The importance of growing slowly: roles for redox active "antibiotics" in microbial survival

>> GOOD AFTERNOON I’M A MEMBER OF THE INTEREST GROUP HOSTING TODAY’S WALS LECTURE, AND IT IS MY GREAT PLEASURE TO INTRODUCE DR. DIANNE NEWMAN C IT NEW MAN IS PROFESSOR OF BIOLOGY AND GEOBIOLOGY AT CAL TECH, AS WELL AS AN INVESTIGATOR OF THE HOWARD HUGHES INSTITUTE SO DR. NEW MAN IS ACTUALLY FROM THIS AREA, FROM VIRGINIA, GREW UP IN ALEXANDRIA AND HAS MOVED SORT OF BETWEEN THE WEST COAST AND EAST COAST SINCE THEN SHE WAS AN UNDERGRADUATE AT STANFORD AND THEN RECEIVED HER PH.D. IN ENVIRONMENTAL ENGINEERING AT MIT MY UNDERSTANDING IS THAT THROUGH SOME CLASSES THAT SHE TOOK AS A GRADUATE STUDENT, DR. NEWMAN BECAME INTERESTED IN BACTERIAL GENETICS SHE CARRIED OUT POSTDOCTORAL WORK WITH DR. ROBERTO COLT IT ER AT HARVARD MEDICAL SCHOOL SINCE THAT TIME, DR. NEWMAN HAS REALLY MELDED HER BACKGROUNDS IN GEOCHEMISTRY AND BACTERIAL GENETICS, EXAMINING ARING METABOLIC REACTIONS CARRIED OUT BY BACTERIA TO SHAPE THE CHEMISTRY OF THEIR ENVIRONMENT RECENTLY THIS WORK HAS SHOWN THAT SOME OF THESE REACTIONS, FOR EXAMPLE, WHICH CAN LEAD TO THE GENERATION OF REDOX ACTIVE ANTIBIOTICS HAVE MEDICAL RELEVANCE DR. NEWMAN’S ABILITY TO SO SUCCESSFULLY BRIDGE TWO FIELDS IS ILL SERRATE ILLUSTRATED BY THE AWARDS S HE HAS RECEIVED PORE EXAMPLE, THE GEOCHEMICAL SOCIETY BEST PAPER AWARD, AND THEN THE NATIONAL ACADEMY OF SCIENCES AWARD IN MOLECULAR BIOLOGY SHE’S ALSO A MEMBER OF BOTH THE AMERICAN SWREE OWE PHYSICAL UNION AND THE AMERICAN ACADEMY OF MICROBIOLOGY AND I THINK THIS BRIDGING OF THESE TWO DIFFERENT FIELDS WILL ALSO BE ILLUSTRATED IN HER TALK TODAY, AND THE TITLE IS THE IMPORTANCE OF GROWING SLOWLY, ROLES FOR REDOX ACTIVE ANTIBIOTICS IN MICROBIAL SURVIVAL PLEASE JOIN ME IN WELCOMING DR. NEWMAN [APPLAUSE] >> THANK YOU VERY MUCH, GIGI AND SUSAN AND EVERYONE ELSE WHO WAS INVOLVED IN INVITING ME TO COME HERE I AM VERY MUCH HONORED TO BE GIVING THIS TALK IN THIS SERIES, AND AS GIGI SAID, I GREW UP IN NORTHERN VIRGINIA AND YET I NEVER HAD THE OPPORTUNITY TO VISIT THE NIH BEFORE AND SO FOR PERSONAL REASONS, THIS HAS BEEN AN EXCITING DAY FOR ME AS WELL AND I’VE REALLY ENJOYED THE CONVERSATIONS AND INTERACTIONS I’VE HAD SO FAR ON THIS VISIT, SO THANK YOU SO WHAT I THOUGHT I WOULD ATTEMPT TO DO, SINCE THIS IS A GENERAL AUDIENCE, IS TALK ABOUT SOMETHING THAT I’VE BECOME FASCINATED BY IN THE PAST DECADE, BUT BEGIN WITH A FRAMEWORK THAT IS BROADER THAN THE PARTICULAR TOPIC OF CHRONIC INFECTION BECAUSE I HOPE IT WILL HELP YOU UNDERSTAND THE WAY I LOOK AT PROBLEMS AND HOW IMPORTANT I THINK A BROAD PERSPECTIVE CAN BE ACTUALLY IN THINKING ABOUT PROBLEMS AND SO THE SPECIFIC PROBLEM THAT I WILL GO INTO IN SOME DEPTH IS HOW IN CHRONIC INFECTIONS, BACTERIAL PATHOGENS SURVIVE THIS IS A VERY IMPORTANT TOPIC ABOUT WHICH WE STILL HAVE MUCH TO LEARN, BUT I THINK IT IS FAIR TO SAY THAT IT’S WELL RECOGNIZED THAT THE STATE OF MANY BACTERIAL PATHOGENS IN CHRONIC INFECTION CAN REALLY CAN UNDERPIN RESISTANCE TO STA STANDARD ANTIBIOTICS, SO LEARNING MORE ABOUT WHAT THIS STATE REALLY COMPRISES IS ONE RATIONAL PATH TO BEING ABLE TO TREAT THESE INFECTIONS BETTER HOWEVER, TO REALLY GROUND THIS SORT OF PROBLEM IN A BIGGER CONTEXT, I WANT TO POINT OUT THAT WHEN WE LOOK AT ORGANISMS IN THIS ENVIRONMENT, AND WHAT YOU’RE SEEING HERE ON THE SLIDE IS AN IMAGE OF SALVE LO STAPHYLOCOCCUS ARREUS IN CYSTIC FIBROSIS, IT’S ACTUALLY POORLY CONSTRAINED IN MANY, MANY WAYS AND OFTEN HOW WE STUDY THESE PATHOGENS IN THE LAB DOESN’T NECESSARILY CAPTURE SO WELL WHAT’S REALLY GOING ON IN SITU

LET ME BEGIN WITH JUST A VERY SIMPLE EXAMPLE OF THIS, AND TAKE THE CONCEPT OF GROWTH RATE I’M GOING TO DO THIS FOR ONE OF MY FAVORITE ORGANISM, PSEUDOMONAS AERUGINOSA SO CONVENTIONALLY, EVEN IN MY OWN LAB, WHEN WE STARTED OFF, WE WOULD OFTEN GROW PSEUDOMONAS IN RICH MEDIUM, SHAKING AEROBICALLY, AND WHEN YOU DO THIS UNDER THESE CONDITIONS, THE GROWTH RATE IS DOUBLING TIMES OF ABOUT A HALF AN HOUR OKAY? AND THAT’S FAIRLY COMMON THERE’S GOOD REASON THAT WE DO EXPERIMENTS LIKE THIS IT CAN BE VERY REPRODUCIBLE, AND INDEED AN INCREDIBLE AMOUNT HAS BEEN LEARNED OVER MANY, MANY DECADES THROUGH TAKING THIS KIND OF APPROACH HOWEVER, I ALSO TEACH INTRODUCTORY BIOLOGY AT CAL TECH, WHICH IS TO AN AUDIENCE OF STUDENTS WHO ARE QUITE QUANTITATIVELY MINDED SO TO HELP THEM GET INTERESTED IN MICROBIAL GROWTH, I HAVE THEM DO A CALCULATION I ASK AT THIS RATE, WHAT WOULD THE VOLUME THAT A SINGLE PSEUDOMONAS CELL OCCUPY AFTER THREE DAYS OF GROWTH, AND TURNS OUT THAT IT WOULD FILL THE VOLUME OF THE EARTH SO HE CAN EXTEN THEY CAN EXTEND THIS AND AFTER A WEEK AT THIS RATE, IT WILL FILL THE VOLUME OF THE UNIVERSE SO OBVIOUSLY WE ALL APPRECIATE THIS ISN’T OCCURRING I THINK IT DOES CONVEY IN A VERY SIMPLE WAY BUT YET A POWERFUL WAY, THE FACT THAT THESE GROWTH RATES ARE REALLY LAB ARTIFACTS, AND IN THE ENVIRONMENT OBVIOUSLY, THIS IS NOT WHAT’S GOING ON FOR MANY, MANY REASONS ONE OF THE REASONS THAT I THINK IS IMPORTANT TO REFLECT ON THAT I PERSONALLY THOUGHT A LOT ABOUT COMING FROM A BACKGROUND IN GEOCHEMISTRY IS THAT IN MANY ENVIRONMENTS, AND FOR MUCH OF MICROBIAL HISTORY, THE EARTH DID NOT HAVE OXYGEN AND SO IF WE JUST LOOK AT THREE IMPORTANT GASES AND THEIR RELATIVE CONCENTRATIONS OVER TIME FOR THE HISTORY OF THE PLANET, WHAT YOU SEE HERE IN BLUE IS THE RISE OF OXYGEN AND IN RED, METHANES, PROFILE, AND IN GREEN CARBON DIOXIDE AND WHILE I THINK MANY OF YOU MAY APPRECIATE THAT THE RISE IS RELATIVELY RECENT ON GEOLOGIC TIME SCALE, DATING TO ABOUT A HALF A BILLION YEARS AGO AT THE EXPLOSION, MICROBES HAVE BEEN DOMINATING THE BIOSPHERE FOR THE MAJORITY OF OUR HISTORY, AND FOR A LARGE PORTION OF THAT TIME, THEY HAVE BEEN EVOLVING AND THRIVING UNDER CONDITIONS THAT WERE EITHER ENTIRELY ANOXIC OR HYPOXIC SO THIS IS PART OF THEIR HISTORY AND SO WHEN WE PUT THESE FACTS TOGETHER THAT GROWTH RATES MUST BE SLOWER THAN WE OFTEN STUDY THEM AND THE FACT THAT THERE’S BEEN THIS INCREDIBLE SPAN OF TIME IN WHICH ORGANISMS HAVE EVOLVED, WE CAN BEGIN TO THINK A LITTLE BIT MORE DEEPLY ABOUT THE — THAT CHARACTERIZE HABITATS TODAY IN MANY, MANY DIFFERENT CONTEXTS SO I’M GOING OH START THIS LECTURE BY TALKING ABOUT AN ENVIRONMENT THAT’S MORE GEOLOGICAL IN RELEVANCE BUT RAPIDLY MOVE INTO ONE THAT HAS IMPORTANT MEDICAL IMPLICATIONS AND LET ME LINK THESE TWO FOR YOU CONCEPTUALLY SO LET’S BEGIN FIRST WITH THINKING ABOUT WHAT HAPPENS IN SEDIMENTS LIKE THIS THESE SEDIMENTS ARE FROM THE HALEY RESERVOIR IN CALIFORNIA THAT SUPPLY A LOT OF WATER TO THE SI OF LOS ANGELES, AND THEY’RE COMPRISED OF IRON OXIDE MINERAL WITHIN THESE SEDIMENTS, YOU HAVE BACTERIA THAT ARE THRIVING, AND THEY’RE THRIVING BECAUSE THEY’RE ABLE TO — THESE ROCKS THAT’S AN AMAZING PROCESS SO HERE’S AN ORGANIZE NI ORGANISM THAT CAN DO THIS, STRAIN MR1, ISOLATED FROM NEW YORK STATE THESE ARE CULTURES FORMING AGGREGATES, WE CALL THESE BIOSOMES, AND THEY’RE FORMING IN THIS PARTICULAR PICTURE ON A PIECE OF ROADING STEEL THAT, WHEN IT CORRODES, IT FORMS A LAYER OF MINERALS THAT ARE THE SAME KIND OF MINERALS THAT YOU SEE IN THIS SEDIMENT SO SOME BACTERIA ARE ATTACHED DIRECTLY TO THESE IRON MINERALS BUT THEN AS THEY FORM THESE CLUMPS, THEY GET AT A DISTANCE FROM THE IRON OXIDE AND SO IF YOU WERE TO MAKE A CARTOON VERSION OF THIS AND FLIP IT AND CUT THESE CLUSTERS, YOU KNOW, IN HALF AND LOOK AT THEM ON THEIR SIDE, WHAT YOU COULD IMAGINE WOULD BE YOU WOULD SEE STACKING, WHERE SOME OF THESE CELLS INDIVIDUALLY ARE ATTACHED TO THE MINERAL SURFACE, AND THEN YOU HAVE SOME EMBEDDED MOUNTAIN CORE OF WHAT I’LL NOW START MORE AND MORE REFERRING OH AS BIOSOMES AND HEN SOME AT THE TOP THAT CAN ACCESS MOLECULAR OXYGEN IN THE WATER ITS ABOVE

