The small acid-soluble proteins of Clostridioides difficile regulate sporulation in a SpoIVB2-dependent manner.

Clostridioides difficile is a pathogen whose transmission relies on the formation of dormant endospores. Spores are highly resilient forms of bacteria that resist environmental and chemical insults. In recent work, we found that C. difficile SspA and SspB, two small acid-soluble proteins (SASPs), protect spores from UV damage and, interestingly, are necessary for the formation of mature spores. Here, we build upon this finding and show that C. difficile sspA and sspB are required for the formation of the spore cortex layer. Moreover, using an EMS mutagenesis selection strategy, we identified mutations that suppressed the defect in sporulation of C. difficile SASP mutants. Many of these strains contained mutations in CDR20291_0714 (spoIVB2) revealing a connection between the SpoIVB2 protease and the SASPs in the sporulation pathway. This work builds upon the hypothesis that the small acid-soluble proteins can regulate gene expression. Author summary: In prior work, we found that the Clostridioides difficile small acid-soluble proteins, SspA and SspB, are important for the UV resistance that is characteristic of dormant spores (as found in many other spore-forming bacteria). Surprisingly, we also found that the combinatorial deletion of sspA and sspB led to a block in the ability of C. difficile to form dormant spores, a phenotype not observed in other sporulating bacteria. Here, we build upon this work and find that this block can be overcome by mutations in spoIVB2 –σF-regulated protease. Our data suggest that the σG-regulated SspA and SspB directly or indirectly activate spoIVB2 expression during late stages of sporulation and that the identified spoIVB2 alleles overcome the absence of sspA and sspB by increasing spoIVB2 translation efficiency and, thus, abundance during late stages of sporulation. Our data build upon the hypothesis that small acid-soluble proteins can regulate gene expression and indicate that other spore-forming bacteria may have unidentified targets that these proteins may regulate during the sporulation process. [ABSTRACT FROM AUTHOR]