I'M REPRESENTING THE LAMBDA LUNCH INTERSEST GROUP HOSTING TODAY'S WALS SPEAKER. IT'S MY PLEASURE TO INTRODUCE DR. LALITA RAMAKRISHNAN WHO RECEIVED HER MEDICAL DEGREE FROM INDIA IN 1983, Ph.D. AT TUFTS UNIVERSITY. SHE DID MEDICAL RESIDENCY AT TUFTS AGAIN IN BOSTON, AND INFECTIOUS DISEASES FELLOWSHIP AT THE UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, AND THEN A POSTDOCTORAL FELLOWSHIP WITH STAN FALCO AT STANFORD UNIVERSITY. SHE THEN JOINED THE FACULTY OF THE UNIVERSITY OF WASHINGTON IN SEATTLE, BUT RECENTLY MOVED TO THE UNIVERSITY OF CAMBRIDGE. DR. RAMAKRISHNAN CARRIED OUT SEMINAL WORK ON THE PATHOGENESIS OF TUBERCULOSIS IN AN EXTREMELY BUT IMPORTANT UNDERSTUDIED DISEASE. ACCORDING TO THE W.H.O., 1.5 MILLION PEOPLE DIED OF TUBERCULOSIS IN 2013, AND I FOUND IT INTERESTING THAT IT'S AMONG THE TOP FIVE CAUSES OF DEATH FOR WOMEN BETWEEN AGES 15 AND 44. MORE THAN 30% OF THE WORLD'S POPULATION HAS LATENTTB, THOUGH A THIRD OF THE PEOPLE IN THIS ROOM CARRY TB, LATENT TUBERCULOSIS. DR. RAMAKRISHNAN IS ADDRESSING QUESTIONS SUCH AS WHY DO PEOPLE DIFFER IN SUSCEPTIBILITY OR RESISTANCE TO TB AND WHY DO THOSE WHO DO NOT CONTRACT TB VARY IN THE SEVERITY OF THE ILLNESS. TO ADDRESS THESE QUESTIONS, HER GROUP HAS DEVELOPED ZEBRAFISH AS A MODEL, AND ZEBRAFISH ARE NATURALLY SUSCEPTIBLE TO TUBERCULOSIS CAUSEDDED BY PIKE ROW BACTERIA MERINUM, A CLOSE RELATIVE OF MICROBACTERIAL TUBERCULOSIS. FISH ARE A GREAT SYSTEM BECAUSE THEY ARE TRANSPARENT SO YOU CAN MONITOR THE INFECTION PROCESS, AND YOU HAVE THE ADVANTAGE OF BEING ABLE TO HAVE BOTH HOST AND BACTERIAL MUTANTS. SO AS WE WILL HEAR TODAY, THE RESEARCH IS SHEDDING LIGHT ON TB PATHOGENESIS AS WELL AS FUNDAMENTAL MECHANISMS OF IMMUNE CELL CHEMO TAX IS, ADHESION, AGGREGATION AND IMMUNE REGULATION. SO FINDINGS FROM THE RAMAKRISHNAN GROUP MADE IN ZEBRAFISH HAVE BEEN BORNE OUT IN HUMAN POPULATIONS AND ARE INFORMING NEW STRATEGIES FOR INTERVENTION. AS YOU MIGHT CORRECT DR. RAMAKRISHNAN HAS RECEIVED NUMEROUS AWARDS, INCLUDING THE NIH DIRECTOR'S PIONEER AWARD AND THIS YEAR WAS ELECTED TO THE NATIONAL ACADEMY OF SCIENCES. SO AS IS SHOWN HERE, THE TITLE OF TODAY'S TALK IS "THE ZEBRAFISH GUIDE TO TUBERCULOSIS." WE'RE LOOKING FORWARD TO HEARING ALL ABOUT THIS GREAT MODEL SYSTEM. [APPLAUSE] >> WELL, THANK YOU. THIS IS REALLY A GREAT HONOR, PROBABLY THE MOST HONORABLE LECTURE I'M ABOUT TO GIVE, AND IT'S -- I'M PARTICULARLY SORT OF THRILLED THAT GIGI IS MY HOST BECAUSE GIGI HAS BEEN FAMOUS FOR VERY LONG TIME, AND I REMEMBER WHEN SHE CAME TO GIVE A SEMINAR AT THE UNIVERSITY OF WASHINGTON WHEN I HAD JUST BECOME -- JUST JOINED THE FACULTY AND I KIND OF WANTED TO TALK TO HER BUT DIDN'T DARE PUT MYSELF ON HER LIST BECAUSE I DIDN'T THINK I HAD ANYTHING INTERESTING OR IMPORTANT TO SAY TO HER, AND IT'S JUST GREAT THAT NOT ONLY DID SHE INVITE ME BUT I FOUND AT BREAKFAST THAT I ACTUALLY DID HAVE SOMETHING TO SAY TO HER, AND I'M HOPING TO EVEN RECRUIT HER AS A COLLABORATOR IN SOME STUFF. THANK YOU AND WE'LL GET STARTED. SO GIGI GAVE A GREAT INTRODUCTION TO MY TALK, AND SHE POINTED OUT TO YOU HOW TB IS STILL WIDESPREAD, AND THE IMPORTANT THING TO NOTE IS THAT IT IS WIDESPREAD AND IS KILLING MORE PEOPLE TODAY THAN IT'S EVER KILLED BEFORE, DESPITE THE FACT THAT WE'VE HAD EFFECTIVE CHEMOTHERAPY FOR IT SINCE THE 1960s, AND THE REGIME THAT WAS DEVELOPED IN THE 1960s IS STILL BEING USED TODAY, TO TREAT TB. AND NOT ONLY IS IT PREVALENT BUT WE NOW HAVE A BURGEONING EPIDEMIC OF DRUG-RESISTANT TB, AND YOU CAN SEE THAT IT'S QUITE WIDESPREAD, AND I LIKE TO SAY THAT IT'S TURNING RUSSIA RED AGAIN, BUT JOKING APART, THIS IS A REALLY SERIOUS PROBLEM BECAUSE DUG-RESISTANT TB, PARTICULARLY EXTENSIVELY RESISTANT DRUG-RESISTANT TB MAKES TB ONCE AGAIN THE LETHAL DISEASE IT WAS IN THE PRE-ANTIBIOTIC ERA. SO IN THE FACE OF FAILING DRUGS AND AN INEFFECTIVE VACCINE, IT DOESN'T TAKE A LOT OF BRAIN TO REALIZE THAT ONE MIGHT HAVE TO FALL BACK ON HAVING TO LOOK AT THE BASIC BIOLOGY OF THE PATHOGEN AND THE HOST, IN OTHER WORDS THE PATHOGENESIS OF THE DISEASE TO SEE IF IT MIGHT TEACH US SOME ASPECT, SOME THINGS ABOUT TB, AND THIS IS WHAT A NUMBER OF LABS HAVE STARTED TO DO OVER THE LAST COUPLE OF DECADES. SO TO INTRODUCE YOU TO THE LIFE CYCLE OF TB AS IT WERE, TB IS TRANSMITTED BY A BACTERIUM, AND FROM THE LUNG OF AN INFECTED INDIVIDUAL WHO HAS TO COUGH IT UP AND IT'S TRANSMITTED IN SMALL AEROSOLS TO THE UNFORTUNATE PERSONS NEAR THEM AND THEN IT GETS INTO MACROPHAGES WHICH ARE PRIMARY DEFENSE CELLS OF THE IMMUNE SYSTEM THAT ARE MEANT TO TACKLE AND KILL PATHOGENS BUT TB LEARNED HOW TO SURVIVE IN THESE, AND INDEED IT SOMEHOW THEN GETS INSIDE THE BODY, SO UNLIKE OTHER PNEUMONIAS, IT'S NOT A PNEUMONIA ON THE SURFACE OF THE LUNG BUT ONE THAT PENETRATES THE EPITHELIUM AND ENTERS THE LYMPHOID TISSUE OF THE LUNG AND CAUSES DISEASE THERE AND FROM THERE IN MANY OTHER ORGANS OF THE BODY AS I'LL SHOW YOU IN A LITTLE BIT. THE CLASSIC -- THE QUINTESSENTIAL STRUCTURE THAT THESE INFECTED MACROPHAGES THAT REACH THE DEEP RECESSES, THE DEEP, DEEP AREAS OF THE LUNG FORM, IS CALLED THE GRANULOMA, OR THE TUBERCLE, FOR WHICH THE DISEASE IS NAMED, AND THE BACTERIA SOMEHOW SURVIVE IN THESE STRUCTURES, AND AT SOME POINT THESE STRUCTURES BREAK OPEN BY THE DEATH OF THE INFECTED CELLS, THE BACTERIA ARE RELEASED, AND IT IS THESE RELEASED BACTERIA FROM THE NECROTIC STRUCTURE THAT CAN THEN TRANSMIT. AND SO WE IN THE LAB HAVE BEEN ABLE TO TAKE A LOOK AT ALL OF THESE VARIOUS PHASES BY STUDYING INFECTION USING THE SURROGATE MODEL THAT GIGI REFERRED TO AND THAT IS MICROBACTERIUM MARENUM, A CLOSE GENETIC RELATIVE OF TUBERCULOSIS, WELL KNOWN TO CLINICIANS ESPECIALLY DERMATOLOGISTS, IT AFFECTS SUPERFICIAL TISSUES OF THE EXTREMITIES, BUT THE THING THAT'S IMPORTANT TO REMEMBER IT DOESN'T JUST INFECT THE HANDS OF POSTDOCS, IT ALSO INFECTS FISH, AND THIS WAS FIRST REALIZED IN 1926 WHEN FISH IN THE PHILADELPHIA AQUARIUM, NOW DEFUNCT, WERE DYING. THEY FOUND THEY HAD TUBERCLES IN THE GILLS, SPLEEN AND SO ON. YES, FISH DON'T HAVE LUNGS, AND THESE -- IT WAS NOT CLEAR WHY THESE FISH WERE DYING. THESE WERE THE FISH IN FACT