Intestinal calcium and bile salts facilitate germination of <i>Clostridium difficile</i> spores

<div><p><i>Clostridium difficile</i> (<i>C</i>. <i>difficile</i>) is an anaerobic gram-positive pathogen that is the leading cause of nosocomial bacterial infection globally. <i>C</i>. <i>difficile</i> infection (CDI) typically occurs after ingestion of infectious spores by a patient that has been treated with broad-spectrum antibiotics. While CDI is a toxin-mediated disease, transmission and pathogenesis are dependent on the ability to produce viable spores. These spores must become metabolically active (germinate) in order to cause disease. <i>C</i>. <i>difficile</i> spore germination occurs when spores encounter bile salts and other co-germinants within the small intestine, however, the germination signaling cascade is unclear. Here we describe a signaling role for Ca²⁺ during <i>C</i>. <i>difficile</i> spore germination and provide direct evidence that intestinal Ca²⁺ coordinates with bile salts to stimulate germination. Endogenous Ca²⁺ (released from within the spore) and a putative AAA+ ATPase, encoded by <i>Cd630_32980</i>, are both essential for taurocholate-glycine induced germination in the absence of exogenous Ca²⁺. However, environmental Ca²⁺ replaces glycine as a co-germinant and circumvents the need for endogenous Ca²⁺ fluxes. <i>Cd630_32980</i> is dispensable for colonization in a murine model of <i>C</i>. <i>difficile</i> infection and <i>ex vivo</i> germination in mouse ileal contents. Calcium-depletion of the ileal contents prevented mutant spore germination and reduced WT spore germination by 90%, indicating that Ca²⁺ present within the gastrointestinal tract plays a critical role in <i>C</i>. <i>difficile</i> germination, colonization, and pathogenesis. These data provide a biological mechanism that may explain why individuals with inefficient intestinal calcium absorption (<i>e</i>.<i>g</i>., vitamin D deficiency, proton pump inhibitor use) are more prone to CDI and suggest that modulating free intestinal calcium is a potential strategy to curb the incidence of CDI.</p>