MECHANISMS UNDERLYING DIARRHEA AND GUT INFLAMMATION MEDIATED BY ENTEROTOXIGENIC AND ENTEROPATHOGENIC E. COLI - PROJECT SUMMARY DIARRHEAL DISEASES MEDIATED BY INFECTIOUS ENTEROTOXIGENIC ESCHERICHIA COLI (ETEC) THAT PRODUCE HEAT-STABLE ENTEROTOXINS (ST) ARE ONE OF THE SIGNIFICANT CAUSES OF MORBIDITY IN DEVELOPING COUNTRIES, WITH CHILDREN BELOW 5 BEING THE MOST SEVERELY AFFECTED. CHILDREN INFECTED WITH THESE STRAINS SHOW MODERATE-TO-SEVERE DIARRHEA BUT FACE THE GREATEST LONG-TERM MORBIDITY AND INCREASED RISK OF DEATH. THERE ARE CURRENTLY NO DIRECTED THERAPIES AGAINST STABLE TOXIN-MEDIATED DIARRHEA, BUT ORAL ADMINISTRATION OF ZINC SALTS HAS PROVEN TO BE BENEFICIAL. THE LONG-TERM GOAL OF THIS PROPOSAL IS TO ELUCIDATE THE MECHANISMS OF HOW CHRONIC CGMP-DRIVEN INCREASES SUSCEPTIBILITY TO ENTERIC BACTERIAL PATHOGENS. ST BINDS TO THE CELL SURFACE RECEPTOR GUANYLYL CYCLASE C (GC-C, ENCODED BY GUCY2C), EXPRESSED IN ALL CELL TYPES IN THE GUT BUT NOT IMMUNE CELLS. WE PREVIOUSLY DESCRIBED AND CHARACTERIZED HYPERACTIVATING MUTATIONS IN THE GUCY2C AND GENERATED KNOCK-IN MICE THAT DISPLAY ELEVATED INTESTINAL CGMP AND DIARRHEA. OUR STUDIES SHOW THAT THESE MICE DISPLAY MSD-LIKE PHENOTYPES. THEREFORE, WE HYPOTHESIZE THAT THESE MICE COULD SERVE AS A PRE-CLINICAL MODEL TO UNDERSTAND THE GUT ENVIRONMENT SEEN IN CHILDREN WITH PROLONGED EPISODES OF ST-MEDIATED DIARRHEA. THESE MICE SHOW AN INTERFERON- DRIVEN INTESTINAL GENE SIGNATURE AND PANETH CELL DEFECTS. WE PROPOSE TO PURSUE THE FOLLOWING OBJECTIVES AND QUESTIONS IN THIS PROPOSAL: (1) ELUCIDATE MECHANISMS BY WHICH THE PANETH CELL DYSFUNCTION SEEN IN TRANSGENIC MICE OCCURS AT THE MOLECULAR LEVEL USING ENGINEERED CELL LINES AND ORGANOID CULTURES; (2) STUDY MECHANISTIC ASPECTS OF STAT1 INDUCTION, AND INFLAMMATION IN TRANSGENIC MICE; (3) MONITOR THE RECRUITMENT AND MATURATION OF THE GUT-ASSOCIATED LYMPHOID TISSUE (GALT) DURING CGMP-MEDIATED DIARRHEA CHARACTERIZE AND (4) ASK WHETHER A PREVAILING CONDITION OF DIARRHEA INDUCES A MORE SEVERE RESPONSE TO FURTHER INFECTION BY ENTEROPATHOGENIC E. COLI (EPEC). ORAL ADMINISTRATION OF ZINC SULFATE IS KNOWN TO SHORTEN DIARRHEAL EPISODES IN AFFECTED CHILDREN. THEREFORE, WE WILL INCORPORATE ZINC ADMINISTRATION IN ANIMALS TO ATTEMPT TO REVERSE INFLAMMATION AND OTHER DETRIMENTAL PHENOTYPES SEEN IN TRANSGENIC MICE. COLLECTIVELY, USING THESE TRANSGENIC MICE TO UNDERSTAND INFECTIOUS SECRETORY DIARRHEA WILL NOT ONLY ELUCIDATE THE UNDERLYING CHANGES THAT OCCUR IN THE GUT DURING PROLONGED SECRETORY DIARRHEA BUT ALSO REVEAL TARGETS FOR THERAPY (E.G., ZINC) IN ALLEVIATING SYMPTOMS. FURTHERMORE, OUR STUDIES WILL IDENTIFY VULNERABILITIES THAT COULD BE TARGETED TO ENSURE THAT CO-INFECTION WITH ANOTHER BACTERIAL PATHOGEN HAS MINIMAL IMPACT.