Project Summary/Abstract Clostridioides difficile infection (CDI) is the most common and costly nosocomial infection in the United States. The responsible pathogen is an extremely resilient bacterium, tolerant of multiple classes of antibiotics. As a result, CDI has a very high 20-35% recurrence rate. While the stringent response (SR) mediated by (pp)pGpp `alarmones' is crucial for survival and virulence in a number of bacterial pathogens, this had not previously been studied in C. difficile. Our preliminary research demonstrated that C. difficile utilizes alarmone signaling to coordinate its response to antibiotic-induced stresses. We further determined that chemical inhibition or genetic knockdown of a clostridial alarmone synthetase enzyme increases C. difficile antibiotic susceptibility. Stationary phase onset, acid stress, and oxidative stress were identified as inducers of alarmone synthesis. Additionally, oxidative stress stimulates C. difficile biofilm formation, a protective mechanism against stressors. As limited nutrient availability triggers the (SR) in a number of pathogens, and stimulates sporulation and toxin synthesis in C. difficile, our hypothesis is that alarmone signaling coordinates clostridial responses to nutrient, immune, and antibiotic stressors and contributes to the extreme resilience of this pathogen. We anticipate that the SR will influence motility, biofilm formation, sporulation, and/or toxin production as well as antibiotic survival. Unexpectedly, we have found that C. difficile was found to exclusively synthesize pGpp rather than canonical (p)ppGpp alarmones and must hydrolyze two phosphate bonds for this synthesis. This is without precedent in bacterial species with characterized SRs. As the challenge of designing therapies against CDI is to balance lethality and specificity in order to avoid damage to beneficial commensal microbiota, the divergence of clostridial synthetases from mechanisms conserved in other organisms presents an attractive target for the design of SR inhibitors specific to C. difficile for reducing antibiotic survival. The Purcell lab has established a robust research program and trained several graduate and undergraduate students. We propose exploring the role of nutrient and stress sensing in regulating C. difficile physiology and behavior within the host while determining the role of the SR in regulating disease-relevant bacterial processes. Further, enzymes that mediate alarmone metabolism in C. difficile will be characterized. This proposal will allow us to expand our training activities in order to involve more students in meaningful research investigating an unexplored mechanism of C. difficile stress survival with implications for CDI persistence and recurrence.