THE KINDS OF FISH THAT WERE DYING, BECAUSE THEY COULDN'T ACTUALLY CULTURE ANYTHING BUT WHEN THEY DID SPECIAL STAINS, THIS GUY CALLED ARONSON, THEY FOUND THAT THE TUBERCLES WERE FULL OF ACID OF BACTERIA THAT LOOKED JUST LIKE MICROBACTERIUM TUBERCULOSIS, AND SO THAT'S WHEN HE HAD THIS BRAIN WAVE THAT SINCE FISH ARE COLD BLOODED, HE MIGHT TRY CULTURING THE TISSUES AT LOW TEMPERATURES AND WHEN HE DID THAT, BINGO, HE WAS ABLE TO CULTURE THE PATHOGENS AND SINCE THEN, 16 S SEQUENCING HAS SHOWN IT'S THE CLOSEST RELATIVE OF THE HUMAN PATHOGEN, CONFIRMED BY SEQUENCING AT THE SANGER CENTER SHOWING THIS PATHOGEN REPRESENTS THE BRANCH POINT IN THE EVOLUTION TO PATHOGENICITY OF THE MICROBACTERIUM GENUS, AND IT IS INDEED THE CLOSEST RELATIVE OF THE TB BUG THAT INFECTS HUMANS. NOW, WHEN I WAS -- A GOOD LESSON FOR PARTICULARLY THE YOUNGER PEOPLE, IT'S ALWAYS -- IT MIGHT BE GOOD TO -- MOST OF MY BREAKTHROUGHS HAVE BEEN BECAUSE I LISTENED TO IDEAS, CLEVER IDEA THAT SOMEBODY'S GIVEN ME, AND WHEN I STARTED TO FIRST WORK WITH MICROBACTERIUM MARENUM ABOUT TO JOIN STAN FALCO'S LAB I WAS TALKING TO THE END THATTING AT UCSF, AND I REMEMBER I WAS SITTING, HE HAD A LOVELY HOUSE WITH A SWIMMING POOL, I WAS SITTING ONE EVENING WITH MY HUSBAND AT -- YOU KNOW, BY HIS SWIMMING POOL. HE SAID WHAT ARE YOU GOING TO DO NEXT YEAR? I THOUGHT OF THIS IDEA, STAN AND I HAVE, WE'RE GOING TO WORK ON THE SURROGATE PATHOGEN. HE SAID, OH, YOU SHOULD EXPLORE THIS IN THE ZEBRAFISH SO YOU GET THE HOST SITE. OF COURSE, I HAD NO IDEA WHAT HE MEANT BUT I WAS TOO EMBARRASSED TO SAY ANYTHING. I WENT HOME AND LOOKED IT UP, AND YEARS LATER I -- WHEN I GOT MY OWN JOB, I DECIDED TO THINK ABOUT WHAT HE SAID AND TO EXPLORE THIS IN THE ZEBRAFISH. FIRST OF ALL, WE HAD TO SHOW THAT THE ZEBRAFISH DID IN FACT HAVE DISEASE THAT LOOKED VERY MUCH LIKE HUMAN DISEASE, SO HERE IS A HUMAN GRANULOMA IN A REAL H AND E SECTION AS OPPOSED TO CARTOON, AND WHAT YOU'LL SEE IN A STAIN THAT ONLY STAINS THE HOST AND NOT THE BUG IS THAT YOU SEE A VERY WELL ORGANIZED STRUCTURE, YOU SEE CELLS AND THIS ACELLULAR AREA, NO NUCLEI, THE NECROTIC AREA. IF YOU LOOK AT THE FISH EQUIVALENT, WHICH I DID, YOU CAN SEE NOW WHEN I STAIN IT WITH A BACTERIAL STAIN, THAT YOU SEE THAT NICE -- THAT SAME WELL CIRCUMSCRIBED STRUCTURE, THERE'S SOME BACTERIA IN THE CELLULAR AREAS BECAUSE THEY CAN CERTAINLY GROW IN THE CELLS TO SOME EXTENT, THE ACELLULAR AREA IS BETTER, THEY CAN GROW IN NECROTIC, SOMETHING GIGI MENTIONED, THE ZEBRAFISH HAS A TRANSPARENCY THE FIRST TWO OR THREE WEEKS OF LIFE, YOU CAN DO ALL KINDS OF WONDERFUL THINGS, THE DEVELOPMENTAL BIOLOGISTS CAN STUDY PROCESSES IN LIVE ANIMALS, WE THOUGHT WE COULD LOOK AT DEVELOPMENTAL PROCESSES IN REAL TIME IN LIVE ANIMALS TOO, I MEAN INFECTION PROCESSES. AND SO WE DID, AND HERE IN A FISH THAT IS ENGINEERED TO HAVE GREEN FLUORESCENT MACROPHAGES, AND RED FLUORESCENT NEUTROPHILS THAT WE'VE INFECTED WITH BACTERIA THAT WE ENGINEERED TO HAVE BLUE CONSTITUENT REPORTER, TIGHT GRANULOMAS FORM, MADE MOSTLY OF MACROPHAGES WITH A BLUE MICROPHIL. THE FISH IS SMALL AND MY TECHNICIAN, KEVIN, WHO DEVELOPED A LOT OF THESE PROCEDURES IN MY LAB WANTS YOU TO SEE IT'S REALLY SMALL ENOUGH TO FIT ON THE CRAVAT OF LINCOLN ON THE PENNY. THAT'S KEVIN. I'M GOING TO SHOW YOU HOW KEVIN INJECTS THESE BACTERIA INTO THE FISH. YOU CAN SOAK THE FISH WITH THE BACTERIA, AND THEY WILL GET INFECTED, BUT IF YOU WANT TO MONITOR INFECTION IN REALTIME, YOU WANT TO KNOW WHEN IT HAPPENED. SO WE INFECT THEM. NOW ONE PLACE TO INFECT THEM IS THE HIND BRAIN VENTRICAL CAVITY, YOU CAN SEE CELL RECRUITMENT HAPPEN AND YOU CAN SORT OF MONITOR THAT VERY FIRST PHASE OF INFECTION. SO TO DO THIS, KEVIN USES A SUCTION DEVICE, HE HAS A LITTLE BIT OF VACUUM THAT HE USES TO HOLD THE FISH, YOU'VE GOT A FINE GLASS NEEDLE HE PULLS, AND HE'S GOT BACTERIA MIXED WITH PHENOL RED SO HE CAN SEE THE DYE GO IN AND YOU CAN WATCH THAT. HE RELEASES THE VACUUM. NOW IF YOU WANT TO BYPASS THAT FIRST STEP BECAUSE WHEN YOU WANT TO PROBE THE INFECTION IN MULTIPLE WAYS, YOU CAN PUT THE BACTERIA DIRECTLY INTO THE BLOODSTREAM AS HE'S DOING HERE, INJECTING TO THE CAWEDDAL VEIN, USING A DIFFERENT APPROACH, SORT OF A HOCKEY STICK TO HOLD THE FISH AND CAN GET IT DONE THAT WAY. PEOPLE IN THE LAB ARE GOOD ENOUGH AT THIS THAT THEY CAN DO 100 OF THESE AN HOUR AND KEEP IT SUSTAINED FOR SEVERAL HOURS, SIX OR SEVEN HOURS AT A TIME SO YOU CAN START TO DO GENETIC SCREENS ON THEM. THE OTHER THING WE'VE DEVELOPED IS A LOT OF LITTLE THINGS WE'VE DONE THAT MAKE THIS MODEL QUITE PROFITABLE, SO I THOUGHT I WOULD SHARE THEM WITH YOU. BECAUSE OF THE LARGE YOLK IT CAN FEED OFF THE YOLK FOR MANY DAYS. HERE AT THE NIH YOU CAN DO THEM IN 38 WELL PLATES BUT WE USE 96 WELLPLATES AND YOU CAN -- THESE FISH, THEY SWIM AROUND, SO YOU HAVE TO IMMOBILIZE THEM FOR MICROSCOPY, SO WHAT KEVIN REALIZED HE COULD DO WAS PLUNK THE PLAYED ON AN ICE BUCKET AND IN TEN MINUTES THE FISH STOP MOVING, AND HE PUTS THEM ON A STAGE THAT MOVES, YOU KNOW, AND HE CAN TAKE IMAGES OF ALL THE FISH WITHIN TEN MINUTES, AND THAT'S ABOUT THE TIME THAT THE FISH STAY ASLEEP. YOU CAN KEEP DOING THIS ALL DAY LONG AND DOESN'T BOTHER THE FISH, YOU CAN DO IT FOR MANY DAYS AND STUDY INFECTION SEQUENTIALLY IN THE FISH AND THAT'S HOW LOW MAGNIFICATION AND INFECTED FISH WOULD LOOK. HERE IS RED FLUORESCENT BACTERIA. I HAD AN UNDERGRADUATE IN THE LAB WHO WROTE SOME CODE AND SHOWED THAT SHE COULD QUANTIFY INFECTION AND CONFIRM THAT IT CORRELATED WELL WITH ACTUAL BACTERIAL PLATE COUNTS. SO BUT NOW, IF YOU WANT TO -- YOU CAN DO THAT AND DO A LOT OF THAT, BUT YOU CAN ALSO STUDY INFECTION IN GREAT DEPTH AND HERE I'M USING OLD-FASHIONED DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPY, PHAGE MICROSCOPY. WE INJECT THE BACTERIUM, WITHIN AN HOUR CELLS ARRIVED, THESE ARE MACROPHAGE, YOU'RE GOING TO WATCH THIS MACROPHAGE EAT THIS BACTERIUM OKAY. WE'VE GOT AN INFECTED MACROPHAGE SITTING AT THE SURFACE, AND WHAT YOU'RE GOING TO SEE NOW ARE SEVERAL DAYS LATER, YOU'RE GOING TO SEE -- WE'VE LEFT BEHIND THE BRAIN, THE CAVITY THERE, AND YOU'RE GOING TO SEE AN INFECTED