The RgaS-RgaR two-component system promotesClostridioides difficilesporulation through a small RNA and the Agr1 system

ABSTRACT The ability to form a dormant spore is essential for the survival of the anaerobic, gastrointestinal pathogen Clostridioides difficile outside of the mammalian gastrointestinal tract. The initiation of sporulation is governed by the master regulator of sporulation, Spo0A, which is activated by phosphorylation. Multiple sporulation factors control Spo0A phosphorylation; however, this regulatory pathway is not well defined in C. difficile . We discovered that RgaS and RgaR, a conserved orphan histidine kinase and orphan response regulator, function together as a cognate two-component regulatory system to directly activate transcription of several genes. One of these targets, agrB1D1 , encodes gene products that synthesize and export a small quorum- sensing peptide, AgrD1, which positively influences expression of early sporulation genes. Another target, a small regulatory RNA now known as SrsR, impacts later stages of sporulation through an unknown regulatory mechanism(s). Unlike Agr systems in many organisms, AgrD1 does not activate the RgaS-RgaR two-component system, and thus, is not responsible for autoregulating its own production. Altogether, we demonstrate that C. difficile utilizes a conserved two-component system that is uncoupled from quorum-sensing to promote sporulation through two distinct regulatory pathways. AUTHOR SUMMARY The formation of an inactive spore by the anaerobic gastrointestinal pathogen, Clostridioides difficile , is required for its survival outside of the mammalian host. The sporulation process is induced by the regulator, Spo0A; yet, how Spo0A is activated in C. difficile remains unknown. To address this question, we investigated potential activators of Spo0A. Here, we demonstrate that the sensor RgaS activates sporulation, but not by direct activation of Spo0A. Instead, RgaS activates the response regulator, RgaR, which in turn activates transcription of several genes. We found two direct RgaS- RgaR targets independently promote sporulation: agrB1D1 , encoding a quorum-sensing peptide, AgrD1, and srsR , encoding a small regulatory RNA. Unlike most other characterized Agr systems, the AgrD1 peptide does not affect RgaS-RgaR activity, indicating that AgrD1 does not activate its own production through RgaS-RgaR. Altogether, the RgaS-RgaR regulon functions at multiple points within the sporulation pathway to tightly control C. difficile spore formation.