Germinant Synergy Facilitates Clostridium difficile Spore Germination under Physiological Conditions

Abstract

is a Gram-positive obligate anaerobe that forms spores in order to survive for long periods in the unfavorable environment outside a host. is the leading cause of nosocomial infectious diarrhea worldwide. infection (CDI) arises after a patient treated with broad-spectrum antibiotics ingests infectious spores. The first step in 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 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. 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 infection (CDI) after ingestion and passage through the stomach. This study investigates the specific conditions that facilitate 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 , 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 spore germination and could provide a biological explanation for why patients taking proton pump inhibitors are associated with increased risk of developing a CDI.

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.…”

“…Interestingly, of the mutants we characterized, only those that were more sensitive to amino acid co-germinants were more sensitive to taurocholate germinant. Amino acid co-germinants were recently reported to act synergistically with divalent cation co-germinants, but neither class of co-germinants synergizes with members of the same class [46]. The authors hypothesized that amino acid co-germinants only synergize with divalent cation co-germinants because the different classes of co-germinants are sensed by different receptors or potentially even different pathways [20, 46].…”

“…Amino acid co-germinants were recently reported to act synergistically with divalent cation co-germinants, but neither class of co-germinants synergizes with members of the same class [46]. The authors hypothesized that amino acid co-germinants only synergize with divalent cation co-germinants because the different classes of co-germinants are sensed by different receptors or potentially even different pathways [20, 46]. Our current data do not distinguish between whether C. difficile spores use two separate receptors, the same receptor, or CspC itself, to sense amino acid and divalent cations, but it is interesting that the G457R mutation increases sensitivity to both classes of co-germinants and that the Gly457, Arg456, and D429 residues all line the edge of a pocket that could bind taurocholate and/or small molecules like amino acids.…”

The impact of pH onClostridioides difficilesporulation and physiology

“…C. difficile is exposed to diverse and changing pH conditions during transit through the gastrointestinal tract. Prior studies showed that the environmental pH impacts spore germination, resulting in inhibition of germination under acidic conditions that is reversed upon exposure to neutral pH (1, 30, 43, 44), which highlights a limiting factor for the germination of spores within the small bowel in the presence of bile acids. In this study, we further assessed the impact of diverse pH conditions on C. difficile growth, sporulation, toxin formation, and motility.…”

Clostridioides difficile Spores: Bile Acid Sensors and Trojan Horses of Transmission

“…21,36,38 As mentioned earlier, C. difficile spore germination occurs in the small intestine, 21 with the ileum supporting the highest levels of spore germination in mice. 40 Although germinant levels are highest in the duodenum because bile salts enter the small intestine at this site, germination is lower in duodenal extracts compared with ileal extracts 40 presumably because the lower pH of the duodenum prevents C. difficile spore germination. 40…”