PROTECTION, UNINOCULATED CONTROL, THIS IS INFECTED SOYBEANS AND THIS IS AFTER PROTECTION WITH BACILLA SERIOUS. SO WE CONVINCED OURSELVES THIS WAS A ROBUST INTERACTION AND UNUSUAL INTERACTION AND THEREFORE PROVIDED A GOOD BASIS FOR STUDY BECAUSE THERE WERE SOLID ROBUST DIFFERENCES TO LOOK AT. SO OUR FIRST APPROACH WAS TO LOOK FOR MUTANTS OF BACILLA SERIOUS THAT DID NOT SUPPRESS DISEASE. EVERY MUTANT WE FOUND NON-SUPPRESSIVE IN THE DISEASE DISEASE TEST ASSAY I SHOWED YOU TURNED OUT NOT TO PRODUCE OWN INHIBITION TESTED AGAINST (INAUDIBLE). SO THESE ARE PLACE, THE WHITE FUZZY IS FYTOTHEUS ON PLACE. EACH ONE CONTAINS A MUTANT AND EVERY MUTANT NEGATIVE HERE WAS ALSO NEGATIVE IN THE PLANT ASSAY. THAT SUGGESTED TO US ONE OF THE MODES OF ACTION OR PART OF THE MODE OF ACTION IS DISEASE SUPPRESSION WAS SOME SORT OF DIFFUSIBLE AGENT OR ANTIBIOTIC THAT AFFECTED THE OMYCETE. WE WORKED QUITE HARD TO PURIFY AND DETERMINE THE STRUCTURE OF THIS MOLECULE, IT WAS CHALLENGING BECAUSE IT'S EXTREMELY POLAR AND NO CHEMIST I TALKED TO FOR YEARS WANTED TO WORK ON IT BECAUSE IT'S POLAR. SO WE STRUGGLED A LONG TIME. A WONDERFUL COLLEAGUE JOHN COLLARTY AT CORNELL AND NOW HARVARD OFFERED FINALLY TO HELP US WITH THE STRUCTURAL PART. IT WAS REALLY CHALLENGING MOLECULE IN A CLASS BY ITSELF SO IT'S A NEW ANTIBIOTIC BUT IT ALSO REPRESENT AS NEW GROUP OF ANTIBIOTICS. SO WE DIDN'T HAVE A LOT OF NMR SPECTRA AND OTHER SPECTRA TO HELP US DISCERN PIECES OF THE MOLECULE WORKING ON THE STRUCTURE, THERE WEREN'T THE TOOLS OUT THERE FOR COMPARISON. SO WE WERE INTERESTED IN FIGURING OUT ONCE WE HAVE THE STRUCTURE AN FOUND IT WAS INHIBITOR TO CERTAIN BACTERIA AND A NUMBER OF EUKARYOTS, WE WANTED TO KNOW TO HEAR ABOUT THIS ANTIBIOTIC MODE OF ACTION I WON'T TALK ABOUT TODAY BUT ALSO BECAUSE IT WAS SO NEW AND WE IMAGINED IT HAD TO REPRESENT A LARGER CLASS, THERE HAVE TO BE OTHER MEMBERS OF THIS GROUP OUT THERE BECAUSE THERE'S SO FEW SINGLETONS OF ANYTHING IN BIOLOGY. IT WOULD BE INTERESTING TO FIND OUT HOW THE ANTIBIOTIC WAS SYNTHESIZED. SINCE THIS WAS DONE IN UTERO WORKING VERY HARD FIGURING HOW ANTIBIOTICS ARE MADE, THEY'RE COMPLEX PATHWAYS AND EVEN THE BELL KNOWN ONES ARE DIFFICULT TO DISCERN. WE WERE A LITTLE BIT HESITANT ABOUT THIS WITH A NEW ANTIBIOTIC STRUCTURE, NEW CLASS OF ANTIBIOTICS AND NOT KNOWING WHERE TO START. SO OUR APPROACH WAS TO DO THE SAFE THING, TO CLONE THE GENE FOR RESISTANCE. THAT WAS SAFE BECAUSE E. COLI HAPPENS TO BE SENSITIVE TO IT SO WE DID A SHOTGUN LIBRARY IN A BACK VECTOR WHICH TAKES LARGE FRAGMENTS OF BACTERIAL CHROMOSOMES. WE PUT THAT IN E. COLI AND THEN SELECTED FOR RESISTANCE. THE THINK HEARING WAS THAT MOST BIOSYNTHETIC PATHWAYS CARRY A RESISTANCE GENE TO THE ANTIBIOTIC BEING SYNTHESIZED BY THAT PATHWAY. BECAUSE IF THEY DIDN'T OFTEN THEY WOULD COMMIT SUICIDE BECAUSE THE PRODUCING ORGANISM IS SENSITIVE TO THE ANTIBIOTIC IT PRODUCES SO THE PREDICTION, IF WE FOUND THE RECESS STANCE GENE WE ALSO WILL FIND THE BIOSYNTHETIC PATHWAYS SO THE IDEA WAS TO SEQUENCE OUT FROM THE RESISTANCE GENE AND DETERMINE WHETHER BIOSYNTHETIC GENES WERE THERE. SO RESISTANCE TURNS OUT TO BE AN INTERESTING STORY IN ITSELF. THE RESISTANCE GENE IS SHOWN HERE, THE MAR AND IT'S PART OF A LARGE CLASS ACETYL TRANSFERASES, THE G MAP GROUP, A VERY LARGE GROUP OF ENZYMES BUT ACTUALLY AT THE TIME WE ISOLATED IT, THAT GROUP HAD NOT BEEN IDENTIFIED AND SO WE DIDN'T KNOW WHAT TO LOOK FOR IN TERMS OF ACTIVITY. WE JUST HAD A SEQUENCE. SO[HAT PROBABLY DATES ME. TODAY THERE ARE THOUSANDS OF G MAP SEQUENCES IN THE DATABASES. SO WE GUESSED IT WAS MOST LIKELY TO BE AN ENZYME MODIFYING ANTIBIOTIC, THE MOST COMMON MECHANISM OF RESISTANCE WITH MOST GENES LOOKED AT. WE DECIDED TO TEST FOR MODIFICATION OF ANTIBIOTIC BY THE GENE, AND WE FOUND AFTER WE TREATED WITH PURE (INDISCERNIBLE) WITH EXTRACT OF ZMAR PRODUCING CELL, WE WOULD IN THE NORMAL CASE PRODUCE OWN INHIBITION, HERE WE'RE DROPPING ANTIBIOTIC, IT'S IN THE FILLER PAPER AND INHIBITS E. COLI QUITE WELL. BUT HERE WE TREATED IT WITH ZMR EXTRACT, WE PURIFIED THE PROTEIN TO PERFORM THE SAME EXPERIMENT AND FOUND THAT THE ANTIBIOTIC HAD LOST ALL ACTIVITY. SO WE DETERMINED THE STRUCTURE OF THE ANTIBIOTIC AFTER BEING TREATED BY -- WITH ZMAR AND SURE ENOUGH, IT HAD BEEN MODIFIED AND SO YOU SEE THIS AMINO GROUP WHERE (INDISCERNIBLE) A IS CRITICAL FOR ACTIVITY AND THIS IS THE GROUP THAT GETS ACETYLATED BY THE ACETYL TRANSFERASE ENCODED BY ZMAR. SO WE DESCRIBED AN EARLY MEMBER OF THIS GROUP OF ACETYL TRANSFERASES THAT TURN OUT TO BE UBIQUITOUS IN THE BACTERIAL WORLD NOT ONLY CONFERRING RESISTANCE BUT MANY OTHER CELLULAR FUNCTIONS AS A REGULATORY FEATURE. SO THAT WAS INTERESTING IN ITSELF, THIS IS AN ACETYL TRANSFERASE THAT WORKING OS ON A COMPOUND THAT IS QUITE DIFFERENT FROM MOST OTHER ANTIBIOTICS WE KNOW OF AND MOST OTHER SUBSTRATES OF THE ACETYL TRANSFERASES. SO THAT LED US TO ASK IF RESISTANCE MECHANISM IS INTRIGUING, WHAT CAN WE LEARN FROM THE GENES FLANKING THE RESISTANCE GENE ABOUT BIOSYNTHESIS? WOULD WE FIND A PATHWAY FOR BIOSYNTHESIS THERE? AND IN FACT, WE DID, WE DID A RANDOM MUTAGENESIS AND LOOKED FOR MUTANTS THAT LACKS