Assessment of chemical methods in the removal of the spore coat and exosporium layers of Clostridioides difficile Spores

Clostridioides difficile spores are essential for initiation, recurrence and transmission of the disease. The spore surface layers are composed of an outermost exosporium layer that surrounds another proteinaceous layer, the spore coat. These spore surfaces layers are responsible for initial interactions with the host and spore resistance properties contributing to transmission and recurrence of CDI. During spore-development, assembly of both layers is tightly interconnected thus studying the surface is essential for understanding the assembly of these layers and identification of potential targets for therapeutics. Several spore coat /exosporium extraction methods have utilized different extraction procedures making comparison across studies difficult and their impact on spore surface layer properties remains unclear. Here, we tested how commonly used chemical methods remove the spore coat /exosporium layers, analyzing treated-spores by phase contrast microscopy, transmission electron microscopy, western blotting, and lysozyme-triggered germination to functionally characterize the extraction efficiency of these treatment on these layers. Our results provide a systematic analysis and offer a platform for future spore coat and exosporium-related studies.