Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feed-forward loop

Clostridioides difficile infections begin when its metabolically dormant spores germinate in response to sensing bile acid germinants alongside amino acid and divalent cation cogerminants 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) cannot germinate when germination is induced with bile acid germinant and amino acid co-germinant alone. However, since the reduced optical density of CaDPA-less spores makes it difficult to accurately measure their germination, 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 found that CaDPA mutant spores germinate in the presence of amino acid co-germinant and bile acid germinant. Notably, we determined that CaDPA mutant spores require higher levels of amino acid co-germinants to induce germination relative to WT spores because CaDPA released during germination can functions in a feedforward loop to potentiate the germination of other spores within a population. Collectively, these data demonstrate that Ca2+ is not essential for C. difficile spore germination because parallel co-germinant sensing pathways activate germination.