Kinetic Evidence for the Presence of Putative Germination Receptors in C lostridium difficile Spores

Abstract

Clostridium difficile is a spore-forming bacterium that causes Clostridium difficile-associated disease (CDAD). Intestinal microflora keeps C. difficile in the spore state and prevents colonization. Following antimicrobial treatment, the microflora is disrupted, and C. difficile spores germinate in the intestines. The resulting vegetative cells are believed to fill empty niches left by the depleted microbial community and establish infection. Thus, germination of C. difficile spores is the first required step in CDAD. Interestingly, C. difficile genes encode most known spore-specific protein necessary for germination, except for germination (Ger) receptors. Even though C. difficile Ger receptors have not been identified, taurocholate (a bile salt) and glycine (an amino acid) have been shown to be required for spore germination. Furthermore, chenodeoxycholate, another bile salt, can inhibit taurocholate-induced C. difficile spore germination. In the present study, we examined C. difficile spore germination kinetics to determine whether taurocholate acts as a specific germinant that activates unknown germination receptors or acts nonspecifically by disrupting spores' membranes. Kinetic analysis of C. difficile spore germination suggested the presence of distinct receptors for taurocholate and glycine. Furthermore, taurocholate, glycine, and chenodeoxycholate seem to bind to C. difficile spores through a complex mechanism, where both receptor homo-and heterocomplexes are formed. The kinetic data also point to an ordered sequential progression of binding where taurocholate must be recognized first before detection of glycine can take place. Finally, comparing calculated kinetic parameters with intestinal concentrations of the two germinants suggests a mechanism for the preferential germination of C. difficile spores in antibiotic-treated individuals.

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

“…Furthermore, mifepristone seems to be a more potent enhancer of C. sordellii spore germination and a better inhibitor of C. difficile spore germination than naturally occurring progesterone or chenodeoxycholate (39). The relevance of these findings to the clinical use of mifepristone in humans remains to be determined.…”

“…Similarly, pregnenolone and dehydroepiandrosterone (DHEA) also behaved as competitive inhibitors of C. difficile spore germination (data not shown). We recently reported that C. difficile spores bind chenodeoxycholate cooperatively (39). Interestingly, inhibition analysis shows no cooperativity for binding of progesterone to C. difficile spores (Fig.…”

“…However, this K i value was obtained for a single chenodeoxycholate concentration and thus cannot be compared directly to the results obtained in our work. Progesterone, just like chenodeoxycholate, was shown to be a competitive inhibitor of taurocholate-mediated germination (39,55), suggesting that taurocholate, chenodeoxycholate, and progesterone bind to the same site in C. difficile spores. However, in contrast to taurocholate and chenodeoxycholate, C. difficile spores show noncooperative binding for progesterone, pregnenolone, or DHEA.…”

“…Furthermore, chenodeoxycholate was shown to be a naturally occurring inhibitor of C. difficile spore germination (54). Work from our laboratory has shown cooperative binding of both taurocholate and glycine, suggesting the presence of unidentified receptors for both germinants (39).…”

In vitro inhibition of Clostridium difficile by commercial probiotics: A microcalorimetric study

“…The germination of spores of C. difficile by taurocholate has also been 11 reported a number of times (Giel et al, 2010;Sorg and Sonenshein, 2008;Wheeldon et al, 2008;Wilson, 1983). Although no specific receptor binding sites have been recognized in C. difficile unlike in Bacillus subtilis, a well-studied spore forming organism, it has been suggested that C. difficile possibly encode unknown receptor proteins to bind sodium taurocholate to trigger germination (Ramirez et al, 2010).…”