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

Ummey Khalecha Bintha AhmedJimmy D Ballard


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 agrB1/agrD1 or deletion of only agrD1 did not significantly alter expression of tcdR or tcdB but did show a minor effect on tcdA expre...Continue Reading


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