Mapping Interactions between Germinants and Clostridium difficile Spores

Abstract

Germination of Clostridium difficile spores is the first required step in establishing C. difficile-associated disease (CDAD). Taurocholate (a bile salt) and glycine (an amino acid) have been shown to be important germinants of C. difficile spores. In the present study, we tested a series of glycine and taurocholate analogs for the ability to induce or inhibit C. difficile spore germination. Testing of glycine analogs revealed that both the carboxy and amino groups are important epitopes for recognition and that the glycine binding site can accommodate compounds with more widely separated termini. The C. difficile germination machinery also recognizes other hydrophobic amino acids. In general, linear alkyl side chains are better activators of spore germination than their branched analogs. However, L-phenylalanine and L-arginine are also good germinants and are probably recognized by distinct binding sites. Testing of taurocholate analogs revealed that the 12-hydroxyl group of taurocholate is necessary, but not sufficient, to activate spore germination. In contrast, the 6-and 7-hydroxyl groups are required for inhibition of C. difficile spore germination. Similarly, C. difficile spores are able to detect taurocholate analogs with shorter, but not longer, alkyl amino sulfonic acid side chains. Furthermore, the sulfonic acid group can be partially substituted with other acidic groups. Finally, a taurocholate analog with an m-aminobenzenesulfonic acid side chain is a strong inhibitor of C. difficile spore germination. In conclusion, C. difficile spores recognize both amino acids and taurocholate through multiple interactions that are required to bind the germinants and/or activate the germination machinery.

Progesterone Analogs Influence Germination of Clostridium sordellii and Clostridium difficile Spores In Vitro

“…Position 17 of the D ring seems to be especially sensitive to deactivation by the presence of a hydroxyl group. This pattern of progesterone analog recognition contrasts with the recognition of bile salts by C. difficile spores, where both germinants and inhibitors require hydroxyl groups at least at positions 3, 7, and/or 12 (24).…”

Clinical update for the diagnosis and treatment ofClostridium difficileinfection

“…Histologically, the rifalazil treated animals had less edema and neutrophil infiltration than the vancomycin treated animals. When vancomycin was discontinued, 65% of the animals developed disease, while none of the rifalazil treated animals had positive toxin assays or disease [202] . Future trials of rifalazil in humans with CDI are eagerly anticipated, especially given the low relapse rate in animal models.…”

“…Since only the vegetative form produces toxin, theoretically prevention of spore germination would prevent symptomatic infection. Howerton et al [202] demonstrated that a cholate meta-benzene sulfonic derivative (CamSA) is a strong competitive inhibitor of taurocholate-mediated C. difficile spore germination. Subsequently, they administered a single 50 mg/kg dose of CamSA to mice infected with C. difficile spores and were able to prevent any signs of CDI [203] .…”

Gastrointestinal dysbiosis and the use of fecal microbial transplantation inClostridium difficileinfection

“…When ingested, the multiple layers of the spore help protect it from stomach acids and digestive enzymes. Spore germination occurs upon interaction with the appropriate germinants within the intestinal tract, which include taurocholic acid, a taurineconjugated bile acid, and glycine [47,48] . The receptor for taurocholic acid on a C. difficile spore, CspC, was only recently discovered [49] , and researchers demonstrated that mutations to this protein …”

“…This complex process has not been well studied in C. difficile but has in related species [51] . Germination normally occurs within the cecum and colon as other factors in the small intestine, such as high concentration of chenodeoxycholic acid, act to suppress widescale germination [48] . Subsequently, C. difficile initiates the activation of the pathogenicity locus (PaLoc) in the genome.…”