THE ABILITY TO PRODUCE (INDISCERNIBLE) AND ALSO SEQUENCED THE GENES AROUND ZMAR WHICH IS HERE. AND FOUND MANY OF THE MUTANTS THAT WE MADE IN A RANDOM MUTANT LIBRARY MAPPED RIGHT NEAR ZMAR IN THIS REGION, THAT YOU SEE HERE. SO EVERY PLACE THERE'S AN ARROW, THOSE ARE MUTANTS FOUND THROUGH A RANDOM MIEW MUTANT SEARCH, WE MADE MUTATIONS IN THE OTHER GENES AND THEY'RE ESSENTIAL SO FAR FOR (INDISCERNIBLE) PRODUCTION. THERE ARE ABOUT 60 GENES IN THIS REGION, IT'S A MASSIVE CLUSTER OF DNA AND WE FOUND THAT AMAZING LOOKING AT THE SMALL PETITE STRUCTURE OF DISWITTER MYOSIN. SO NO ONE WAS PREPARED FOR THE COMPLEXITY OF THE BIOSYNTHETIC PATHWAY. AS WE WERE FINISHING THE MUTANT ANALYSIS AND MAPPING THE GENES THAT WE HAD FOUND WE STARTED TO COLLABORATE WITH MICHAEL THOMAS WHO IS A PROFESSOR AT THE UNIVERSITY OF WISCONSIN WHO STUDIES ANTIBIOTIC BIOSYNTHETIC PATHWAYS. HE LOOKED AT WHERE OUR MUTANTS MAPPED AND THE GENES THAT THEY FELL IN AND THE STRUCTURES OF ZWITER MYOSIN AND HE DEVISED THIS PATHWAY FOR THE INTERMEDIATES IN ZWITTERMICIN BIOSYNTHESIS AND I CONTINUE THE FIND THAT ONE OF THE GREAT FEATS OF MY COLLABORATORS BECAUSE IT INVOLVED TWO THINGS, FIRST IT INVOLVED ONLY SOME OF THE GENES HAD BEEN IDENTIFIED. SO HE WAS WORKING WITH PARTIAL INFORMATION ABOUT A DOZEN GENES IN THE WHOLE PATHWAY HAS CLOSE TO 60. AND SECOND, EVEN MORE IMPORTANT, WAS THAT HE HAD TO PROPOSE THAT THERE WERE INTERMEDIATES THAT HAD NOT ACTUALLY BEEN OBSERVED IN ANTIBIOTIC BIOSYNTHETIC PATHWAYS BEFORE. SO THE PATHWAY THAT HE PROPOSED WAS RELATED TO THE POLYKETIDES. I'LL EXPLAIN THAT IN A MINUTE. THOSE MOLECULES ARE MADE FROM REPEATING UNITS AND THERE ARE -- THERE WERE FOUR WHEN WE DID THIS WORK, THERE WERE FOUR REPEATING UNITS KNOWN TO BE USED IN POLYKETIDE SYNTHESIS. AND THE AMINO ALLELE AND HYDROXY MA ALLELE HAS NOT BEEN DISCOVERED YET, THIS WAS THE FIST SYSTEM THEY WERE DESCRIBED SO THERE'S A BOLDNESS IN WHAT MICHAEL DID THAT NOT ONLY WORKING WITH A NOVEL GROUP OF MOLECULES WITHOUT ALL THE GENES KNOWN, AND HAVING TO PROPOSE TWO NEW INTERMEDIATES IN THE BIOSYNTHESIS. HE PUT THAT TOGETHER IN A PATHWAY THAT THEN WENT ON TO SHOW TO DEMONSTRATE IN VITRO AND IN VIVO WAS IN FACT THE WAY THAT ZWITTERMICIN WAS MADE. SO IT'S MADE ON A VERY INTERESTING LARGE PROTEIN, THE POLYKETIDES ARE A LARGE CLASS OF ANTIBIOTICS THAT DON'T LOOK MUCH LIKE ZWITTERMICIN BUT SHARE STRUCTURAL FEATURES IN THE POLYKETIDE SPBTSIS THEY START WITH EXTENDER UNITS AND THE EXTENDER YIEWBTS ARE PIECED TOGETHER IN A SEQUENTIAL WAY LENGTHENING THE CHAIN PRODUCING THE POLYKETIDE AND ANTIBIOTICS ARE MODIFIED SOMETIMES CYCLIZED AND OFTEN DECORATED WITH IMPORTANT MODIFICATIONS. SO AN EXAMPLE OF A POLYKETIDE IS E REIT THROW MYOSIN, A FAIRLY COMPLEX ONE AND REQUIRES 125 KB OF DNA FOR SYNTHESIS AND THAT'S BEEN ACCOMPLISHED IN A LET ROG GOWS SYSTEM SO -- HETEROLOGOUS SYSTEM. SO THAT'S BETTER UNDERSTOOD. AND YOU CAN SEE THE FEATURES THE KETO GROUPS THUS THE POLYKETIDE NAME. SO THIS WAS EXIEDING BECAUSE -- EXCITING BECAUSE MICHAEL HAD BEEN CORRECT THAT IN FACT THERE WERE TWO GENES INVOLVED IN THE BIOSYNTHETIC PATHWAY FOR THESE TWO NEW EXTENDER UNITS, THE AMINO MALONEL AND HYDROXYL COA SO INSTEAD OF FOUR IT HAS SIX EXTENDER UNITS TO USE IN THE PRUX OF SYNTHETIC POLYKETIDE, THAT'S AN AREA OF INTEREST TO PIECE TOGETHER SUBUNITS IN NEW COMBINATIONS AND PERMUTATIONS TO CREATE NEW ANTIBIOTICS. SO THE MODEL MICHAEL DEVELOPED WAS CORRECT. ANTIBIOTICS START WITH SERE REN, INVOLVES THE PIECING TOGETHER OF THE POLYKETIDE PIECES AN HZ A NUMBER OF FEATURES, ENZYMES THAT LOOK LIKE THE ENZYMES INVOLVED IN NON-RIBOSOMAL PEPTIDE SYNTHESIS. AND SO THIS IS THE COMPLEX PATHWAY, I WON'T GO INTO ALL THE DETAILS, THIS PART HAS BEEN PUBLISHED. BUT IT REALLY -- I THINK WAS A VERY EXCITING FEATURE OF MICHAEL'S WORK TO MAKE THE THEORETICAL PREDICTION AND REDUCE IT TO PRACTICE AND TO REALITY USING COMBINATION OF MUTANTS WE IDENTIFIED, SEQUENCED AND A LOT OF TOUGH BIOCHEMISTRY. NCI AT THIS POINT WORK ON SYMBIOSIS WE FOUND A BACILLA SERIOUS WITH A DRAMATIC DISEASE SUPPRESSIVE ACTIVITY ON PLANTS. WE FOUND A NEW ANTIBIOTIC RESPONSIBLE FOR THAT ACTIVITY BUT THE NEW ANTIBIOTIC IS MUCH MORE INTERESTING THAN JUST FOR ITS BIOLOGICAL ACTIVITY BECAUSE I HAVE POWERFUL RESISTANCE MECHANISM DUE TO ACETYL TRANSFERASE. AND MORE IMPORTANTLY SYNTHESIZED BY A VERY NOVEL HYBRID PATHWAY INVOLVING BOTH PEPTIDE AND POLYKETIDE ELEMENTS. FINALLY THAT PATHWAY PROVIDED MEANS TO SYNTHESIZE TWO NEW EXTENDER UNITS WE NOW KNOW ARE INVOLVED IN ANTIBIOTIC SYNTHESIS IN NATURE MAKING THE ZWITTERMICIN PATHWAY UNIQUE BECAUSE IT WAS THE FIRST AND ONE OF FEW THAT USES THE EXTENDER UNITS BUT CERTAINLY IS A MAJOR CONTRIBUTION TO THE COMMONTORIAL APPROACHES TO SYNTHESIZE NEW ANTIBIOTICS. SO I WOULD LIKE TO EXPLAIN NOW HOW THAT WORK RELATES TO OUR WORK WITH BACILLA SERIOUS AND BACILLAS THERGENSUS AND HOW THEY COME TOGETHER TO INSECT HOST. SORT OF BY ACCIDENT WE FOUND ZWITTERMICIN HAD A DIFFERENT AFFECT IN THE DIFFERENT BIOLOGICAL SYSTEM FROM THE PLANT SYSTEM WE STUDIED. SO THIS IS BACILLAS (INAUDIBLE) ONE OF THE TRIO WE MENTIONED THAT HAVE DRAMATIC EFFECTS ON THEIR