PEOPLE HAVE BEEN STUDYING BIOSOMES IN SO MANY DIFFERENT ORGANISMS AND ONE OF THE THINGS THAT’S VERY WELL APPRECIATED IS THAT AFTER YOU REACH A CERTAIN CRITICAL SIZE OF THESE FILMS, THE RATE OF OXYGEN CONSUMPTION OUTPACES THE RATE OF DIFFUSION SO LARGE PORTIONS OF THESE BIOSOMES ARE ANOXIC OR HYPOXIC SO SOMETHING THAT REALLY INTERESTED US IS, HOW WOULD A CELL IN THE MIDDLE OF THESE BIOSOMES METABOLICALLY SURVIVE? BECAUSE IT WOULDN’T BE ABLE TO ACCESS THE IRON OXIDE DIRECTLY, NOR COULD IT ACCESS MOLECULAR OXYGEN, AN ALTERNATIVE ELECTRONIC SECTOR IN THE BROAD SCHEME OF METABOLISM, AND SO HOW MIGHT IT RESPIRE? SO IF WE THINK ABOUT THIS AT THE LEVEL OF A SINGLE CELL, IF OXYGEN CAN DIFFUSE ACROSS — I’M SHOWING THE CELL STRUCTURE OF A GRAM-NEGATIVE BACTERIUM, THIS ISN’T SUCH A HARD THING TO ENVISION, WE KNOW OXYGEN IS PERMEABLE BUT FOR AN IRON OXIDE MINERAL, DESPAIRINGLY SOLUBLE, IT SIMPLY CAN NOT DIFFUSE ALL THE WAY IN HERE, SO THERE HAS TO BE ANOTHER ANSWER AND OVER THE PAST AT THIS POINT I WOULD SAY EVEN ALMOST THREE DECADES OF RESEARCH, MANY DIFFERENT GROUPS, I HAD SOME INVOLVEMENT BUT MANY OTHER GROUPS HAVE BEEN INVOLVED IN UNDERSTANDING THE PATHWAY OF ELECTRON TRANSFER FROM INNED SIDE THE CELL WHERE ELECTRONS ARE GENERAT GENERATED BY THE BURNING OF CARBON SOURCES GOING THROUGH CENTRAL METABOLIC PATHWAYS THAT ULTIMATELY WIND UP BEING DUMPED ON TO IRON OXIDE OUTSIDE OF THE CELL VIA A SERIES OF INTERESTING PROTEINS, MANY OF WHICH CONTAIN C-TYPE CYTOCHROMES SOME ARE EVEN ON THE OUTSIDE OF THE CELL AND CAN COME IN CLOSE ENOUGH CONTACT WITH IRON MINERALS TO REDUCE THEM DIRECTLY HOWEVER, AS I SHOWED YOU IN THAT FIRST CONCEPTUAL CARTOON, IF THE CELL ISN’T CLOSE ENOUGH TO THE IRON MINERAL, THERE HAS TO BE SOME OTHER WAY OF DUMPING THOSE ELECTRONS SO RESEARCHERS IN THIS AREA OF ENVIRONMENTAL MICROBIOLOGY IN THE 90s REALLY ELABORATED ON THIS IDEA THAT THERE WERE THINGS CALLED ELECTRON SHUTTLES THAT COULD BE A COMPOSITION OF COMPOUNDS, IT SIMPLY HAS THE ABILITY TO INTERFACE WITH THE CELL WHERE THEY PICK UP ELECTRONS FROM CELLULAR METABOLISM, AND THEN TRANSFER OUTSIDE OF THE CELL THOSE ELECTRONS TO AN EXTRACELLULAR OXIDE, IN HAD CASE, IRON YOU HAVE VERY SMALL AMOUNTS OF THESE BECAUSE THEY’RE CATALYTIC, THEY CAN BE RECYCLED SO THIS COULD BE SOMETHING THAT COULD HELP EXPLAIN HOW THINGS WORK AT A DISTANCE SO WHEN MY LAB GOT INVOLVED IN THINKING ABOUT THIS PROBLEM, SOMETHING THAT WE WREC RECOGNIZED WAS THAT ENVIRONMENTALLY IMPORTANT ELECTRON SHUTTLES, COMPOUNDS CALLED HUMIC SUBSTANCES, LARGE EXOINDLARGECOMPOUNDS, QI KNOWNS THESE WERE WELL APPRECIATED TO SERVE AS STIMULATING IRON REDUCTION IN GROUNDWATER AQUIFERS, VARIOUS PLACES AROUND THE EAST COAST BUT WHAT WE RECOGNIZED WAS THAT STRUCTURALLY, WE HAD QUITE A LOT IN COMMON WITH OTHER SMALL MOLECULES KNOWN AS ANTIBIOTICS YET WHEN YOU LOOK AT THESE PARTICULAR TYPES OF ANTIBIOTICS, THEY’RE UNITED WITH THESE ELECTRON SHUTTLES BY VIRTUE OF THE FACT THAT THEY ARE ALL REDOX ACTIVE THEY CAN ALL GAIN AND GIVE UP ELECTRONS, OFTEN COUPLED TO PROTON TRANSFER AS WELL SO POSSIBLY A SOLUTION TO HOW MICROBES MIGHT EXIST IS IF THEY WERE ABLE TO SELF-GENERATE, WHO I LIKE TO IT REFER TO AS AN ENDOGENOUS ELECTRON SHUTTLE, AND WE MIGHT HAVE THOUGHT ABOUT THESE THINGS AS BEING ANTIBIOTICS, BUT UNDER OTHER CONTEXTS, THEY MIGHT BE SERVING A IMPORTANT PHYSIOLOGICAL FUNCTION FOR THE CELLS THAT PRODUCE THEM SO THAT’S THE BROAD BACKGROUND THAT NOW LEADS ME INTO THE DEPTHS OF WHAT I’D LIKE OH TELL YOU ABOUT TODAY, WHICH IS HOW THIS MIGHT WORK, HOW WE TESTED THIS NOTION IN A SPECIFIC CONTEXT AND THAT CONTEXT IS THE MUCOUS ENVIRONMENT THAT COLLECTS FROM THE SURFACE OF THE EPITHELIA IN PATIENTS WITH CYSTIC FIBROSIS CH WITHIN THESE LUNGS, OVER TIME, A COMPLEX MICROBIAL COMMUNITY CAN ACCUMULATE AND THIS COMPOSITION OF THE COMMUNITY CAN BE VERY DIFFERENT FROM PATIENT TO PATIENT, YET IT’S FAIR TO SAY THAT AMONGST THE PATHOGENS THAT OVER TIME GROWS TO BE VERY SUCCESSFUL IN ITS HABITAT IS THE

ORGANISM PSEUDOMONAS AERUGINOSA IT’S ARING FOR MANY REASONS, BUT INTERESTI NG FOR MANY REASONS BUT FOR ME, IT SELECTED THESE BEAUTIFUL MOLECULES CALLED PHENAZINE, WHOSE STRUCTURE UR SEE HERE, THIS NICE RING THAT HAS THESE NITROGEN MOIETIES WHERE YOU CAN EXCHANGE ELECTRONS ON AND OFF DEPENDING UPON THE PARTICULAR PEN SEEN THAT YOU’RPHENAZINE THAT YOU’RE C AN CONSIDERING, THEY COME IN LITERALLY EVERY COLOR OF THE RAINBOW, YOU WILL HAVE DIFFERENT CHEMISTRY AT THESE POSITIONS DECORATING THE RING AND THE PROPERTY OF THESE MOLECULES UP AND DOWN THE REDOX SPECTRUM IN TERMS OF THEIR POTENTIAL, IN TERMS ALSO OF THEIR HYDROSOBICITY AND HYDROSOLICITY SO TO ILLUSTRATE THE REDOX ACTIVITY, LET ME SHOW YOU THIS FILM SO WHAT YOU’RE LOOKING AT HERE IS A CULTURE OF PSEUDOMONAS AERUGINOSA THAT HAS COME OUT OF A SHAKER AND WHAT WE CAN DO IS PLAY WITH IT O ON THE BENCH, WHERE WE HAVE A LITTLE VORTEX MACHINE DOWN BELOW, AND YOU CAN JUST SIT HERE, TAKE YOUR CULTURE, IT’S VERY RELAXING, YOU CAN AIR RATE ITAIR ATE IT OVER ON THIS SIDE, WHAT I’M SHOWING IS THE REACTION WHAT’S GOING ON IS THERE IS ONE PARTICULAR — CALLED PIOCYANINE, IT’S EXCRETED OUTSIDE THE CELL IN ITS REDUCED STATE AND WHEN IT ENCOUNTERS OXYGEN, THIS REDUCED GETS OXIDIZED, AT WHICH POINT IT TURNS BLUE AND IT TRANSFERS ITS ELECTRONS ON TO OXYGEN GENERATING REACTIVE OXYGEN SPECIES THEN THE OXIDIZED PHENAZINE IS TAKEN UP BY THE CELL, IT GETS REDUCED INSIDE THE CELL AND EXCRETED OUT, AND THIS IS A VERY REGULATED PROCESS WE UNDERSTAND WHAT THE TRANSPORT MACHINERY IS TO SOME DEGREE AND HOW THE CELL — THESE MOLECULES AND REGULATES RESPONDENT NOW BECAUSE THEY REACT WITH OXYGEN, FOR GOOD REASON, FOR MANY, MANY YEARS, THEY WERE CONSIDERED TO BE TOXIC SECONDARY METABOLITE AND THE MAJOR DRIVER OF THE TOXICITY HAS BEEN ATTRIBUTED TO THE ABILITY TO GENERATE SUPER OXIDE DUE TO THIS PROCESS, PHENAZINE HAS BEEN SHOWN TO HARM NEUTROPHILS AND OTHER BACTERIA IN THEIR ENVIRONMENT, AND TO CONTRIBUTE TO VIRULENCE IN A VARIETY OF INFECTION MO