Combined and Distinct Roles of Agr Proteins in Clostridioides difficile 630 Sporulation, Motility, and Toxin Production

The Clostridioides difficile accessory gene regulator 1 (agr1) locus consists of two genes, agrB1 and agrD1, that presumably constitute an autoinducing peptide (AIP) system. Typically, AIP systems function through the AgrB-mediated processing of AgrD to generate a processed form of the AIP that provides a concentration-dependent extracellular signal. Here, we show that the C. difficile 630 Agr1 system has multiple functions, not all of which depend on AgrB1. CRISPR-Cas9n deletion of agrB1, agrD1, or the entire locus resulted in changes in transcription of sporulation-related factors and an overall loss in spore formation. Sporulation was recovered in the mutants by providing supernatant from stationary-phase cultures of the parental strain. In contrast, C. difficile motility was reduced only when both AgrB1 and AgrD1 were disrupted. Finally, in the absence of AgrB1, the AgrD1 peptide accumulated within the cytoplasm and this correlated with increased expression of tcdR (15-fold), as well as tcdA (20-fold) and tcdB (5-fold), which encode the two major C. difficile toxins. The combined deletion of agrBliagrD1 or deletion of only agrD1 did not significantly alter expression of tcdR or tcdB but did show a minor effect on tcdA expression. Overall, these data indicate that the Agr1-based system in C. difficile 630 carries out multiple functions, some of which are associated with prototypical AIP signaling and others of which involve previously undescribed mechanisms of action. IMPORTANCE C. difficile is a spore-forming, toxigenic, anaerobic bacterium that causes severe gastrointestinal illness. Understanding the ways in which C. difficile senses growth conditions to regulate toxin expression and sporulation is essential to advancing our understanding of this pathogen. The Agr1 system in C. difficile has been thought to function by generating an extracellular autoinducing peptide that accumulates and exogenously activates two-component signaling. The absence of the peptide or protease should, in theory, result in similar phenotypes. However, in contrast to longstanding assumptions about Agr, we found that mutants of individual agri genes exhibit distinct phenotypes in C. difficile. These findings suggest that the Agr1 system may have other regulatory mechanisms independent of the typical Agr quorum sensing system. These data not only challenge models for Agr's mechanism of action in C. difficile but also may expand our conceptions of how this system works in other Gram-positive pathogens.