HOST, BACILLUS CARRIES ONE OR MORE PLASMIDS THAT ENCODE THE ABILITY TO PRODUCE -- O I THINK YOU CAN SEE IT HERE THIS ROM BOYD SHAPED CRIST LYNN PROTEIN SO IT FORMS A VISIBLE CRYSTAL IN THE CELL, THIS IS THE SPORE AND THE CRYSTAL TOXIN IS THERE. THIS CRYSTAL PROTEIN HAS TREMENDOUSLY AGGRESSIVE ACTIVITY AGAINST CERTAIN GROUPS OF INSECTS, THE ONE WE WORK ON ACTUALLY AFFECTS LEPADOPRIN INSECONDS AND BUTTERFLIES AND AS A RESULT OF THAT IT'S VERY HIGH ACTIVITY OF KILLING INSECTS IN A NARROW SPECTRUM MANNER BEEN USED IN AGRICULTURE FOR MORE THAN HALF A CENTURY TO CONTROL INSECT PESTS. THE DISCOVERY OF BACILLUS SERE NEXT GENSUS WAS IN 1904. VERY WELL STUDIED. WE STARRED WITH A GIPSY MOTH BECAUSE IT WAS OF INTEREST TO A COLLEAGUE, TURNED OUT TO MEET MANY REQUIREMENTS FOR A SIMPLE HOST MODEL SYSTEM. FOR MICROBIAL COMMUNITIES AND I'LL SHOW YOU THE FEATURES AND SUSCEPTIBLE TO BACILLAS THEREGENUS AND THE THIRD WAS FROM THE ALFALFA ROOT. LY TALK ABOUT THE GIPSY MOTH ON THE LAST SLIDE AND THIS IS THE TOBACCO HORN WORM, THIS REALLY IS A REAL PICTURE NEXT TO A MOUSE. ONE OF THE THINGS THAT WE WANTED TO HAVE ACCESS TO WAS THE BLOOD OF THESE INSECTS AND YOU CAN SEE THIS INSECT IS LARGE ENOUGH THAT WE CAN GET ABOUT A ML OF HEMALIN FOR BLOOD OUT OF A SINGLE LARVAE, SO THEY GROW FAST, EASY TO MAINTAIN, NOT SUSCEPTIBLE TO TOO MANY LAB BASED DISEASES, AND MUCH CHEAPER AND YOU DON'T HAVE TO GO THROUGH THE ANIMAL HEALTH CERTIFICATIONS THAT YOU HAVE TO WITH MICE. BECAUSE THEY DON'T HAVE BACKBONES. THESE INSECTS ARE PARTICULAR INTEREST TO US BECAUSE OF THE PROMINENCE OF THE GUT IN THEIR PHYSIOLOGY AND ANATOMY. THIS IS A CAT SCAN WITH A BARENE STAIN THAT'S FED TO THE INSECT. YOU CAN SEE THIS WHOLE DARK AREA IS THE GUT. IT FILLS UP ALMOST THE ENTIRE BODY CAVITY OF THE O INSECT. AND THE REST IS ESSENTIALLY HEMOLIN AROUND IT, AROUND THE GUT. SO THE TWO THINGS THAT WE'RE INTERESTED IN IS GUT HEALTH AND HOW HEMOLIN INTERFACES WITH THAT. THOSE ARE THE TWO FEATURES THAT MAKE UP MOST OF THE INSECT BODY. IT'S VERY EASY SYSTEM TO MANIPULATE BECAUSE THE INSECT WILL EAT MOST THINGS THAT WE FEED IT IF WE HIDE THEM IN THE NORMAL FOOD. WE CAN PUT ANYTHING FROM ZWITTERMICIN TO PLANT MATERIAL AND OTHER ANTIBIOTICS AND THE INSECTS DON'T SEEM TO REJECT THEM. SO THIS SYSTEM AND THE GIPSY MOTH SYSTEM IS THE TWO I'LL TALK ABOUT AND WE FOUND SYSTEM RESULTS, I'LL POINT OUT A FEW OF THE PLACES WHERE THEY DIVERGE BUT IF I'M TALKING ABOUT A GIPSY MOTH EXPERIMENT YOU'LL SEE A GIPSY MOTH, AND A TOBACCO HORN WORM YOU'LL SEE A PICTURE OF ONE. THE THING WE FOUND SOMEWHAT BY ACCIDENT WAS THAT THIS VERY INTERESTING ANTIBIOTIC ZWITTERMICIN WITH ANTI-BACTERIAL ACTIVITY ALSO HAD TREMENDOUSANT TO SYNERGIZE OR POTENTIATE THE ACTIVITY OF BT TOXIN. SO HERE WE'RE LOOKING AT INSECT MORTALITY WITH INCREASING AMOUNTS OF ZWITTERMICIN. A CONSTANT AMOUNT OF BT TOXIN SO WITHOUT ANY ZWITTERMICIN THERE'S 20% MORTALITY IN THE EXPERIMENT BUT AS WE INCREASE THE CONTENT OF THE FOOD, WE SEE DRAMATIC INCREASE IN THE KILLING BY BT. THIS IS STILL TO THIS DAY WE DISCOVERED THIS A NUMBER OF YEARS AGO, TO THIS DAY THERE'S NO SYNERGIST WE CAN FIND OR IN THE LITERATURE THAT COMES CLOSE TO THE POTENTIATING ABILITY OF ZWITTERMICIN. WE LOOKED FAR AND WIDE FOR OTHER COMPOUNDS THAT DO THE SAME THING TO BEGIN TO UNDERSTAND WHY COMPOUNDS SYNERGIZE WITH BT AND HAVEN'T FOUND THEM SO WE HAVE AN ON GOING STUDY LOOKING AT MODE OF ACTION OF ZWITTERMICIN. I'LL TELL YOU HOW WE STARTED DOWN THE PATH AND GOT DISTRACTED BUT WE HAVE GONE BACK TO THAT. SO THIS ANTIBIOTIC IS ACTIVE AT NANOMOLAR LEVELS AS A SYNERGIST, IT'S MUCH BETTER AS SYNERGIST QUANTITATIVELY THAN IT IS AS AN ANTIBIOTIC. BUT YET THE ONLY THING WE KNEW AT THE TIME WE DID THESE EXPERIMENTS ABOUT THE ACTIVITIES OF ZWITTERMICIN, IT IS AN ANTIBIOTIC SO THE SIMPLEST HYPOTHESIS WE COULD COME UP WITH FOR ITS ACTIVITY WAS IT WAS AFFECTING THE PHALANX AGAIN. SO WE IMAGINED AS WE DID ON PLANT ROOTS THAT THE INSECT GUT HAS A MICROBIAL COMMUNITY THAT PROTECTS IT FROM INFECTION BY PATHOGENS SUCH AS BACILL BACILLSTHERNGENSUS, THE IDEA IS IF ZWITTERMICIN IS KILLING THAT COMMUNITY THE INSECT NO LONGER HAS THAT BARRIER OR PHALANX AND IT BECOMES SUSCEPTIBLE TO BT, SO THAT WAS A REASONABLE HYPOTHESIS TO TEST. SO WE WANTED TO FIND OTHER WAYS, FIRST LOOK AT THE EFFECT OF ZWITTERMICIN ON POPULATIONS OF THE GUT AND OTHER WAYS OF ACCOMPLISHING THE SAME END AND SEE WHETHER WE ACCOMPLISH THE SYNERGY. SO THE FIRST EXPERIMENT WE DID WAS SIMPLE. WE WANTED TO USE A BATTERY OF ANTIBIOTICS, WIPE OUT AS MANY MEMBERS OF THE GUT IMMUNITY AS WE COULD IN ONE TREATMENT AND SEE IF WE GOT THE SAME SYNERGY THAT WE DID WITH BT. SO THE FIRST PART WAS TO DETERMINE WHAT ORGANISMS WERE IN THE GUT. WE DID A CULTURE BASED ANALYSIS THAT DEMONSTRATED IN THE GIPSY MOTH THERE WERE SEVEN ORGANISMS, ALL MEMBERS OF THE FERMICUTES WHICH ARE THE LOW GC GRAM POSITIVE ORGANISMS AND THE GAMMA PROTEOBACTERIA. SO VERY SIMPLE AT THE PHYLUM LEVEL, THEN WE DID SEE SOME VARIABILITY AND VARIATIONS BETWEEN INDIVIDUALS AND BETWEEN POPULATIONS OF GIPSY MOTHS AT THE GENUS AND SPECIES LEVEL. BUT IN GENERAL COC CRURKSS AND STAFF