I INFEK MOTIOUS MO DELS AS WE WERE THINKING ABOUT THE BIOSYNTHETIC PATHWAY IS THAT WITH THE EXCEPTION OF THE PHENAZINE PYO — ANY OTHER CAN BE MADE UNDER STRICTLY ANOXIC CONDITIONS MOREOVER, THE REGULATION OF THE BIOSIPT CYSOF THEBIOSYNTHESIS OF THESE MOL ECULES OCCURS AT LATE STAGES OF GROWTH, SO PHENAZINES WERE WELL-KNOWN TO BE COMING HE END OF GROWTH AS SORT OF THE END STAGE OF THE REGULATORY CASCADE AND YOU CAN SEE THIS HERE WHEN YOU GROW THESE CULTURES YOU LOOK AT THESE IT FIGURES IN BLACK, HERE AT THE POINT WHERE THEY REACH THE TAIL END OF WHAT’S CALLED EXPONENTIAL GROWTH AS THEY’RE TRANSITIONING INTO APPARENT STATIONARY PHASE, WHERE YOU SEE THE CULTURE MAINTAINING ITS HIGH LEVELS OF CELLS BUT NO LONGER MULTIPLYING AT THE POPULATION LEVEL THIS IS WHEN PHENAZINES ARE MADE IN COINCIDING WITH THIS TIME POINT IS WHEN OXYGEN, YOU MEASURED IN THESE CULTURES, IS NO LONGER DETECTABLE SO WE HAD AN ALTERNATIVE COMPLEMENTARY HYPOTHESIS, WHICH IS AT THIS STAGE OF THEIR EXISTENCE, AT HIGH CELL DENSITY, AND UNDER LOW OXYGEN TENSION, WHICH I WOULD LIKE TO UNDERSCORE ARE CONDITIONS THAT CHARACTERIZE BIOSOMES IN ANY HABITAT, WHETHER IT’S ON THE SURFACE OF A ROCK OR WITHIN AGGREGATES EMBEDDED WITHIN CF PATIENTS’ MUCUS THESE ARE TWO ATTRIBUTES THAT YOU SEE OVER AND OVER AGAIN AND THAT THEY MIGHT BE PLAYING IMPORTANT PHYSIOLOGICAL FUNCTIONS AT THIS IT STATE SSTATE SO WHAT WE PUT OUT AS AN IDEA IS THAT THEY ARE SECONDARY METABOLITES ONLY IN TERMS IT OF TIME SIMPLY BECAUSE THEY’RE PRODUCED AT A LATER STAGE NO WAY IMPLIES ANYTHING ABOUT THEIR ABILITY TO BE IMPORTANT PHYSIOLOGICALLY FOR THE CELLS THAT PREDICT THEM, IT JUST MEANS THAT WE HAVE TO

REFLECT ON THE CONDITIONS OF THE CULTURES AT THIS TIME TO FIND POTENTIAL PHYSIOLOGICAL FUNCTION SO THAT’S WHAT WE SIGHT OUT TO DO THAT WAS THE OVERALL CONTEXT THAT WE JUST TRANSPOSED THINKING ABOUT THIS PROBLEM IN THE CONTEXT OF IRON MINERALS, YOU HAVE THE SAME SITUATION HERE WHERE YOU HAVE AN INFECTED EPITHELIAL SURFACE AND YOU HAVE HYPOXIC MUCUS THE CHALLENGE AT THE CELLULAR LEVEL FOR THIS CELL IS ESSENTIALLY THE SAME FROM THAT ASPECT IT’S LIMITED FOR OXIDANT SO HOW DOES IT SURVIVE? NOW FOR THE REST OF THIS TALK, I WANT TO SHOW YOU THE DATA FROM OUR STUDIES THAT HELPED US EXPLORE AND TEST THIS IDEA TO DO THIS REVERSELY, MUCH OF WHAT FRAMED OUR WORK WAS THE LUNGS OF CF PATIENTS AS I MENTIONED THERE WERE THREE QUESTIONS WE ADDRESSED THE FIRST SIMPLE QUESTION IS HOW FAST ARE THE PATHOGENS GROWING WITHIN THIS ENVIRONMENT? AMAZINGLY, WE KNOW VERY LITTLE ABOUT HOW FAST BACTERIA ARE GROWING IN ANY ENVIRONMENT SO THIS SEEMS LIKE A GOOD PLACE TO START FOR REASONS I’LL MENTION IN A MOMENT SECOND, WE WANTED TO THINK HARDER ABOUT WHAT MIGHT BE LIMITING THESE GROWTH RATES, AND ONCE WE UNDERSTOOD THAT, BEGIN TO EXPLORE COPING MECHANISM SO AS I MENTIONED, WE’VE BEEN DOING THIS WORK DEVELOPING OUR METHODOLOGIES FOR ASKING AND ANSWERING THESE QUESTIONS MOUNTAIN CONTEXT OF THE SPUTUM IT COLLECTS OF INDIVIDUALS WITH CYSTIC FIBROSIS THE REASON THIS WAS AN ATTRACTIVE ENVIRONMENTAL SAMPLE TO WORK WITH FROM THE PERSPECTIVE OF BEING AMENABLE TO ADDRESSING THE QUESTIONS WE THOUGHT TO ADDRESS WAS BECAUSE IT WAS SO EASY TO ACQUIRE INDIVIDUALS WITH CF EVERY DAY COUGH UP THEIR MUCOUS, WE CAN LOOK AT IT IN THE HOSPITAL OF WE’VE BEEN COLLABORATING WITH CHILDREN’S HOSPITAL IN LOS ANGELES SO AT THE C CAN HLA, CF PEDIATRIC CLINIC, WE HAVE A LITTLE SPACE WHERE WE CAN HARVEST THE SPUTUM AS IT’S COUGHED UP, AND WE CAN TAKE IT AND MANIPULATE IT IMMEDIATELY AFTER EXPECTORATION SO TO ADDRESS THE FIRST QUESTION, HOW FAST ARE THEY GROWING, WE NEEDED TO DEVELOP A METHOD TO MEASURE GROWTH RATE THIS WAS ACTUALLY A VERY HARD THING TO DO WHAT I WILL NOW SHOW YOU IS HOW WE APPROACH THIS FOR MEASURING MEAN GROWTH RATE AND MEASURING THE DISTRIBUTION OF GROWTH RATE THAAT THE SINGLE CELL LEVEL SO ONE OF THE PRIVILEGES OF WORKING AT CAL TECH IS THAT I GET VERY TALENTED STUDENTS AND POSTDOCS TO JOIN MY GROUP, AND I HAD A BRILLIANT PH.D. STUDENT IN COLLABORATION WITH MY COLLEAGUE WHO IS AN OR BAC ORGANIC GEOCHEMIST THE STRATEGY ESSENTIALLY WORKS AS FOLLOWS WE MAKE USE OF THE FACT THAT WE CAN INCUBATE THESE SPUTUM SAMPLES FOR VERY SHORT TIME POINTS, ALLOWING THE PATHOGENS WITHIN THE SPUTUM TO BE EXPOSED TO H2, HEAVY WATER, TANNED TO INCORPORATE A CERTAIN AMOUNT AS THEY GROW INTO NEWLY SYNTHESIZED FATTY ACIDS NOW, THE HARD PART COMES IN ANALYZING THEM WE HAVE TO BE ABLE TO PICK A FATTY ACID THAT IS CAN DIAGNOSTIC FOR A PATHOGEN WE’RE INTERESTED IN THIS FATTY ACID HAS TO HAVE PARTICULAR PROPERTIES OF BEING SYNTHESIZED DE NOVO, NOT RECYCLING THINGS IN ITS ENVIRONMENT THERE ARE MANY CONTROLS I’M NOT GOING TO GO INTO NOW, BUT I’M HAPPY TO EXPLAIN LATER, BUT THE BOTTOM LINE IS WHAT COMES OUT OF THIS IS WE CAN EXTRACT VERY SELECTIVE FATTY ACIDS THAT MEET THE CRITERIA WE NEED TO DO THIS QUANTITATIVELY AND ANALYZE THEM USING GC PYROLOSIS, AND MASS SPEC TOM IT TREE IN ORDER TO MEASURE VERY TRACE AMOUNTS OF UP TAKE SO WHEN WE DO THIS AND CAL GREAT THE AMOUNT OF UPTAKE UNDER DIFFERENT GROWTH RATE CONDITION, WE CAN COME TO A PLACE WHERE WE ARE ACTUALLY APPROXIMATE THE GROWTH RATE FROM REAL HUMAN CLINICAL SAMPLES AND THIS IS WHAT WE FOUND SO WHAT I’M PLOTTING FOR YOU NOW ARE SAMPLES THAT HAVE BEEN TAKEN FROM MANY PATIENTS IN A CROSS-SECTIONAL STUDY, I THINK WE HAVE 37 SAMPLES FROM 16 PATIENTS SHOWN HERE IN THIS GRAPH TO BEGIN, LET ME NOTE THAT UNDER

STANDARD LABORATORY CULTURE, THE WAY WE GO AFTER DRUG DEVELOPMENT, NORMALLY THE GROWTH RATES ARE IN THIS ZONE AND WHAT’S IMMEDIATELY APPARENT WHEN YOU LOOK AT THIS IS THAT THE AVERAGE GROWTH RATES THAT WE’RE LOOKING AT THAT’S INFERRED AS A WEIGHTED AVERAGE OF TWO VERY SPECIFIC FATTY ACIDS IN THE PATHOGEN USED IN THESE STUDY, ALL OF THEM HERE, THE CIRCLES, ARE WELL BELOW THE STANDARD REGIME ACROSS ALL OF THESE PATIENTS, THERE’S CONSIDERABLE VARIATION, OVER ALL THE AVERAGE RATE OF DOUBLING IS APPROXIMATELY TWO DAYS NOW THAT – MAY SLOW BUT THE GOOD NEWS IS THIS IS ABSOLUTELY TRACKABLE — IN ORDER TO CONTROL THE EXPOSURE OF THE ORGANISMS TO NUTRIENTS AT A SLOW ENOUGH RATE THAT YOU’RE ABLE TO CHANNEL THEIR GROWTH INTO RATES THAT ARE RELEVANT FOR THE ACTUAL IN SITU ENVIRONMENT AND I’LL COME BACK TO THAT SO THIS IS THE BULK RATE WHAT ABOUT AT THE SINGLE CELL LEVEL? AGAIN MAKING USE OF THE INTERDISCIPLINARY DEPARTMENTS AT CAL TECH IT, WE WERE ABLE TO GO TO THE GEOLOGICAL DEPARTMENT WHERE THEY HAD A SECONDARY MASS SPECTROMETER THAT ALLOWS YOU TO DETECT ISOTOPES WITHIN CELLS AT INCREDIBLY SMALL SCALES OF RESOLUTION ACCURATELY AND RESOLVE THEIR COMPOSITION ISOTOAPICALLY SO ONE CAN IDENTIFY PARTICULAR ORGANISMS FROM THE SAMPLE, HERE YOU’RE AGAIN LOOKING AT ONE OF THESE SECTIONS FROM A VERY SMALL BIT OF ACTUAL SPUTUM FROM A CYSTIC FIBROSIS PATIENT, AND HERE WE’RE STAINING NOW FOR A PARTICULAR CELL