Analyzing the Early Stages of Clostridium difficile Spore Germination

Infections caused by Clostridium difficile have increased steadily over the past several years. While studies on C. difficile virulence and physiology have been hindered, in the past, by lack of genetic approaches and suitable animal models, newly developed technologies and animal models allow for improved experimental detail. One such advance was the generation of a mouse-model of C. difficile infection. This system was an important step forward in the analysis of the genetic requirements for colonization and infection. Equally important is understanding the differences that exist between mice and humans. One of these differences is the natural bile acid composition. Bile acid-mediated spore germination, a process whereby a dormant spore returns to active, vegetative growth, is an important step during C. difficile colonization. Mice produce several different bile acids that are not found in humans (muricholic acids) that have the potential to impact C. difficile spore germination. In order to understand potential effects of these different bile acids on C. difficile physiology, we characterized their effects on C. difficile spore germination and growth of vegetative cells. We found that the mouse-derived muricholic acids affect germination similarly to a previously-described inhibitor of germination, chenodeoxycholic acid. Chenodeoxycholic acid was previously demonstrated to be a competitive inhibitor of C. difficile spore germination, though with what the inhibitors or activators of germination interacted was unknown. However, the inhibitory property of chenodeoxycholic acid was used in a screen to identify potential germinant receptors and led to the identification of the germination-specific, pseudoprotease, CspC, as the bile acid germinant receptor. Based on the hypothesized location of CspC within the C. difficile spore (cortex rather than inner membrane), we hypothesized that there may be differences between the order of the stages of C. difficile and Bacillus subtilis spore germination. Germination in B. subtilis, a well-studied spore-former, is divided into two genetically separable stages. Stage I is characterized by the release of dipicolinic acid (DPA) from the spore core. Stage II is characterized by cortex degradation, and stage II can be activated by the DPA released during stage I. Thus, DPA release precedes cortex degradation during B. subtilis spore germination. To understand how the different location of the C. difficile germinant receptor affects the order of DPA release and cortex degradation, we first investigated the timing of DPA release and cortex degradation during C. difficile spore germination and found that cortex degradation precedes DPA release. Based on this result and work with SpoVAC in B. subtilis, we then investigated germination under high osmolyte concentrations. Because both cortex degradation and DPA release during C. difficile spore germination are dependent on the presence of the germinant receptor and cortex degradation, the release of DPA from the core may rely on the swelling of the core upon cortex degradation