New antibiotics from the microbial dark matter

New antibiotics from the microbial dark matter


KIM GREW UP IN MOSCOW AND APPARENTLY MET SOME MEMBERS OF OUR AUDIENCE AT A MUCH YOUNGER AGE THERE, OBTAINED HIS PHD IN CHEMISTRY AT MOSCOW UNIVERSITY. HE’S BEEN AT NORTHEASTERN UNIVERSITY IN BOSTON SINCE 2001 WHERE HE’S NOW THE DIRECTOR OF ANTI-MICROBIAL DISCOVERY CENTER AND A UNIVERSITY DISTINGUISHED PROFESSOR. SO, AS MOST OF YOU PROBABLY KNOW, INCREASING THE RESISTANCE OF BACTERIAL PATHOGEMS HAS BEEN ON THE FRONT PAGE OF JOURNALS AND EVERYBODY’S ATTENTION. WE’RE FORTUNATE TO HAVE DR. LEWIS HERE TODAY TO TELL US ABOUT HIS WORK BECAUSE HE’S BEEN AT THIS FOREFRONT OF ATTACKING THAT PROBLEM. I THINK I FIRST BECAME AWARE OF HIS WORK AND HIS INTERESTS WHEN HE STARTED TALKING ABOUT PERSISTERS. A SMALL POPULATION OF CELLS ARE NOT KILLED BY ANTIBIOTICKIC TREATMENT BUT REMAIN READY TO GROW OF THEY GET THE CHANCE AND REESTABLISHING INFECTION WE HAVE A SECOND TALK TOMORROW AT 11:00 A.M. IN BUILDING 37 TOMORROW MORNING IN THE SIXTH FLOOR CONFERENCE ROOM. IF YOU NEED MORE INFORMATION ABOUT WHERE AND WHAT THAT IS, LET ME KNOW. THE TITLE IS CHANGES IN ENERGY LEVELS PRODUCED DRUG TOLERANT PERSISTENT CELLS. TODAY’S TALK, HE WILL BE DISCUSSING HIS EXCITING WORK ON THE WORK FOR NOVEL ANTIBIOTICS AND DEVELOPING INTERESTING TO GROWING UNCULTURAL BACTERIA AND THE TITLE TO THE TALK TO ANTI- MICROBIAL DARK MATTER. PLEASE US KIM LEWIS TO THE STAGE.>>THANK YOU VERY MUCH, SUSAN, FOR A KIND INTRODUCTION AND THANK YOU FOR THE INVITATION TO THIS REALLY GREAT PLACE. I GET TO SEE IT FROM THE OTHER SIDE WHICH IS ALSO A PLEASURE. SO WHAT I WILL TELL YOU ABOUT TODAY IS SOME OF OUR BASIC SCIENCE AND HOW THAT INFORMS OUR DRUG DISCOVERY EFFORTS, AND I WILL START WITH THIS SORT OF A TEXTBOOK PRESENTATION OF BACTERIAL RESISTANCE. WHAT YOU WILL SEE HERE IS THERE ARE LOTS OF DECREASED MECHANISMS, OR TARGET MODIFICATION. AND REALLY EACH AND EVERY LOGICAL POSSIBILITY HAS BEEN REALIZED IN NATURE AND IT’S BECAUSE OF THIS PLURALLALITY OF MECHANISMS. WE FIND OURSELVES IN THE ANTI-MICROBIAL RESISTANCE CRISIS THAT SUSAN MENTIONED. APART FROM JUST THE THREAT OF INFECTIOUS DISEASES, PER SAY, ONE OF THE THINGS THAT’S BECOMING INCREASINGLY CLEAR AND I’M SURE THAT EXPERIENCED A NUMBER OF CASES WITH RESISTENT NOT SO LONG AGO. WITHOUT DEFECTIVE ANTIBIOTICS WE DO NOT HAVE THE ENTERPRISE OF MEDICINE THAT WE ARE USED TO HAVING, SO YOU CANNOT DO CHEMOTHERAPY OR SURGERY IF YOU DON’T HAVE EFFECTIVE ANTIBIOTICS. BUT THIS PROBLEM IS COMPOUNDED BY ANOTHER PHENOMENON WHICH IS RELATED TO CHRONIC INFECTIONS. ANOTHER WAY THAT BACTERIA USED TO AVOID BEING KILLED BY ANTIBIOTICS AND IT COMES BY THIS PARADOX OF CHRONIC INFECTIONS. ANTIBIOTICS ARE INFECTIVE AGAINST ANTIBIOTIC SUS SUSCEPTIBLE PATHOGENS. THESE CHRONIC INFECTIONS OFTEN ASSOCIATE WITH — AND THERE’S A LONG LIST OF THEM, TUBERCULOSIS, AND ONE COMMONALITY AMONG THOSE TWO IS THE FACT THAT THE IMMUNE SYSTEM IS NOT VERY EFFICIENT IN ATTACKING THE PATHOGEN. THEN THE PATHOGEN WHICH IS OTHERWISE SUSCEPTIBLE TO TESTS ON A PETRIE DISH, THAT SAME ANTIBIOTIC IS . WE FOUND THIS PRETTY SIMPLE KILLING PHENOMENON. YOU SEE THAT THE BULK OF THE CELLS ARE EASILY KILLED, NOTHING SPECIAL ABOUT THEM. THEN YOU HIT UPON THE POPULATION OF CELLS THAT DO NOT DIE. SO THAT SEEMS TO BE THE CULPRIT. THIS CULPRIT, OUR PERSISTER CELLS DISCOVERED ORIGINALLY IN 1934 BY JOSEPH BIGGER WHO WAS UNABLE TO CULTURE A STAPH WITH PENICILLIN. HE WAS VERY FRUSTRATED HE COULD NOT DO THAT. HE PUBLISHED A MARYP AND THAT PAPER WAS LARGELY FORGOTTEN FOR A COUPLE OF DECADES. IF YOU WILL REDISCOVER PERSTERS AND REALIZE THAT IS VERY IMPORTANT, PROBABLY VERY IMPORTANT MEDICALLY, AND SO WE WANTED TO OF COURSE FIGURE OUT WHAT IS IT THAT MAKES THESE CELLS ESSENTIALLY INVULNERABLE TO ANTIBIOTICS. NOW TODAY I WILL GIVE YOU SOME HIGHLIGHTS OF OUR WORK IN THIS FIELD. I WILL TALK IN MUCH MORE DETAIL TOMORROW AND TODAY I WILL GIVE YOU SUFFICIENT BACKGROUND TO THEM AND EXPLAIN WHAT WE’RE DOING. OKAY. SO FIRST OF ALL, WE WANTED TO KNOW IN PRINCIPAL WHAT IS IT ABOUT THESE CELLS THAT ALLOWS THEM TO STAY ALIVE. WE THOUGHT THAT PERHAPS THEY ARE DORMANT BECAUSE THEY NEEDED TO GROW