TYPE, AND THEN HERE IN THIS CHANNEL, WHAT YOU’RE LOOKING AT IS THE IONIC COMPOSITION, SO WE HAVE A POSITIVE CONTROL OF PARTICULAR IONS WE EXPECT TO SEE IN EVERY LIVING CELL NOW HERE WE SEE THE CHANNEL THAT WILL ONLY SHOW INCORPORATION OF — SO BY MAKING — DOING A WHOLE VARIETY OF CONTROLS THAT AGAIN I WON’T SHOW HERE, BUT I’D REFER YOU TO PUBLICATIONS THAT YOU’RE INTERESTED IN, WE WERE ABLE TO BE QUANTITATIVE AND TO ASSESS AT THE SINGLE CELL LEVEL THE I DENSITY DISTRIBUTION FUNCTION OF GROWTH RATE FOR DIFFERENT POPULATIONS WITHIN THIS ACTUAL MUCUS I THINK WHAT’S ASTOUNDING IS THE VARIATION IT’S REALLY STRIKING, A VERY SMALL SPATIAL SCALE SO AS AN EXAMPLE, YOU SEE FOUR DIFFERENT PATIENTS IN RED ARE SPECIES TYPE 1, AND YOU CAN SEE THESE DENSITY DISTRIBUTION FUNCTIONS, WHILE THEY OVERLAP BETWEEN PATIENTS, YOU HAVE DIFFERENT SKEWS TO THE DISTRIBUTION FUNCTION THAT’S QUITE HETEROGENEOUS AND YOU CAN RESOLVE DENSITY DISTRIBUTION FUNCTION GROWTH RATE DIFFERENCES BETWEEN SPECIES AT THE SINGLE CELL LEVEL IN SITU SO THIS IS VERY EXCITING AND OPENS UP ALL SORTS OF EXPERIMENTS ONE CAN BEGIN OH DO TO BEGIN TO UNDERSTAND MORE DEEPLY WHY IT IS THAT STUDENT THAT CERTAIN POPULATIONS ARE GROWING FASTER OR SLOWER AND WE CAN DISCUSS THAT NOW LATER BUT RETURN NOW TO THE BULK ENVIRONMENT AND ASK THE QUESTION, WHAT IS LIMITING, WHAT ARE ONE OF THE HINGES THAT COULD BE LIMITING, WE RETURN TO OUR THINKING ABOUT OXYGEN, AND IT’S IMPORTANT TO MANY ENVIRONMENTS AND WHAT ARE ITS REAL CONCENTRATIONS AT THE MICRO SCALE? SO THE GREAT IRONY, GIVEN THAT THE ENVIRONMENT THAT WE’RE LOOKING AT HERE IS THE LUNG, IS THAT WITHIN THE SPUTUM THAT COLLECTS IN THE LUNG, WHERE WE ACHE IN OXYGENTAKE IN OXYGEN, WE FIND VERY GOOD EVIDENCE THAT I’M ABOUT TO SHOW YOU FOR SUBSTANTIAL — ANOXIA WHERE THESE ORGANISMS ARE THRIVING SO HERE IS A SCHEMATIC DEPICTING DIFFERENT PARTS OF THE PULMONARY SYSTEM, AND WE CAN MODEL DIFFERENT REGIONS, WHERE IT’S AN ALVEOLAR SACK OR WITHIN A VERY SMALL BRONCHIOLE COLLECTING IN ONE OF THESE PIPES, THERE ARE DIFFERENT WAYS TO BEGIN TO CALIBRATE AN EXPECTATION FOR OXYGEN AT SPATIAL SCALES THAT ARE RELEVANT I’M GOING TO GIVE YOU ONE ILLUSTRATIVE DATASET HERE FROM MODELING WE IT DID LOOKING AT A BRONCHIOLE WHERE WE ESTIMATED A DISTANCE OF AROUND 500-MICRONS, AND ASK WHAT WOULD THE OXYGEN CONCENTRATIONS BE IN HAD MUCOUS? NOW IF WE CUT THIS PIPE AND WE LOOK IN TWO DIMENSIONS WITHIN SUCH A SMALL TUBE, UNDER DIFFERENT CONCENTRATIONS OF SELF, GIVEN RESPIRATION RATE FOR OXYGEN, SO THIS IS WHAT YOU’RE LOOKING AT HERE THIS IS A THICKNESS THAT WE’RE ASSUMING FOR MUCUS AS IT BEGINS BEGIN

TO CLOG THESE BRONCHIOLES IN HERE, ACROSS THE DIFFERENT ESTIMATES THE IMPORTANT THING TO APPRECIATE IS THAT AS YOU GO FROM PURPLE TO RED ON THE SCALE, WE’RE GOING FROM OXYGEN CONCENTRATIONS WITH THE AMBIENT AIR TO REGIME HAS ARE TOTALLY ANOXIC WHAT THIS VERY SIMPLE SIMULATION PREDICTS IS, OF COURSE, INTUITIVE, THE THICKER THE MUCUS CLOGGING AND THE HIGHER THE CONCENTRATION OF CELLS RESPIRING, THE FASTER YOU WILL DRIVE TOWARDS A STATE OF EQUILIBRIUM WHERE YOU WOULD EXPECT VERY LITTLE OXYGEN SO HOW DO YOU ACTUALLY MAP TO MEASUREMENTS? SO WE WENT AFTER THIS BY MAKING USE OF MICRO ELECTRODES HERE’S A TECH IN MY LAB NOW GOING ON TO IT GRAD SCHOOL, AND WHAT SHE DID WAS SET OFF A CAMP WHERE SHE WAS ABLE TO TAKE THE SPUTUM IMMEDIATELY UPON EX-PECK TRAITION AND MEASURE OXYGEN CONCENTRATIONS, REDOX, SULFIDE, AND PH FOR ALL OF THESE, YOU ALSO NEED A REFERENCE ELECTRODE AS SAMPLE, AS SOON AS WE WERE ABLE TO TRANSFER THEM TO HER SYSTEM SO THIS IS WITHIN ABOUT FIVE MINUTES OF EXPECTORATION AND REFLECTS AS BEST WE CAN IN VIVO ENVIRONMENT IN PROFILE AFTER PROFILE IN COLLECTING THE SPUTUM AFTER IT’S COUGHED UP, WHAT WE FIND IS THERE IS A VERY STEEP INCLINE WHERE WE GO THROUGH A HYPOXIC REGION INTO A REGIME THAT’S DEVOID OF OXYGEN THIS IS NOT SURPRISING FROM THE MODEL WHAT WAS SURPRISING AND FASCINATING WERE THE EXTREMES OF REDOX OFFICIAL CHANGES THAT WE OBSERVED CONCOMITANT WITH THIS SHIFT IN OXYGEN WHAT I’D LIKE TO DRAW YOUR ATTENTION TO HERE IS THAT THESE LOWER RANGES OF REDOX POTENTIAL THAT YOU’RE SEEING ARE COMPATIBLE WITHIN THE RANGE THAT’S KNOWN TO TYPIFY VARIOUS PHENAZINES PRODUCED IN THIS ENVIRONMENT IT’S ALSO CONSISTENT WITH SULFIDE, WHICH WEECH DETECTED HERE, SO THERE CAN BE MULTIPLE THINGS THAT CONTRIBUTE, BUT FOR NOW LET’S FOCUS ON THE POTENTIAL RELEVANCE OF PHENAZINE IN HELPING UNDERPIN SURVIVAL IN AN AKNOCKS IX REALM WITHIANANOXIC REALM WITHI N THIS CONTEXT THE FIRST THING WE HAD TO ASK IS IT PRESENT AT SON 10 TRAITIONS CONCENTRATI ONS THAT ARE IMPORTANT CHILDREN’S HOSPITAL OF BOSTON, WE COLLECTED A DAY A SET THAT WE REPLICATED THAT SHOWS A CORRELATION BETWEEN THE CONCENTRATION OF THE PHENAZINE — AND THE LUNG FUNCTION, WHICH IS MEASURED BY A METRIC CALLED THE FORCED EXPIRATORY VO VOLUME 12TH THIS IS OBSERVABLE IN THESE TOMOGRAMS THAT SHOW YOU THE RANGE OF VALUES FOR AN INDIVIDUAL WHOSE LUNG FUNCTION IS MILD TO MODERATE TO SEVERELY IMPAIRED AND WHAT I HOPE YOU SEE CLEARLY ON THIS SLIDE IS THAT AS LUNG FUNCTION DECLINES, THE CONCENTRATION OF THE PHENAZINE RISES VERY SIGNIFICANTLY IN DAY A THAT I’M NOT SHOWING YOU HERE, WHAT WE ALSO SAW WAS A VERY STRIKING CORRELATION BETWEEN THE CONCENTRATION OF PYOCYANIN PREDICTING THE LUNG FUNCTION DECLINE SO THAT SEEMS TO BE A VERY USEFUL PARAMETER THAT’S DIAGNOSTIC FOR WHERE ONE WOULD EXPECT LUNG FUNCTION HEALTH TO BE GOING IN ANY EVENT, THIS SATISFIED CRITERION NUMBER ONE THE QUESTION IS, ARE ORGANISMS GROWING IN BIOSOMES WITHIN THESE LUNGS WHERE THE ENVIRONMENT COULD BE HYPOXIC? THERE HAD BEEN VERY GOOD DATA FROM GREENBERG’S LAB MANY YEARS AGO REGARDING — PRODUCED IN THIS HABITAT THAT’S ALSO CORRELATED WITH INFORMATION VERY SPHRONGLY, BUT WE WANTED TO BE ABLE TO DEVISE A METHOD TO ACTUALLY PERCEIVE AND SEE THESE BIOSOMES IN SITU, IN ORDER TO UNDERSTAND THEM MORE DEEPLY AND ULTIMATELY GET TO IT A PLACE WHERE WE COULD LOOK EVEN FOR THE EXPRESSION OF PARTICULAR GENES IN SITU THAT OUR LABORATORY EXPERIMENTS HAD SUGGESTED MIGHT BE IMPLICATED IN SURVIVAL SO TO DO THIS, WE TEAMED UP WITH A GREAT COLLEAGUE OF MINE AT CAL TECH WHO COMES FROM THE BIOENGINEERING FIELD AND NEUROSCIENCE IN PARTICULAR, AND SHE WAS PART OF A GROUP AT STANFORD THAT DEVELOPED A METHOD CALLED CLARITY THAT’S BEEN USED IN BRAIN IMAGING, AND WHAT IT DOES IS ALLOWS FOR TISSUES TO BE CLEARED, TO BECOME OPTICALLY TRANSPARENT, AND WE THOUGHT MAYBE WE WOULD BE ABLE TO ADAPT

