The RgaS-RgaR two-component system promotes Clostridioides difficile sporulation through a small RNA and the Agr1 system

Abstract

The ability to form a dormant spore is essential for the survival of the anaerobic 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 SpoZ, impacts later stages of sporulation through a small hypothetical protein and an additional, 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.

KipOTIA detoxifies 5-oxoproline and promotes the growth ofClostridioides difficile

“…C. difficile strains were grown in BHIS, genomic DNA extracted and prepared for whole genome sequencing as previously detailed (A. N. Edwards et al, 2016; Edwards and McBride, 2023; Harju et al, 2004). Library preparation and Illumina sequencing was performed by SeqCenter (Pittsburgh, PA) as previously described (Edwards and McBride, 2023).…”

“…C. difficile strains were grown in BHIS, genomic DNA extracted and prepared for whole genome sequencing as previously detailed (A. N. Edwards et al, 2016; Edwards and McBride, 2023; Harju et al, 2004). Library preparation and Illumina sequencing was performed by SeqCenter (Pittsburgh, PA) as previously described (Edwards and McBride, 2023). Demultiplexing, quality control, and adapter trimming was performed with bcl-convert (v3.9.3 for MC2375, v4.1.5 for MC2519 and MC2520; https://support-docs.illumina.com/SW/BCL_Convert/Content/SW/FrontPages/BCL_Convert.htm).…”

The impact of orphan histidine kinases and phosphotransfer proteins on the regulation of clostridial sporulation initiation

“…The function of another OHK, CD630_19490, is unknown but does not impact spore formation ( 59 ). Another OHK, CD630_05760, now known as RgaS, has recently been identified as the cognate sensor kinase to the orphan response regulator, RgaR ( 73 ). RgaR directly activates the transcription of several operons, including agrB1D1 , which encodes the gene products necessary to produce the AgrD1 quorum-sensing peptide, and spoZ , encoding a regulatory small RNA ( 73 , 74 ).…”

The pH-responsive SmrR-SmrT system modulatesC. difficileantimicrobial resistance, spore formation, and toxin production

“…As shown in To determine if the increase in sporulation observed for the 630∆erm smrR mutant extends to other C. difficile isolates, we examined the impact of smrR on sporulation in the epidemic UK1 strain (027 ribotype). Using a CRISPR interference (CRISPRi) approach (23,24), we knocked-down transcription of the smrR ortholog in UK1 (CDR20291_2397)…”

“…Genomic DNA (gDNA) was isolated from C. difficile strains using a modified Bust 'N' Grab protocol (44,45). The Benchling CRISPR Guide RNA Design tool was used to create the sgRNA targeting CDR20291_2397, which was generated by PCR (23,24). The details of vector construction are provided in Supplemental Table S1.…”