NOR DIE IN THE PRESENT OF ANTIBIOTIC. SO HERE’S OUR VIEW OF THESE TWO FACES OF THREAT. THESE ARE RESISTANCE MECHANISMS. ALL OF THEM DO ESSENTIALLY THE SAME THING, THEY PREVENT FROM BINDING TO THE TARGET. TOLERANCE WHICH PERSTERS EXHIBIT, WE NEED TO APPRECIATE KILL NOT BY INHIBIT INHIBITING. THAT IS WHAT KILLS THE CELL, NOT BECAUSE STOPPED TURNING OUT PROTEINS. IF YOUR TARGETS ARE INACTIVE IN A DORMANT CELL OR LARGELY INACTIVE, THEN THE TARGET IS NOT CORRUPTED AND THE CELL SURVIVES. SO THIS SEEMED TO US LIKE A REASONABLY GOOD GUESS. WE LEARNED HOW TO ISOLATE PERSTERS, GOT THEIR TRANSCRIPT. AND WE WERE LOOKING FOR PROTEINS THAT HAVE THIS ABILITY TO STOP IMPORTANT FUNCTIONS IN THE CELL. AND ONE OF THE FIRST THINGS THAT WE NOTICED WAS THE OVEREXPRESSION OF A NUMBER OF TOXIN, ANTI-TOXIN SYMPTOMS. HAVE BEEN KNOWN FOR A WHILE AS A MECHANISM OF MAINTENANCE. IF THE DAUGHTER CELL BY ACCIDENT LOSES, THE ANTI-TOXIN GETS DEGRADED. DEPENDING ON WHAT THAT TARGET IS, KILLS THE CELL OR CAUSES STATION IS. BUT THEN PEOPLE STARTED SEQUENCING GENOMES AND COPIOUS NUMBERS. SO OUR SIMPLE PROPOSITION WAS THAT WHAT THESE THINGS DO INTO THE CHROMOSOME IS THEY ARE PERSISTER GENES, AT LEAST SOME OF THEM. HERE I’LL GIVE YOU A SUMMARY GROUPS OVER THE YEARS, SO WHAT THEY FOUND IS DIFFERENT TOXINS, ANTI-TOXINS WILL SUPPORT PERSIST INTERESTING MECHANISM THAT TOBEY IN MY LAB DISCOVERED WHERE A STRESS RESPONSE TURNS DOWN THE B TOXIN WHICH CAUSES THE DECREASE AND ATP. SO THESE ARE HIGHLY REDUNDANT AND PARALLEL PATHWAYS OF PERISTER INFORMATION. SO ARMED WITH THAT INFORMATION FROM E.COLI, WE THEN DECIDED THAT IT WOULD BE USEFUL TO FIND OUT WHAT’S HAPPENING IN OTHER BACTERIA. AT THAT TIME JOINED MY LABORATORY AND HE CAME FROM OUR STAPH LAB SO HE WANTED TO FIGURE OUT WHAT’S HAPPENING. THE FIRST THING THAT HE DID IS HE KNOCKED OUT TOXINS, ANTI-TOXINS AND HAS ONLY THREE, UNLIKE 80 IN TUBERCULOSIS. SO SO HE KNOCKED OUT THOSE THREE TOXINS, ANTI-TOXINS AND ASKED A SIMPLE QUESTION, IF YOU EXPOSE THESE GROWING CELLS, WHAT WILL HAPPEN? AND ABSOLUTELY NOTHING HAPPENS. THERE’S NO DIFFERENCE IN PERISTER FORMATION IN THE WILD TYPE OR KNOCK-OUT. SAME IN STATIONARY CULTURE. SO THAT EXPERIMENT IMMEDIATELY TOLD US THAT WHAT WE LEARNED IN THE PREVIOUS DECADE WAS COMPLETELY USELESS, AND WE NEED TO START — USELESS FOR STAFF. AND THAT WE NEED TO START FROM SCRATCH. WE WENT LOOKING FOR CLUES, AND ONE OF THE CLUES THAT WE THOUGHT WOULD POINT US IN THE RIGHT DIRECTION WAS THE FACT THAT STAPH BECOMES HIGHLY TOLERANT IN STATIONARY STATES. SO THEN WE THOUGHT, WELL, MAYBE PERSISTERS IN THE GROWING POPULATION OF STAPH THAT WENT INTO STATIONARY EARLY. HERE’S A SIMPLE TEST HER THAT PROPOSITION. WE HAVE A STATIONARY STATE MARKER CLONED WITH GFB. THE CULTURE GROWS FROM LACK IN STATIONARY STATE, AND YOU SEE THAT THERE ARE STATIONARY CELLS SHOWING UP IN THE GROWING CULTURE. IF YOU NOW TAKE THESE CELLS FROM A GROWING CULTURE AND SORT THEM OUT, THOSE THAT ARE EXPRESSING THE STATIONARY MARKER AND EXPOSED TO ANTIBIOTIC, THOSE ARE THE ONLY CELLS THAT SURVIVE, SO THOSE ARE CLEARLY ENRICHED IN PERSISTERS SO WE HAVE THIS EFFECT HAPPENING THAT A GROWING CULTURE PRODUCES STATIONARY STATE. I ALWAYS THOUGHT THESE ARE TWO DIFFERENTLY DIFFERENT THINGS. BY DEFINITION, STATIONARY ARE NOT GROWING AND YET THE GROWING CULTURE PRODUCES STATIONARY CELLS. SO WHAT WE’RE THINKING MAY BE HAPPENING IN PRODUCING PERSISTER CELLS IS THAT OF COURSE WE NOTED THE STATIONARY CULTURE THE LEVELS OF ATP ARE CONSIDERABLY LOWER. SO WE WERE THINKING THAT MAYBE PERSTERS ARE CELLS THAT HAVE LOW LEVELS OF EXPRESSION OF KEY ELEMENTS THAT ARE RESPONSIBLE FOR ATP PRODUCTION, AND ONE OF SUCH KEY ELEMENTS IS ALPHAKETOGLUTER. TARATE. IN WHICH IS COATED BY THE SUCA. TESTED THIS AND DID THE VERY SIMPLE EXPERIMENT WHERE HE HAS A GFP REPORT OF SUCA PROMOTERS AND LOOKING AT THE DISTRIBUTION OF EXPRESSION OF THIS GENE IN THE POPULATION. AND NOW SORTING OUT CELLS SHOWS THOSE THAT HAVE LOW LEVELS OF EXPRESSION OF KETO GLUTTERATE. WE’RE PRETTY HAPPY ABOUT IT BECAUSE THE ALTERNATIVE WAS THAT THERE WAS NO MECHANISM BECAUSE LOTS OF THINGS ARE GOING TO EFFECT THE LEVELS OF ATP. BUT NOW WE KNOW THERE ARE SOME KEY ELEMENTS THAT