MACROPHAGE, CARRYING BACTERIA IN, AND YOU CAN SEE PLENTY OF BACTERIA IN THERE, THEY ARE DOING PRETTY WELL. IT TURNS OUT THESE ARE NOT THE SAME MACROPHAGES. THEY ARE COMPLETE -- THEY ARE NOT EVEN THE SAME TYPE OF MACROPHAGE. AND THIS HAS ALL COME TO LIGHT IN THE LAST FEW MONTHS. THIS WAS MY FIRST GRADUATE STUDENT WHO SHOWED ALL THIS, AND THE NEXT -- THE PERSON WHO'S DETERMINED WHAT'S GOING ON IS MY MOST RECENT GRADUATE CJ CAMBIER, WHO DECIDED TO TACKLE THE QUESTION, WHAT'S HAPPENING IN THE FIRST VERY PHASE? AND SO WHAT HE DID WAS TO SAY IF I PUT JUST GARDEN VARIETY BACTERIA INTO THE HIND BRAIN VENTRICLE, WHAT DO I SEE? AND HE SHOWED THAT HE COULD SEE MACROPHAGES COMING TO PICK UP THESE BACTERIA, AND AS PREDICTED, HE COULD SHOW THAT THIS -- THE RECOGNITION OF THESE BACTERIA WAS MEDIATED BY TOLL-LIKE RECEPTORS BECAUSE IF YOU KNOCKED OUT THE ADAPTER FOR TOLL-LIKE RECEPTORS YOU NO LONGER GOT MACROPHAGES COMING, BUT WHEN HE LOOKED AT MICROBACTERIUM HE COULD SEE MACROPHAGES COMING BUT ABSOLUTELY NO DEPENDENCE ON TOLL-LIKE RECEPTORS. THIS WAS VERY ODD, BECAUSE NOT ONLY DO MICROBACTERIA HAVE TOLL-LIKE RECEPTORS, SOMEBODY IDENTIFIED 99 OF THEM, NOT SURE HE GOT THAT NUMBER BUT THE DISCOVERY OF TOLL-LIKE RECEPTORS WAS FACILITATED BY THE FACT CHARLIE JANES REALIZED TO BE AN EFFECTIVE VACCINE, OR TO MOUNT AN INFECTED IMMUNITY YOU NEED AN ADJUVANT, AND ADJUVANTS, THE BEST ADJUVANT KNOWN, IS BCG, WHICH IS -- OR PPD, WHICH IS NOTHING BUT KILLED MICROBACTERIAL CELLS. AND SO IF THERE ARE ALL THESE BACTERIAL PATHS PRESENT, WHY AREN'T THEY RECOGNIZED BY THE BODY? WHAT'S GOING ON? AND HERE C.J. HAD A CLUE BECAUSE HE USED A NON-PATHOGENIC ANCESTOR, SOIL-DWELLING ANCESTOR OF THE MICROBACTERIA MARINUM, SOMETHING HAD CHANGED DURING THE EVOLUTION OF PATHOGENICITY THAT MADE THE PATHOGEN NO LONGER RECOGNIZED BY TOLLLIKE RECEPTORS, AND WHAT HE REALIZED WAS HAPPENING THERE WAS A LIPID, A FATTY COAT, CALLED PDIM THAT MASKS THE PAMP AND HE DID EXPERIMENTS TO SHOW IT'S JUST A PHYSICAL MASK OF THE PAMP, SO THE PATHOGENS HAVE PAMPS THAT MASK THEM, NOT PATHOGENIC VARIETY AND GARDEN VARIETY DON'T HAVE THESE PAMPS. THERE'S A GREAT SIGNIFICANCE TO HAVING THIS COAT. IF YOU DON'T HAVE THE PAMP, MASKING LIPID AND SIGNAL THROUGH TOLL-LIKE RECEPTORS, YOU'RE BRINGING IN -- SOMEHOW ENDING UP IN A MACROPHAGE THAT KILLS YOU BECAUSE THE MACROPHAGE IS ABLE TO MAKE INDUCIBLE NITRIC OXIDE SYNPHASE, THIS IS NOT THE BUGS, SIMPLY A GREEN FLUORESCENT MACROPHAGES, WITH AN ANTIBODY, INOS-PRODUCING MACROPHAGES, ON THE OTHER HAND IF YOU HAVE THE MASKING LIPID YOU DON'T MAKE THE INOS AND WE ALSO SHOWED THE INOS IS WHAT KILLS THE BACTERIA, OKAY? THEY HAVE A MASKING LIPID THAT PREVENTS THE BAD MACROPHAGES FROM EATING THEM AND KILLING THEM. HOW ARE THEY GETTING? THE PDIM HAS A PARTNER, IT'S A SIDE ARM, AND PERMITTING THE MICRO -- MACROPHAGES TO COME IN AS WELL. IF YOU'RE NOT PATHOGENIC YOU RECRUIT TOLL LIKE RECEPTORS AND BRING IN THE MACROPHAGES, BUT THE PATHOGENIC ONES HAVE THESE MASKING LIPIDS, AND THEN THEY HAVE A RECRUITING LIPID, SOMEHOW BRINGING IN PERMISSIVE MACROPHAGES, HE SHOWED THIS WAS THROUGH A HOST -- SPECIFIC HOST CHEMOKINE, CCL 2, THAT THIS WAS HAPPENING. SO THIS LED US TO A CONUNDRUM. TB UNLIKE MOST INFECTIONS ARE THE BIGGER THE INOCULUM THE GREATER THE CHANCE OF GETTING INFECTED. TB CAN ONLY INFECT YOU IN THE BOTTOM OF THE LUNG, IT'S A PARADOX, IN THE ALVEOLAR SPACES. BECAUSE OF THAT IT CAN ONLY BE TRANSMITTED BY SMALL DROPLETS THAT CONTAIN 1-3 BACTERIA. I'VE BEEN TEACHING THIS TO MEDICAL STUDENTS FOR YEARS, USING THIS VERY CARTOON. LOOK YOU HAVE TO GO WAY DOWN IN THERE IN ORDER TO INFECT, AND OF COURSE I HAVEN'T KNOWN WHY, AND SOMETIMES DOCTORS DON'T WORRY ABOUT WHY. AND THIS HAS BEEN SHOWN NOT ONLY VERY NICELY IN EPIDEMIOLOGIC STUDIES, THE BEST ONE ACTUALLY BEING DONE IN A NAVAL SHIP HERE OFF THE COAST OF BALTIMORE, USS CONSTITUTION, WHERE THEY WERE ABLE TO TRACK WHO GOT INFECTED AND WHO DIDN'T AND SHOWED THAT ONLY PEOPLE CONNECTED BY VERY FINE DUCTWORK WERE INFECTED BY PEOPLE WHO -- CASES OF TB, BUT IT'S ALSO BEEN SHOWN IN ANIMAL STUDIES WHERE WELLS WORKING AT HOPKINS INFECTED RABBITS WITH EITHER BIG DROPLETS OF 10,000 BACTERIA HE COULD SHOW BY TRANSSECTION GOT STUCK IN THE TRACHEA VERSUS SIGNEE DROPLETS OF 1-3 BACTERIA WHICH HE COULD SHOW LANDED DEEP INSIDE. YOU CAN SEE NICELY HERE, ONLY THE RABBITS WHO GOT A FEW BACTERIA GOT INFECTED SO YOU HAVE TO GO TO THE BOTTOM OF THE LUNG. THIS MADE US COME UP WITH A MODEL, WE THOUGHT THIS MIGHT BE BECAUSE IF WE HAVE THE MICROBACTERIA, THEY HAVE A STRATEGY TO AVOID DETECTION IN THE UPPER AIRWAYS, BUT THE UPPER AIRWAYS ARE REPLETE WITH OTHER BACTERIA, PESKY BACTERIA THAT EXPRESS PAMP AND BRING IN THE WRONG MACROPHAGE, MICRO BACTERIA MIGHT BE CAUGHT IN THE CROSS-FIRE AND ESSENTIALLY BE GOBBLED UP BY THESE MACROPHAGES. AND SO THE WAY WE TESTED THAT WAS SIMPLY BY TAKING MICROBACTERIA AND CO-INFECTING THEM WITH A PAMP-EXPRESSING BACTERIA AND LOOKING AT THE PROGRESSION OF THE MICROBACTERIUM. HERE WE HAVE MICROBACTERIUM THAT WE AFFECTED ALONE OR CO-INVENTED WITH PSEUDOMONIS. IF IT'S ALONE IF THREE DAYS YOU GET GROWTH, IF YOU'VE GOT A CO-INFECTING BACTERIUM YOU DON'T SEE ANY GROWTH AND INDEED WE SHOW THIS WAS DEPENDENT, THIS INHIBITION, BY THE PAM-EXPRESSING BACTERIA WAS DEPENDENED ON THE HOST HAVING IDN 88. IT HAS TO HAVE THE MASKING LIPID AND RECRUITING LIPID BUT THAT ONLY WORKS IF THESE ARE -- IF THERE'S A SMALL DROPLET SIZE AND SO THE STRATEGY DOESN'T WORK IN THE UPPER AIRWAY BUT IN THE LOWER AIRWAY WHERE THERE ARE FAR FEWER COMMENSALS, THEN YOUR STRATEGY OF BRINGING IN THE RIGHT MACROPHAGE CAN WORK AND SO YOU CAN THINK OF IT AS THE MICROBIOME IS PROTECTING US BECAUSE THE FACT IT HAS TO GO IN SMALL DROPLET MAKES IT LESS INFECTIOUS THAN MEASLES AND COLDS. YOU CAN THINK OF IT AS TB SURVIVAL STRATEGY ON THE OTHER HAND, DESPITE THIS IT'S BEEN AROUND 70,000 YEARS AND HAS SEEN A LOT OF CHANGES IN HUMAN BEHAVIOR. IT'S SEEN ITS WAY THROUGH THE NEOLITHIC TRANSFORMATION AND MANAGED TO SURVIVED, CO-LEFT USING OF PATHOGEN AND HOST. NORMALLY I WOULD END THIS PART HERE BUT