Intestinal calcium and bile salts facilitate germination of Clostridium difficile spores.

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.

Updates to Clostridium difficile Spore Germination.

Germination of Clostridium difficile spores is a crucial early requirement for colonization of the gastrointestinal tract. Likewise, C. difficile cannot cause disease pathologies unless its spores germinate into metabolically active, toxin-producing cells. Recent advances in our understanding of C. difficile spore germination mechanisms indicate that this process is both complex and unique. This review defines unique aspects of the germination pathways of C. difficile and compares them to those of two other well-studied organisms, Bacillus anthracis and Clostridium perfringensC. difficile germination is unique, as C. difficile does not contain any orthologs of the traditional GerA-type germinant receptor complexes and is the only known sporeformer to require bile salts in order to germinate. While recent advances describing C. difficile germination mechanisms have been made on several fronts, major gaps in our understanding of C. difficile germination signaling remain. This review provides an updated, in-depth summary of advances in understanding of C. difficile germination and potential avenues for the development of therapeutics, and discusses the major discrepancies between current models of germination and areas of ongoing investigation.

Germinant Synergy Facilitates Clostridium difficile Spore Germination under Physiological Conditions.

Clostridium difficile is a Gram-positive obligate anaerobe that forms spores in order to survive for long periods in the unfavorable environment outside a host. C. difficile is the leading cause of nosocomial infectious diarrhea worldwide. C. difficile infection (CDI) arises after a patient treated with broad-spectrum antibiotics ingests infectious spores. The first step in C. difficile pathogenesis is the metabolic reactivation of dormant spores within the gastrointestinal (GI) tract through a process known as germination. In this work, we aim to elucidate the specific conditions and the location within the GI tract that facilitate this process. Our data suggest that C. difficile germination occurs through a two-step biochemical process that is regulated by pH and bile salts, amino acids, and calcium present within the GI tract. Maximal germination occurs at a pH ranging from 6.5 to 8.5 in the terminal small intestine prior to bile salt and calcium reabsorption by the host. Germination can be initiated by lower concentrations of germinants when spores are incubated with a combination of bile salts, calcium, and amino acids, and this synergy is dependent on the availability of calcium. The synergy described here allows germination to proceed in the presence of inhibitory bile salts and at physiological concentrations of germinants, effectively decreasing the concentrations of nutrients required to initiate an essential step of pathogenesis.IMPORTANCEClostridium difficile is an anaerobic spore-forming human pathogen that is the leading cause of nosocomial infectious diarrhea worldwide. Germination of infectious spores is the first step in the development of a C. difficile infection (CDI) after ingestion and passage through the stomach. This study investigates the specific conditions that facilitate C. difficile spore germination, including the following: location within the gastrointestinal (GI) tract, pH, temperature, and germinant concentration. The germinants that have been identified in culture include combinations of bile salts and amino acids or bile salts and calcium, but in vitro, these function at concentrations that far exceed normal physiological ranges normally found in the mammalian GI tract. In this work, we describe and quantify a previously unreported synergy observed when bile salts, calcium, and amino acids are added together. These germinant cocktails improve germination efficiency by decreasing the required concentrations of germinants to physiologically relevant levels. Combinations of multiple germinant types are also able to overcome the effects of inhibitory bile salts. In addition, we propose that the acidic conditions within the GI tract regulate C. difficile spore germination and could provide a biological explanation for why patients taking proton pump inhibitors are associated with increased risk of developing a CDI.