THIS METHODOLOGY FOR LOOKING AT MICROBIAL POPULATIONS WITHIN THE SPUTUM, OR FRANKLY, WITHIN ANY SAMPLE I THINK THIS HAS A LOT OF POTENTIAL EXTENSION FOR PEOPLE HERE IN THIS AUDIENCE WHO MIGHT BE STUDYING THE HUMAN MICROBIOME THIS IS A METHOD I’D BE VERY DELIGHTED TO SHARE SO MOUNTAIN CONTEXT OF TH IN THE CONTEXT O F THE LUNG, WE TAKE SPUTUM THAT HAS BEEN EXPECT TRAITED, AND WE SUBJECT THEM THROUGH A PROCESSING THAT HELPS US GO FROM A SAMPLE WHERE YOU CAN’T SEE ANYTHING IN IT TO ONE THAT IS OPTICALLY TRANSPARENT THEN AT THIS POINT, WE’RE ABLE TO IMAGE THEM, THEN USING THE METHODS THAT AMPLIFY THE EXPRESS GENE WE WANT TO TRACK OR RIBOSOME RNA GENE THAT MIGHT GIVE US THE ABILITY TO TRACK A PARTICULAR MICROBE IN A COMMUNITY, WE CAN BEGIN TO FIND THEM IN THIS BIG MASS OF SAMPLE SO IN ORANGE, YOU SEE THE LERTIN MUCUS, IN GREEN, THESE ARE BACTERIA THAT WE’VE IDENTIFIED USING A BROAD PROBE FOR BACTERIAL RIBOSOMOL RNA THAT’S SPECIFIC FOR ALL BACTERIA FROM THIS, WE ARE ABLE IT TO SCAN LARGE VOLUMES AT A TIME, IDENTIFY WHERE THE BACTERIA ARE AND THEN BECOME MORE ABLE TO ASK QUESTIONS SUCH AS WHAT ARE THE VOLUME THAT THEY OCCUPY? SO HERE I JUST THRESHOLDED ABOVE 100 CUBIC MICRONS THRESHOLD FOR SIZE IN THIS PARTICULAR SUPPLIES LOU THE SPUTUM, AND WHAT I THINK WHAT’S IMPORTANT TO APPRECIATE IS THAT IN VIVO, THE VAST MAJORITY OF THE BACTERIA WE SEE ARE IN THIS CLASS WHERE THEY’RE CLUSTERED IN AGGREGATE DEFINING THE MAJORITY OF CELLS IN THIS HABITAT AND THAT’S VERY DIFFERENT FROM HOW WE OFTEN DPROA HEMII WE OFTEN GROW THE M IN THE LAB RI, AND THEY TYPICALLY OCCUPY CLUMPS OF MUCH SMALLER SIZE IF YOU RECALL, ONE OF THE INITIAL IDEAS WAS THAT AS A CELL IS GROWING IN SIZE AND MASS AND NO LONGER HAS ACCESS TO OXYGEN, THE ABILITY TO UTILIZE THESE REDOX ACTIVE SHUTTLES MIGHT ENABLE IT TO BE FUNCTIONAL SO NOW RETURNING TO A SIMPLE IN VITRO SYSTEM WHERE WE CAN DO EXPERIMENTS THAT ARE REDUCTIONIST AND MORE WELL CONTROLLED, WE ASK THE QUESTION, COULD PHENAZINE PLAY A ROLE IN BIOFILM DEVELOPMENT SO HERE YOU’RE SEEING AN EXPERIMENT WHERE WE ARE COMPARING THE WILD TYPE TO A MUTANT THAT OVEREXPRESSES PHENAZINE, TO ONE THAT IS UNABLE TO MAKE IT AT ALL, AND WE’RE GOING TO NOW FOLLOW JUST SIMPLY THE MORPHOLOGICAL DEVELOPMENT OF THESE COLONY BIOSOMES OVER THE COURSE AFTER WEEK SO AFTER ONE DAY, THEY BASICALLY LOOK THE SAME AFTER DAYS — THREE DAYS AT THE LAB, THEY START TO DIVERGE AND BY DAY SEVEN, THEY LOOK QUITE DIFFERENT INDEED, WITH THE PHENAZINE MUTANT OCCUPYING A MUCH LARGER SURFACE AREA AND CREATING HE’S BEAUTIFUL WRINKLES, WHEREAS THE MUTANT THAT OVEREXPRESSES PHENAZINES REMAINING NICE AND TIGHT AND SMOOTH AND BECOMING EVER MORE BLUE IN COLOR DUE TO THE GROWTH OF THE PHENAZINE SO RETURNING TO THE CONCEPTUAL MODEL FROM THE BEGINNING, WE WANTED TO ASK THE QUESTION WHETHER OR NOT, AS WE MIGHT PREDICT FROM OUR MODEL, COULD IT BE THAT THE WILD TYPE IS ABLE TO OCCUPY A VOLUME WITHIN THESE BIOSOME STRUCTURES THAT LACK OXYGEN BECAUSE OF THE PRESENCE OF PHENAZINE, WHEREAS THE PHENAZINE MUTANT WOULD BE RESTRICTED ONLY TO ZONES WHERE OXYGEN COULD BE DETECTED AGAIN, WE USE OUR MICRO IT — SYSTEM TO HELP US MAKE THE MEASUREMENTS TO ANSWER THIS QUESTION SO A FORMER POSTDOC IN MY LAB NOW PROFESSOR AT COLUMBIA WHO’S NOW CONTINUING THIS PROJECT DID THIS BEAUTIFUL WORK I’M SHOWING YOU HERE WE HAD THE ADVANTAGE OF A WONDERFUL DIGITAL MIKE SOAP THAT ALLOWEMICROSCOPETHAT ALLOWED US TO VIEW TH ESE AT A LEVEL THAT’S QUANTITATIVE AND IN THREE DIMENSIONS YOU’RE LOOKING AT A TOPOGRAPHIC RENDERING OF THE SURFACE OF HE’S BIOSOMES, AND WHAT’S SO GREAT IS THAT IT’S ON THE SCALE SO WE CAN MAKE VERY PRECISE MEASUREMENT OF DEPTHS WITHIN THESE VUK YOU’RES AND WHESTRUCTURES.WHEN WE DO THIS AND COUP LE THESE QUANTITATIVE MEASUREMENTS OF THE SIZE AND WIDTH OF THESE STRUCTURES, TWO MEASUREMENTS WHERE IN THE SAME WRINKLES HERE OR AT THE BASE, WE STICK MICRO ELECTRODES CONCOMITANTLY AND SEE HOW LOW CAN WE G GO TO DETECT OXYGEN, HERE YOU SEE THE SETUP, IT’S FAIRLY SIMPLE, IT’S JUST GROWING IN THE PETRI PLATES, WE

CAN ASK WHERE WITHIN THESE COLONIES ARE THE OXIC ZONES AND DOES THAT CORRELATE WITH THE CELL AS WE WOULD EXPECT SO WE IT DID AN EXPERIMENT WHERE WE MEASURED IN BOTH THE WRINKLE AND THE BASE UNDER WELL CONTROLLED CONCENTRATIONS OF AMBIENT OXYGEN AND I CAN’T SEE ANY OF YOU BECAUSE OF THE SHINING LIGHT BUT MAYBE YOU SHOULD DO THIS EXPERIMENT BEFORE I SHOW YOU THE DATA WHAT WOULD YOU ANTICIPATE AS YOU CRANK UP THE CONCENTRATION OF OXYGEN IN THE ATMOSPHERE? WOULD YOU IMAGINE THAT THE WRINKLED STRUCTURE WOULD GET THICKER OR THINNER? IT’S A HARD ENVIRONMENT OH DO A Q & A SO I’LL TELL YOU AND SHOW YOU THE DATA, BUT THINK ABOUT WHAT YOU WOULD PREDICT I’LL GIVE YOU A MOMENT AND THEN I’LL SHOW YOU THE EXPERIMENTAL RESULTS WHAT WOULD HAPPEN WHEN CRANK IT DOWN, AND THE OTHER KEY QUESTION IS, WOULD THERE BE A DIFFERENCE BETWEEN THE WILD TYPE AND THE MUTANTS SO WHAT WE SAW, THE INTUITION I HOPE YOU HAD WAS AS YOU CRANK UP THE OXYGEN, THE WIDTH WOULD BE EXPECTED TO INCREASE, AND THIS WOULD BE TRUE REGARDLESS FOR THE WILD TYPE OR THE MUTANTS AS SHOWN HERE, SO WHAT YOU’RE LOOKING AT NOW IS FOR OXYGEN AMBIENT CONCENTRATIONS OF 15, 21 AND 40% THE MEASURED DEPTH OF COLONIZATION WITHIN THESE STRUCTURES IN GRAY THE WILD TYPE AND IN WHITE THE PHENAZINE MUTANT, AND IN ALL CASES YOU CRANK UP THE OXYGEN, THAT INCREASES WHICH WAS PREDICTED WHAT WAS VERY SATISFYING AND EXCITING TO US, HOWEVER, WAS TO RECOGNIZE THAT THE DIFFERENCE THAT ONE SAW BETWEEN THE WILD TYPE AND THE PHENAZINE MUTANTS, THE DEPTHS WHERE OXYGEN BECAME IMMEASURABLE REALLY LIMITED THE GROWTH OF THE PHENAZINE MUTANT, WHEREAS THE WILD TYPE WAS ABLE TO OCCUPY SPACE AND VOLUME THAT EXCEEDED THE REGIME TO WHICH WE COULD MEASURE THE OXYGEN SO THAT WAS VERY IMPORTANT BECAUSE IT HELPED VALIDATE WITHIN THIS CONTEXT OF BIOSOMES THAT CAN — MAY BE ABLE TO PROMOTE ANAEROBIC SURVIVAL SO WE WANTED TO UNDERSTAND THAT IN GREATER DEPTH, HOW DOES IT PROMOTE ANAEROBIC SURVIVAL, THROUGH WHAT METABOLIC PATHWAY IS IT EXERTED ITS EFFECT TO ANSWER THIS, WE ACTUALLY SHIFTED INTO A DIFFERENT WAY OF SETTING UP THIS EXPERIMENT, WHERE WE ACTUALLY BEGAN THE GROWTH CURVE AT THE PLACE WHERE MANY PEOPLE END THE EXPERIMENT, AND THAT IS AT STATIONARY PHASE, UNDER ANOXIC CONDITIONS SO WE DID THESE EXPERIMENTS IN ANAEROBIC CHAMBERS, WHERE WE COULD GROW CULTURES IN SYSTEMS LIKE THESE WHERE WE WERE ABLE TO HAVE AN ELECTRODE SERVING AS THE WORKING ELECTRODE TO OXIDIZE PHENAZINES THAT WE ADDED AT CAN DISCRETE CONCENTRATIONS BASED UPON WHAT WE HAD MEASURED IN THE CF LUNG AND BEING TYPICAL VALUES, AND TO MAKE THINGS SIMPLE, WE COULDN’T USE THE MUTANT TO PRODUCE ITS OWN PHENAZINE SO WE WERE ABLE TO BE QUANTITATIVE IN INPERRING HOW MANY TIMES A CELL WOULD BE CYCLING A PHENAZINE BASED UPON THE AMOUNT OF CURRENT BEING GENERATED AT THE ELECTRODE SO KNOWING THE CELL DENSITY AND MEASURING THE CURRENT, WE COULD INFER UNDER THESE CONDITIONS THAT RELS WER CELLS WERE CYCLING THESE PHENAZINES APPROXIMATELY 50 TIMES SO THE KEY FINDING HERE IS THAT UNDER THESE CONDITIONS IN THE PRESENCE OF GLUCOSE, WHAT WE FOUND WAS THAT WE COULD OBSERVE PHYSIOLOGICAL SURVIVAL AS MEASURED BY COLONY FORMING UNITS WHEN YOU TAKE CELLS FROM THIS CHAMBER