C.J. HAS JUST GOT SOME BEAUTIFUL RESULTS THAT I THINK ARE WORTH SHARING TO COMPLETE THIS STORY, BECAUSE AT THE END OF THAT WORK, WHICH WAS PUBLISHED IN "NATURE," CJ'S COMMITTEE TOLD HIM HE -- HE HAD TWO PAPERS, THE SECOND WAS A "NATURE" PAPER, THEY SAID WHY DON'T YOU JUST GRADUATE? C.J. WAS HAPPY WITH THAT, I DON'T KNOW WHY, BUT HE LIKED THAT IDEA A LOT BUT GOT A RESULT THAT MADE HIM STAY ANOTHER SEVEN OR EIGHT MONTHS, AND THE RESULT WAS THIS. SO HE HAD ENVISIONED THAT THE WAY THE RECRUITING LIPID WORKED TO MAKE THE HOST MAKE CCL2 WAS TO INDUCE CCL 2 PRODUCTION IN THE EPITHELIUM BECAUSE THE BACTERIUM FIRST CONTACTED THE EPITHELIUM. THAT MAKES SENSE BECAUSE THE EPITHELIUM MAKES CCL 2. THE EXPERIMENT WAS HE KNOCKED OUT MACROPHAGES IN THE FISH AND FULLY EXPECT THE CCL 2 PRODUCTION WOULD REMAIN INTACT, IN FACT HE FOUND IT DROPPED. HE COULD GET NO CCL 2 IF THERE WERE NO MACROPHAGES, SO WHAT'S GOING ON? THE CCL 2 IS REQUIRED TO RECRUIT MACROPHAGES, YET MACROPHAGES ARE REQUIRED TO MAKE CCL2. THIS WAS THE PARADOX THAT C.J. STAYED ON TO HAVE A LOOK AT. AND WHAT HE REALIZED RIGHT AWAY WAS THAT, YES, WHILE CCL 2 WAS REQUIRED TO RECRUIT MACROPHAGES IF YOU COMPARED TO MOCK INFECTION THERE WAS STILL MACROPHAGE RECRUITMENT IN THE ABSENCE OF CCL 2, THIS MEANT THERE WAS SOME SUBPOPULATION OF MACROPHAGES COMING IN, IN THE ABSENCE OF CCL 2. SO HERE HE USED THE CLEVER TRICK AND THE TRICK IS THAT EVEN AT THIS YOUNG AGE THESE FISH HAVE A BLOOD-BRAIN BARRIER, AND THIS MIGHT MAKE -- PARTICULARLY MAKE SENSE FOR A CREATURE FREE SWIMMING IN THE GANGES AT THE ENDER AGE OF 3 BETTER HAVE GOOD THINGS IN PLACE. IN YOU INJECT DYE INTO THE CAUDAL VEIN, THE ONLY WAY IT WILL GET IN IS IN MACROPHAGES FROM THE CIRCULATION BRING IT IN. SO WHEN HE LOOKED AT INFECTION NOW, HE COULD SEE SOME MACROPHAGES DIDN'T HAVE THE DYE, OTHERS DID. IT TURNED OUT THESE GUYS THAT DIDN'T HAVE THE DYE WERE BRAIN-RESIDENT MACROPHAGES, SO JUST LIKE THE LUNG THE BRAINING HAS RESIDENT MICROGLIA, AND SO HE SAW COMBINATION OF PERIPHERALLY RECRUITED CIRCULATING MONOCYTES, AND MACROPHAGES. AND WHAT HE FOUND WAS THAT IN THE -- HE COULD SEE THAT THE BRAIN -- THE RESIDENT MACROPHAGES GET RECRUITED BEFORE THE CIRCULATING ONES, AND HE COULD SHOW THE FIRST RECRUITMENT WAS NOT DEPENDENT ON THE CCL2, SO THEY WERE DEFAULT RECRUITMENT, BUT THE SECOND ONE WAS. SO THE MODEL WOULD BE THAT THE BACTERIA GET INTO THE FIRST RESIDENT MACROPHAGES THAT ARE RECRUITED IN A DEFAULT WAY TO ALL PATHOGENS, THOSE ARE MAKING THE CCL2 RECRUIT THE NEXT ROUND. AND HE WAS ABLE TO SHOW THIS BY USING A MUTANT IN THE FISH THAT DOESN'T HAVE THE BRAIN-RESIDENT MACROPHAGES BECAUSE NOW HE WAS ABLE TO SHOW THAT THE RECRUITMENT OF THE SECOND CIRCULATING MACROPHAGES WENT AWAY COMPLETELY SO THE BRAIN RESIDENT MACROPHAGES WERE REQUIRED TO RECRUIT THAT NEXT ROUND. AND IN FACT, WHAT HE SHOWED WAS THAT THE BUGS GET IN TO THE DEFAULT -- TO THE RECRUITMENT -- TO THE RESIDENT MACROPHAGES THAT ARE RECRUITED IN THE DEFAULT FASHION. AND THIS IS A UBIQUITOUS RECRUITMENT TO ALL BACTERIA, THE MICROBACTERIUM DOESN'T KNOW WHAT TO DO ABOUT IT AND HAS TO GET INTO THEM. IT'S NOW REPROGRAMMING THESE CELLS TO MAKE CCL 2, THE VERY CELLS THAT COME TO IT ARE MAKING THE CCL 2, IT'S RECRUITING A NEW CELL, AND THE BACTERIA ARE TRANSFERRING INTO THAT. SO YOU CAN SEE HERE THIS IS AN INFECTED CELL THAT'S A RESIDENT MACROPHAGE BECAUSE THERE'S NO BLUE IN IT AND YOU'LL SEE IF YOU'RE GOING TO SEE IT FUSE TO A NEW CELL THAT'S JUST COMING IN. THESE ARE ALL DONE IN SEQUENTIAL IMAGING THAT LASTS, OH, 20, 30 HOURS, SO THERE IT IS. THERE'S THE BLUE GUY. YOU CAN SEE THAT THEY COME AND THEY FUSE. AND IF THAT DOESN'T HAPPEN, AND YOU CAN SEE THAT ALL THE TRANSFER EVENTS HAPPEN WITHIN THE FIRST -- BETWEEN TWO DAYS AND ABOUT FIVE DAYS OF INFECTION, THEY ARE COMPLETELY DEPENDENT ON THE BACTERIUM HAVE THE PHENOLIC GLYCOLIPIDS, IF THEY DON'T SEE IT YOU DON'T SEE TRANSFER UNTIL LATE. IN THE NEXT SLIDE YOU'LL SEE THAT THE RESIDENT MACROPHAGE IS VERY WELL CAPABLE OF KILLING THE BACTERIA, AND HERE IS A BACTERIUM IN THE PROCESS OF BEING KILLED BY THE CELL OVER SEVERAL HOURS. BOOM, IT'S GONE. WHAT'S HAPPENING HERE IS THAT THE BUG IS -- FINDS ITSELF IN A RESIDENT MACROPHAGE, IF IT DOESN'T HAVE THE PHENOLIC GLYCOLIPID THEN IT'S SLOWLY KILLED BY THE RESIDENT MACROPHAGE. IF IT DOES HAVE THE PHONE ROLLIC GLYCOLIPID, IT'S ABLE TO TRANSFER. ONE-THIRD OF THE HUMAN RACE THAT GIGI WAS TALKING ABOUT, DEPENDS ON WHETHER THE RESIDENT MACROPHAGE IS ABLE TO KILL US BEFORE IT MANAGES TO ESCAPE. THAT'S THE FIRST BIT, HOW DOES TB BEGIN. I HOPE I'VE TOLD YOU A LITTLE BIT ABOUT THIS. NOW LET'S LOOK AT THIS NEXT PHASE AND SEE WHAT WE'VE LEARNED FROM THE FISH ABOUT THIS PHASE. SO LET'S GO BACK TO LOOKING AT WHAT WE FIRST SAW BEFORE WE STARTED TO DELVE INTO IT. SO THAT NEXT STAGE AFTER THE MACROPHAGE GOES IN, YOU CAN ALREADY SEE THE GRANULOMAS FORMING AS I ALREADY SHOWED YOU BEFORE WITH FLUORESCENCE MICROSCOPY. YOU SEE AN UNINFECTED MACROPHAGE JOINING THIS, RECRUITING MACROPHAGES TO COME TO IT. HERE IS THE NEW MACROPHAGE, IT'S COMING IN, YOU'RE GOING TO SEE IT'S KIND OF SQUEEZING ITS WAY IN HERE. OKAY. AND THEN THIS IS THE KIND OF TIME LAPSE MOVIE THAT MADE US QUESTION A VERY OLD DOGMA IN THE FIELD, AND THAT WAS THAT THE TUBERCULOSIS GRANULOMA IS A CRITICALLY IMPORTANT HOST PROTECTING STRUCTURE THAT WALLS OFF THE BACTERIA. THAT'S THE LANGUAGE OF MEDICAL TEXT BOOKS AND IMMUNOLOGY TEXT BOOKS, AND RESTRICTS THEIR GROWTH. AND WHAT WE FOUND WAS THAT IT WAS A HIGHLY DYNAMIC STRUCTURE, AND WHAT WAS HAPPENING WAS THAT BACTERIA WOULD FILL UP A MACROPHAGE BECAUSE THEY WOULD BE ABLE TO GROW IN IT, AND THEN THEY WOULD KILL THE MACROPHAGE BY APOPTOSIS, AND THIS IS THAT BASELINE CELL-DEATH PATHWAY THAT'S OPERANT. THIS IS DYING WITHIN IT, A NEW MACROPHAGE COMES AND ENGULFS THIS MACROPHAGE SO THAT THE BACTERIA TRANSFER FROM ONE CELL TO ANOTHER. NOW, THIS IS DISTINCT FROM THE FIRST EVENT I SHOWED YOU TRANSFERRED FROM A LIVE BACTERIUM TO A DEAD ONE, TO A NEW ONE. HERE A DEAD CELL IS GIVING UP ITS BACTERIA TO A NEW CELL, AND THAT YOU CAN SORT OF SEE WHY