DO THAT. TOMORROW I’LL TELL ABOUT YOU SOME ADDITIONAL MECHANISMS THAT WE FOUND. OKAY. SO HERE’S OUR ADDITIONAL VIEW OF WHAT’S HAPPENING WITH PERISTER FORMATION. I THINK THIS IS A GENERAL MECHANISM. SUMMER SPECIFIC AND PERHAPS SELECTIVE OR GIVEN BACTERIA. THIS IS A GENERAL AND IMPORTANT MECHANISM. THAT IS THAT ALL OF THE ANTIBIOTICKIC TARGETS REQUIRES ATP OF COURSE. YOU GET CORRUPTION OF THESE TARGETS BY ANTIBIOTICS AND DEATH. AND THEN IF STATISTICALLY THE LEVEL OF THE TARGET DECREASES, YOU GET DORMANT CELLS, AND YOU GET THE TARGET SHUTDOWN. SO THAT VERY SIMPLY EXPLAINS THE NATURE OF PERSISTENS. IT’S PRETTY SIMPLE EXPLANATION. I’M GOING TO SAY THAT WE OVERLOOKED IT. WE COULD HAVE DISCOVERED THIS TEN YEARS AGO BUT WE DIDN’T FOR REASONS I DON’T UNDERSTAND. OKAY. SO APART FROM THAT, APART FROM THE BASIC BIOLOGY OF PERSISTERS THERE’S ANOTHER THING I WANTED TO TELL YOU ABOUT, AND THAT IS THE IMPORTANT QUESTION OF THE CLINICAL RELEVANT RESISTANCE OF PER ABC SISTERS. SO A FRIEND OF MINE, LOU RICE, WHO IS HEAD OF MEDICINE AT BROWN. HE TOLD ME A NUMBER OF YEARS AGO THAT IT IS VERY INTERESTING THAT IF YOU GUYS ARE DOING WITH PERSISTER CELLS AND I’M SURE AMUSING BUT I’M SURE IT HAS NOTHING TO DO WITH CLINICAL MANIFESTATION OF DISEASE THAT’S ALL THERE IS TO IT. SO IT’S DIFFICULT TO ADDRESS THAT QUESTION. WE WERE HOPING TO DO AN EXPERIMENT WHERE WE ISOLATED PERSISTERS. WE COULD INTRODUCE THEM INTO AN ANIMAL AND SEE IF THAT ANIMAL BECOMES TOLERANT TO KILLING BY ANTIBIOTICS. AT THE MOMENT YOU INTRODUCE PERSISTERS INTO AN ANIMAL, THEY WAKE UP. THAT WAS QUITE A BIG PROBLEM FOR US AND FOR OTHERS IN THE FIELD, AND THE CLUE TO SOLVING IT CAME FROM AN EXPERIMENT THAT WE WERE DOING ON A COMPLETELY DIFFERENT SUBJECT. SO A GRAD STUDENT IN MY LAP WAS TRYING TO CATEGORY ALL POTENTIAL PERSISTER JEANS IN E.COLI. THE WAY SHE DID THAT WAS PERFORM THIS PRETTY SIMPLE SELECTION EXPERIMENT WHERE YOU HIT A CULTURE WITH ANTIBIOTIC COLLECT SURVIVING PERSISTERS REPEATED A COUPLE OF TIMES, BUT THEN YOU GET AN ENRICHMENT IN MUTANTS THAT MAKE MORE PERSISTENS. FIGURE OUT WHERE THOSE POTENTIALLY PERSISTER GENES ARE. BUT IF YOU LOOK AT THIS EXPERIMENTS, WHAT YOU REALIZE IS THIS IS EXACTLY WHAT HAPPENS WHEN WE PEOPLE ARE TREATED WITH ANTIBIOTICS. WE GET PERIODIC HIGH DOSES OF ANTIBIOTICS AND BEER PERSISTER IS USEFUL FOR THE PATHOGEN, THEN THERE WILL BE SOLUTIONS IN THE COURSE OF ANTIBIOTICKIC TREATMENT. IN THE WAY MILLIONS OF PEOPLE PARTICIPATED AND THE EXPERIMENT INCLUDING PROBABLY EVERYONE PRESENT IN THIS AUDIENCE, SO WE SIMPLY NEED TO LOOK AT THE RESULTS OF THIS EXPERIMENT AND DECIDE WHETHER THEY ARE — BUT THAT EXPERIMENT WE DID BUT FIRST I’LL SHOW YOU WHAT WE FOUND IN THE INVITRO EXPERIMENT. MOST OF THE HIGH PERSISTER MUTANTS TURN OUT TO BE MUTANTS IN DIFFERENT POSITIONS OF THIS HIPPA TOXINS WHICH PRO DUNES 100 TO 1,000 TIMES MORE PERSISTERS. ENORMOUSLY STRONG FHELPO TYPE. SO THEN SCREENED A LARGE COLLECTION OF E.COLI ISOLATE AND FOUND THAT IN HALF OF THESE STRAINS THERE ARE MUTATIONS IN THE HIPA LOCUSTS. SO WE WANTED TO UNDERSTAND MECHANISMISTICLY. A STUDY WE DID A NUMBER OF YEARS AGO, WE WERE TRYING TO UNDERSTAND WHAT IS IT THAT HIPA DOES IN THE CELL TO STOP IT FROM GROWING. WE DIDN’T HAVE ANY LEADS . AT WHICH POINT I GAVE A CALL TO EUGENE AND I ASKED HIM TO TAKE A LOOK AT HIPA WHICH DOESN’T LOOK LIKE ANYTHING IN THE DATA BASE. SO EUGENE RELUCTANTLY AGREED TO DO THAT, AFTER SOME ARM TWISTING. AND IN THE COUPLE OF DAYS I WAS SENT AN E-MAIL WHICH SAID HIPA WAS A KINASE. I THOUGHT IT WAS NONSENSE BECAUSE THERE ARE NONE OF THOSE IN BACTERIA. THE ACTIVE CITES LINED UP VERY WELL AND WE FOUND INDEED HIPA IS A PROTEIN KINASE. THAT WAS A GOOD CLUE. SO HIPA IS A PROTEIN KINASE. NORMALLY STOPS TRANSLATION, BUT NORMALLY IN THE CELL PRESENT IN THE FORM OF A DIMER. SO NORMALLY HIPA IS LARGELY INAT THIS POINT ALL IMITATIONS, THEY HAPPENED AT THE INTERFACE BETWEEN THESE DIMEERS. SO THIS IS THE CASE WHEN WE CAN UNDERSTAND AND WE BY THEN ASK THE NUMBER OF OTHER PATHOGENS. SO ALL PATHOGENS WE LOOKED AT AND THESE WITH TUBERCULOSIS, FROM CYSTIC FIBROSIS PATIENTS. IN THOSE CASES WE FIND AND CYSTIC FIBROSIS ABOUT HALF OF ISOLATES. THERE ARE THESE TWO