Single-spore germination analyses reveal that Clostridioides difficile uses parallel pathways to sense distinct classes of co-germinants during spore germination.

Clostridioides difficile infections begin when its metabolically dormant spores germinate in response to sensing bile acid germinants alongside amino acid and divalent cation co-germinants in the small intestine. While bile acid germinants are essential for C. difficile spore germination, it is currently unclear whether both co-germinant signals are required. One model proposes that divalent cations, particularly Ca2+, are essential for spore germination, while another proposes that either co-germinant class can induce germination. The former model is based on the finding that spores defective in releasing large stores of internal Ca2+, in the form of calcium dipicolinic acid (CaDPA) do not germinate when germination is induced with amino acid co-germinant and bile acid germinant alone. However, accurately measuring germination in CaDPA-less spores using standard optical density-based germination assays is challenging because these spores are less optically dense. To overcome this issue, we developed a novel automated, time-lapse microscopy-based germination assay to analyze CaDPA mutant germination at the single-spore level. Using this assay, we show that CaDPA mutant spores still germinate in the presence of amino acid co-germinant and bile acid germinant, although they require higher levels of amino acid co-germinants to induce germination relative to WT spores. This requirement for elevated amino acid co-germinant levels is because CaDPA released during germination can function as a public good to potentiate the germination of other spores within a population. Collectively, these data demonstrate that Ca2+, is not essential for C. difficile spore germination, indicating that parallel co-germinant sensing pathways activate germination ### Competing Interest Statement A. Shen is a paid consultant for LIV Process.