Study of Atypical Alarmone Synthesis in Clostridioides difficile

Clostridioides difficile is a Gram-positive anaerobic bacterium that causes infections in humans that costs healthcare systems billions per year. C. difficile infection has high rates of recurrence due to multiple antibiotic resistance. When bacteria are in stressful environments, they produce hyperphosphorylated guanosine ribonucleotide signaling molecules called alarmones. The accumulation of alarmones activates the stringent response (SR), in which bacterial cells induce transcription of stress survival genes to delay growth and replication. The C. difficile SR is regulated by enzymatic activity of a bifunctional synthetase/hydrolase, RelA/SpoT homolog (RSH), and a monofunctional small alarmone synthetase (RelQ). Additionally, the SR is potentially regulated by a third putative synthetase (RelC) which has an uncharacterized domain. Unlike other characterized SR-utilizing bacteria, C. difficile exclusively produces a single triphosphate alarmone through atypical mechanisms despite high active site sequence homology. The first goal of this project was in silico mutational analysis of RSH and RelQ to highlight important residues potentially responsible for the unique alarmone metabolism and the second goal was to predict the functionality of RelC. Together, these aims provide the theoretical framework to identify the structural basis of unusual SR activity in C. difficile.