ANDBACTER AND PANTORIA WERE THE ORGANISMS WE SAW CONSIST EBTSLY IN THE GIPSY MOTH GUT. WHEN WE DID A CULTURE INDEPENDENT ANALYSIS LOOKING AT THAT TIME 16S RNA DISTRIBUTION IN THE INSECT GUT, WE FOUND THREE ADDITIONAL ORGANISMS THAT DIVERGED REASONABLY DEEPLY FROM KNOWN SPECIES, THEY SEEM TO BE NEW SPECIES BUT KNOWN GENRE. ONCE AGAIN THEY FELL IN THE PROTEOBACTERIA AND FERMICUTES PHYLA BUT IN THIS CASE WE ADDED THE ALPHA PROTEOBACTERIA WITH BACTERIUM WHICH COMES AN GEESE, AND SOMETIMES IN THE -- GOES, SOMETIMES IN THE GUT, SOMETIMES NOT. I'LL TALK NOW ABOUT TWO ORGANISMS WE STUDIED THE MOST, ONE IS COCCUS FATALES AND ANTERCOCCUS IS GRAM POSITIVE, AND BACT TEAR IS NEGATIVE THEY'RE WELL DISTRIBUTED IF NOT UBIQUITOUS IN THE GUTS OF ANIMALS THAT I CAN FIND THAT HAVE BEEN LOOKED AT. SO WE THOUGHT THESE WERE NICE MODELS, IN ADDITION THEY HAVE SIMILAR EFFECTS IN THE EXPERIMENTS I'M GOING TO TELL YOU ABOUT. SO WE WENT BACK TO NOW THAT WE KNEW WHO WAS THERE BACK TO REDUCING THE POPULATIONS USING ANTIBIOTICS AND ASKING IF YOU ELIMINATE THE BACTERIAL COMMUNITY AT LEAST AS FAR AS IT CAN BE DETECTED WHAT HAPPENS TO THE SUSCEPTIBILITY OF THE LARVAE TO BT. SO ON THIS SLIDE YOU'RE SEEING AN CREASEN CONCENTRATION OF ANTIBIOTICS. AND WHICH PRESUMABLY, I'LL SHOW YOU DATA TO SUPPORT THIS, WOULD BE CORRELATED WITH A REDUCTION IN THE BACK BACTERIAL COMMUNITY OF THE GUT AND THE LARVAL MORTALITY. AND YOU MIGHT NOTICE THAT THE CURVE HERE GOES IN THE OPPOSITE DIRECTION FROM WHAT WE PREDICTED. WE PREDICTED THAT AS YOU INCREASE ANTIBIOTICS CONCENTRATION, THE PHALANX OR PROTECTIVE BACTERIA DROP IN POPULATION AND THE INSECOND WOULD BECOME MORE SUSCEPTIBLE. INSTEAD WE SAW REDUCTION IN MORTALITY. THIS IS ANTIBIOTICS WITH NO BT AND LITTLE EFFECT ON MORTALITY WITH ANTIBIOTICS WITHOUT BT. WITH BT THERE IS VERY DRAMATIC DROP IN ACTIVITY AS ANTIBIOTIC CONCENTRATION INCREASED. AT THE POINT WE FOUND NO MORE CULTURABLE BACTERIA, THAT WAS AT 125 MICROGRAMS EACH OF A COCKTAIL OF FOUR ANTIBIOTICS, THAT WAS THE POINT WHICH THE CURVE CONVERGED WITH NO LARVAL MORTALITY. SO WE FOUND OUR PHALANX HYPOTHESIS WAS ABSOLUTELY NOT CORRECT. THERE WAS NO WAY THE DATA WERE SUPPORTIVE OF IT. SO WE HAD TO GO BACK TO TO THE DRAWING BOARD AND RECONSIDER OUR RESULTS, THAT IF ZWITTERMICIN IS NOT WIPING OUT THE COMMUNITY AS THIS LAST EXPERIMENT DID WELL, IF THAT IS NOT THE MECHANISM OF SYNERGY, WHAT ELSE COULD BE GOING ON. SO WE TUSHED MORE TO EMPHASIZE THE BACILLA THERE JENSIS COMMUNITY AND TURNED TO THE TRADER HYPOTHESIS. AND THE BACTERIA IN THE GUT TURN ON THE HOST AND ASSIST BACILLAS THERENGENSIS KILLING THE INSECT HOST. SO WE PROPOSE IN FACT THE NORMAL GUT BACTERIA THAT DO NOT CAUSE ANY HARM TO THE INSECT UNDER NORMAL CIRCUMSTANCES, COLLABORATED WITH BT THAT LED TO DEATH OF THE CATERPILLAR. IF WE RESTORE INHAS BEEN TANS, WE SHOULD RESTORE BT KILLING SO WE SAW IN THE PREVIOUS SLIDE WE COULD REDUCE THE BACTERIAL COMMUNITY TO UNDECKABLE AND LOSE BT ACTIVITY. SO THE NEXT STEP WAS TO RESTORE MEMBERS OF THE COMMUNITY AND WE TRIED THIS WITH INTERBACTER AND COCCUS. I'LL SHOW YOU DATA FOR BOTH. THIS IS ON THE GIPSY MOTH AND WITH NO ANTIBIOTICS WE SEE BT, 100% MORTALITY SO IN THIS ASSAY WE USE LIE HIE LEVEL OF BT. IN THIS CASE WE DON'T SEE EFFECT OF ADDING MORE BACTERIA BECAUSE THE GUT ALREADY CONTAINS BACTERIA SO THERE'S ESSENTIALLY NO EFFECT ON MORTALITY. ON ANTIBIOTICS WE SEE REDUCTION OF BT KILLING WHEN NO BACTERIA MEASURABLE IN THE GUT BUT IF WE ADD BACK THE ANERA BACTER TO BT WITH ANTIBIOTIC TREATED ANIMALS WE SEE A DRAMATIC PATTERN OF RESTORATION OF KILLING. SO THIS WAS REPEATED MANY TIMES ON GIPSY MOTHS AS WELL AS OTHER INSECTS AND AMONG A WIDE DIVERSITY OF LAPADOPTRIN INSECTS. WE SAW THE SAME TRENDS, THAT ANTIBIOTIC TREATMENT REDUCED BT ACTIVITY, SOMETIMES COMPLETELY WIPED IT OUT, OTHER TIMES DROPPED IT. AND RESTORING THE GUT COMMUNITY OR CERTAIN MEMBERS OF IT RESTORED KILLING. SO THIS IS AN EXAMPLE OF MENDUKA, THE TOBACCO HORN WORM WE WORK ON MOSTLY NOW, YOU CAN SEE HERE THE ONLY REAL DIFFERENCE IS THAT BT ALONE DOES ACTUALLY KILL THE ANTIBIOTIC TREATED ANIMAL. SO WE DON'T COMPLETELY WIPE OUT BT ACTIVITY BY REDUCING THE MICROBIAL COMMUNITY TO UNDETECTABLE. BUT IT'S CLEARLY MUCH WEAKER ACTIVITY THAN WE WOULD SEE IN THE PRESENCE OF THE NORMAL COMMUNITY. WHEN WE ADD ANERACOCCUS THE SCRIP SI MOTH AND TOBACCO HORN WORM DUCKS WE INCREASE BT ACTIVITY QUITE WELL AND BACK TO WHAT IT WOULD BE WITH THE NATIVE COMMUNITY THERE. SO THIS IS NOW CONSISTENT STORY WITH A NUMBER OF INSECTS GETTING RID OF THE GUT BACTERIA REDUCES BT ACTIVITY AND WITH TWO SPECIES OF BACTERIA, ONE GRAM POSITIVE, ONE NEGATIVE THAT RESTORE ACTIVITY BT ACTIVITY TO THE KILLING THE INSECT. SO WE'RE CURIOUS ABOUT THIS. BT HAS BEEN STUDIED FOR AS I MENTIONED WELL OVER 100 YEARS, IT WAS DISCOVERED IN 1-9D 04 -- 1904 IN JAPAN AND GERMANY AND HAS BEEN ONE OF THE GREAT CURIOSITIES OF INSECT MICROBE INTERACTIONS AND THERE'S ENORMOUS AMOUNTS OF WORK ON THE MODE OF ACTION OF THE TOXIN, HOW THE TOXIN FORMS PORES IN THE EPITHEME YUM -- EPITHELIUM OF THE GUT. THAT'S ASSUMED TO BE THE MODE OF ACTION OF HOW IT KILLS. NO ONE