A network of small RNAs regulates sporulation initiation in Clostridioides difficile

Abstract

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. image RIL‐seq has revealed the RNA chaperone Hfq‐dependent interactome of small RNAs and their targets in gram‐negative bacteria. Here, application of RIL‑seq in the human pathogen C. difficile uncovers the first Hfq‐dependent RNA interactome in a gram‐positive bacterium and identifies a pair of small regulatory RNAs that modulate sporulation initiation. RIL‐seq in C. difficile strain 630 identifies Hfq‐associated sRNA‐mRNA pairs. RIL‐seq analysis identifies the master regulator of sporulation, Spo0A, as a central target of sRNA‐based regulation. The small RNAs SpoY and SpoX act as negative and positive regulators, respectively, of sporulation by base‐pairing with the spo0A mRNA. Deletion of either SpoY or SpoX affects gut colonization in a mouse model of C. difficile ‐induced colitis.