MACROPHAGES ARE CALLED MACROPHAGES, OR BIG EATERS. IT'S REALLY KIND OF -- MAYBE WE SHOULD GO INTO OBESITY RESEARCH OR SOMETHING HERE. SO WE'VE GOT A NEW INFECTED MACROPHAGE BUT IN REALITY YOU'RE GOING TO SAY, WELL, OKAY, THESE TRANSFERRED FROM ONE TO THE OTHER, WHY WOULD THE GRANULOMA BE A BACTERIA EXPANDING STRUCTURE. MOST OF THE TIME A SINGLE DEAD MACROPHAGE PACKED UP IS EATEN BY TWO TO THREE NEW MACROPHAGES SO THE CONTENTS ARE DISTRIBUTED AND NOW THEY CAN GROW AGAIN. SO THEY CAN -- THEY USE THE MACROPHAGES TO EXPAND THEMSELVES INTRACELLULARLY, AND THIS IS -- THIS STRATEGY IS DEPENDENT ON A VERY FAMOUS VIRULENCE SYSTEM IN TB, THAT IS A SECRETION SYSTEM THAT HAS A PARTICULAR EFFECTOR CALLED ESAT6, SECRETED BY THE SECRETION SYSTEM, AND DANA, HANNAH AND MUSE IN THE LAB WERE ABLE TO SHOW THE EXPANSION, THAT RECRUITMENT OF CELLS AND EXPANSION INTO THEM, WAS DEPENDENT ON THE ESAT6 STRUCTURE. HERE USING FLUORESCENCE MICROSCOPY, THEY CAN SHOW YOU HOW IN THE WILD TYPE VERY RAPIDLY NEW MACROPHAGES ARE BEING BROUGHT IN, AND ARE BEING INFECTED BECAUSE ONLY INFECTED MACROPHAGES ARE SHOWING UP ON THIS. IN CONTRAST, IF YOU HAVE AN ESATMUTANT IT'S PERFECTLY WELL ABLE TO GROW WITHIN THE MACROPHAGE, BUT THE PROBLEM IS THAT IT'S NOT ABLE TO RECRUIT NEW MACROPHAGES EVEN THOUGH IT REALLY, REALLY FILLS UP. THE PEDIATRIC INFECTIOUS DISEASE DOCTOR ASKED WHAT HOST PARTNER IS INDUCEDOME WHEN THE ESAT6 DETERMINANT IS PRESENT, SO USING WILD TYPE BACTERIA VERSUS ESAT6 MUTANT BACTERIA COMPARING THE DIFFERENCE IN HOST GENE EXPRESSION CAME UP WITH MMP9. PEOPLE SAY IT'S THE PROBLEM WITH DOING SCREENS, THESE KINDS OF ARRAYS AND RNA-SEQ IS YOU GET TOO MANY CANDIDATES. I HAVE A GOOD TRICK FOR YOU, TO DO IT BADLY BECAUSE THEN YOU ONLY GET ONE. IN FACT WE LITERALLY ONLY GOT TWO GENES, MMP9 AND ITS REGULATOR, A TEMP2. SO WE SAW MMP9 DIFFERENTIALLY INDUCED BUT THE PROOF OF THE PUDDING, DOES IT MATTER, AND THE WAY TO TEST THAT IS YOU KNOCK OUT MMP9 AND YOU NOW ASK, IF I PUT WILD TYPE BACTERIA INTO AN MMP9 MUTANT AM I GOING TO GET AN ATTENUATED INFECTION AS I DO WITH THE ESAT6 MUTANT. WE HAVE THE WHOLE FISH SHOWING THE ESAT6 MUTANT. HERE IS WILD TYPE, HERE IS THE ESAT6 MUTANT, ALL IN WILD TYPE FISH, NOW LOOK AT THE MMP-9 SYSTEM, HERE NOW WILDTYPE BACTERIA IN WILD-TYPE FIRST VERSUS MMP9 MUTANT, YOU CAN SEE HOW IT REPRODUCES THAT. THIS PART TELLS YOU WHAT'S HAPPENING IS THAT ONCE THE BUG GETS IN, NOW REMEMBER WE'VE ALREADY SHOWN YOU THAT IT MOVED FROM THE NASTY BAD MACROPHAGES INTO A PERMISSIBLE MACROPHAGE SO NOW IT WANTS TO RECRUIT MORE OF THESE BECAUSE IT'S HAPPY TO STAY IN THESE, AND SO WHAT IT'S DOING IS TO USE -- IT SWITCHED FROM USING ITS LIPIDS TO USING SECRETED PROTEIN THAT'S NOW INDUCING MMP9, USING THE EPITHELIUM, WE COULDN'T FIND IN THE FIRST STEP NOW COMING INTO PLAY USING THE LUNG EPITHELIUM TO -- OR IN THE FISH SOME OTHER EPITHELIUM, TO RECRUIT, TO INDUCE THE MMP9, TO BRING PERMISSIVE MACROPHAGES IT CAN THEN TRANSFER INTO. OKAY. SO THERE WAS -- SO AT THIS POINT, WE FOUND OURSELVES INADVERTANTLY GETTING INTO A VERY BIG QUESTION IN TB, WHICH IS THE QUESTION OF WHY DOES IT TAKE SO LONG TO TREAT TB, AND AS I TOLD YOU, THERE HAVE BEEN PERFECTLY EFFECTIVE DRUGS FOR TB SINCE THE 1960s, AND YET THERE'S A LOT OF TB. I DIDN'T TELL YOU WHY BECAUSE I WAS SAVING IT TILL NOW. THE REASON FOR THAT, EVERYONE AGREES, IS BECAUSE TREATMENT TAKES TOO LONG. IT TAKES SIX MONTHS TO NINE MONTHS TO TREAT DRUG SENSITIVE TB WITH THREE TO FOUR DRUGS. AND THIS IS REALLY NOT FEASIBLE, WOULDN'T BE FEASIBLE HERE, LET ALONE IN ECONOMICALLY UNDERPRIVILEGED AREAS WHERE YOUR LIVELIHOOD DEPENDS ON YOUR NOT GOING TO A CLINIC TO GET DRUGS. SO THE QUESTION HAS BEEN WHY, WHY IS IT THAT IT TAKES SO LONG? IF YOU PUT TB INTO -- IN A LAB CULTURE 99% ARE KILLED WITHIN 24 HOURS, JUST LIKE ANY RAPIDLY GROWING BUG. SO IT ISN'T SLOW GROWTH THAT'S THE PROBLEM IN CULTURE. THE MODEL HAS BEEN THAT IN VIVO, AS A COROLLARY TO THE PROTECTIVE GRANULOMA MODEL THAT WE DON'T THINK IS QUITE RIGHT ANYMORE, HAS BEEN THAT THE BUGS GET INTO THIS AND THEY ESSENTIALLY ARE PUT TO SLEEP. THEY BECOME DORMANT. AND THEY -- AND BACTERIA THAT ARE IN STATIONARY PHASE, AS WE ALL KNOW, ANY BACTERIUM IS NOT SENSITIVE TO ANTIBIOTICS BECAUSE ANTIBIOTICS TARGET THINGS THAT ARE REALLY ESSENTIAL FOR GROWING BACTERIA, NOT NON-GROWING BACTERIA SUCH AS CELL WALL AND RIBOSOMES AND RNA POLYMERASE AND EVERYTHING. SO THAT HAS BEEN THE MODEL THAT YOU'VE GOT TO HAVE, THAT THERE ARE THESE NON-GROWING, NON-REPLICATING BACTERIA THAT CAUSE DRUG TOLERANCE. BUT STARTING WITH THE ZEBRAFISH AND GETTING FURTHER, WHAT WE DISCOVERED WAS THAT IN FACT, AND I'M GOING TO TELL YOU THE PUNCHLINE FIRST, IS THAT WHAT'S HAPPENING IS THAT WHEN BACTERIA ENTER MACROPHAGES, THEY NOW TURN ON EFFLUX PUMPS, BACTERIAL EFFLUX PUMPS, THEY HAVE MANY AND WE'VE IDENTIFIED A FEW. AND THESE EFFLUX PUMPS ALLOW THE BACTERIA TO GROW IN THE MACROPHAGE, REMEMBER HOW I TOLD YOU THAT ESAT6 WAS NEEDED TO BRING IN NEW MACROPHAGES BUT NOT REQUIRED TO GROW IN THE MACROPHAGES? THE EFFLUX PUMPS ARE REQUIRED FOR THE BACTERIA TO GROW IN THE MACROPHAGE. BUT THESE EFFLUX PUMPS ALSO INDUCE DRUG TOLERANCE, SO THESE -- SIDE EFFECT IS THEY ARE PUMPING OUT THE ANTIBIOTICS WE USE. AND SO AS A RESULT OF THAT, YOU HAVE BACTERIA THAT ARE THE MOST RAPIDLY GROWING BECAUSE THEY ARE ABLE TO TOLERATE THE -- THEY ARE ABLE TO ACTUALLY GROW IN THE MACROPHAGE BECAUSE THEY HAVE EFFLUX PUMPS, THAT ARE ALSO THE MOST TOLERANT. SO KIND OF TURNING THE OLD MODEL TOPSY-TURVY. AND SO KRISTIN IDENTIFIED IF YOU USED EFFLUX PUMP INHIBITORS IT WOULD PREVENT THEM FROM GROWING AND ACT AS IN VIVO ANTI-MICROBIAL AND ALSO PREVENT REPAMPACIN FROM BEING PUMPED OUT AND KRISTIN MADE THE INITIAL DISCOVERY AND WAS JOINED BY JOHN WHO IS TAKING THIS WORK TO A CLINICAL TRIAL, AND KEVIN TAKAKI WHO DID MUCH OF THE WORK THAT INITIALLY STARTED IN THE FISH. SO THE WORK STARTED IN THE FISH, BECAUSE PEOPLE ASKED US WHAT HAPPENS WHERE ANTIBIOTICS IN IF THE FISH, AND WHEN WE USED CLASSIC TB DRUGS IN THE FISH, WE COULD SHOW THAT INDEED THEY KILLED THE BACTERIA BUT NOT ALL OF