AND THEN PLATE THEM, CAN THEY GROW UP, ARE THEY STILL VIABLE WHEN THE ELECTRODE WAS TURNED ONTO RECYCLE THE PHENAZINE, AND THEN THE CELL REDUCES IT, EX-CREATES IT AND THEN IT GETS REOXIDIZED HERE, THAT’S THE REASON WHY YOU NEED THE ELECTRODE ON; TO RECYCLE IT BUT YOU TURNED OFF THE ELECTRODE OR DIDN’T ADD THE PHENAZINE, THEN THESE CELLS WOULD DIE MUCH MORE RAPIDLY THAN HERE IN THE PRESENCE OF PHENAZINES WHERE CYCLINE WAS INEN ABLED SO THAT WAS AN EXCITING FINDING, AND WHAT MADE US THINK THAT THIS WAS ACTUALLY BIOLOGICALLY REALLY SIGNIFICANT AND POTENTIALLY PART OF THE EVOLUTIONARY HISTORY OF THIS PATHOGEN WAS THE OBSERVATION THAT ALL NATIVE PHENAZINES PRODUCED BY PSEUDOMONAS AERUGINOSA PROMOTE SURVIVAL YET SYNTHETIC ELECTRODE SHUFFLES DO NOT STRUCTURE FUNCTION REQUIREMENTS FOR TRANSPORT AND FOR CHAPERONING THE MOLECULES WITHIN THE CELL IN A WAY WHERE THEY FACILITATE SURVIVAL BUT DON’T CAUSE TOXICITY AND I’M HAPPY TO IT ELABORATE ON IT LATER WHAT IS IT THAT THE CELL IS GETTING FROM THE PROCESS OF REDUCING THESE PHENAZINES THAT ALLOWS IT TO DO BETTER? SO AS A CLUE, A STUDENT IN MY LAB STARTED MEASURING THE METABOLITES THAT WERE PRODUCED IN THESE SURVIVAL CHAMBER EXPERIMENTS AT DIFFERENT STAGES SOMETHING THAT HE OBSERVED WAS HA IN THE PRESENCE OF PHENAZINES

PHENAZINES, HE SAW AN INCREASE IN A PEAK THAT WE KNEW TO CORRESPOND TO THAT OF THE METABOLITE ACETATE NOW, IT’S BEEN KNOWN FOR A LONG TIME AND I’VE LEARNED FROM SOME OF THE CONVERSATIONS I’VE HAD TODAY THAT THIS IS A PATHWAY MANY OF YOU IN THE AUDIENCE THINK ABOUT TOO AND FIND INTERESTING, WHICH MADED ME HAPPY, AND IT’S ALSO KNOWN — IT’S BEING REPORTED IN OTHER ORGANISMS, E. COLI AMONGST SEVERAL, THAT DURING SURVIVAL AND DURING INFECTION, ONE OF THE WAYS A CELL CAN GENERATE ENERGY, OF COURSE, IS THROUGH LIMITATION SO WE WERE DOING OUR EXPERIMENTS UNDER CONDITIONS WHERE WE GAVE THE CELLS GLUCOSE GLUCOSE CAN BE CONVERTED TO PYRUVATE I’M SIMPLIFYING A LOT OF METABOLIC PATHWAYS SO IT’S NOT TOO BUSY AND SHOWING YOU REALLY THE PARTS THAT ARE THE MOST IMPORTANT SO AS GLUCOSE IS OXIDIZED TO PYRUVATE, WHEN WE GET TO PYRUVATE, CELLS CAN TAKE THAT AND FERMENT, AND THERE ARE DIFFERENT WAYS THIS WORKS ONE DAY IT CAN WORK WITH PSEUDOMONAS IS BY REDUCING THE PYRUVATE TO LACTATE, THUS ENABLING THE OXIDATION OF NADH TO NAD PLUS, AND THAT IS CATALYZED BY THE ENZYME LACTATE D.LDHA, AND THEN THAT PI PYRUVATE, THIS REDUCTIONTIVE ARM IS CAN COUPLED TO — WHERE IT GETS OXIDIZED TO ACETATE A VERY IMPORTANT STEP ALONG THIS PATHWAY IS THE LAST ONE, WHERE ACA IS ABLE TO TAKE AN INTERMEDIATE AND FROM THAT INTERMEDIATE TRANSFER PHOSPHATE GROUP IN ORDER TO GENERATE ATP AND THIS STEP IS CRITICAL IN PROVIDING ATP THAT CAN SUPPORT ENERGY GENERATION NOW WHAT IS PREDICTED BY THIS IS THAT IN THE PRESENCE OF PHENAZINES, THE ABILITY TO GO FROM PYRUVATE AND MAKE LACTATE MAY NOT BE NECESSARY BY VIRTUE OF DUMPING ELECTRONS, PHENAZINE COULD BE REOXIDIZED, ALLOWING THIS TO TURN OVER AND ENABLE — PATHWAY AND WHAT WE KNEW INTERESTINGLY AND I’M NOT GOING TO GO INTO THE REASONS NOW, UNDER THE CONDITIONS OF OUR EXPERIMENT, THIS PARTICULAR ENZYME IS NOT AK ITTIVE AND SO WE HYPOTHESIZE THAT IN OUR SURVIVAL ASSAY, WE COULD MAKE A MUTANT DEFECTIVE IN THIS AND IT WOULD SURVIVE JUST FINE IN THE PRESENCE OF PHENAZINE, BUT NOT MOUNTAIN ABSENCE OF PHENAZINE WHEREAS IF WE MADE A MUTANT IN THIS PATHWAY, IN PARTICULAR, IN ANY OF THESE STEPS, AND I’M JUST GOING OH FOCUS HERE — THEN IT WOULD BE DEFECTIVE IN SURVIVAL WE SPECULATED THE ENERGY COMING FROM THIS STEP WAS KEY IN HELPING IT SURVIVE SO THAT’S NOW THE DATA I’M GOING TO SHOW YOU SO LET’S FIRST COMPARE THE SURVIVAL CURVE FOR THE WILD TYPE THIS IS AGAIN UNDER ANAEROBIC CONDITIONS IN THE PRESENCE OF GLUCOSE AND PHENAZINE THERE’S A CONTROL WITH THE WILD TYPE IN THE ABSENCE OF PHENAZINE AND WHAT WE SEE CLEARLY AS PREDICTED WITH THE ABSENCE OF THE ENZYME ACA, THE CELLS CAN’T SURVIVE, EVEN IF THEY’RE GIVEN GLUCOSE AND PHENAZINE AND YES THE MUTANT THAT DOESN’T MAKE LDHA JUST DOES FINE, IN THE PRESENCE OF PHENAZINE MONOW, ONE WOULD PREDICT THAT ITS NADH TO NAD PLUS RATIO WOULD BE OFF IN THE ABSENCE OF PHENAZINE AND THAT IN THE PRESENCE OF PHENAZINE, IT WOULD BE HIGHER, AND THAT’S WHAT WE’VE FOUND SO THE LDHA IN THE ABSENCE OF PHENAZINE HAS A MUCH LOWER OXIDIZED NAD PLUS AND NADH RACE YOE IN THE PRESENCE OF 15 DEAN AND THIS CORRELATES VERY BEAUTIFULLY WITH VIABILITY IN THIS ASSAY SO WHAT THAT SUGGESTED WAS THAT WE WOULD SEE GREATER ACETATE PRODUCTION IN THIS LDHA STRAIN, THE PRESENCE OF PHENAZINE, THAN IN THE ABSENCE AND THAT THIS WOULD TRANSLATE DIRECTLY INTO THE AMOUNT OF ATP WE WITH WOULD FIND IN THESE CULTURES IN THE PRESENCE OF 1 PHENAZINE IN COMPARED TO THE IN THE ABSENCE SO TO ME, THE REASON THAT I THINK THIS IS INTERESTING AND POTENTIALLY IMPORTANT IS THAT IT EXPANDS OUR THINKING ABOUT THE METABOLIC PATHWAYS AVAILABLE TO THE CELL IN THIS ENVIRONMENT, AND OFTENTIMES IT’S HARD TO PRECAN DICTIONARY JUST FROM A BIOCHEMICAL MAP FROM THE SWREE NOME WHAGENOMEWHAT METABOLIC PATHWAYS MAY BE OCCURRING, AND SO IT WILL BE

INTERESTING TO SEE THE EXTENT TO WHICH THIS TYPIFIES REACTIONS THAT MIGHT BE MORE BROADLY IMPORTANT IN BOTH NATURE AND SURVIVAL PHYSIOLOGY IN THE CONTEXT OF INFECTION AND SO BRINGING THIS BACK NOW TO THE GROWTH RATE EXPERIMENTS I SHOWED YOU EARLIER, WHAT MOTIVATES ME TO THINK THAT THIS IS A MODE OF METABOLISM THAT BEARS EXPERIMENTAL FOCUS IS THE FACT THAT THE RATES OF DOUBLING THAT WE’RE ABLE TO MEASURE UNDER THESE CONDITIONS ARE VERY RELEVANT TO THE REGIME THAT WE SAW AS BEING IMPORTANT WHEN WE MADE THESE IN SITU GROWTH RATE STUDIES SO WHAT RAISED — THE PARTICULAR KIND OF REDOX METABOLITES SUPPORTING SURVIVAL BUT REALLY A MUCH MORE PROFOUND QUESTION IS WHAT UNDERPINS ANAEROBIC SURVIVAL IN GENERAL I THINK THERE’S A LOT OF ROOM FOR FUNDAMENTAL DISCOVE DISCOVERY HERE I’LL GIVE YOU ONE CASE AND WITH THAT,LY END THE TALK THAT IS TO TAKE A STEP BACK AND TAKE AN AGNOSTIC POINT OF VIEW AND ASK, UNDER THESE CONDITIONS, WHAT ARE THE CELLS DOING? AS I SAID EARLIER, OFTENTIMES EXPERIMENTS ARE DONE UNDER CONDITIONS WHERE CELLS ARE GROWING RAPIDLY, OFTEN IN THE PRESENCE OF OXYGEN OF COURSE THERE’S A LOT OF GREAT WORK DONE UNDER ANAEROBIC CONDITIONS AS WELL BUT THERE’S NOT A LOT OF WORK DONE UNDER SLOW GROWTH RATES, PARTICULARLY UNDER ANAEROBIC CONDITION, WHICH MIGHT BE VERY IMPORTANT SO WE TOOK THIS CONDITION OF THE SURVIVAL ASSAY — BY PHENAZINE AND WE ASKED WHAT IS BEING EXPRESSED WITH THE PROTEOME SO I ACTUALLY NEED TO CORRECT THAT WE DID IT ON THE PYRUVATE SURVIVAL CONDITION AS A FERMENTATION, NOT IN THE PRESENCE OF PHENAZINE UNDER THESE CONDITIONS OF THIS PARTICULAR EXPERIMENT, AND IF YOU’RE INTERESTED, THIS PAPER, I THINK, IS COMING OUT IN JUST A COUPLE WEEKS SO IN COLLABORATION WITH MY COLLEAGUE DAVE CHURELL, WHOSE LAB THE IN THE DIVISION