HAS TAKEN THAT TO THE NEXT STEP OF SO THERE ARE PORES IN THE GUT WALL BUT WHY DOES THAT ACTUALLY KILL? AND THE ANSWER TO WAS, I THINK REMAINS STILL, NOT COMPLETELY COMPLEER, BUT I THINK WE HAVE SOME HINTS ABOUT WHY THAT'S IMPORTANT N. SOME CONDITIONS WE FIND PARTICULARLY WHEN WE GOT RID OF THE GUT COMMUNITY THE PORES OCCUR, BT INSERT INTO THE EPITHELIUM AND FORM PORES BUT THEN THEY CAN HEAL. THE INSECOND HAS TREMENDOUS ABILITY TO HEAL OVER THE GUT EPITHELIUM AN REPLACE THE CELLS DAMAGED. SO THE INDOESN'T NECESSARILY DIE BECAUSE IT HAS PORES IN ITS GUT. SO WE WANTED TO EXAMINE WHAT HAPPENS TO THE GUT EPITHELIUM WITH BT AND THE GUT BACTERIA. WE WANTED TO EXAMINE THE HEMOLIN. THE INSECT -- THE HEMOLIN DOESN'T CARRY OXYGEN BUT ACTS AS THE IMMUNE ORGAN, THE FULL PRETTY MUCH FULL LOCATION OF HEMOCYTES THE MIEWN LOGICALLY ACTIVE CELLS -- IMMUNOLOGICALLY ACTIVE CELLS IN THE IP SECOND. SO -- INSECT. SO WE WANTED TO KNOW WHAT THE HEMOCYTES WERE DOING IN TERMS OF MOUNTING IMMUNOLOGIC RESPONSE. SO FIRST STEP WAS THE LOOK AT THE GUT WALL WHICH WAS DONE MANY TIMES WITH BT BUT HAD NEVER BEEN DONE WITH AND WITHOUT ANTIBIOTICS. YOU CAN SEE THAT IN ABSENCE OF TREATMENT OR JUST WITH ANTIBIOTICS ALONE, THE EPITHELIUM HAS A NICE REGULAR STRUCTURE, THE EPITHELIAL TISSUE HAS MICROVILLEI AND WE CAN SEE THE CRIPS THAT ARE TYPICAL OF THE MAMMALIAN GUT AS WELL SO A LOT OF FEATURES IN COMMON WITH ALL OTHER GUTS STUDIED. AND IT HAS A VERY REGULAR AND DISCERNIBLE STRUCTURE. WHEN WE INOCULATE WITH BT AND ANDER COCCUS WE FIND DISORGANIZATION IN THE SAME AMOUNT OF TIME. WE LOSE CELL INTEGRITY, VISIBLE CELL STRUCTURE AND CELL WALLS. AND THERE'S NO MICROVILLE LAY STRUCTURE LEFT SO A HARD TIME TAKING UP NUTRIENTS. BT WILL CAUSE A SMALL EFFECT LIKE THIS BUT NOTHING AS DRAMATIC. AND AND ESHRCOCCUS HAS NO EFFECT ON THE GUT WALL. SO IF GUT WALL WAS BECOMING SO DISSENT GRATED AS A RESULT OF BEING TREATED WITH BT AND ANDERCOCCUS, WHAT'S HAPPENING IN THE BLOOD? NOW THERE ARE HOLES IN THE GUT WALL THAT MAKE FOR REGULAR CONVERSATIONS BETWEEN THE HEMOLINS AN GUT. SO IN THIS CASE WE FED INTERCOCUS ALONE OR WITH BT AN WITHDREW BLOOD SAMPLES FROM THE INSECTS WITH THE TOBACCO HORN WORM WHICH IS EASIER TO BLEED THAN MOST INSECTS SO YOU CAN SEE WHEN WE COULD FRURRED FROM THE HEMOLIN IN ABSENCE OF WE JUST FED ANERCOCCUS ALONE, 3 OR 20 HOURS AFTER FEEDING SAW NO BACTERIA IN THE BLOOD. SO LIKE US THE INSECT HAS WHAT APPEARS TO BE NEARLY STERILE BLOOD. BUT IF WE FED THE INTERCOCCUS WITHBT, THOSE ARE SHOWN HERE, WE SAW MASSIVE POPULATIONS, 10 TO THE 8TH ML OF BLOOD INTERCOCCUS COLONY FORMING UNITS. SO THIS WAS A DRAMATIC DIFFERENCE, THIS SUGGESTED THAT THE INTERCOCCUS COULD ENTER THE INSECT THROUGH THE GUT INTO THE GUT THROUGH THE GUT WALL IF BT WAS THERE SUGGESTING THAT BT WAS NOT REALLY THE KILLER IN THIS CASE BUT WAS PROVIDING A ROUTE FOR ANDERCOCCUS TO REACH BLOOD AND BE THE PATHOGEN. SO WE PROPOSED THAT IF THIS WERE THE CASE YOU WERE TO IMAGINE INTERCOCCUS INJECTED INTO THE HEMOLIN HAS THE SAME IMPACT. WHEN WE FED, WE SAW 100% SURVIVAL, NO EFFECT OF INTERCOCCUS. NO BT INVOLVED AND WHEN WE INJECTED ANERCOCCUS YOU SEE RAPID MORTALITY THAT OCCURS WITHIN A DAY OF INJECTION. SO THIS SUGGESTED FROM THE SHAPE OF THE CURVE AND NATURE OF THE DEATH THAT THIS WAS SIMILAR TO WHAT WE OBSERVED WHEN WE TREATED WITH BT AND THE NATIVE COMMUNITY AND THAT SUGGESTED THE HYPOTHESIS THAT THE GUT WALL BEING BREECHED SIMPLY ALLOWS THE GUT BACTERIA, THE NORMAL AND BENIGN BACTERIA TO REACH THE BLOOD. THIS IS A MECHANISM WHICH IT'S PEST MATED ABOUT HALF OF THE SEPSIS CASES IN HUMANS OCCURS THAT ABOUT HALF PROBABLY ORIGINATE WITH BACTERIA THAT LIVE NORMALLY IN THE GUT AND ARE BENIGN BUT WHEN THE GUT WALL IS DAMAGED FOR SOME REASON OR EITHER IMMUNOLOGICALLY OR THROUGH PHYSICAL PERFORATION THEN BACTERIA CAN REACH THE BLOOD AND THAT LEADS TO SEPTICEMIA WHICH MEANS BACTERIA IN THE BLOOD AND ULTIMATELY SEPSIS WHICH MEANS DEATH BY OR SERIOUS DISEASE BY HAVING BACTERIA IN THE BLOOD. SO WE WANT TO PROPOSE THIS MODEL AS A MODEL FOR SEPSIS, ONE OF THE REASONS WE FOUND THAT EXCITING IS THAT SEPSIS IS A VERY RAPID DISEASE, IT TAKES 24 TO 48 HOURS FOR AN UNCHECKED CASE OF SEPSIS TO KILL A PERSON. SO IT'S VERY DIFFICULT TO DO RESEARCH ON. PHYSICIANS ARE USUALLY JUST STRUGGLING SO HARD TO KEEP THEIR PATIENTS ALIVE THEY'RE NOT GOING TO TRY EXPERIMENTAL TREATMENTS AND THERE'S VERY LITTLE TIME TO LOOK AT THE PARAMETERS, THE CHANGES IN HOST PHYSIOLOGY OR BACTERIAL BEHAVIOR DURING THE CASE OF SEPSIS. STRANGELY, THERE ARE NOT VERY GOOD MODELS FOR SEPSIS IN OTHER MAMMALS SUCH AS RODENTS. THERE'S PECULIAR ONES WHERE THEY USE FOOT PAD INJECTIONS OR PERFORATIONS TO INTRODUCE BACTERIA. BUT APPARENTLY ACCORDING TO PEOPLE WHO KNOW SEPSIS SYMPTOMS IN HUMANS, NOT A VERY GOOD MODEL. SO WE WERE PRETTY ENTHUSED THAT THIS COULD BECOME A MODEL FOR FIGURING OUT HOW SEPSIS OCCURS AND HOW TO PREVENT IT AND OUR MORE RECENT WORK PERTAINS TO THAT. SO BASED ON THE HUMAN SEPSIS MODEL WE PROPOSE THE KEY WAS KILLING BY INCITING AN OVERBLOWN INFLAMMATORY RESPONSE THAT'S RESPONSIBLE FOR KILLING THE HOST. SO THIS IS THE MODEL IN HUMANS