THEM. WE SAW THE SAME PHENOMENON OF DRUG TOLERANCE, BACTERIUM ALWAYS IN MACROPHAGES, THEY WERE NEVER DRUG RESISTANT. IF YOU CULTURED THEM OUTSIDE THE FISH, THEY WERE COMPLETELY SUSCEPTIBLE. IT WAS JUST THE CLASSIC MECHANISM, THE CLASSIC PHENOMENON OF TOLERANCE, WHICH WAS INDUCED BY THE ENVIRONMENT. AND SHE SHOWED -- KRISTIN NOW SHOWED VERY NICELY THAT THIS WAS INDUCED BY MACROPHAGE GROWTH, IF YOU PUT THE BUGS IN HUMAN MACROPHAGES IN VITRO IN CULTURE INITIALLY THEY ARE KILLED VERY NICELY, BUT IF THEY HAVE LIVED IN THE MACROPHAGE FOR A BIT THEN THEY ARE NOT KILLED VERY NICELY AT ALL. SO THIS IS TOLERANCE, AND AGAIN I EMPHASIZE THIS IS NOT RESISTANCE. AND THE WAY WE FOUND OUT THAT THIS WAS THE MOST RAPIDLY GROWING BACTERIA WAS TO USE A PLASMID THAT SEGREGATES FAITHFULLY WITH REPLICATION. SO IF THE OLD MODEL WAS CORRECT, YOU WOULD PREDICT THE BACTERIA THAT HAD LOST THE PLASMID -- THAT RETAINED THE PLASMID BECAUSE THEY HADN'T REPLICATED WOULD BE THE ONES ENRICHED FOR THE TOLERANT POPULATION. BUT IN FACT, WE FOUND JUST THE OPPOSITE. IT WAS THE BACTERIA THAT HAD LOST THE PLASMID THAT WERE THE MOST ENRICHED FOR DRUG TOLERANCE. AND THIS THEN TOLD US THAT WE MIGHT BE REALLY THINKING ABOUT A DIFFERENT MECHANISM AND THIS IS HOW WE THEN FIGURED OUT THIS WAS MEDIATED BY EFFLUX PUMPS, AND THIS PARTICULAR CASE WE IDENTIFIED AN EFFLUX PUMP THAT MEDIATES TOLERANCE TO ONE OF THE FRONT LINE DRUGS RIFAMPICIN, AND THIS IS WILD TYPE AND YOU CAN SEE AFTER GROWTH IN A MACROPHAGE IT'S BECOME TOLERANT, BUT IF YOU KNOCK OUT THE PUMP TOLERANCE GOES AWAY, AND NOT ONLY THAT BUT EVEN IN THE ABSENCE OF ANTIBIOTICS AS I ALREADY TOLD YOU IN THE MODEL, THIS PUMP IS RESPONSIBLE FOR INTRACELLULAR GROWTH. WILD TYPE GROSS IN THE MACROPHAGE IN 9 6 HOURS, AND THE MUTANT DOESN'T GROW. THEN WE LOOKED AT THE EFFLUX PUMP LITERATURE, VERAPIMIL CAN BE USED -- MIGHT BE A GOOD CANDIDATE TO TRY TO REDUCE TOLERANCE AND INDEED WE WERE ABLE TO SHOW IT REDUCED TOLERANCE OF RIFAMPICIN BUT ALSO THE OTHER FRONT LINE DRUG, WE USED ONE DRUG TO REDUCE TOLERANCE OF TWO AGENTS. WITHOUT DRUGS VERAMIL THEY REDUCE GROWTH, EVEN IN THE ABSENCE OF ANTIBIOTICS, AND INDEED VERAPIML MAGICALLY CONVERTS IN VITRO, THE PREDICTED DOUBLE WHAMMY. BASED ON THIS WE GOT A COLLEAGUE OF OURS, BILL, TO TEST THIS IN MICE, TO SEE IF VERAPIMIL WOULD REDUCE RELAPSE OF SHORT-TERM TREATMENT IN A MOUSE MODEL OF TB AND HE WAS ABLE TO SHOW NICELY THAT IT DID, BASED ON THAT WE WERE ABLE TO GET A TRIAL FUNDED WHICH IS BEING DONE AT THE TB RESEARCH CENTER IN INDIA WHICH IS WHERE INITIALLY ALL THE DRUG TREATMENT PROTOCOLS THAT WE USE TODAY WERE ACTUALLY DEVELOPED THAT BY DANNY, THE SAME PLACE IN OUR TESTING WHETHER VERAPAMIL CAN BE USED AS A TREATMENT SHORTENING AGENT, GREAT LUCK IT CONCENTRATES IN IT THE LUNG, SO WE MAY BE IN GOOD SHAPE FOR THIS TRIAL. SO THIS IS SOMETHING THAT'S JUST BEGUN. OKAY. SO IN THE LAST BIT, I WANT TO TELL YOU, I'VE WALKED YOU THROUGH HOW TB CHEATS THE IMMUNE SYSTEM TO ENTER, I'VE SHOWN YOU HOW IT NOW YET AGAIN TRICKS AND CAN SPREAD FROM CELL TO CELL, USES EFFLUX PUMPS TO GROW WITHIN THE MACROPHAGES AND THIS LED US TO UNDERSTAND A LITTLE BIT ABOUT DRUG TOLERANCE, AND IN THE LAST FEW MINUTES I WANT TO TELL YOU HOW WE'VE COME TO UNDERSTAND HOW IT RUPTURES OUT OF THE GRANULOMA, A CRITICAL STEP IN TRANSMISSION. IF YOU DON'T HAVE A NECROTIC GRANULOMA, NO MATTER HOW INFECTED YOU ARE, YOU DON'T REALLY TRANSMIT VERY WELL SO IT WOULD BE A DEAD END FOR THE ORGANISM WITHOUT THIS LAST STEP. THIS UNDERSTANDING AGAIN CAME BY SERENDIPITY, FROM DAVID TOBIN, WHO NOW HAS HIS OWN LAB AT DUKE UNIVERSITY, AND AT THE TIME CECELIA MOENS WAS DOING A FORWARD GENETIC SCREEN LOOKING AT DETERMINANTS OF MOTOR MIGRATION AND WE PIGGYBACKED TO SEE IF THE FISH COULD TELL US WHAT MADE IT MORE SUSCEPTIBLE AND RESISTANT TO INFECTION. WE WOULD SIMPLY TAKE HER FISH, MUTANT FISH, WE DIDN'T KNOW THE MUTATION AND COULD SCREEN THEM PHENOTYPICALLY. HERE IS A WILDTYPE FISH, HERE IS THE MUTANT FISH, HIGHLY INFECT THE AS COMPARED TO WILD TYPE SIBLING. SO WHEN DAVID MASKED MUTATION MY HEART SANK BECAUSE IT WAS IN THE ARACIDONIC, I REMEMBERED SOMETHING TO DO WITH ASPIRIN AND ASTHMA. TURNS OUT THE MUTANT, THAT THE FISH WAS MUTANT IN THE LEUKOTRIENE ENZYME THAT CONCERTS UNSTABLE LTA4 INTO A HIGHLY STABLE MOLECULE, LEUKOTRIENE B4, YOU WOULD THINK THIS WOULD LEAD TO THE ABSENCE OF THE MOLECULE, THE REASON THE FISH SUCCUMBED TO TB BUT IN FACT IT TURNS OUT THAT WE DON'T NEED LEUKOTRIENEB4, THE FISH WAS UNSUSCEPTIBLE, IT NOW HAD A DOMINANT ANTI-INFLAMMATORY MILIEU SO THE HOST SIMPLY COULDN'T MOUNT AN APPROPRIATE RESPONSE TO TB. YOU DON'T MOUNT A KEY INFLAMMATORY MOLECULE, TNF, AND GET UNCONTROLLED MICROBACTERIAL GROWTH. HE FOUND IF YOU HAD TOO MUCH OF THE ENZYME AND MADE TOO MUCH OF THE PRO INFLAMMATORY MOLECULE YOU HAD THE SAME UNCONTROLLED BACTERIAL GROWTH WHICH DIDN'T MAKE ANY SENSE BECAUSE WHY SHOULD INFLAMMATION BE BAD? AND HE SHOWED THAT WAS MEDIATED BY HAVING TOO MUCH TNF. WHAT COULD BE GOING ON? BEFORE WE PROBE WHAT'S GOING ON, WE REALIZE THIS WAS ALL DONE IN FISH EGGS AND WE NEEDED TO HAVE SOME RELEVANCE, AND SO DAVID SEARCHED THE THOUSAND GENOMES PROJECT THAT JUST MATURED AND WAS ABLE TO FIND A POLYMORPHISM IN THE HUMAN LTA4-H PROMOTER, HE WAS ABLE TO SHOW THAT THIS TRANSITION WAS FUNCTIONAL IN THAT IT MEDIATED LEVELS OF LTA4-H, SO IT WAS THE CAUSAL VARIANT THAT WAS A COMMON CAUSAL VARIANT, AND IN A NUTSHELL BEFORE I SHOW YOU THE DATA, WE FOUND IN A HUMAN CLINICAL STUDY THAT IT REGULATED TB SEVERITY IN EXACTLY THE WAY PREDICTED BY THE FISH, TOO LITTLE AND TOO MUCH, BOTH CAUSED SEVERE TB, WHERE AS THE MIDDLE WAS THE ONE THAT CAUSED THE LEAST SEVERE TB. SO LET'S HAVE A LOOK AT THE DATA. HERE IS THE POLYMORPHISM. HERE IS THE LEVEL OF PROTEIN IN RELATION TO THE POLYMORPHISM, THE CCs MAKE THE LEAST, TTs MAKE THE MOST, HETEROZYGOTES IN THE MIDDLE. WE SWITCHED TO PEOPLE NOW. THIS IS ALL PEOPLE. THE COHORT WE LOOKED IN IS TB MENINGITIS, A VERY SEVERE -- THE SEVEREST FORM OF TB, AND IN THE BEST HANDS AS IS THE CASE AT THE WELCOME TRUST UNIT IN VIETNAM, IT CARRIES A MORTALITY OF