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

Clostridium difficile is a Gram-positive, rod-shaped, spore-forming, obligately anaerobic bacterium. Under stress conditions, vegetative C. difficile cells differentiate into infectious spores (26). Like other bacilli and clostridia, C. difficile spores are metabolically inactive and resistant to most environmental insults. Spores can revert to toxin-producing bacteria (a process called germination) in nutrient-rich environments, such as the mammalian host (25, 33). C. difficile spores are carried asymptomatically by up to 5% of healthy human adults (15). In the absence of antibiotics, normal intestinal microflora interferes with colonization, and C. difficile remains in its quiescent spore state (8, 31). Following antimicrobial treatment, however, the normal bacterial flora is disrupted, and it is believed that C. difficile spores germinate in the intestines. The resulting vegetative cells fill empty niches in the depleted microbial community, where they multiply and produce toxins (12). Germination of C. difficile spores in the gastrointestinal (GI) tract of immunocompromised patients is required for C. difficile-associated disease (CDAD), a condition responsible for approximately 25% of all cases of antibiotic-associated diarrhea (39). Prompt empirical antimicrobial treatment is mandatory for cancer patients with severe neutropenia caused by antineoplastic treatment (22) and in the postoperative period after organ transplantation (5). Unfortunately, these antimicrobial regimes also favor colonization by C. difficile (41). Furthermore, C. difficile is the most common identifiable cause of diarrhea in HIV patients (7). Thus, CDAD is a major complication that increases morbidity and mortality in cancer chemotherapy, transplant, and AIDS patients. Even though germination of C. difficile spores is the first required step in CDAD establishment, little is known about this process. The mechanisms of spore germination have been studied mainly in bacilli. In these cases, the germination process is commonly triggered by the initial detection of low-molecular-weight germinants by proteinaceous germination (Ger) receptors (25, 33). Ger receptors generally consist of three membrane-bound proteins encoded by tricistronic operons (16, 19). Each Ger receptor recognizes a cognate germinant, such as amino acids, nucleosides, sugars, or salts (25, 33). Proteins involved in germination are remarkably conserved in both bacilli and clostridia. Basic Local Alignment Search Tools (BLAST) searches of spore-specific proteins reveal analogs in all sequenced sporulating bacteria. Interestingly, C. difficile genes encode analogs for most spore-specific proteins, except for Ger receptors and coat proteins (32). Since C. difficile spores must germinate, C. difficile germination receptors may be too divergent from other sporulating bacteria. Alternatively, C. difficile spores may use a different protein set to detect its germinants (32). Indeed, a novel germination mechanism that does not require Ger receptors has been recently discovered (34). In this case, Bacillus subtilis spores use a serine/threonine protein kinase (PrkC) instead of Ger receptors to germinate in the presence of peptidoglycans (34). A search of genome sequences reveals the presence of a putative prkC homologue in C. difficile (28). Whether PrkC-like proteins contribute to the germination of C. difficile spores has not been determined. Although the mechanism of C. difficile spore germination has not been elucidated, previous studies have shown that addition of bile salts increases the recovery of vegetative cells from C. difficile spores (21, 44). Furthermore, a recent article showed that C. difficile spores recognize glycine (an amino acid) and taurocholate (a bile salt) as germinants (36). Furthermore, another bile salt, chenodeoxycholate, was shown to inhibit taurocholate-induced C. difficile spore germination (37). Neither glycine nor taurocholate has been previously described as germinants for spores of bacilli or clostridia. Taurocholate could induce germination by two alternate mechanisms. Since bile salts are involved in the emulsification of fats (9), taurocholate could trigger germination by nonspecifically permeabilizing spore membranes. This type of germination mechanism has been previously described for nisin-type antibiotics (23). On the other hand, taurocholate could be specifically recognized as a germinant by unknown receptors. Activation of receptors by small molecules is the most common germination mechanism utilized by Bacillus and Clostridium spores (16, 19, 27). Transposon mutagenesis has been used in Bacillus species to identify germination receptors and their cognate ligands (6, 11). However, access to C. difficile mutants has been impeded by the lack of appropriate genetic tools (24). Conjugative transposons Tn916 and Tn5397 have been used to mutagenize C. difficile. Unfortunately, Tn916 tends to integrate multiple copies of the transposon in every bacterium genome (20). Furthermore, Tn5397 nonrandomly targets specific sites in the C. difficile genome (42). More recently, a mariner-based transposon system has been reported for the in vivo mutagenesis of C. difficile (10). This new system could allow single transposon insertion at random genomic loci. However, phenotypic screening of a C. difficile random mutant library yielded a single sporulation/germination defective clone and a single pyrimidine auxotroph (10). As an alternative to genetic approaches, we have used kinetic methods and molecular probes to study Bacillus and Clostridium spore germination (1, 3, 14, 30). These techniques allow proposing possible mechanisms for spore germination even when the identity of the germination receptors is not known (30). In the present study, we examined C. difficile spore germination kinetics to distinguish whether taurocholate activates unknown germination receptors or causes spore membrane disruption. Kinetic analysis was consistent with a receptor-mediated mechanism for C. difficile spore germination. The kinetic data obtained can be best fitted to a complex ordered sequential mechanism where taurocholate must be recognized first, before binding of glycine can take place. Finally, comparing calculated kinetic parameters with intestinal concentrations of taurocholate and glycine suggested a mechanism for the difference in germination profile of C. difficile spores in healthy and antibiotic-treated individuals.