PHENOMENON, ONE IS HERITABLE TOLERANCE. TO MAKE MATTERS MORE DIFFICULT FOR US. THERE WAS A PAPER THAT JUST CAME OUT IN SCIENCE THAT SHOWED THAT IN THE COURSE OF ANTIBIOTIC TREATMENT, PATHOGEN FIRST REQUIRES TOLERANCE MUTATION THAT’S HELP THE PATHOGENS SURVIVE HIGH LEVELS OF ANTIBIOTICS AND ON THAT BASIS RESISTANCE MUTATIONS ARE THEN ACQUIRED. SO THESE TWO MECHANISMLY DISTINCT, THEY COLLABORATE, IF YOU WILL, IN PRODUCING RESISTENT MUTANTS. WE NEED TO WORRY ABOUT BOTH OF THESE PHENOMENON AND TAKE CARE OF BOTH OF THEM. SO WHAT IS IT THAT WE LEARNED HOW DOES THAT INFORM OUR DRUG DISCOVERY EFFORTS. SO THE PATHWAYS OF PERSISTER FORMATION IN THE MODEL E.COLI ARE MULTIPLE AND REDUNDANT. MANY OF THESE PERSISTERS ARE GOING TO BE LOW IN ATP. SO IF YOU HAVE REDUNDANT PATHWAYS, IT MEANS YOU DON’T HAVE A TARGET. SO WHAT WE FOUND TELLS YOU VERY SIMPLY THAT DRUG DISCOVERY IS RATHER HOPELESS. SO FROM THAT UNPROMISING BASIS, WE WERE THINKING OF HOW TO GET SOMETHING THAT’S GOING TO KILL PERSISTERS. WHEN MOTHER NATURE COMES UP WITH A PERFECT DEFENSE, WHAT WE KNOW IS THAT IT ALSO PROVIDES A RESPONSE TO THAT PERFECT DEFENSE. AND SO WE WERE WONDERING IF AMONG NATURAL PRODUCTS THERE MAY BE SOMETHING THAT EVOLVED BECAUSE PRODUCERS VERY SIMILAR PROBLEM TO US. THEY WANT TO KILL THEIR NEIGHBORS INCLUDING THEIR PERSISTERS. WHAT WE’RE LOOKING FOR. WE’RE LOOKING FOR A COMPOUND THAT WILL IN A DORMANT TARGET CORRUPT IT AND KILL THE CELL AND THAT’S NOT GOING TO REQUIRE ANY ATP. THAT’S THE KIND OF A MAGICAL COMPOUND WE ARE LOOKING FOR. ONCE YOU FORMULATE THAT, THEN INDEED THERE IS A COMPOUND THAT COMES TO MIND. THIS IS A PEPTIDE, A NATURAL COMPOUND DISCOVERED BY A GROUP FROM ELI LILY. APPARENTLY SOMEONE WAS VACATIONING IN HAWAII AND BROUGHT BACK THIS STRAIN THAT PRODUCE THIS IS ANTIBIOTIC. SO LILY HAD A LOOK AT THIS COMPOUND, AND AT THE TIME, THIS WAS 195, EVERYBODY WAS LOOK FOR A BROAD SPECTRUM ANTIBIOTICS. THIS COMPOUND ONLY KILLS ONE BACTERIA SO LILY DROPPED IT. ABOUT 20 YEARS LATER WE WERE EXPERIENCING AN EPIDEMIC OF MRSA WHICH IS STILL ONGOING. SO A GROUP FROM BAYER TOOK ANOTHER LOOK AT THIS COMPOUND, THEY MADE A MORE ACTIVE DERIVATIVE AND THEY ALSO DROPPED THE COMPOUND, BUT BEFORE DROPPING IT, THEY DETERMINED THE PRINCIPAL MECHANISM OF ACTION. IT IS VERY INTERESTING THAT THAT’S WHAT ATTRACTED OUR ATTENTION. SO THE PROTEASE WILL NORMALLY RECOGNIZE THE PEPTIDES. IN THE PRESENCE OF ADEP, ATP IS NO LONGER NEEDED. SO THAT’S THE KEY, OF COURSE. THAT’S EXACTLY WHAT THEY WERE LOOKING FOR. THEY ARE LOOKING FOR A FORGET THAT CAN BE CORRUPTED WITHOUT ANY REQUIREMENT FOR ATP. IT SEAMED LIKE A PERFECT CANDIDATE FOR AN ANTI-PERSISTENT COMPOUND, BUT THERE WASN’T A PROBLEM, ACTUALLY TWO PROBLEMS. THESE TWO PAPERS, ONE OF THEM REPORTED THAT IN THE PRESENCE OF ADEP, THEY ARE GETTING OFF RIBOCELL. THE OTHER PAPER IDENTIFIED THE PRIMARY TARGET. IT FORMS THE RING AND THAT IS ALSO ONLY REQUIRED IN DIVIDING CELLS, NOT IN DORMANT CELLS. SO THE QUESTION IS WHAT DO YOU DO WHEN FACTS CLEARLY CONTRADICT YOUR BEAUTIFUL HYPOTHESIS. YOU HAVE TWO BASIC OPTIONS. ONE IS YOU DECIDE THAT YOUR HYPOTHESIS IS WRONG. OTHER YOU DECIDE THAT FACTS ARE WRONG. SO I TOOK A VERY CAREFUL LOOK AT THESE TWO PAPERS AND I DIDN’T FIND ANY PROBLEM WITH THEM. THEY ARE VERY WELL EXECUTED STUDIES. BUT LIKE MOST BIO CHEMISTRY STUDIES, THEY ARE PERFORMED ON A FAIRLY SHORT TIME SPAN OF ABOUT 10 TO 15 MINUTES. AND THAT IS AN ISSUE BECAUSE ANTIBIOTICS ACT AT A TIME FRAME OF HOURS AND DAYS, NOT A TIME FRAME OF MINUTES. WE DECIDED TO REPEAT THIS EXPERIMENT AT THE MORE REALISTIC TIME FRAME OF 24 HOURS WITH NONE GROWING STATIONARY STATE. AND SO HERE IS THE FULL PROTEAM AND YOU SEE MASSIVE DEGRADATION. IT LOOKS LIKE ADEP FORCES THE CELL TO CELL DIGEST. WE HAD A LOOK AT WHAT HAPPENS WITH KILLING. THIS IS A CONTROL. THESE ARE GROWING. THAT WAS THE FIRST TIME WE SAW COMPLETE STERILIZATION WITH ANYTHING THAT WAS NOT BLEACH. THAT WAS ENCOURAGING AND THEN WE RECALLED WHY BAYER DROPPED THE COMPOUND. THEY HAD A REASON TO DO THAT. YOU GET HIGH PROBABILITY RESISTENT MUTANTS THAT ARISE WHEN THERE’S A MAL MUTATION. CLIP MUTANTS BECOME COMPLETELY RESISTENT. HAS THIS POTENTIAL SIMPLY COMBINE IT WITH ANOTHER