OF CHEMISTRY AND CHEMICAL ENGINEERING AT CAL TECH PIONEERED A TECHNIQUE CALLED BONCAT, WHICH STANDS FOR THE AMINO ACID TAGGING APPROACH, WHAT THAT ALLOWS YOU TO DO IS TO IDENTIFY PROTEINS THAT ARE NEWLY SYNTHESIZED AS A PARTICULAR POINT IN TIME YOU COULD EVEN DO THIS AT A PARTICULAR PLACE IN SPACE, BUT I WON’T TALK ABOUT THAT RIGHT NOW AND WE ADAPTED IT TO OUR ANAEROBIC SURVIVAL CONDITION SO WHAT WE IT DID WAS AN EXPERIMENT COMPARING THE PROTEOME DHAFS BEING ACTIVELY SYNTHESIZED AT HA TIME BETWEEN AROBE BICLY GROWN AND THOSE UNDER ANAEROBIC CONDITIONS WE FOUND PROTEINS HA WERE EITHER UNIQUE IN ONE CONDITION OR THE OTHER AND THEN A LARGE NUMBER THAT OVERLAP OF HAD LARGE OVERLAP, WE FOUND 41 THAT WERE SIGNIFICANTLY HIGHER IN THEIR PRODUCTION UNDER ANAEROBIC CONDITIONS BUT ALSO SEEM TO HAVE SOME RELEVANCE UNDER AEROBIC CONDITIONS, SO WE DECIDED TO FOCUS ON ONE BECAUSE IT WAS COMPLETELY UNKNOWN IT WAS A SMALL PROTEIN OF UNKNOWN FUNCTION, AND WE THOUGHT WELL, THIS WOULD BE AN INTERESTING ONE TO PURSUE SO TWO PHENOTYPES THAT MADE US INTERESTED I IT ARE THE FACT THAT THIS PARTICULAR PROTEIN, HERE I’M CALLING SUTA, THE CELLS ARE NOT ABLE TO MAKE BIOSOMES THIS IS A METRIC FOR BIOSOME FORMATION BASED ON A CRYSTAL VIOLET ASSAY, COMPARED TO THE WILD TYPE, IF YOU OVEREXPRESS THIS PROTEIN, THEY’RE ABLE TO MAKE MUCH THICKER BIOSOMES AND SIMILARLY HERE, WE SAW VERY SIGNIFICANT DIFFERENCES IN THE RATIO FOR THE MUTANT OVEREXPRESSING THIS PROTEIN COMPARED TO THE STRAIN THAT WAS UNABLE TO PRODUCE IT MOREOVER, BECAUSE BONCAT IS A PROTEOMIC METHOD, WE WERE ABLE TO DISTINGUISH BETWEEN AND SEE CHANGES FOR THINGS THAT WERE POST TRANSCRIPTIONALLY REGULATED AS OPPOSED TO TRANSCRIPTIONALLY REGULATED JUST IN GENERAL, I THINK THERE’S LESS KNOWN ABOUT THIS SO WE DECIDED FOR THESE VARIOUS REASONS THAT WE WANTED TO UNDERSTAND WHAT IT DID AND SO BEFORE I’M GOING TO SHOW YOU OUR BEGINNING INSIGHT INTO THE MOLECULAR MECHANISM OF WHAT THIS THING DOES, WHAT I WANT TO GO BACK TO IS WHY DO WE CALL THIS THING SUTA AND THE FACT THAT WE SELECTED IT, BECAUSE WE SAW IT EXPRESSIO EXPRESS, SYNTHESIZED AND MADE IN AEROBIC AND ANAEROBIC CONDITIONS WHAT WE FOUND WAS THAT IN SOME PATIENTS, WE WOULD SEE REALLY AMAZING SWINGS IN REDOX, AND IF YOU THINK ABOUT IT, OF COURSE THERE’S GOING TO BE

INCREDIBLE — EVERY TIME SOME INDIVIDUAL COST, YOU CAN IMAGINE, THERE HAVE BEEN SOME PERTURBATIONS OF THE OXYGEN CONTENT SO IT’S NOT ENOUGH TO JUST THINK ABOUT SOMETHING IN A STATIC ENVIRONMENTAL STATE, IT’S ALWAYS GOING TO — THE DYNAMICS ARE VERY IMPORTANT AND SO WE THOUGHT WHAT IF WE WERE TO VACILLATE AND CHANGE FROM AEROBIC TO ANAEROBIC SURVIVAL CONDITIONS BACK TO AEROBIC, ANAEROBIC SURVIVAL, AND DO THIS MANY TIMES IN TRANSFER, WOULD WE SEE A DIFFERENCE IN FITNESS FOR THIS INTERESTING MUTANT THAT WE HAVE FOUND AND SURE ENOUGH, WE SAW A SIGNIFICANT DIFFERENCE THE WILD TIME WAS WHEN COMPETED WITH THIS, MUCH MORE COMPETITIVE UNDER THIS DYNAMIC CHANGE OF ENVIRONMENTAL STATE AND THAT ACTUALLY WINDS UP MAKING A GREAT DEAL OF SENSE, BECAUSE WHEN WE WENT THE NEXT STEP TO TRY TO FIGURE OUT WHAT THIS PROTEIN DOES, WHAT WE FOUND IS THAT THIS SMALL PROTEIN, I GUESS SMALL IS RELATIVE AS THIS IS ONLY 105 AMINO ACIDS, IT WAS COMPLETELY UNKNOWN, IT WAS INTERESTING, IT HAD THIS VERY LARGE ACIDIC AND TERMINAL REGION, AND TO OUR GREAT SURPRISE, AND KIND OF DELIGHT, WHEN WE DID A COI.P. EXPERIMENT WHERE WE WERE LOOKING TO SEE WHAT DID THIS PROTEIN PHYSICALLY INTERACT WITH, SO WHAT REALLY CAME OUT VERY STRONGLY AND VERY SIGNIFICANTLY WAS RNAP, THE RNA PPOLYMERASE THAT STRONGLY ASSOCIATE WITH SUTA, WHICH STANDS FOR SURVIVAL UNDER TRANSITION SO THE DETAILS OF HOW IT INTERACTS WITH RNA POLYMERASE ARE ASPECTS THAT WE’RE NOW ACTIVELY PURSUING AND I’M HAPPY TO TALK ABOUT LATER SUFFICE IT TO SAY FOR NOW THAT THE IMPACT THIS LITTLE PROTEIN HAS IS ACTUALLY QUITE BROAD GLOBALLY, SO IN DOING RNA SEQ EXPERIMENTS, WE COULD SEE WHERE THIS PROTEIN WAS HAVING AN EFFECT GENOME-WISE, AND I THINK THE MOST IMPORTANT ASPECT AT LEAST FOR ME THAT CAME OUT OF THIS WAS THAT WHEN WE LOOKED AT THE COMPARISON BETWEEN OUR OVEREXPRESSION STRAIN AND OUR COLLEAGUES STRAIN, AND WE WERE LOOKING AT THE RNA FROM THESE TWO DIFFERENT STRAINS AND WE WERE LOOKING AT WHICH CLASSES OF GENES ARE ENRICHED IN BEING UPREGULATED OR IN BEING REPRESSED AS A FUNCTION OF THIS LITTLE SUTA PROTEIN WHAT WE FOUND IS THAT COMPARED NOW THE WAY YOU READ THIS IS HERE ARE THESE DIFFERENT CATEGORIES OF CELLULAR FUNCTIONS AND IN BLACK IS THE FRACTION OF THE GENOME HA CAN B THAT CAN BE BROADLY CLASSIFIED IN THESE CAN DIFFERENT WAYS IN COLOR IS THE AMOUNT OF TRANSCRIPTS THAT WE WERE ABLE TO SEE CORRESPONDING TO THESE CLASSES, AND I THINK WHAT’S REALLY IMPORTANT TO NOTE HERE IS THAT THERE WAS A REAL ENRICHMENT OF GENES THAT ARE ANNOTATED AS BEING INVOLVED IN MAINTENANCE AND SECONDARY METABOLISM, THIS LITTLE PROTEIN HELPS TO UPREGULATE, AND THERE’S A SIGNIFICANT DOWNREGULATION IN CLASSES OF GENES INVOLVED IN MOTILITY DEFENSE AND SIGNALING OF COURSE IN BOTH CASES, WE HAVE A LARGE FRACTION OF GENES OF UNKNOWN MOTION, BUT WE’D LIKE TO STIPULATE ARE UNKNOWN BECAUSE WE’VE BEEN DOING EXPERIMENTS ONLY IN A VERY LIMITED WINDOW OF CONDITION AND AS WE EXPAND OUR THINKING ABOUT WHAT THE ACTUAL ENVIRONMENT IS HA WE SEEK TO UNDERSTAND IN THE LABORATORY, WE’LL MORE AND MORE REDUCE THIS UNKNOWN GROUP AND BETTER APPRECIATE THE PHYSIOLOGY OF CELLS IN CONTEXT SO AS ONE EXAMPLE, IT BRIRNTION ME BACK TBRINGS MEBACK TO THE IMPORTANT PO INT, IN THE CONTEXT OF SEDIMENTS IN A RESERVOIR, HOLDING POTABLE WATER SUPPLIES OR WITHIN THE LUNGS OF INDIVIDUALS WITH CYSTIC FIBROSIS, ORGANISMS IN THESE ENVIRONMENTS, BACK TIER WRA AND BACTERIA O F ALL FLAVORS CAN — IN WHICH TO IT ADAPT STRATEGIES THAT ALLOW THEM TO BE SUCCESSFUL SO I THINK WE ACTUALLY HAVE A LOT TO LEARN SOMETIMES BY MAKING THESE JUMPS IN OUR THINKING AND TRYING TO ASK, WHAT IS IT THAT THESE ENVIRONMENTS HAVE IN COMMON AND HOW MIGHT ORGANISMS THAT ARE KNOWN TO TRANSITION FROM THE SOIL INTO THE HUMAN HOST HAVE EVOLVED THAT ALLOW THEM TO BE SO SUCCESSFUL SO THAT, I’LL POINT OUT THEIRS IS A REALLY COMPLEX SYSTEM, AND IN ORDER TO UNDERSTAND ANY OF THESE SYSTEMS, WE MUST EMBRACE BOTH THE COMPLEXITY OF OUR ENVIRONMENT, TRY TO CHARACTERIZE IT, AT THE SAME TIME WE EMBRACE THE POWER OF REDUCTIONIST