BUT NOT THE BACTERIA THAT KILLS US BUT THE MASSIVE INNATE IMMUNE RESPONSE THAT WE MOUNT IN RESPONSE TO THE BACTERIA THAT IS THE TOXIC AND ULTIMATELY FATAL ELEMENT IN THE DISEASE. INSECTS ARE NICE IN THAT WE HAVE AN INNATE IMMUNE RESPONSE, A NUMBER OF ELEMENTS OF THE INNATE IMMUNE SYSTEM OF PEOPLE WERE DISCOVERED IN DROSOPHILA IN INSECTS. SO THE ANALOGIES ARE QUITE POWERFUL. BUT THE INSECTS DON'T HAVE THE ADAPTIVE IMMUNE RESPONSE, THE ABILITY TO PRODUCE ANTIBODIES TO COMPLICATE THINGS. SO IT'S A STREAMLINE INFLAMMATORY RESPONSE THAT I THINK IS MUCH EASIER TO STUDY BECAUSE IT IS IN THE ABSENCE OF THE MORE COMPLEX IMMUNORESPONSE OF HUMANS. HERE WE HAVE PROGRESSION OF THE DISEASE, WE LOOKED AT THE INSECTS AT THE MACROSCOPIC LEVEL, THE STRUCTURE OF THE THE WALL OF THE EPITHELIUM, WE LOOKED AT THE BLOOD IN THE HEMOLIY, COULD WE SEE BACTERIA, AND WE LOOKED AT THE HEMOCYTES IN THE BLOOD. THIS IS THE HEALTHY ANIMAL YOU CAN'T SEE ANY BACTERIA IN THE BLOOD. THE HEMOCYTES ARE CIRCULATING AS SINGLE CELLS, THEVILLELY ARE IN GOOD HEALTH AND STRUCTURE. BY 24 HOURS WE'RE STARTING TO SEE A LITTLE DISCOLORING OF THE INSECT AT A MACRO SCALE AND THE VILI ARE BEGINNING TO SHOW SOME GAPS AND SPACES BETWEEN THEM AND GET A LITTLE DISORGANIZED LOOK THAT WE SAW SO DRAMATICALLY IN THE PREVIOUS SLIDES. WE STILL SEE NO BACTERIA IN THE HEMOLIN BUT WE START SEEING INDICATION OF AN IMMUNE RESPONSE WHICH IS USUALLY INDICATED FURTHER IN INSECTS BY THE CLUMPING OR AGGREGATION OF THEIR HEMOCYTES SO THE CELLS CLUMP LANGUAGE BEFORE ACTUAL EXPOSURE OF THE CELLS TO THE BACTERIA. SO THAT'S CIRCULATEING A CHEMICAL INDUCING RESPONSE. TEN DAYS AFTER INNOCULATION THE INSECT IS LARGELY MELOIZED SO THE MELANIN IS AN INDICATOR OF THE INNATE IMMUNE SYSTEM BEING TURNED ON SO USUALLY YOU SEE FIRST THE BEGINNINGS OF AGGREGATION OF HEMOCYTES AND THE DARKENING OF THE BODY AND THAT'S AN INDICATION OF INNATE IMMUNE RESPONSE. WE COULDN'T DO MUCH WITH THE GUT WALL IN THIS CASE BECAUSE THE INSECT UNDER THESE CONDITIONS WAS PRETTY SQUISHY INSIDE. THERE WASN'T ENOUGH INTEGRITY TO GET SAMPLES BUT WE WERE ABLE TO TAKE BLOOD SAMPLES AND THESE DID HAVE -- MIGHT BE A LITTLE TOO LIGHT TO SEE BUT THEY DID HAVE ABUNDANT BACTERIA IN THEM AT A VERY,VERY HIGH TITER. AND THE AGGREGATES OF THE HEMOCYTE CONTINUED TO INCREASE IN SIZE. SO WE HAD ALL THE MASHINGS OF THE INNATE IMMUNE RESPONSE OF THE LEPADOPRIN HOST, THE HEMOCYTE AGGREGATION AND THEN LOOK AT T THE OTHER ELEMENTS SO THE COX AND THE TOLL REGULATED GENES ARE CRITICAL IN MAMMALS AN INSECTS FOR EXPRESSION OF THE TOTAL IMMUNE RESPONSE, PART OF THAT RESPONSE IS THE PRODUCTION OF NITRIC OXIDE AND REACTIVE OXYGEN SPECIES. ANOTHER OUTCOME OF THE COX AND TOLL RECEPTORS BEING TURNED ON IS THE PRODUCTION, THE ANTI-MICROBIAL PEPTIDES WHICH IS ONE OF THE MECHANISMS BY WHICH IMMUNE RESPONSE REDUCES BACTERIAL LOAD. TO FOLLOW-UP ON THE VISUAL MARKETINGS -- MASHINGS OF THE IMMUNE RESPONSE, WE TURN TO A PHARMACO LAJCAL APPROACH, AND ASK WHETHER SUPPRESSORS OF THE INNATE IMMUNE RESPONSE HAD ANY EFFECT ON BT KILLING. AND WHAT WE FOUND WAS NOT ALL, DEPENDED ON THE TARGETS BUT SOME IMMUNE SUPPRESSORS LIKE FOR EXAMPLE ENENDOMETHSIN SHOWN HERE PROLONGED THE LIFE OF THE INSECT. SO HERE WE SEE LARVAL SURVIVAL SO ANYTHING ABOVE THE DASHED LINE INDICATES THE TREATMENT IS INCREASING SURVIVAL COMPARED WITH A CONTROL. SO ENDOMETHISIN AT VARIOUS CONCENTRATIONS CLEARLY HAS STATISTICALLY SIGNIFICANT EFFECT ON SURVIVAL AND BOTH INCREASES THE LENGTH OF THE DURATION OF THE INSECT LIFE AND THEN ALSO KEEPS MANY OF THEM ALIVE. SOME TREATMENTS WE TRIED OF THIS TYPE ONLY INCREASE DURATION OF LIFE BUT ULTIMATELY THEY ALL DIED ANYWAY. ANOTHER ONE IS GLUTATHIONES THAT AFFECTS ASPECTS OF THE INNATE IMMUNE SYSTEM AND THIS ONE WAS ONE THAT HAS GIVEN VARIABLE RESULTS BUT IS FAMILIAR ENOUGH ANTI-INFLAMMATORY AGENT, ASPIRIN WE WERE INTRIGUED TO TEST AND THIS ONE SEEMS TO BE HOST-SPECIFIC, IT SEEMS TO HAVE MORE OF AN AFFECT ON REDUCING THE BT KILLING AN RATE OF KILLING IN SOME INSECTS AND OTHERS AND WE DON'T HAVE A MECHANISM TO EXPLAIN THAT YET. SO THIS WAS CERTAINLY CONSISTENT WITH THE INFLAMMATORY RESPONSE BEING RESPONSIBLE FOR KILLING. WE HAVE COMPARED THE AFFECTS TOBACCO HORN WORM AND GIPSY MOTH AND ONCE AGAIN, YOU CAN SEE IN THE TOBACCO HORN WORM EMPERIMENT WE HAVE 40% SURVIVAL. AFTER THREE DAYS OF TREATMENT WITH BT ALONE AND BOTH IBUPROFEN AN ASPIRIN GIVES STATISTICALLY SIGNIFICANT INCREASE IN SURVIVAL. SO NOW WITH TWO INSECT SPECIES AND QUITE A FEW DIFFERENT IMMUNE INFLAMMATORY SUPPRESSANTS WE FOUND IN FACT INCREASING SURVIVAL, REDUCING BT TOXICITY, WHICH IS CERTAINLY CONSISTENT WITH THE INNATE IMMOON RESPONSE OR INFLAMMATORY RESPONSE BEING RESPONSIBLE FOR KILLING. SO THE COROLLARY OF THAT EMPERIMENT WAS IF WE COULD INDUCE THE INNATE IMMUNE SYSTEM TO HAVE FULL BLOWN INFLAMMATORY RESPONSE, WITHOUT BACTERIA COULD WE OBSERVE BT AS WE DID WITH BACTERIA. SO THESE WERE LARVAE TREATED WITH ANTIBIOTIC. WHEN WE TREAT WITH BT ALONE, THERE WERE -- THEY WERE KILLED IN 6.6 DAYS, A TIME TO DEATH, TIME TO 50% DEATH ANERABACTER, IN THIS CASE GIPSY MOTH SO WE USED THAT AS THE COOPERATIVE