Clostridioides difficile is a Gram-positive anaerobic bacterium that causes infections in humans that costs healthcare systems billions of dollars per year. People at high risk for C. difficile infection include the elderly, immunocompromised, and individuals who have recently been on antibiotics. When bacteria are in stressful environments, stimuli like antibiotic stress or nutrient starvation trigger the accumulation of hyperphosphorylated guanosine ribonucleotide signaling molecules called alarmones. This activates the stringent response (SR), in which bacterial cells induce transcription of stress survival genes and delay further cellular development. Chemical or genetic interference with the SR increases antibiotic susceptibility. The C. difficile SR is regulated by enzymatic activity of a dual function Rel-Spo alarmone synthetase-hydrolase (RSH), a monofunctional small alarmone synthetase (RelQ), and possibly by a third putative synthetase (RelC). The alarmones created by all other Gram-positive bacteria are pentaphosphate (pppGpp), tetraphosphate (ppGpp), and triphosphate (pGpp). However, C. difficile exclusively produces pGpp and its mechanism of synthesis is different from all previously studied. The first objective of this project is mutational analysis of RSH and RelQ to determine the structural basis of their unusual activity. We have identified sites where these enzymes diverge from conserved synthetase sequences and will generate point mutants to see if restoring the sequences allows for synthesis of longer alarmones. The other objective of this project is to determine whether RelC is a functional alarmone synthetase despite low sequence homology to other synthetases. The results of this project will inform future studies of atypical alarmone synthesis in C. difficile.

Declan Butler

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