Project Grant F31AI200143

ROLE OF LIPOTEICHOIC ACID BIOGENESIS IN REGULATING CLOSTRIDIOIDES DIFFICILE PHYSIOLOGY - PROJECT SUMMARY C. DIFFICILE INFECTION (CDI) IS THE LEADING CAUSE OF HEALTHCARE-ASSOCIATED DIARRHEA IN THE UNITED STATES, RESULTING IN SUBSTANTIAL MORBIDITY AND ECONOMIC COSTS. CDIS OFTEN OCCUR FOLLOWING ANTIBIOTIC TREATMENT, WHICH DISRUPTS THE HEALTHY GUT MICROBIOME AND ALLOWS FOR THIS BACTERIUM TO OVERGROW. PARADOXICALLY, ANTIBIOTICS ARE ALSO THE STANDARD TREATMENT FOR THIS DISEASE, WHICH PERPETUATES GUT DYSBIOSIS AND OFTEN LEADS TO RECURRENT INFECTIONS. THUS, THERE IS AN URGENT NEED TO DEVELOP NARROW-SPECTRUM TREATMENTS THAT TARGET C. DIFFICILE WITHOUT FURTHER DISRUPTING THE REST OF THE GUT MICROBIOME. MANY ANTIBIOTICS TARGET CELL ENVELOPE BIOGENESIS DUE TO ITS VITAL ROLES IN CELL GROWTH AND DIVISION AS WELL AS PROTECTING BACTERIA FROM EXTRACELLULAR STRESSORS. IN MANY GRAM-POSITIVE BACTERIA, LIPOTEICHOIC ACID (LTA) PLAYS VITAL ROLES IN CELL WALL HOMEOSTASIS, INCLUDING IN REGULATING PEPTIDOGLYCAN HYDROLASES THAT ENABLE GROWTH AND DIVISION. AS A RESULT, THERE HAS BEEN CONSIDERABLE INTEREST IN DEVELOPING INHIBITORS OF LTA BIOSYNTHESIS. DISRUPTING LTA PRODUCTION IN C. DIFFICILE HAS BEEN PROPOSED AS A PROMISING STRATEGY FOR DEVELOPING NARROW- SPECTRUM ANTIBIOTICS AGAINST THIS CLINICALLY IMPORTANT PATHOGEN BECAUSE THIS CELL WALL GLYCOPOLYMER HAS A UNIQUE STRUCTURE IN C. DIFFICILE. HOWEVER, IT HAS NOT BEEN POSSIBLE TO DETERMINE WHETHER LTA IS CRITICAL FOR C. DIFFICILE GROWTH BECAUSE THE GENES ENCODING LTA BIOSYNTHESIS PROTEINS HAVE REMAINED ELUSIVE FOR DECADES. TO ADDRESS THE ROLE OF LTA IN REGULATING C. DIFFICILE PHYSIOLOGY, I RECENTLY IDENTIFIED AN ESSENTIAL OPERON THAT IS REQUIRED FOR LTA BIOSYNTHESIS. KNOCKING DOWN THE EXPRESSION OF THESE GENES REDUCES LTA LEVELS, INHIBITS GROWTH, AND CAUSES SEVERE MORPHOLOGICAL DEFECTS AND CELL LYSIS. IMPORTANTLY, SOME OF THE MORPHOLOGICAL DEFECTS OBSERVED UPON LTA DEPLETION ARE RESCUED BY CONDITIONS THAT INHIBIT PEPTIDOGLYCAN HYDROLASE ACTIVITY. SUPPORTING A MODEL WHERE LTA REGULATES PEPTIDOGLYCAN HYDROLASE ACTIVITY. CONSISTENT WITH THIS MODEL, I ALSO FOUND THAT DEPLETING COMPONENTS ENCODED BY THIS OPERON LEADS TO CELL SEPARATION DEFECTS. TO DETERMINE HOW THIS OPERON REGULATES LTA BIOSYNTHESIS AND C. DIFFICILE GROWTH AND DIVISION, THIS FELLOWSHIP TRAINING PROJECT WILL SYSTEMATICALLY DETERMINE THE FUNCTIONS OF THE COMPONENTS ENCODED BY THIS OPERON USING GENETIC AND BIOCHEMICAL APPROACHES. AIM 1 WILL DETERMINE HOW PREDICTED GLYCOSYLTRANSFERASES ALTER LTA PRODUCTION AND AFFECT PEPTIDOGLYCAN HYDROLASE ACTIVITY DURING CELL ELONGATION. THE SECOND AIM WILL DEFINE HOW REGULATORY PROTEINS WITH STRUCTURAL HOMOLOGY TO REGULATORS OF CELL SEPARATION IN OTHER GRAM-POSITIVE BACTERIA INFLUENCE CELL SEPARATION. COMPLETING THESE AIMS WILL PROVIDE FUNDAMENTAL INSIGHTS INTO C. DIFFICILE CELL WALL PHYSIOLOGY AND PROVIDE A BASIS FOR INVESTIGATING LTA AS A TARGET FOR NARROW-SPECTRUM ANTIBIOTICS THAT PRESERVE THE GUT MICROBIOME AND BREAK THE RECURRENT CYCLE OF CDIS.