ANTIBIOTIC. DOESN’T MATTER WHICH TO PRESENT THE RISE. YOU SEE INSTEAD OF AN ATTEMPT TO KILL, CONVENTIONAL ANTIBIOTICS WHICH IS NOT DOING VERY WELL. WE ALSO HAVE A LOOK IN VIETNAM ROW. ASTRAZENECA DEVELOPED A MODEL. HERE YOU SEE THE GROWTH OF A MASSIVE, INCURABLE INFECTION. REGULAR ANTIBIOTICS DO VERY WELL IN THIS MODEL. THEY KILL GROWING CELLS BUT THEN YOU HIT UPON GROWING PERSTERS AND THIS IS AN INCURABLE INFECTION. AGAIN, IT STERILIZES IN THIS INFECTION. THAT GIVES US AN SCAN PAL OF A COMPOUND THAT TELLS US IT DID EVOLVE. THAT WAS THE FIRST BUT I’M SURE NOT THE LAST, AND A BIO TECH COMPANY IS NOW DEVELOPING THIS INTO A DRUG. OKAY. SO IN THE REMAINING TIME, I WILL TELL YOU ABOUT OUR EFFORTS TO DISCOVER NEW ANTIBIOTICS AND I WILL START WITH THIS BRIEF INTRODUCTION INTO WHERE THE FIELD IS AT THE MOMENT. SO THE FIELD STARTED WITH THE FOLDEN ERA WHEN THE MAIN CLASSES OF TOO BYTES AND THEY WERE DISCOVERED. THIS IS BASED ON AN EXTREMELY SIMPLE SCREENING DEVELOPED AND GOT A NOBLE PRIZE. THE SCREEN ESSENTIALLY EMULATES THE ACCIDENTAL DISCOVERY MADE BY FLEMMING OR WHEN FLEMMING FOUND THAT IF HE HAD A PLATE WITH STAPH THAT HE WAS GROWING AND THEN HE GOT AN ACCIDENTAL CONTAMINATION. THERE WAS A ZONE OF INHIBITION AROUND THAT CONTAMINANT. REALIZED THAT YOU CAN USE THAT SYSTEMATIC SCREEN AND HE HAD SORT OF THE GENIUS TO HIT UPON THE WORLD’S BEST PRODUCERS OF ANTIBIOTICS. HOW HE MANAGED THAT, I DON’T KNOW. BUT HE DID.>>AND SO THIS VERY SIMPLE SCREEN PRODUCES THE MAJOR CLASSES OF ANTIBIOTICS. THAT GOES ON FOR A WHILE VERY SUCCESSFULLY, AND THEN SOMETHING HAPPENS AROUND THE 1960s AS THE MECHANISM OF DISCOVERY IS TURNED OFF. WE GO INTO THE DARK AGES AND THEN THERE’S A BIT OF A RESURGENCE OF ACTIVITY. THIS PRIMARILY DEFAULTS BACK TO THE GOLDEN ERA MEANING THAT WE’RE PIPELY TAKEN INITIALLY FAILED LEADS AND TRYING TO RESUSCITATE THEM BECAUSE WE DON’T HAVE — WE HAVE A PRETTY GOOD UNDERSTANDING AROUND WHAT HAPPENED AROUND 1960 AND THAT IS THIS RESOURCE, THE SOIL, MICRO MICROORGANISMS WAS IN THE WAY LARGELY OVER-MINED. BY SCREENING BECAUSE ONLY 1% OF THEM WILL GROW. THE REST OF THE MICROBIAL DARK MATTER THAT DOESN’T GROW. SO WITH MY COLLEAGUE AT NORTHEASTERN, WE DECIDED THAT WE WILL TRY TO REVIVE THE PLATFORM BY GOING AFTER THIS MICROBIAL DARK MATTER. SO THIS IS A SIMPLE EXPERIMENT THAT IS SOMETIMES PERFORMED IN OUR UNDERGRADUATE LABORATORY WHICH ILLUSTRATES WHAT UNCULTURED BACTERIA ARE. YOU TAKE A SAMPLE FROM THE ENVIRONMENT LIKE MARINE SEDIMENT OR SOIL, AND YOU PUT BUN DROPLET UNDER THE MICROSCOPE AND YOU COUNT THE NUMBER OF CELLS, ANOTHER GOES ON THE PETRIE DISH, AND YOU TRY TO FIGURE OUT HOW MANY COLONIES THESE CELLS PRODUCE, AND THE DIFFERENCE IN THE COUNTS ARE BETWEEN THE NUMBER OF CELLS AND THE NUMBER OF COLONIES THAT THEY PRODUCE IS KNOWN AS THE GREAT PLATES COUNT ANOMALY. THAT’S THE OLDEST PROBLEM AND DISCOVERED IN 18 EU8. SO WINTERBERG THERE’S BEEN SIGNIFICANT TO PUSH THE NUMBERS UP AND THAT DID NOT WORK. SO WE DECIDED TO DO SOMETHING DIFFERENT. INSTEAD OF GROWING BACTERIA ON THE PETRIE DISH, GROW THEM WHERE WE KNOW THEY DO GROW, AND THAT’S THEIR NATURAL ENVIRONMENT. SO WE NEEDED TO COME UP WITH A GADGET WHERE BACTERIA CAN GROW IN PURE CULTURE. SO HERE YOU TAKE A SAMPLE FROM MARINE SEDIMENT, DILUTE IT AND PLACE BETWEEN THESE TWO SEMI PERMEABLE AND THIS CONTRAPTION KNOWN AS A DEFUSION CHAMBER AND GOES BACK IN THE NATURAL ENVIRONMENT. THEY ARE DRINKING BACTERIA. NOT SURPRISING. LOTS OF STUFF GROWS. WE GET RECOVERY CLOSE TO 50% IN THE DEFUSION CHAMBER AS AS OPPOSED TO THE PETRIE DISH WHICH IS REALLY LOW. JUST A PIECE OF PLASTIC WITH LOTS OF LITTLE HOLES WHICH YOU DIP IN THE SUSPENSION OF BACTERIA. EACH CAPTURES APPROXIMATELY ONE CELL. YOU COVER THIS WITH SEMI PERMEABLE MEMBRANES AND YOU GET MICRO COLONIES IN THESE WELLS. SO OF COURSE WE WERE VERY INTERESTED IN TRYING TO FIGURE OUT WHY UNCULTURED BACTERIA DO NOT GROW IN THE PETRIE DISH. WHAT IS THE MOLECULAR MECHANISM? SO THIS IS FROM MY LAB AND WE DECIDED TO START WITH THIS — WITH BEACH SAND AS A MODEL WHERE I SUSPECTED THAT ON THESE PARTICLES OF SAND WILL BE MICROORGANISMS. SO THIS IS WHAT YOU ACTUALLY WORK ON OF YOU WALK ON THE BEACH. YOU WALK ON THIS. AND