EXPERIMENTAL LABORATORY WORK SO THAT WE CAN DESIGN OUR EXPERIMENTS IN WAYS THAT ULTIMATELY HAVE THE MOST MEANING SO I’LL END BY THANKING A PHENOMENAL GROUP OF PEOPLE WHO HAVE — THROUGHOUT THE TIME OF MY LAB, CONTRIBUTED TO THE DEVELOPMENT OF THESE IDEAS HERE I’M TELLING YOU PHOTOS OF SWRUS THE MOST RECENT KEY FIGURES THIS IS THE TEAM AT CHOA THAT WE WORKED WITH AT THEIR PULMONARY CLINIC THAT HAS PROVIDED THE MOST WELCOME OF HOMES AND BEEN JUST A WONDERFUL GROUP OF PEOPLE WITH WHOM TO COLLABORATE BUT THE VAST MAJORITY OF THE EXPERIMENTAL WORK I’VE SHARED WITH YOU TODAY WAS REALLY DONE BY FOUR VERY TALENTED PEOPLE, THREE OF THEM WHO ARE STUDENTS, A GEOCHEMIST STARTING HIS OWN LAB AT U.C. BOULDER THIS FALL, MY STUDENT, NATE GLASSER, AND BRETT BABIN, WHO I JOINTLY MENTORED WITH DAVE, AND MY POSTDOC, MEGAN BERGKESSEL BECAUSE I’M HERE AT THE NIH, I WANWANT TO SAY ONE ADDITIONAL THING, WHICH IS A LOT OF WHAT I TALKED ABOUT TODAY CAME FROM BEING FORTUNATE TO RECEIVE ONE OF THESE TRANSFORMATIVE RESEARCH AWARDS ABOUT FOUR YEARS AGO, AND I’M REALLY, REALLY GRATEFUL TO THE SELECTION COMMITTEE WHO GAVE US THE OPPORTUNITY TO TRY TO ADAPT SOME OF THESE TOOLS FROM THE WORLD OF GEOCHEMISTRY INTO THIS CONTEXT AT LEAST FOR ME PERSONALLY, IT’S BEEN A VERY EXCITING JOURNEY AND HAS ALLOWED US TO DEVELOP METHODS THAT HOPEFULLY ARE MORE BROAD IN THEIR APPLICATION AND RELEVANT WELL BEYOND THE SPECIFIC CONTEXT IN WHICH WE DEVELOP THEM SO I’M HAPPY TO ANSWER ANY QUESTIONS AND THANK YOU VERY MUCH AGAIN FOR THE HONOR OF BEING INVITED HERE TO GIVE THIS SEMINAR [APPLAUSE] >> THAT WAS REALLY GREAT WITH RESPECT TO ANOTHER MODEL SYSTEM, THERE IS A LOT OF WORK BEING DONE ON ORGANISMS THAT LIVE WITHIN OTHER ORGANISMS AND THESE ENDOSYMBIANTS ARE OFTEN BACTERIA, SOMETIMES FUNGI, BUT THEY MAKE A HOME IN ANOTHER ORGANISM AND THERE’S A VERY COMMON BACTERIA, WOBAKIA, THAT’S PROBABLY IN 50% OF THE INSECTS, AND THERE ARE MANY STRAINS OF THIS BUT THESE ENDOSYMBIANTS HAVE TO SURVIVE BY THE GRACE OF THE HOST, AND ONE OF THE THINGS THEY’VE LEARNED TO DO, IN COOPERATION WITH A HOST PROBABLY, IS TO GROW VERY SLOWLY AND AS ALWAYS, THERE’S A LOT OF WORK ON WHY DO THEY GROW SO SLOWLY SOMETIMES, THE HOST MAKES PEPTIDES OR MICRO RNAs OR — BUT THE ORGANISMS THEMSELVES, THE ENDSYMBIANTS ALSO PARTICIPATE IN THIS, SO THAT WOULD BE AN INTERESTING — I’M SURE THERE ARE A LOT OF PEOPLE THAT ARE WORKING ON IT THAT WOULD BE AN INTERESTING MODEL THE INSECTS ACTUALLY HAVE AN ORGAN CALLED A BACTERIOME, YOU CAN JUST PLUCK THAT OUT AND LOOK WITHIN THAT ORGAN >> YEAH, I HINGE HEA THINK HEAS THAT’S A FANTASTIC EXAMPLE IT’S INCREDIBLE HOW MANY EXAMPLES YOU CAN ACTUALLY THINK ABOUT WHERE SLOW GROWTH PHYSIOLOGY IS SO IMPORTANT, WHETHER EVEN IN WASTEWATER TREATMENT, THERE’S SO MANY CONTEXTS, AND YET I THINK AT A DETAILED MOLECULAR LEVEL, HOW IT IS THAT THE CELL IN THESE SLOW GROWTH STATES ACTUALLY ACHIEVE BASIC PROCESSES FOR TRANSCRIPTION AND TRANSLATION AND MEMBRANE MAINTENANCE THERE’S A LOT MORE TO LEARN THERE STILL >> WHEN YOU WERE SPEAKING OF BIOFILMS AND YOU WERE TALKING ABOUT, IF I UNDER STOOND UNDERSTOOD RECTAL LY, CERTAIN TYPES OF MUTANTS IN MAKING BACTERIA SO THEY CAN’T PRODUCE BIOFILMS, THIS WOULD BE OF FURTHER SIGNIFICANT THAN WHAT YOU’RE IMPLYING BECAUSE OF THE IDEA OF MEDICAL DEVICES, ET CETERA, THE BIOFILMS IS A HUGE PROBLEM THERE’S A MILLION AND ONE NOW PAPERS OUT ON SOLUTIONS FOR THIS IF YOU COULD FIGURE OUT A WAY TO MAKE BIOFILMS MUTANT AS IN CONTRAST TO TRY TO COMBAT THEM WITH ANTIBIOTICS WHICH ISN’T EFFECTIVE BECAUSE A LOT OF THE NEW HYPOTHESES SAY THEY’RE IN LAYERS, ONE LAYER PROTECTS THEM, THE OTHER IS ACTIVE, YOU CAN GO THROUGH THE LITERATURE AND HAVE

THAT, BUT GEE, WE’RE GOING TO FIGURE OUT A WAY TO MAKE THE BIOFILM IN THIS GUY WHO’S GOT A PACEMAKER INFECTION, GO AWAY BY INTRODUCING A MUTANT STRAIN OF WHAT YOU’VE CULTURED OUT OF THEM THEM, AND I JUST — >> I’M JUST SMILING BECAUSE I’M WORKING ON FINISHING AN R01 APPLICATION THAT’S VERY RELATED TO THAT, THINKING ABOUT HOW ONE MIGHT, YOU KNOW, UNDERMINE BIOSTRUCTURE BY GOING AFTER THESE TYPE OF METABOLITES THAT CAN HELP SUSTAIN AS A NOVEL THERAPEUTIC APPROACH I THINK THAT THERE’S A LOT STILL WE HAVE TO DO IN ORDER TO KNOW IF THAT’S A VIABLE STRATEGY, BUT I THINK RATIONALLY MAKES SENSE, IT’S VERY DIFFERENT THERE ARE MANY DIFFERENT WAYS ONE CAN BEGIN TO THINK CREATIVELY WHEN YOU EMBRACE THE IMPORTANCE OF METABOLISM SUSTAINING THESE, AND THEN THINKING ABOUT HOW CAN YOU PERTURB METABOLISM OR HOW CAN YOU INTERFERE WITH WHATEVER IS SUSTAINING THAT MODE OF METABOLISM AND CREATE A NEW WAY THAT CAN EITHER DISINTEGRATE BIOFILMS OR — THAT ALLOWS FOR CONVENTIONAL ANTIBIOTICS TO BE MORE EFFECTIVE I THINK THESE ARE REALLY IMPORTANT ASPECTS TO TRY WITH A LOT OF POTENTIAL >> THANK YOU >> SO MAYBE IT’S RELATED BUT — SO OBVIOUSLY IF YOU HAVE A METABOLITE NOT ONLY THE GUY HA PUT ITHATPUT IT OUT CAN USE IT BUT OTHER GUYS CAN AND VICE VERSA, DO YOU HAVE A SENSE FROM ANY OF YOUR LOCALIZATION OR ANYTHING ELSEWHERE WHETHER HOW MUCH SHARING THERE IS, AND IF BACTERIA A DIDN’T MAKE THIS, WOULD SOMEBODY ELSE? >> YES WE HAVE THOUGHT A LOT ABOUT THAT SO I DON’T HAVE ANY EVIDENCE FROM THAT LOCALIZATION BECAUSE THAT’S SO NEW WE HAVEN’T YET BEEN ABLE TO IDENTIFY WHO’S MAKING WHAT, BUT WHAT WE DO HAVE IS FROM BULK SEQUENCING, IN THE — WHERE WE LOOKED AT — CONCENTRATION OVER TIME IN CF LUNGS, WE SAW A REAL — IN RESTRICTING OF THE MICROBIAL DIVERSITY IN PATIENTS, AND THOUGH THERE’S NOT ONE PARTICULAR ORGANISM THAT ALWAYS TRACKS WITH THE RISE OF PSEUDOMONAS, ONE THING THAT IS CLEAR IS THAT THERE’S A RISE IN ANAROBES THAT OCCUPY THE SAME NICHE ONE SMALL INROAD WE’VE MADE INTO UNDERSTANDING ONE WAY THEY MIGHT BENEFIT IS TO ISOLATE FROM THE SOIL MICRO BACTERIA THAT CAN EAT PHENAZINES AS A CARBON SOURCE AND SOME OF THESE ARE THE KIND OF MICRO BACTERIA THAT YOU FIND WITHIN CF, IT’S ACTUALLY A GROWING PATHOGEN, SOME OF THE RAPIDLY GROWING NON-TUBERCULOSIS MICRO BACTERIA INCREASINGLY ARE FOUND AT LEAST IN THE C F-16 TER THAT WE COLLABORATCF CENTERTHAT WE COLLABO RATE WITH SO IT’S AN AREA I WOULD LOVE TO LEARN MORE ABOUT AND I THINK ONE OF THE NEXT FRONTIERS OF RESEARCH >> DO YOU HAVE ANY IDEA WHAT THE PRIMARY CAR UPO CARBON SOURCE IS FOR THE PSEUDOMONAS IN THE LUNG? >> THE PRIMARY CARBON SOURCE I WISH I COULD SAY THERE WAS A PRIMARY CARBON SOURCE IN EXPERIMENTS THAT HAVE BEEN DONE TO MEASURE ALL THE DIFFERENT FORMS OF CARBON, THERE IS A LARGE DIVERSITY OF CARBON FORCES WHAT I THINK IS FAIR TO SAY IS THAT GLUCOSE IS PROBABLY NOT THE MOST IMPORTANT AT ALL AND SOME VERY GOOD WORK I CAN REFER YOU TO FROM MARVIN WHITELY’S GROUP, BEGAN TO MEASURE SOME OF THE METABOLITES AND CONSTRUCT SYNTHETIC CF SPUTUM MEDIUM I KNOW THAT LACTATE IS FOUND AT REASONABLY HIGH ABUNDANCE, AND I’D I HAVE TO GO BACK AND CHECK CONCENTRATIONS OF OTHER FATTY ACIDS AND OTHER CARBON SOURCES, BUT IT’S QUITE COMPLEX I WOULDN’T THINK IT’S JUST ONE, I THINK THERE’S A LOT OF DIFFERENT CARBONS >> THANK YOU >> I JUST WANT TO MAKE WOULD QUICK ANNOUNCEMENTS ONE, THERE WILL BE A RECEPTION IN THE LIBRARY FOLLOWING THIS TALK,, IMMEDIATELY FOLLOWING THIS TALK SECONDLY, DR. NEW MAN HAS KINDLY AGREED TO TBIF ANOTHER TALK TOMORROW FOR THE NORMAL LUNCHTIME AT 11:00 IN THE SIXTH FLOOR CONFERENCE ROOM IN BUILDING 37 NOW I JUST WANT TO THANK DR. NEWMAN FOR SUCH A VERY INTERESTING TALK [APPLAUSE]