ABOUT 20% BUT MORE IN NORMAL PLACES AVERAGE PLACES ABOUT 40%, AND THIS IS DRUG SENSITIVE TB WITH FULL TREATMENT. SO IF DAVID IS RIGHT, THEN YOU WOULD PREDICT THE PEOPLE WHO LIVE WOULD BE THE PEOPLE WHO HAVE THE -- THE HETEROZYGOTES SHOULD FIVE AND HOMOZYGOTES SHOULD DO BADLY, THAT'S WHAT DAVID FOUND. HETEROZYGOTES LIVE, HIGHS AND LOWS BOTH DO WELL. BUT THE IMPLICATIONS OF THIS GO DIRECTLY TO TREATMENT, AND THIS IS BECAUSE OF THE FOLLOWING. BECAUSE TB MENINGITIS IS SUCH A SEVERE DISEASE AND BECAUSE YOU SEE ALL THAT GOOEY STUFF AT THE BASE OF THE BRAIN THAT SUGGESTS A LOT OF INFLAMMATION PEOPLE HAVE IN THE HAND WAVING WAY USE POTENT ANTI-INFLAMMATORY TREATMENT AS AN ADJUVANT TO ANTIBIOTIC TREATMENT. THEY HAVE USED DEXA METHOZONE, A GLUCOCORTICOID FOR AGES, MY MEDICAL COLLEGE -- THAT WAS ANCIENT TIMES, WE WERE ALREADY USING IT. THIS STUDY IN VIETNAM MADE IT THE STANDARD OF CARE SHOWING IN A PLACEBO CONTROLLED DOUBLE BLINDED STUDY THAT DEXOMETHOZONE SHOWED A SLIGHT SLIGHT IMPROVEMENT. YOU'RE NOT ALLOWED TO USE YOUR JUDGMENT ANYMORE, YOU JUST USE IT. SO DAVID MADE THE PREDICTION THAT THIS SMALL IMPROVEMENT WAS BECAUSE THE LOW INFLAMMATORY GROUP IS LOOKED HIGH STEROIDS AND ONLY THE HIGH INFLAMMATORY GROUP IS GOING TO BE HELPED BY IMMUNOANTI-INFLAMMATORY TREATMENT, HE LOOKED AT THE HISTORICAL COHORT. LET'S HAVE A LOOK OURSELVES. HIGH, WITHOUT DEXO, DYING A LOT, CLOSE TO 40%. IN THE ADMITTEDLY SMALL COHORT, THERE'S NOT A SINGLE DEATH WITH DEXAMETHOZONE. THE LOWS ARE BEING HARMED BY THE TREATMENT. AND SO TO SUMMARIZE THIS PART, WE STARTED OFF WITH THE SCREEN, WITH THE FISH THAT THE FISH COME FROM THE HOLY WATERS OF THE GANGES, I LIKE TO SAY THAT'S WHY THEY ARE CLEAR. WE WENT TO VIETNAM AND WE FOUND THAT OUR MUTANT WAS RELEVANT THERE, AND THEN WE ACTUALLY LOOKED AT A COHORT OF LEPROSY PATIENTS IN NEPAL, THE VARIANTS WITH BOTH HOMOZYGOTES GIVING YOU SEVERE LEPROSY, HETEROZYGOTES BEING PROTECTED, DONE WELLCOME TRUST WITH HELPFUL PEOPLE, LEPROSY WORK DONE WITH DEANNA WHO RAN THE MISSION HOSPITAL, AND MARY-CLAIRE KING WAS A HUMAN GENETICIST INTERESTING IN BREAST CANCER AND SCHIZOPHRENIA, WE ROPED HER INTO TB AND SHE WAS HELPFUL. NOW WE KNOW WE SHOULD ONLY TREAT THE PEOPLE WHO HAVE THE HIGH CONDITIONS WITH STEROIDS. IN FACT THAT'S JUST BEEN VERIFIED IN A REPEAT STUDY AND NOW THERE'S A TRIAL GOING ON TO DO THIS IN A PROSPECTIVE MANNER AND SHOW DEXOMETHOZONE IS HARMING THOSE WITH LOW INFLAMMATORY GENOTYPE. WHY DOES THE HIGH INFLAMMATORY STATE, WHY IS IT BAD FOR YOU IF YOU HAVE TB? BECAUSE EVOLUTIONARILY YOU WOULD PREDICT THAT INFLAMMATION IS GOOD FOR INFECTION, AND BAD FOR MODERN DAY CONDITIONS LIKE HEART DISEASE AND ARTHRITIS AND SO ON BUT HERE WE'RE SAYING IT'S NOT SO SIMPLE, IT'S BAD FOR INFECTION TOO IN EXCESSIVE AMOUNTS. FRAN, A POSTDOC IN THE LAB, NOW A FACULTY TO CAMBRIDGE, FIGURED THIS OUT. SO IF YOU HAVE OPTIMAL LEVELS OF TNF, YOU GROW BY INTRACELLULAR SPREAD IN THE GRANULOMA, THAT'S NOT SO GREAT FOR YOU BUT IT'S NOT TERRIBLE. AND IF YOU HAVE TOO LITTLE TNF, THEN THE BUG, THE CELL, CANNOT CONTROL THE BACTERIA. AND SO THE BACTERIUM OVERGROSS AND CELL RUPTURES AND THAT'S EVEN WORSE BECAUSE EXTRACELLULARLY THE BACTERIA HAVE NOTHING, THE HOST HAS NO DEFENSES AGAINST EXTRACELLULAR BACTERIA, SO THEY REALLY GROW LIKE GANG BUSTERS IN THAT CASE I SHOWED YOU. WHAT ABOUT IN THE HIGH TNF STATE, WHAT FRAN FOUND WAS INITIALLY THE MACROPHAGE CONTROLS THE BUG EVEN BETTER, AND YET SUDDENLY THE MACROPHAGE POPS OPEN, AND THE FEW BACTERIA THAT GET RELEASED RAPIDLY CATCH UP AND THEY GROW. SO WHY IS THAT? SO HERE IS THE BARE ELEMENTS OF THE PATHWAY HE FIGURED OUT. IF YOU MAKE TOO MUCH TNF BECAUSE YOU MAKE TOO MUCH OF THE LEUKOTRIENE B4 WHEN YOU BIND YOUR RECEPTOR IN THE SORT OF EXCESS, THEN YOU TRIGGER A BUNCH OF KINASES, RIP 1, RIP 3, AND THEN A BUNCH OF OTHER PROTEINS THAT CAUSE THE MITOCHONDRIA TO MAKE REACTIVE OXYGEN. THEN THE REACTIVE OXYGEN TRIGGERS THE TRANSLOCATION OF REDOX PROTEIN TO COME FROM THE MATRIX TO THE MEMBRANE WHERE IT PARTICIPATES IN THE FORMATION OF THE MITOCHONDRIAL PORE COMPLEX, THIS CAUSES THE LEAKAGE OF VOLTAGE POTENTIAL, AND THE CELL IS KAPUT, ITS MEMBRANES LIES LYS OPEN, AND IT IS RELEASED. REACTIVE OXYGEN ALSO TRIGGERS -- DIFFUSES OUT AND TRIGGERS TO OVERPRODUCE A LIPID CALLED SERAMIDE THAT BIOMECHANISM NOW FULLY IDENTIFIED CAUSES -- HEALTH TO BURST OPEN THE MITOCHONDRIAU M. FRAN MADE THE PREDICTION IF YOU INTERRUPT THIS DOWNSTREAM OF REACTIVE OXYGEN YOU GET BENEFIT OF REACTIVE OXYGEN AND BE ABLE TO KILL THE BACTERIA WITHOUT KILLING THE CELLS BECAUSE IT'S ESSENTIALLY A RACE. YOU'RE IN THE PROCESS BY THE REACTIVE OXYGEN OF KILLING THE BACTERIA WHEN YOU POP OPEN AND NOW THE BACTERIA COME OUT AND THE GAME IS OVER FOR THE HOST. AND SO HE MADE THAT PREDICTION AND HE WAS ABLE TO TEST THAT GENETICALLY AND IT CAME TRUE, THE PREDICTION, BUT THEN HE WENT AND FOUND TWO DRUGS, ONE IS IN CLINICAL TRIAL, DISCOVERED FOR MITOCHONDRIAL MUSCULAR DYSTROPHIES, IS IN CLINICAL TRIALS FOR HEPATITIS C, AND ANOTHER IS A VERY OLD ANTI-DEPRESSANT THAT TARGETS THIS PATHWAY, HE SHOWED BY USING THESE TWO DRUGS THAT HE WAS NOW ABLE TO TAKE THE BAD GENOTYPE OF TB AND MAKE IT BETTER, YOU'RE BETTER OFF HAVING THIS GENOTYPE IF -- NOW YOU HAVE DRUGS IT, THERE'S AN ANALOGY FOR THIS IN THE CANCER WORLD BECAUSE NOW, YOU KNOW, FOR EXAMPLE IF YOU HAVE BREAST CANCER THAT'S EGF POSITIVE, HER-2 NEW POSITIVE, YOU'RE ALMOST BETTER OFF THAN IF YOU HAVE ONE THAT IS TRIPLE-NEGATIVE BECAUSE WE'VE GOT DRUGS TO TREAT THESE AND THIS IS COMING THROUGH IN A BUNCH OF CANCERS NOW WHERE AN AGGRESSIVE CANCER YOU'RE BETTER OFF WITH IT. THE VERY LAST THING I WANT TO TELL YOU IS THAT WE'VE NOW COME UP, WE'VE COME UP WITH A LOT OF CURES FOR THE HIGH END HIGH TNF, WE ALREADY HAVE STEROIDS WHICH WERE GIVING YOU 100% CURE, BUT WHAT ABOUT THE POPULATION THAT HAS THE LOW LTA4-H, ACTUALLY SLIGHTLY THE MAJORITY, IT'S A 70/30 DISTRIBUTION AT LEAST IN ASIA. SO WHAT ABOUT THEM? AND SO WE WERE RACKING OUR BRAINS AND WE REALIZED THAT IF YOU WENT UPSTREAM AND INHIBITED WITH A WELL-KNOWN DRUG FOR ASTHMA YOU WOULD BLOCK LIPOXIN AND