THIS IS A — I DON’T KNOW WHAT THIS IS BUT IT LOOKS VERY IMPOSING. SO THEN WE HAD THE SIMPLE HYPOTHESIS, AT LEAST SOME OF THEM DO NOT GROW BECAUSE THEY REQUIRE GROWTH FACTORS FROM THEIR NEIGHBORS. SO HERE’S A TEST OF THIS YOU GET A WHOLE ZOO OF CALL KNEES GROWING. THEN YOU SAY MAYBE THIS SMALL TOWN GREW BECAUSE IT HAPPENED TO BE IN THE VICINITY OF THIS BIGGER ONE. IT WAS RELEASING A GROWTH FACTOR. HERE’S A TEST OF THAT, OF THAT IDEA. HERE IS A SIMPLE TEST WHERE YOU PATCH ONE OF THEM, THE BIGGER ONE AS IT GROWS IN THE COLONY. THE OTHER ONE YOU WITH SPREAD ON A PETRIE DISH AND IT ONLY GROWS AROUND THIS BIGGER ONE. THAT FIRST OF ALL TELLS YOU THE IDEA IS CORRECT IN PRINCIPAL AND YOU CAN PURIFY THE GROWTH FACTOR BY SIMPLY GROWING UP THE BACTERIA. WHAT WE GOT ARE THESE GROWTH FACTORS. IF YOU DROP ONE OF THEM ON A PETRIE DISH, IT RECAPITULATES THE HELPING ORGANISM. SO NORMALLY IRON EXISTS IN THE FORM OF INSOLUBLE IRON THREE. UNCULTURED BACTERIA INTERESTINGLY LOST THE ABILITY TO MAKE THEIR OWN. THEY COME FROM A SECONDARY LOSS. WHY THEY LOSE THEIR ABILITY TO MAKE AND WITH IT THEY LOSE THEIR LIBERTY, THEY ALWAYS DEPEND ON SOMEONE, THAT IS A FASCINATING QUESTION. BUT NOW THEY HAVE IS TO STEAL FROM THEIR NEIGHBORS AND THAT IS HOW THEY GROW. SO WE GOT VERY EXCITED WHEN WE FOUND THIS BECAUSE I THOUGHT THAT WE’LL JUST KEEP FINDING ADDITIONAL GROWTH FACTORS AND WE’LL CLOSE THE GAP. THAT DID NOT HAPPEN. WE CAN EXPLAIN APPROXIMATELY 10% OF UNCULTUREABILITY. THE NEXT GROWTH FACTOR GAVE US 1% AND THEN NOTHING. I DON’T UNDERSTAND AT THE MOMENT, BUT I HAVE A POST DOC IN MY LAB WHO IS SUPPOSED TO FIND AN ANSWER TO THIS QUESTION BY THE END OF THIS YEAR. [ LAUGHTER ]>>SO ONE USEFUL THING THAT WE FOUND IS THAT ONCE GROWN IN THE DEFUSION CHAMBERS, ESPECIALLY IF YOU REINOCULATE, WITH HIGH PROBABILITY YOU GET GROWTH ON A REGULAR PETRIE DISH. SO THAT OF COURSE GIVES YOU NOW ACCESS. AND SO FAR WE HAVE 2 NEW COMPOUNDS. SO I’LL TELL YOU ABOUT TWO OF THEM. SO ONE COMPOUND CAME FROM AN EFFORT TO SOLVE A VERY TOUGH PROBLEM, A NATIONAL PRODUCT DISCOVERY AND THAT’S THE ENORMOUS BACKGROUND OF KNOWN COMPOUNDS IN JUNK. THE WAY CHEMISTS TRY TO SOLVE THAT PROBLEM IS TO ASK THE QUESTION WHETHER YOU HAVE A NEW MONTHLY CULTURE IN YOUR EXTRACT. THAT TAKES TIME AND ENERGY AND THE ANSWER IS NOT ENTIRELY SATISFACTORY BECAUSE YOUR NEW MONTHLY CULTURE CAN BE A NEW DETERGENT WHICH IS NOT TERRIBLY EXCITING. WE DECIDED ONE WAY TO SOLVE THAT PROBLEM IS IF YOU GO AFTER SPECIES OR GROUP SELECTIVE COMPOUNDS. SO NATURE HAS VERY FEW, IF ANY, COMPOUNDS THAT HAVE BEEN KNOWN TO HIT A PARTICULAR GROUP OF BACTERIA. LIKE MICHAEL BACTERIA, FOR EXAMPLE. SO THEN IF NATURE MAKES COMPOUNDS, THEY ARE SPECIFIC, THEN YOU CAN DO A VERY SIMPLE THING. YOU TAKE AN EXTRACT AND YOU’RE LOOKING FOR THIS PROPER. YOU’RE LOOKING FOR INHIBITOR ACTIVITY BUT NOT AGAINST, FOR EXAMPLE, IF YOU CAN DO THAT, THEN CAN YOU IMMEDIATELY SAY IT CONTAINS A NEW COMPOUND. BY DOING THAT WE DISCOVERED THIS NEW ANTIBIOTIC. IT DOESN’T HIT BACTERIA — AND IT HAS AN INTERESTING PROPERTY. IF YOU LOOK AT STATIONARY IT’S VERY TOUGH TO KILL THEM. THIS IS OUR BEST KILLING COMPOUND. WITH SURVIVING PERSTERS. WE DON’T SEE ANY INDICATION OF SURVIVING PERSISTERS. SO WE GOT VERY INTERESTED IN THIS COUNT DOWN IN THE POST DOC. GOT RESISTENT MUTANTS FOR. WE DON’T DO MAPPING ANYMORE BUT SEQUENCING SHOW. THAT IS THE UNIT OF THE MICRO BACTERIAL CLIP, WHICH IS FAIRLY SPECIFIC FOR MICRO BACTERIA. IT’S ALSO ESSENTIAL. THAT’S WHY WE GAVE IT THE SAME LASSOMYCIN BECAUSE IT LOOKS LIKE A LASSO. BUT THEN ALSO THERE’S STRONG ACTIVATION. SO IF YOU LOOK AT WHAT HAPPENS IN WHOLE CELLS, LASSOMYCIN COLLAPSES MIEVMENT TB. WHAT I THINK IS HAPPENING IS THERE’S THIS MECHANISM I JUST TOLD YOU ABOUT WHICH IS ADP DEPLETION BUT NOT THE ZERO, AND IT LOOKS LIKE LASSOMYCIN PUSH IT’S DOWN AN EXTRA STEP IN THE ABYSS OF IRREVERSIBLE DORMANCY. I THINK IT’S A SNEAKING MECHANISM, SORT OF PIGGY-BACKING ON THE NATURAL WAY TO MAKE PERSISTERS TO GET RID OF THEM. SO IT LOOKS LIKE WE HIT UPON PERSISTERS. IT’S THIS PARTICULAR MACHINE WHICH IS UNDER ATTACK, SO IN BACTERIAL, FORCES THE PART OF THIS MACHINE TO DEGRADE PROTEINS AND THAT’S HOW IT KILLS DORMANT