AGENT AND THAT CERTAINLY REDUCED TIME TO DEATH. KNIGHT NICE VIRULENT TREATMENT. INSTEAD OF ANERABACTER WE TRIED A NUMBER OF FROM CELL WALLS, IN THIS CASE THE GLYCAN BT THAT SPEEDED UP DEATH EVEN MORE THAN THE ANERABACTER DID. SO THIS IS AN EXAMPLE IN THE ABSENCE OF LIVE BACTERIA WE HAVE A CHEMICAL TREATMENT FRAGMENTS FOR CELL WALL THAT INSIGHT AN IMMUNE RESPONSE AND ALSO CAUSED DEATH. SUGGESTING THAT ONE OF THE MECHANISMS BY WHICH BT KILLS IS ENABLING THE BACTERIA FROM THE GUT TO REACH THE HEMOLIN AND PRODUCE THE CLASSIC INFLAMMATORY RESPONSE SYMPTOMS. WE'RE IN THE PROCESS OF TESTING OTHER ELEMENTS OF THIS. SO I'M RUNNING OUT OF TIME SO I'LL QUICKLY MENTION A NEW DIRECTION THAT WE HAVE BEEN TAKING THE LAST COUPLE OF YEARS, THAT I THINK IS BEGINNING TO YEEL VERY EXCITING DATA. ONE CHALLENGE IN THESE EXPERIMENTS IS THE BACTERIA THAT WE INTRODUCE HAVE TO INVADE AN ACTIVE COMMUNITY. SO IT'S A COMMUNITY THAT HAS A STRUCTURE AND A FUNCTION AND INTERACTIONS AND IT'S AN EXISTING COMMUNITY. WE'RE ADDING BACTERIA THAT HAVE TO SURVIVE. WE CERTAINLY KNOW FROM PROBIOTICS EMPERIMENTS IN HUMANS THAT INTRODUCE BACTERIA ARE HARD TO ESTABLISH IN THE HUMAN GUT AND THAT'S TRUE IN INSECTS AS WELL. SO WE WANT TO LOOK AT THE GENETIC BASIS FOR INVASION ABILITY. SO WHAT MAKES A BACTERIUM ABLE TO INVADE ACTIVE NORMAL INTACT COMMUNITY AND ON THE OPPOSITE SIDE WHAT MAKE AS COMMUNITY ABLE TO RESIST OR NOT INVASION BY A NEWCOMER. SO WE DID AN ANALYSIS OF GENES INDUCED IN THE GIPSY MOTH GUT ASKING IN THIS CASE WE USE ENTERCOCCAS FATALIS ASKING WHICH WERE TURNED ON IN THE GUT AND WE USE AD GENETIC SYSTEM NAME, WE CAN TALK ABOUT THAT LATER IF ANYONE IS INTERSDZ. SO WE FOUND A NUMBER OF GENES AND THE ONES WE KEPT COMING UP WITH OVER AND OVER, OVERP REPRESENTED FROM THE LIBRARIES IN THE GUT WERE THE ONES WE STUDIED FIRST AND WE FOUND A FEW THAT WE IMMEDIATELY PICKED THEM UP FOR EXAMPLE 36 TIMES IN THE SCREEN, WE THOUGHT THEY WERE LIKELY TO BE GOOD CANDIDATES FOR IMPORTANT PHENOTYPES IN THE GUT SWOA KNOCKED OUT THOSE GENES. AND IN THIS CASE THE RED BAR IS ONE OF REAL INTEREST HERE SO THIS IS A MUTANT IN RED A SO IT'S A DNA RECOMBINATION PROTEIN. WHEN THE BACTERIA IN THE MUTANT IS INOCULATED ALONE, IT DOESN'T SURVIVE QUITE AS WELL, THOUGH IT'S NOT BAD COMPARED TO THE WILD TYPE. WHICH IS THE BLACK CHECKERED BARS. SO WE FEED THE BACTERIA THE SAME WAY, THERE IS A SMALL DIFFERENCE IN THIS MUTANT. THE INTERESTING DATA WHEN WE INOCULATED THE RAD A MUTANT ALONG WITH A WILD TYPE STRAIN, IN THAT CASE THE MUTANT YOU CAN SEE HERE IS ALMOST GONE BY THE END OF THE FIFTH DAY. SO THIS DOES SEEM LIKE THE GENES THAT WE ISOLATED FROM THE EMPERIMENT TURNED ON IN THE GIPSY MOTH GUT ARE APPARENTLY REQUIRED FOR COMPETITION AGAINST ANOTHER MEMBER OF THE GUT COMMUNITY. SO A CLEAN GUT, THIS MUTANT DOES REASONABLY WELL, BUT PRESENCE OF COMPETITION FALLS OFF QUICKLY. THE MORE EXCITING EXPERIMENT, I'LL MENTION THIS DATA WE GOT LAST WEEK, I CAN'T RESIST TALKING ABOUT IT, WAS A SIMILAR EXPERIMENT WITH THE SAME TYPES OF MUTANTS, THIS IS A MUTANT ALSO FOUND IN THE RABBIT EXPERIMENT BUT IN THIS CASE MAPS IN A GENE THAT DOESN'T HAVE GOOD HOMOLOGY TO ANYTHING BUT LOOKS LIKE IT MIGHT BE INVOLVED IN CENTRAL METABOLISM. IT'S VERY PECULIAR SEQUENCE. SO WE'RE STILL WORKING ON SEQUENCE ANALYSIS. IN THIS CASE WE USED DROSOPHILA AS THE HOST. AND DROSOPHILA HAS A HO ROBUST AND SIMPLE GUT COMMUNITY, TWO MAJOR GENRE, THAT'S IT BUT SEEMS LIKE ANYTHING WE DO TO IT IT BOUNCES BACK, ELASTIC COMMUNITY. WHEN WE INOCULATED THIS MUTANT IN METABOLISM GENE, MUTANT 1918, WHEN WE INOCULATED INTO NORMAL GUT COMMUNITY OF DROSOPHILA IT WAS DRA GNATICALLY REDUCED COMPARED TO THE WILD TYPE BY THE BLACK CHECKERED BARS SO THAT WE HAVE TAKE THN OUT LONGER NOW, THE DATA CAME IN TODAY AND IT LOOKS LIKE THE MIEW ABOUT THE IS CONTINUING TO DECREASE. SO WE'RE EXCITED NOW BECAUSE WE HAVE ABILITY TO LOOK AT HOST GENETICS IN DROSOPHILA AND MANIPULATE THE ENVIRONMENT WHICH THIS IMMUNITY EXISTS AND WE HAVE IDENTIFIED AT LEAST ONE GENE THAT SEEMS TO BE IMPORTANT FOR INVIEDING THE -- INVADING THE NORMAL COMMUNITY. WHEN WE TREATED WITH ANTIBIOTICS AND GAVE THIS MUTANT 1918, EVERY ADVANTAGE IN TERMS OF COLONIZING THE HOST WITHOUT HAVING TO INVADE THE COMMUNITY, IT WAS JUST LIKE THE WILD TYPE. SO AS FAR AS I CAN SEE THIS IS THE FIRST INDICATION THAT IS POSSIBLE TO IDENTIFY MUTANTS IN THISW INVASION PHENOTYPE. THERE AREN'T JUST UNABLE TO GROW IN THE HOST OR UNABLE TO ATTACH BUT THEY'RE SPECIFICALLY AFFECTED IN THE ABILITY TO COMPETE WITHIN THE COMMUNITY. SO HOPE THIS IS THE BEGINNING OF BEGINNING TO UNDERSTAND THE NATURE OF COMMUNITIES AND THE DYNAMICS THAT GOVERN INVASION AND RESISTANCE TO INVASION. SO I HAVE SHOWN YOU TODAY A NUMBER OF INTERACTIONS, THE BACCILAS SERIOUS AND OMYCES INTERACTION MEDIATED BY ZWITTERMICIN IS ANTAGONISTIC. AND THAT WITH BACILLES THEREGENIS WORK TOGETHER TO KILL THE HOST IT THRIVES IN. WE DISCOVERED AN INDIRECT MEANS THIS VERY UNUSUAL ANTIBIOTIC THAT HAS NOT ONLY UNUSUAL STRUCTURE WHICH STILL HAS ONLY -- THE ONLY ONE OF ITS KIND KNOWN BUT ALSO HAS A UNIQUE MODE OF SYNTHESIS AND LED TO THE