The small acid-soluble proteins of Clostridioides difficile are important for UV resistance and serve as a check point for sporulation

Clostridioides difficile is a nosocomial pathogen which causes severe diarrhea and colonic inflammation. C. difficile causes disease in susceptible patients when endospores germinate into the toxin-producing vegetative form. The action of these toxins results in diarrhea and the spread of spores into the hospital and healthcare environments. Thus, the destruction of spores is imperative to prevent disease transmission between patients. However, spores are resilient and survive extreme temperatures, chemical exposure, and UV treatment. This makes their elimination from the environment difficult and perpetuates their spread between patients. In the model spore-forming organism, Bacillus subtilis, the small acid-soluble proteins (SASPs) contribute to these resistances. The SASPs are a family of small proteins found in all endospore-forming organisms, C. difficile included. Although these proteins have high sequence similarity between organisms, the role(s) of the proteins differ. Here, we investigated the role of the main α/β SASPs, SspA and SspB, and two annotated putative SASPs, CDR20291_1130 and CDR20291_3080, in protecting C. difficile spores from environmental insults. We found that SspA is necessary for conferring spore UV resistance, SspB minorly contributes, and the annotated putative SASPs do not contribute to UV resistance. In addition, the SASPs minorly contribute to the resistance of nitrous acid. Surprisingly, the combined deletion of sspA and sspB prevented spore formation. Overall, our data indicate that UV resistance of C. difficile spores is dependent on SspA and that SspA and SspB regulate/serve as a checkpoint for spore formation, a previously unreported function of SASPs.Author summary: C. difficile infections remain problematic and elimination of spores within an environment is essential to limit person-to-person spread. A deeper understanding of how spores resist cleaning efforts could lead to better strategies to eradicate the spores in a contaminated environment. The small acid-soluble proteins (SASPs), found in all endospore-forming organisms, are one mechanism that allows for spore resilience. In the model organism, B. subtilis, the SASPs are thought to non-specifically bind to DNA to force the DNA into a conformation that prevents the formation of thymidine dimers and, instead, result in the formation of the spore photoproduct. Here, we investigated the roles of the SASPs in C. difficile find that C. difficile SspA and SspB protect against UV light. Unexpectedly, we found that these SASPs also regulate spore formation, a role not described for any SASP to date. Our data suggest that C. difficile SASPs may bind specific DNA sequences to regulate spore formation.