A network of small RNAs regulates sporulation initiation in Clostridioides difficile

The obligate anaerobic, enteric pathogen Clostridioides difficile persists in the intestinal tract by forming antibiotic-resistant endospores that contribute to relapsing and recurrent infections. Despite the importance of sporulation for C. difficile pathogenesis, environmental cues and molecular mechanisms that regulate sporulation initiation remain ill-defined. Here, by using RIL-seq to globally capture the Hfq-dependent RNA-RNA interactome, we discovered a network of small RNAs that bind to mRNAs encoding sporulation-related genes. We show that two of these small RNAs, SpoX and SpoY, regulate translation of the master regulator of sporulation, Spo0A, in an opposing manner, which ultimately leads to altered sporulation rates. Infection of antibiotic-treated mice with SpoX and SpoY deletion mutants revealed a global effect on gut colonization and intestinal sporulation. Our work uncovers an elaborate RNA-RNA interactome controlling the physiology and virulence of C. difficile and identifies a complex post-transcriptional layer in the regulation of spore formation in this important human pathogen.

Automated summary

The anaerobic pathogen Clostridioides difficile forms antibiotic-resistant endospores to persist in the intestinal tract and cause recurrent infections. Through RIL-seq, we identified a network of small RNAs that bind to sporulation-related genes, and discovered that SpoX and SpoY regulate the translation of the master regulator of sporulation, Spo0A, in an opposing manner, leading to altered sporulation rates. Deletion mutants of SpoX and SpoY showed global effects on gut colonization and intestinal sporulation in antibiotic-treated mice. Our study uncovers a complex RNA-RNA interactome controlling the physiology and virulence of C. difficile and identifies a complex post-transcriptional layer in the regulation of spore formation in this important human pathogen.