DISCOVERY OF TWO NEW EXTENDER UNITS FOR POLYKETIDE SYNTHESIS. FINALLY, BT KILLS MANY DIFFERENT LAPADOPTRIN SPECIES IN COOPERATION WITH THE GUT MICROBIOTA AND THAT IS ASSOCIATED WITH OVERBLOWN OR PERNICIOUS INFLAMMATORY RESPONSE AND THAT'S PARTLY SUPPORTED BY THE FACT THAT ANTI-INFLAMMATORIES REDUCE MORTALITY. THERE ARE MORE QUESTIONS RAISED THAN ANSWERED BY THIS WORK AS IS ALWAYS TRUE IN SCIENCE. WE'RE CERTAINLY INTERESTED IN THE NO LEK LAR TRIGGERS OF THE INNATE IMMUNE RESPONSE IN THIS INSECT SYSTEM. WE'RE INTERESTED IN OTHER INTERACTIONS BETWEEN MEMBERS OF THE MICROBIAL COMMUNITY AND BETWEEN THE COMMUNITY AND PATHOGENS AND WE CONTINUE TO WORK THE MECHANISMS WHICH ZWITTI RERKSMI CRIRKS N HELP IT IS SYNERGISTIC AFFECT AND I WOULD LIKE TO THANK THE WONDERFUL PEOPLE WHO DID THIS WORK. THE INSECOND WORK WAS PIONEERED BY ANY COLE BRODERICK, UNDERGRADUATE AND GRADUATE STUDENT IN THE LAB WHO DID ALL THE GIPSY MOTH WORK I SHOWED AND JOHN HOLT WHO HELPED DEVELOP AND GENERATED A LOT OF DATA FOR MENDUCA SYSTEM AND ALSO SINGLE HANDEDLY SET UP THE RIBOT SYSTEM AND DEVELOPED THE MUTAGENESIS APPROACH. THE ZWITTERMICIN WORK DEPENDENT ON JOHN CLARTY THE CHEMIST POST DOC DETERMINED THE STRUCTURE AND BIOSYNTHESIS WORK WAS DONE BY MICHAEL THOMAS, AND A GRADUATE STUDENT ELAN SHUN. FINALLY I WOULD LIKE TO THANK MY FUNDERS, NIH, MOST APPROPRIATELY HERE, IT'S REALLY NICE THE THANK THE PEOPLE THAT MAKE THIS WORK POSSIBLE T. HOWARD HUGHES MEDICAL INSTITUTE AND NSF CONTRIBUTED TO THE WORK DONE HERE. WITH THAT, I'LL STOP AND SORRY WE RAN OVER SO MUCH. [APPLAUSE] >> DO YOU KNOW WHEN THE INNATE IMMUNE RESPONSE REQUIRE AS TOXIN? THE BT TOXIN? >> WE CAN INDUCE AN INNATE IMMUNE RESPONSE WITHOUT THE TOXIN BY A NUMBER OF DIFFERENT MEANS AND THE TOXIN DOES SEEM TO CONTRIBUTE TO IT. SO IT'S NOT EITHER WAY. IT'S A LITTLE BIT OF EACH. EXACTLY. IT HAS SLIGHTLY DIFFERENT FEATURES WHEN THE TOXIN IS THERE. >> THAT WAS VERY NICE. AS I'M SURE YOUR AWARE BUT I WAS UNAWARE UNTIL A YEAR OR SO AGO, MANY DROSOPHILA AND ACTUALLY 70% OF INSECTS ARE FELT TO HAVE AN ENDOGENOUS -- ENDOSYMBIANT, INTRACELLULARLY IN THEIR OWN ORGAN CALLED A BIOTONE OR WHATEVER AND THAT IS LIKE A WOBACIA WHICH HAS AN EXAMPLE OF A FREE LIVING INSECT. ARE YOU AWARE OF ANY INTERACTION BETWEEN ENDOGENOUS ENDOSYMBIANT AND THE GUT WHICH ARE TRULY OUTSIDE THE ANIMAL AND NOT IN A CELL? >> YEAH, IT'S A REALLY GOOD QUESTION. WE HAVE NOT SEEN, PHYSICALLY SEEN THE ENDOSYMBIA NCIS IN THE HOST BUT WE ASSUME THEY'RE THERE BECAUSE THEY'RE UBIQUITOUS. IT'S HARD TO FIND CURED INSECTS THAT HAVE LOST THOSE SIMBIANS SO IT'S A HARD QUESTION TO ADDRESS, I'M NOT SURE HOW WE WOULD DO THAT. ONE SYSTEM THAT COULD BE USED IS ONE NANCY MORAN DEVELOPED WHERE THE GENOME OF THE HOST AND SIMBIANT HAS BEEN SEQUENCED. THAT MIGHT GIVE SOME CLUES IF THERE ARE INTERACTION, WE AT LEAST KNOW THE GENES TO LOOK FOR IN THAT INTERACTION. >> I'M SURE THERE ARE DROSOPHILA THAT HAVE LOST AND THEY TREATED SOME AND MOVE IT BUT THE DROSOPHILA GENOME CONTAINS THE FULL GENOME OF THIS WOBAKIA—X DISCOVERED IN 2005. THEY DEPOSITED THEIR WHOLE GENOME INTO THE >> THE ZWITTERMICIN AND BT RESULTS, THESE RESULTS SUGGEST THAT THE ZWITTERMICIN IS KILLING BACTERIA IN THE COMMUNITY PROTECTING THEM FROM ANERBACTER WHEN YOU TREAT THEM WITH NORMALLY JUST BT ALONE, DO YOU HAVE AN IDEA WHAT HAPPEN THE BACTERIA PROTECTING AGAINST ANERABACTER? >> I'M NOT SURE I SEE EVIDENCE FROM KILLING BACTERIA. I DON'T THINK THAT'S THE MODE OF ACTION BECAUSE WE CAN KILL ALL OF THE BACTERIA WITH A VERY HARSH ANTIBIOTIC BT VERSUS ZWITT RERKSMYCIN YOU GET ADDITIVE AFFECT F. YOU ADD BT WITH ANERBACTER YOU GET SYNERGISTIC EFFECT ON KILLING. THAT SUGGESTS SOMEHOW THAT THE ORGANISM IS BEING PROTECTED AGAINST THE ANERBACTER THERE WHEN YOU NORMALLY FEED THEM BT. NO? >> I DON'T THINK SO. WE CAN KILL OFF THE BACTERIA AND SEE NO KILLING UNLESS WE ADD AN IMMUNOINCITE SO THAT GETS AROUND THE BACTERIA, MY BET IS AN INTERACTION BETWEEN ZWITTERMICIN AND BT TOXIN. >> SO IF YOU TAKE A CELL WIPED OUT FOR BACTERIA AND ADD BT PLUS ZWITTERMICIN YOU'RE SEEING THE EASKT OF ADDING IT? >> I SEE, YES, WE HAVE DONE THAT, RIGHT. >> OKAY. ALL RIGHT. >> (INDISCERNIBLE) JUST HAVE A QUESTION ABOUT YOUR -- THE MICE MODEL YOU SHOWED. COULD YOU IN OTHER OBESE MICE DO WE FIND -- CAN WE IDENTIFY BACTERIA COMMUNITIES THAT CAUSE OBESITY IN THE OTHER MICE THAT YOU SHOW SO IT TURNS OUT MORE COMPLICATED SINCE THAT INITIAL DISCOVERY WAS MADE. THERE ISN'T A SINGLE BACTERIUM BUT IT'S RELATED TO THE RATIO OF TWO OF THE THREE MAYOR PHYLA, BACTERIA DEDES AND FERMICUTES. A LOT OF NUTRITIONAL ASPECTS ASSOCIATED WITH THE GUT ARE LINKED TO THE FATTY ACID METABOLISM, PARTICULARLY THE FERMICUTES WHICH PRODUCE SHORT CHAIN FATTY ASITS THE HUMAN BODY CAN USE. SO THE MORE EFFICIENT THEY ARE CONVERTING THE FOOD WE TAKE INTO FATTY ACIDS THE FATTER WE GET. SO WE SHOULD END HERE. I WILL REMIND YOU THAT THERE IS A RECEPTION TODAY IN THE LIBRARY. THANKS TO THE FAES, SOME OF YOU MAY HAVE NOTICED THERE HASN'T BEEN FOR A FEW WEEKS BUT WE'RE BACK IN BUSINESS SO INVITE YOU TO COME IN AND CONTINUE THE CONVERSATION WITH JO. THANK YOU VERY MUCH.