LTB4, WE DON'T NEED EITHER TO FIGHT TB, SO WHEN WE DO THAT WE CAN SHOW THE LOW -- THIS IS WILD TYPE, THIS IS THE LOW STATE, WHICH IS HYPER SUSCEPTIBLE, IF YOU GIVE THESE ANIMALS ZILEUTON, YOU CAN BRING THEM DOWN, SOMETHING THE WELLCOME TRUST IS TRYING, ASTHMA IS A DISEASE OF THE LUNG AND IT WILL BE PARTICULARLY ADVANTAGEOUS FOR US TO TRY THIS FOR EXAMPLE IN PULMONARY TB. OKAY. SO I'VE SHOWN YOU FOR THE LAST BIT, I'VE SHOWN YOU A WHOLE BUNCH OF PATHWAYS, AND IF YOU'RE LIKE ME YOU FORGOTTEN ALL OF THEM SO I'M GOING TO GIVE YOU A PUNCHLINE MESSAGE. SO THIS IS NOT THE GANGES RIVER. DO YOU KNOW WHAT RIVER THIS MIGHT BE? THIS IS THE BIOLOGY RIVER IN YELLOWSTONE NATIONAL PARK. AND IT'S ON THE MONTANA-WYOMING BORDER. IF YOU A HOT DAY'S HIKING IN YELLOWSTONE YOU WANT TO GO AND TAKE A BATH IN THIS RIVER. AND IN THE OLD DAYS THEY DIDN'T ALLOW IT BUT PEOPLE USED TO SNEAK IN SO THEY MADE AN ENTRANCE. YOU HAVE TO WALK ALL THIS WAY, HALF A KILOMETER BEFORE YOU GET IN, AND AS YOU WALK ALONG YOU CAN'T HELP BUT NOTICE THAT EVERYBODY'S IN A STRAIGHT LINE IN THE RIVER. AND YOU ENTER THE RIVER AND YOU CAN REALLY SEE IT. YOU ENTERED ABOUT HERE AND YOU CAN REALLY SEE IT. THERE'S A COUPLE KIDS THAT WANDER OUT A BIT BUT EVERYBODY SITTING HERE, WHY MIGHT THAT BE? (OFF MIC). >> TOO HOT. THIS IS COLD RIVER. FED BY A HOT SPRING. SO IF YOU STEP HERE, IT'S 95 DEGREES AND MORE. IF YOU STEP HERE, IT'S WAY TOO COLD. AND SO I WOULD ARGUE THAT TB IS A DISEASE OF BEING JUST RIGHT. AND IF YOU'RE GENETICALLY JUST RIGHT YOU DON'T NEED OUR HELP, YOU CAN CAN YOU MUDDEL THROUGH WITH ANTIBIOTICS, IF WE'RE TOO HOT WE WITH HELP YOU, WE HAVE ZEL OTRON THAT WE'RE GOING TO TRY. COMING BACK, I THINK I'VE SHOWN YOU THAT USING THESE LITTLE LARVAE WE'VE COME UP WITH THINGS THAT MIGHT BE RELEVANT TO HUMAN TB, STARTING WITH HOW INFECTION BEGINS, HOW THE BACTERIA EXPAND IN GRANULOMAS, HOW THAT LEADS US TO UNDERSTAND A LITTLE BIT ABOUT DRUG TOLERANCE, HOW THEY EXPLOIT THE GRANULOMA TO FIRST SPREAD WITHIN CELLS AND THEN HOW UNDER CERTAIN CIRCUMSTANCES OF HOST-GENETICS, THEY ARE MORE PRONE TO RUPTURE OUT OF THE GRANULOMA, WHICH IS REQUIRED FOR THEIR FINAL TRANSMISSION. THANK YOU. [APPLAUSE] >> COULD YOU TELL US MORE ABOUT THE EFFLUX PUMP IN TUBERCULOSIS THAT SEEMS TO HAVE AN EFFECT BOTH ON ANTIBIOTICS AND ON THE SURVIVAL? >> YES. IT'S THE SPECIFIC ONE FOR -- SO WE KNOW THAT EVERY SINGLE ANTIBIOTIC WE TESTED, AND WE'VE TESTED MOST OF THE TB ANTIBIOTICS, PRACTICALLY ALL OF THEM, FIRST LINE, SECOND LINE, THIRD LINE DRUGS, THEY ALL HAVE MACROPHAGE INDUCED EFFLUX. INCLUDING THE ONE THAT CLIFF BARRY HAS BEEN PROMOTING QUITE EFFECTIVELY OVER HERE, ALSO HAS EFFLUX. WE'VE IDENTIFIED THE PUMPS FOR TWO OF THEM, FOR RIFAMPICIN AND [ INAUDIBLE ] THERE ARE TWO PUMPS. EACH OF WHICH IS REQUIRED TO MEDIATE TOLERANCE SO WE SUSPECT THEY WORK TOGETHER. THE FIRST IS A MFS PUMP, IT'S OF THE MFS FAMILY, 1258C, BUT THAT DOESN'T MEAN ANYTHING. APARTMENT THE OTHER IS AN RSB, A TRANSPORTER, AN RSB. WELL, WHAT'S REALLY INTERESTING IS THAT THESE PUMPS ONLY -- RIFAMCIPIN IS HYDROPHOBIC, YOU COULD THINK IT WOULD PUMP OUT THIS LITTLE HYDROPHYLIC MOLECULE BUT IT DOESN'T, SO REMOVING THIS PUMP COMPLETELY PRESERVES ISONAZID TOLERANCE BUT REMOVES RIFAMPICI NEXT TOLERANCE, VIRAMPIL REMOVES BOTH, SOMEHOW ACTING ON BOTH THE RIFAMPICIIN N PUMP. >> DO YOU KNOW THE SUBSTRATES FOR THE PUMPS? >> NO. BECAUSE THE RIFAMPICIN PUMP IS THE ONE THAT IS ALSO REQUIRED FOR GROWTH IN THE MACROPHAGE, WE'RE WONDERING IF IT'S SOMETHING LIKE AN ANTI-MICROBIAL PEPTIDE WHICH IS HYDROPHOBIC BUT WE DON'T KNOW WHAT THE ENDOGENOUS SIGNAL OR SUBSTRATE IS. IF YOU THINK ABOUT IT A LITTLE BIT, AS I DID MANY MONTHS AFTER DISCOVERING ALL THIS, IF YOU GO BACK AND LOOK AT ALL THE SPECIES AND MICROBACTERIUM GENUS THEY ALL HAVE EFFLUX PUMPS INCLUDING 1258C, IN THE SOIL-DWELLING ONE AS WELL YOU WONDER IF IT WAS THERE FOR BACTERIAL WARFARE OR ORGANISMAL WARFARE, GOT REPURPOSED TO ALLOW IT TO GROW WHEN IT ENTERED A EUKARYOTIC CELL. BEN, WHO IS ONE OF THE WORLD EXPERTS IN SOLVING STRUCTURES AT BACTERIAL TRANSFERS IS AT CAMBRIDGE, NOW TACKLING THIS PUMP, WE HOPE IF WE CAN GET SOME PROTEIN, LET ALONE A STRUCTURE, WE CAN START TO ASK QUESTIONS ABOUT THE SUBSTRATE SO WE'RE ACTIVELY WORKING ON IT NOW WITH MORE. >> ANOTHER QUESTION. >> YEAH, MAYBE YEARS AGO I WORKED AT -- GREAT PRIVILEGE OF WORKING ON THE TEX-MEX BORDER, CREATING MANY CASES OF TUBERCULOSIS, ALL THE DRUG SENSITIVE ONES YOU WERE TALKING ABOUT HOW DIFFICULT IT IS, THE SYSTEM IN PLACE WAS. [SPEAKING SPANISH] WAS EXCELLENT, SPECIAL NURSES, 30 YEARS AGO. >> YEP. >> INH, IN SOME CASES STREPTOMYCIN, I SAW THEM ON THE COAST AND IN THE TEX-MEX BORDER, WITH ROMA, TEXAS, AND I SAW DRAMATIC CURES WITH SCRUFOLISIS, I'VE SEEN EVERYTHING. I THINK IN INDIA PERHAPS YOU HAVE A GROUP, I'M NOT TRYING TO BE PRESUMPTUOUS, THEY DON'T GET THE DRUGS DISTRIBUTED PROPERLY, THEY DON'T GIVE THEM PROPERLY, OR WHATEVER, VIETNAM ALSO. BECAUSE IT'S DIFFICULT FOR ME TO BELIEVE THAT MANY YEARS AGO THAT WE WERE ABLE TO BE THAT EFFECTIVE AND THAT GOOD. THESE OTHER COUNTRIES CAN'T IF THEY HAVE THE PROPER MEDICINE AND THEY KNOW HOW TO -- >> YOU HIT THE NAIL ON THE HEAD. TB CONTROL PROGRAM IN INDIA IS A DISASTER, THAT IS TRUE FOR ALL THE WORLD WHERE THERE IS TB, BUT THE BOTTOM LINE IS LOOK, LET'S BE HONEST, IF YOU'VE BEEN PRESCRIBED ANTIBIOTICS FOR TEN DAYS EVEN, HOW MANY OF YOU CAN TELL ME THAT YOU'VE NEVER MISSED A DOSE? WE'RE PRIVILEGED PEOPLE. IF YOU HAVE DIRECTLY OBSERVED THERAPY, AND YOU HAVE NURSES TO GO OUT THERE AND CHASE AFTER PEOPLE, YES, YOU CAN GET TREATMENT. MY MOTHER HAD, YOU KNOW, TB AND EVENTUALLY SHE GOT CURED WHEN RIFAMPICIN CAME INTO BEING, BUT IT'S NOT EASY, AND TB IS A SOCIAL PROBLEM. IN FACT, IF WE DIDN'T HAVE POVERTY I WOULD BE OUT OF BUSINESS. >> MAYBE WE CAN TAKE THIS DISCUSSION TO A RECEPTION. I'VE BEEN TOLD WE NEED TO CUT OFF FOR TIMING, BUT THERE WILL BE A RECEPTION IN THE LIBRARY, CO-SPONSORED -- SPONSORED BY THE PARTNER FAES BUT I WANT TO END BY THANKING YOU FOR SUCH A STIMULATING AND INTERESTING TALK. THANK YOU. >> THANK YOU. [APPLAUSE]