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. ImportanceC. difficile is easily spread through the production of highly resistant spores. Understanding how spores are formed could yield valuable insight into how the sporulation process can be halted to render spores that are sensitive to cleaning methods. Here, we identify another protein involved in the sporulation process that is seemingly controlled by the small acid-soluble proteins (SASPs). This discovery allows us to better understand how the C. difficile SASPs may bind to specific sites on the genome to regulate gene expression.