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>> 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 *** 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 *** 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 WOBAKIAĆ³X
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.