Kinetic evidence for the presence of putative germination receptors in Clostridium 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.

FIG. 1.

Compounds tested as agonists and antagonists of C. difficile spore germination. Taurocholate (compound I), cholate (compound II), taurine (compound III), glycine (compound IV), sodium dodecyl sulfate (SDS; compound V), Triton X-100 (compound VI), N-methylglycine (compound VII), glycine methyl ester (compound VIII), chenodeoxycholate (compound IX), taurochenodeoxycholate (compound X), glycochenodeoxycholate (compound XI), and 7-ketolithocholate (compound XII).

FIG. 2.

C. difficile spore germination in the presence of taurocholate and glycine mixtures. (A) C. difficile spores were incubated with 8 mM taurocholate and titrated with 8 (•), 9 (○), 10 (▪), 13 (□), and 16 (⧫) mM glycine concentrations. (B) C. difficile spores were incubated with 8 mM glycine and titrated with 6 (•), 7 (○), 9 (▪), 12 (□), and 15 (⧫) mM taurocholate concentrations.

FIG. 3.

Kinetics of Clostridium difficile spore germination in the presence of taurocholate and glycine. Germination rates were calculated from the linear segment of optical density changes over time. (A) Lineweaver-Burk plots of C. difficile spore germination at various concentrations of taurocholate (8, 10, 14, 20, and 30 mM) and 8 (•), 9 (○), 10 (▪), 13 (□), and 16 (⧫) mM glycine concentrations. (B) Squared Lineweaver-Burk plots of C. difficile spore germination at various concentrations of taurocholate (8, 10, 14, 20, and 30 mM) and 8 (•), 9 (○), 10 (▪), 13 (□), and 16 (⧫) mM glycine concentrations. (C) Hill plot for taurocholate binding at the saturating glycine concentration. (D) Lineweaver-Burk plots of C. difficile spore germination at various concentrations of glycine (8, 10, 12, 14, and 18 mM) and 6 (•), 7 (○), 9 (▪), 12 (□), and 15 (⧫) mM taurocholate concentrations. (E) Squared Lineweaver-Burk plots of C. difficile spore germination at various concentrations of glycine (8, 10, 12, 14, and 18 mM) and 6 (•), 7 (○), 9 (▪), 12 (□), and 15 (⧫) mM taurocholate concentrations. (F) Hill plot for glycine binding at the saturating taurocholate concentration.

FIG. 4.

Kinetic analysis of chenodeoxycholate inhibition of C. difficile spore germination. Germination rates were calculated from the linear segment of optical density changes over time. (A) Lineweaver-Burk plots of C. difficile spore germination at various concentrations of taurocholate (4, 6, 8, and 12 mM) and 0.2 (•), 0.4 (○), 0.5 (▪), 0.6 (□), and 0.8 mM (⧫) chenodeoxycholate concentrations. (B) Squared Lineweaver-Burk plots of C. difficile spore germination at various concentrations of taurocholate (4, 6, 8, and 12 mM) and 0.2 (•), 0.4 (○), 0.5 (▪), 0.6 (□), and 0.8 mM (⧫) chenodeoxycholate concentrations. (C) Dixon plot of C. difficile spore germination at various concentrations of chenodeoxycholate (0.2, 0.4, 0.5, 0.6, and 0.8 mM) and 4 (•), 6 (○), 8 (▪), and 12 (□) mM taurocholate. (D). Replot of Dixon plot x intercepts versus taurocholate concentrations was fitted to a straight line and yielded a K_i of 1.3 mM² for chenodeoxycholate binding.

FIG. 5.

Proposed kinetic mechanism for C. difficile spore germination. Spores are represented by gray circles. Cooperative germination receptors are represented by white split circles. Solid arrows represent fast processes. Dashed arrows represent slow processes. K_m(T) is the apparent Michaelis-Menten constant for taurocholate binding, and a is the interaction factor between taurocholate binding sites. K_m(G) is the apparent Michaelis-Menten constant for glycine binding, and b is the interaction factor between glycine binding sites. Dormant spores (A) must first detect a taurocholate molecule to start the germination process (B). The cooperative behavior of taurocholate binding rapidly saturates other taurocholate binding sites (C). The resulting taurocholate-spore complex is then competent to bind glycine (D). Cooperative binding of glycine allows rapid saturation of glycine binding sites to form a taurocholate-spore-glycine complex (E) that is activated for germination.