CELLS, WITHOUT ANY REQUIREMENT FOR ATP. A VERY DIFFERENT COMPOUND HITS A DIFFERENT PART OF THE SAME MACHINE FORCING IT TO DIGEST ADP. OF COURSE THAT DOESN’T REQUIRE ATP EITHER. IT JUST MOPS UP WHATEVER IS IN THE CELL. WHY THIS PARTICULAR MACHINE IS UNDER ATTACK BY COMPLETELY UNRELATED NATURAL PRODUCTS IS A FASCINATING QUESTION. WHILE I’M SURE THAT THERE ARE OTHER ANTI-PERSISTER COMPOUNDS TO BE FOUND, I WILL TELL YOU ABOUT ANOTHER COMPOUND THAT WE FOUND SORT OF RELATED TO THE GENERAL PROBLEM OF ANTIBIOTIC RESISTANCE. THIS WAS DISCOVERED FROM THIS UNUSUAL CULTURED BACTERIA. THAT PARTICULAR GROUP IS NOT KNOWN TO MAKE ANTIBIOTICS. IT IS A GRAM NEGATIVE BACTERIA. IT FORMS A NEW GENUS AND WE HAVE TO GIVE IT A NAME WHICH IS FREE EARTH. THIS NAME IS A MIXTURE OF GREEK AND LATIN WHICH YOU’RE NOT SUPPOSED TO DO WHEN GIVING NAMES TO CREATURES BUT I FIGURED THAT NOBODY CARES ABOUT THESE THING ANYMORE, SO THIS IS THE NAME OF THE CREATURE. IT’S AN INTERESTING COMPLEX MONTHLY CULTURE, AND WE CHECKED IT OUT FOR ACTIVITY AND FOUND THAT IT HITS ALONG LIST OF BACTERIA INCLUDING SOME NASTY PATHOGENS LIKE MRSA AND TUBERCULOSIS. WE TRIED TO GET RESISTENT MUTANTS TO THIS COMPOUND AND FAILED WHICH WAS REALLY FRUSTRATING BECAUSE FIRST OF ALL, YOU DO NOT HAVE AN EASY PATH TO DETERMINE THE COURSE OF ACTION BUT MORE IMPORTANTLY IN OUR EXPERIENCE, IF YOU HAVE NO RESISTANCE DEVELOPMENT, THAT MEANS THAT YOU DISCOVERED A NEW DETERGENT. BUT THEN PARALLEL, WE WERE — THIS IS AGAINST HUMAN CELLS. THERE’S NO ACTIVITY AGAINST HUMAN CELLS, SO THIS WAS VERY TRILLION. THIS IS THE FIRST TIME WE SAW A COMPOUND WHERE THERE’S NO RESISTANCE DEVELOPMENT, AND SO SIGHT OF EXISTING. SO WE EXAMINED IN THE NUMBER OF ANIMAL MODELS. I’M NOT GOING TO GO OVER THIS IN ANY DETAIL JUST TO TELL YOU SEVERAL SIMPLE MODELS, PROTECTION INFECTION AND MOUSE LUNG IN PNEUMONIA. THIS IS ALL MOUSE. SO THAT GAVE US SOME ADDITIONAL ENCOURAGEMENT THAT THIS COMPOUND IS WORTHY OF INVESTIGATING. SO THEN WE TOOK PARALLEL INVESTIGATE RESISTANCE DEVELOPMENT. THERE’S A PROTOCOL HOW TO DO IT CORRECTLY, SO WE DID IT CORRECTLY. HERE’S A CONTROL. YOU ADD THE ANTIBIOTIC AND EACH DAY YOU UP THE CONCENTRATION. SO IF YOU HAVE A MUTANT WITH ANY INCREASE IN RESISTANCE, EVEN MINIMAL, IT WILL TAKE OVER THE POPULATION. YOU ENCOURAGE RESISTANCE DEVELOPMENT. THEN AFTER ABOUT THE MONTH YOU’RE SUPPOSED TO DO IT FOR ABOUT A MONTH, YOU GET AN ENORMOUS INCREASE IN RESISTANCE, AND THIS IS PRETTY MUCH HOW IT WILL LAND ANTIBIOTICS IN CURRENT CLINICAL PRACTICE BEHAVIOR. WE DID NOT SEE ANY CHANGES. SO THAT WAS PRETTY SURPRISING. NOW PERFORMED A SPECIFICITY TEST TO SEE. THE TEST IS VERY SIMPLE. YOU SIMPLY FOLLOW THE RATES OF LABEL INCORPORATION TO THESE MAJOR POLY EMERGENCIES AND THEN THIS EXPERIMENT TAKES IT BACK AND SELECTIVELY SO WE KNOW THIS IS A SOLO ACTING INHIBITOR. AND THERE’S NO RESISTANCE DEVELOPMENT TO IT SO WE CONCLUDED THIS COMPOUND DOES NOT HAVE A PROTEIN TARGET. PROTEINS WILL ALWAYS MUTATE. YOU WILL ALWAYS HAVE A MUTANT PROTEIN THAT DOES NOT BIND YOUR ANTIBIOTIC. A REALISTIC TARGET WHICH IS NOT PROTEIN. SO WE TARGETED WITH A SIMPLE TEST. WE HAD GROWING STAFF OR I COULDN’T SAY WHICH IS NOT GROWING IN THE PRESENCE. IF YOU ADD LIPID, YOU RESTORE GROWTH. IF YOU’RE COLLABORATING WITH TANJA SCHNEIDER WHERE YOU CAN ISOLATE LIPID TWO AND SEE IT LATER, BUT YOU CAN NO LONGER EXTRACT LIPID TWO FROM SOLUTION. SO USING THAT METHOD, THEN ADDING DIFFERENT VERSIONS OF LIPID TWO, YOU CAN THEN ZERO IN. AND IT IS THE LIPID SUGAR PART. THIS IS WHAT TAKES IT BACK. THE NATURE OF THE SUGAR IS NOT IMPORTANT SO THAT TOLD US THAT IT WILL ALSO PROBABLY BUY AND INDEED IT DOES, TO THE IMPORTANT POLYMER. IT HAS THE SAME STRUCTURE, JUST A DIFFERENT SUGAR. SO THIS INVESTIGATION TELLS US AN INTERESTING THING. IT INDICATES THAT NATURE EVOLVED THE COMPOUND TO BE FAIRLY FREE OF RESISTANCE. IT HITS TWO TARGETS. NEITHER OF THESE TARGETS IS MUTABLE. THESE THINGS ARE NOT CODED BY GENES, AT LEAST NOT DIRECTLY. THE TARGET ITSELF IS EXPOSED ON THE SURFACE OF THE CELL SO YOU CANNOT PUMP IT OUT. IT’S ALREADY OUTSIDE. AND IT APPEARS THAT THE MAIN DOING MA THAT WE’VE BEEN OPERATING UNDER WHICH IS BACTERIA WILL DEVELOP RESISTANCE TO EVERYTHING AND IN MANY CASES RAPIDLY IS PROBABLY INCORRECT. THEY ARE PROBABLY COMPOUNDS THAT EVOLVED TO BE LARGELY FREE OF RESISTANCE DEVELOPMENT. SO GOING AFTER THOSE COMPOUNDS I THINK MAKES A LOT OF SENSE. SO WITH THAT I WILL THANK MY TEAM. I WILL THANK MY COLLABORATORS IN SUPPORT WHICH HAS BEEN SUPPORTING THE WORK OF MY TEAM OVER THE YEARS, AND I THANK YOU FOR LISTENING. [ APPLAUSE ]>>IF THERE’S A QUESTION, I THINK YOU NEED TO GO TO THE MICROPHONE.