| dc.description.abstract | Obligate anaerobe Clostridioides difficile relies on the formation of spores to survive in aerobic environments. Despite the role of C. difficile spores in transmission of the pathogen there remains much unknown about the sporulation and germination pathways of Clostridioides difficile. Outer spore layers are essential for interaction with the environment and spore resistance to decontaminants. The outer spore layers of C. difficile spores are complex and our understanding of them lacking due to a low number of orthologs shared with model gram positive spore forming bacteria species like Bacillus subtilis. Mutations in the outer spore layer genes cdeC and cotL render spores susceptible to lysozyme-triggered germination due to defects in spore coat formation. The aim of this study was to develop a genetic screen to identify determinants of spore coat formation in C. difficile spores. Therefore, we first defined conditions for screening for resistance to lysozyme-triggered germination in C. difficile cdeC::CT mutant spores. We observed that heat is required to kill lysozyme-germinated cdeC::CT spores. Next, we tested whether a genetic screen of Ethyl methanesulfonate (EMS) mutagenized cdeC::CT was able to identify suppressor mutants with restored wild type resistance to lysozyme-triggered germination. Our results demonstrate that our screen was not sensitive enough to identify suppressor mutants. Since our screen was unsuccessful, we then tested if there were optimal heat and mutagenesis conditions that might be utilized in future screens. Overall, these results indicate that the same genetic screen in a mutant background with a more sensitive phenotype of lysozyme-triggered germination, such as cotL, may identify suppressor mutants of spore coat permeability. | |
