Imaging Clostridioides difficile spore germination and germination proteins

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. ImportanceGermination by C. difficile spores is the first step in the establishment of potentially life-threatening 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 is non-conducive to the establishment of colonization / disease.