>>YOU WERE SPEAKING ABOUT BIO FILMS. YOU DIDN’T SAY ANYTHING ABOUT THE STRUCTURE OF BIO FILM. SUPPOSEDLY THEY HAVE AN OUTER LAYER OF PROTECTORS AND INNER LAYER OF GUYS KIND OF PROTECTED AND LOWER LAYER OF SOMETHING ELSE. CAN THE TYPE OF THING YOU’RE TALKING ABOUT ACTUALLY BEAT ALL OF THE LAYERS OF A BIO FILM OR PARTICULARS THAT ARE LAID OUT FLAT. THAT IS SIGNIFICANT BECAUSE WHAT YOU’RE 3 TALKING ABOUT IS IT MEDICAL DEVICES THAT ARE GOING TO BE IN DWELLING AND THEY DO DEVELOP ALL OF THOSE DIFFERENT LAYERS OF THINGS.>>SURE. OF COURSE THIS IS AN IMPORTANT QUESTION AND THERE ARE TWO ASPECTS TO IT. ONE IS THE ABILITY OF THE COMPOUND TO PENETRATE THROUGH. AND THOUGH THERE’S THE ABILITY TO KILL CELLS, SO THE ABILITY OF COMPOUNDS TO PENETRATE INTO THE BIO FILM HAS BEEN WELL DEMONSTRATED WITH FEW EXCEPTIONS. MOST COMPOUNDS PENETRATE FAIRLY WELL. THERE’S SOME PROBLEM, THERE’S SOME RETARDED PENETRATION. THE MAIN PROBLEM IS NOT PENETRATION INTO BUT THE ABILITY TO KILL CELLS IN THE BIO FILM WHICH ARE DORMANT. WHAT WE SHOWED PREVIOUSLY IS THE MOST DIFFICULT CELLS TO KILL ARE NOT CELLS IN THE BIO FILM BUT A SIMPLE STATIONARY CULTURE. SO ADEP KILLS CELLS IN STATIONARY CULTURE AND IN — INVITRO.>>THANK YOU.>>THE FIRST PART OF YOUR LECTURE, YOU SPOKE ABOUT DIFFERENT KINDS OF MECHANISMS BEHIND PERSISTER FORMATION. AND THEN THERE ARE SPECIFIC MECHANISMS BROUGHT ABOUT BY TOXINS, SO COULD YOU ELABORATE UNDER WHAT KIND OF CIRCUMSTANCES, IN WHAT SITUATIONS THESE SPECIFIC MECHANISMS ARE INVOLVED.>>MY ELABORATION IS LIMITED BY MY IGNORANCE SO I WILL ONLY TALK ABOUT SOME CLEAR EXAMPLES THAT WE HAVE AT THE MOMENT. A GOOD EXAMPLE THAT WE HAVE IS THE INDUCTION TO B TOXIN. DURING DNA DAMAGE, DNA REPAIR. THAT RESPONSE ACTIVATES AND YOU HAVE ALSO AN ACTIVATION OF THAT EXPRESSION WITH THEM. WHICH CONTROLS THE ENTIRE SOS RESPONSE. SO GETS ACTIVATED, INSERTS ITSELF IN THE MEMBRANE. IT IS A VERY STRANGE MONTHLY CULTURE THAT DIMINISHES, DECREASES ATP, AND THAT’S HOW YOU GET PERSISTENCE UNDER THOSE VERY SPECIFIC CONDITIONS. SO IN OTHER WORDS, UNDER TYPES OF STRESS, YOU WOULD IMPLICATE ALL TOXINS BUT UNDER GENERAL CONDITIONS THE SPONTANEOUS –>>MY FEELING IS THAT PROBABLY UNDER CONDITIONS WHERE THE CULTURE IS NOT NECESSARILY, STRESS IS PRIMARILY GUIDED BY ADEP LEVELS.>>LET ME ASK TWO QUESTIONS ABOUT THE ENVIRONMENTAL MICROBIOLOGY THAT COMES OUT OF SOME OF THE THINGS THAT YOU DO. THAT YOU SEE BEING BORROWED BY WHAT WAS OTHERWISE NON-CULTUREABLE ONES, ARE THOSE GOING TO BE SPECIFIC TO WHOSE IN THE NEIGHBORHOOD IF YOU TRIED AN UNCULTUREABLE SOME PLACE ELSE?>>THAT’S A GREAT QUESTION AND WE EXAMINED THAT, AND WHAT WE FOUND IS THAT THERE’S EVERY LOGICALLY POSSIBLE SCENARIO. SO WE FOUND SOME BACTERIA THAT WILL ONLY TAKE IT FROM A PARTICULAR NEIGHBOR AND THEN WE FOUND THOSE THAT WE COULD INDUCE GROWTH WITH THAT WE COULD BUY COMMERCIALLY AND EVERYTHING IN BETWEEN.>>SO WE’LL EVENTUALLY UNDERSTAND IT BUT NOT YET AND PROBABLY THE ANSWER TO THE OTHER QUESTION SAID THE BACTERIA THAT GAVE YOU YOUR ANTIBIOTIC, ANYTHING KNOWN ABOUT WHY IT WOULD BE MAKING THIS AND PRESUMABLY KILLING OFF ALL ITS GROUND POSITIVE NEIGHBORS?>>THAT’S AN EXCELLENT QUESTION. IN ONE SIMPLE SCENARIO, YOU WANT TO GET RID OF YOUR NEIGHBORS. THAT’S A COMMON SENTIMENT, AT LEAST OF WILD CREATURES. SO PUT THERE’S ONE PARTICULAR PROPERTY THAT KIND OF TELLS US WHY THAT COMPOUND, SO WHAT WE FOUND ABOUT, IT HAS A TERRIFIC ACTIVITY TO Y CELLS, BETTER THAN ANY OTHER CELL. PROBABLY BECAUSE IT HITS THESE TWO TARGETS. AND SO I THINK THAT THIS CREATURE IS A HUNTER. I THINK IT HUNTS AND EATS THEM.>>CAN THERE BE DRAWN A PARALLEL — WHICH OCCUR WHEN RECEPTORS WON’T RESPOND –>>WHY NOT. [ APPLAUSE ]

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