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.

Germination by C. difficile spores is the first step in the establishment of potentially life-threatening C. difficile infection (CDI). A deeper understanding of the mechanism by which spores germinate may provide insight for how to either prevent spore germination into a disease-causing vegetative form or trigger germination prematurely when the spore is either in the outside environment or in a host environment that does not support the establishment of colonization/disease.

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.

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 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 SASPs do not contribute to UV resistance. In addition, none of these SASPs contribute to the resistance of tested chemicals. 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.

Clostridioides difficile spores are the infective form for this endospore-forming organism. The vegetative cells are intolerant to oxygen and poor competitors with a healthy gut microbiota. Therefore, in order for C. difficile to establish infection, the spores have to germinate in an environment that supports vegetative growth. To initiate germination, C. difficile uses Csp-type germinant receptors that consist of the CspC and CspA pseudoproteases as the bile acid and co-germinant receptors, respectively. CspB is a subtilisin-like protease that cleaves the inhibitory pro-peptide from the pro-SleC cortex lytic enzyme thereby activating it and initiating cortex degradation. Though several locations have been proposed for where these proteins reside within the spore (i.e., spore coat, outer spore membrane, cortex, inner spore membrane), these have been based, mostly, on hypotheses or prior data in C. perfringens. In this study, we visualize the germination process using TEM and SEM, and, using immunogold labeling of the spore proteins, find that these proteins are localized to the spore cortex, consistent with the observed, rapid, changes to the spore structure during germination