Intestinal calcium and bile salts facilitate germination of Clostridium difficile spores

Abstract

Clostridium difficile (C. difficile) is an anaerobic gram-positive pathogen that is the leading cause of nosocomial bacterial infection globally. C. difficile 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. C. difficile 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 C. difficile 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 Cd630_32980, 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. Cd630_32980 is dispensable for colonization in a murine model of C. difficile infection and ex vivo 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 C. difficile germination, colonization, and pathogenesis. These data provide a biological mechanism that may explain why individuals with inefficient intestinal calcium absorption (e.g., 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.

Hierarchical recognition of amino acid co-germinants during Clostridioides difficile spore germination

“…C. difficile spore germination cannot occur without a co-germinant (historically an amino acid, but calcium may play an unknown role [22]) and the amino acid germinant receptor is still unknown. Amino acids are frequently used as germinants.…”

The CspC pseudoprotease regulates germination of Clostridioides difficile spores in response to multiple environmental signals

“…Recently, Kochan et al . determined that divalent cations, particularly calcium, potentiate taurocholate-induced germination and that amino acid and calcium co-germinant signals can synergize to further enhance taurocholate-induced germination[46, 47]. It is currently unknown how C. difficile spores sense and respond to these co-germinants.…”

Vegetative Cell and Spore Proteomes ofClostridioides difficileshow finite differences and reveal potential biomarkers.

“…These are alkanesulfonate and taurine binding proteins, respectively and SsuA is involved in sulfur metabolism (52). Interestingly, the taurine side chain of taurocholate selectively binds its potential receptor site(53) and taurine itself is an alkane sulfonate, thus higher levels of SsuA and CD2365 in spores could indicate potential taurine interaction during germination, similar to CD3298(54). Identified protein CD0114 shares 25% identity with CD3298, making it worth studying for a possible role in spore germination.…”