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

The accessory gene regulator 1 () locus consists of two genes, and , 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 630 Agr1 system has multiple functions, not all of which depend on AgrB1. CRISPR-Cas9n deletion of , a, 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, 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 (15-fold), as well as (20-fold) and (5-fold), which encode the two major toxins. The combined deletion of / or deletion of only did not significantly alter expression of or but did show a minor effect on expression. Overall, these data indicate that the Agr1-based system in 630 carries out multiple functions, some of which are associated with prototypical AIP signaling and others of which involve previously undescribed mechanisms of action. is a spore-forming, toxigenic, anaerobic bacterium that causes severe gastrointestinal illness. Understanding the ways in which senses growth conditions to regulate toxin expression and sporulation is essential to advancing our understanding of this pathogen. The Agr1 system in 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 genes exhibit distinct phenotypes in 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 but also may expand our conceptions of how this system works in other Gram-positive pathogens.