IMPACT OF YERSINIA YOPS ON M CELL POPULATION DYNAMICS AND HOST IMMUNITY - PROJECT SUMMARY/ABSTRACT YERSINIA PSEUDOTUBERCULOSIS (YPTB) IS A GRAM-NEGATIVE PATHOGEN THAT ASSOCIATES WITH MICROFOLD (M) CELLS AND TARGETS THEM TO INVADE THE UNDERLYING PEYER'S PATCHES (PPS) IN THE SMALL INTESTINE. YPTB THEN COLONIZES THE PPS AND MESENTERIC LYMPH NODES, CAUSING SELF-LIMITING MESENTERIC LYMPHADENITIS. M CELLS, WHICH ARE EXPLOITED BY MANY PATHOGENS IN ADDITION TO YPTB, PLAY A KEY ROLE IN MUCOSAL IMMUNITY BY DELIVERING ANTIGENS TO B AND T CELLS IN THE PPS; M CELL-DEFICIENT HOSTS HAVE BEEN SHOWN TO HAVE DEFECTIVE RESPONSES TO MUCOSAL ANTIGENS. HISTORICALLY, PATHOGEN-M CELL INTERACTIONS HAVE BEEN STUDIED USING LIGATED LOOP INFECTIONS, WHICH HAVE FOUND THAT YPTB INVASIN BINDS TO APICAL B1 INTEGRINS ON M CELLS. HOWEVER, LIGATED LOOP MODELS LACK THE ABILITY TO OBSERVE CELL DYNAMICS ASSOCIATED WITH YPTB INFECTION. TO OVERCOME THIS, THE MECSAS LAB DEVELOPED AN ORGANOID-BASED MODEL OF INFECTION WITH A DIFFERENTIATED EPITHELIAL MONOLAYER. YPTB INFECTION IN THESE ENTEROIDS SHOWS THAT M CELLS WITH APICALLY BOUND YPTB ARE EXTRUDED FROM THE MONOLAYER IN A BACTERIAL TYPE 3 SECRETION SYSTEM-DEPENDENT MANNER. SPECIFICALLY, THE EFFECTOR PROTEIN YOPE IS NECESSARY FOR M CELL EXTRUSION. THIS RESULT LED US TO TEST IF M CELL POPULATIONS CHANGE DUE TO INFECTION IN MICE. PRELIMINARY QRT-PCR DATA SHOWS THAT EXPRESSION OF M CELL MARKERS GP2 AND SPIB DECREASED AFTER 72 HOURS WT YPTB INFECTION, BUT SPECIFIC BACTERIAL AND HOST FACTORS BEHIND THIS PHENOMENON AND THE IMMUNE IMPACTS OF DEPLETION ARE YET UNKNOWN. THE GOAL FOR THIS PROJECT THEN IS TO DETERMINE THE FACTORS INVOLVED IN M CELL DEPLETION AND THE IMPACTS OF DEPLETION ON HOST IMMUNE RESPONSES. BUT HOST TARGETS OF YOPE CANNOT BE KNOCKED OUT IN MICE AS THEY ARE EMBRYONICALLY LETHAL. THUS, I MUST TAKE A MULTI-FACETED APPROACH USING BOTH MURINE INFECTION AND ENTEROID MODELS. I HYPOTHESIZE THAT TRANSIENT M CELL DEPLETION OCCURS IN A YOPE-DEPENDENT MANNER IN MICE, CAUSING A DEFECT IN THE HOST'S INNATE IMMUNE RESPONSE TO SUBSEQUENT ENTERIC INFECTIONS. TO TEST THIS HYPOTHESIS, I WILL TAKE A DUAL-PRONGED APPROACH OF ANIMAL INFECTION AND ENTEROID MANIPULATION, WHICH WILL ALLOW ME TO STUDY BOTH BACTERIAL AND HOST FACTORS INVOLVED IN M CELL DEPLETION. AIM 1 WILL USE QRT-PCR, FLOW CYTOMETRY, AND IMMUNOFLUORESCENCE TO DETERMINE THE BACTERIAL EFFECTORS NECESSARY FOR M CELL DEPLETION AND THE IMPACT THAT DEPLETION HAS ON T CELL ACTIVATION AND DIVISION. AIM 2 WILL USE CRISPR/CAS9 TECHNOLOGIES TO CREATE AN INDUCIBLE GENE KNOCK-OUT SYSTEM THAT IS SPECIFIC TO M CELLS IN THE EXISTING ENTEROID SYSTEM. AFTER CRISPR/CAS9 IMPLEMENTATION, AIM 2 EXPERIMENTS WILL DETERMINE THE ROLE OF HOST TARGET PROTEINS IN M CELL EXTRUSION. TOGETHER, THESE AIMS WILL ELUCIDATE THE BACTERIAL EFFECTORS RESPONSIBLE FOR M CELL DEPLETION, THE HOST TARGETS OF THESE PROTEINS, AND THE IMMUNE SYSTEM IMPACTS OF M CELL DEPLETION. ADDITIONALLY, THIS PROPOSAL WILL DEVELOP NEW SYSTEMS FOR THE STUDY OF PROTEIN TARGETS IN ENTEROIDS, WHICH HAS POSITIVE IMPLICATIONS FOR FURTHER YPTB STUDIES AND BROADER APPLICATIONS FOR INDUCIBLE KNOCKOUT ORGANOID SYSTEMS. THE PROPOSED WORK WILL FURTHER OUR UNDERSTANDING OF THE ENTERIC IMMUNE SYSTEM, ITS DISRUPTION BY INTESTINAL PATHOGENS, AND ITS IMPLICATIONS FOR HUMAN HEALTH.