Clostridioides difficile is a Gram-positive, obligately anaerobic microorganism that is of considerable medical concern as a nosocomial and community-acquired human pathogen. C. difficile is an endospore-forming organism and spores are commonly introduced into a host via fecal-oral route. The spore is metabolically dormant and exhibits considerable resistance to environmental conditions (e.g., oxygen, heat, UV, and commonly used disinfectants). The toxin-producing, vegetative form develops from the germinated spore and the toxins damage the lower gastrointestinal tract epithelium. Spore germination is an irreversible and tightly regulated process. Spore germination begins upon the detection of small-molecule germinants and ends with the release of the large depot of calcium dipicolinic acid (DPA) from the core, core rehydration and the resumption of metabolic activities. Here, using EM immunolabeling, we determined the location of the CspB, CspA, and CspC, and SleC proteins. Also using a split-luciferase system, we determined that the products of the spoVA operon, which are responsible for DPA packaging into the spore core during sporulation, interact and are localized to the inner spore membrane. The combined data is the first to determine how the SpoVA proteins interact and to determine the location of the C. difficile 'germinosome' components.
