Inhibiting the Initiation of Clostridium difficile Spore Germination using Analogs of Chenodeoxycholic Acid,a Bile Acid

To cause disease, Clostridium difficile spores must germinate in the host gastrointestinal tract. Germination is initiated upon exposure to glycine and certain bile acids, e.g., taurocholate. Chenodeoxycholate, another bile acid, inhibits taurocholate-mediated germination. By applying Michaelis-Menten kinetic analysis to C. difficile spore germination, we found that chenodeoxycholate is a competitive inhibitor of taurocholate-mediated germination and appears to interact with the spores with greater apparent affinity than does taurocholate. We also report that several analogs of chenodeoxycholate are even more effective inhibitors. Some of these compounds resist 7α-dehydroxylation by Clostridium scindens, a core member of the normal human colonic microbiota, suggesting that they are more stable than chenodeoxycholate in the colonic environment.Clostridium difficile is a Gram-positive, spore-forming, anaerobic bacterium that is pathogenic for both humans and animals (33, 44). Infections caused by C. difficile range from mild diarrhea to more life-threatening conditions, such as pseudomembranous colitis (33). In the classic case, prior antibiotic treatment that disrupts the normally protective colonic flora makes patients susceptible to C. difficile infection (CDI) (35, 53). Other antibiotics, such as vancomycin and metronidazole, are the most commonly used treatments for CDI (54). However, because these antibiotics also disrupt the colonic flora, 10 to 40% of patients whose symptoms have been ameliorated suffer from relapsing CDI (15, 24). The annual treatment-associated cost for CDI in the United States is estimated to be between $750 million and $3.2 billion (8, 9, 16, 31). Moreover, the number of fatal cases of CDI has been increasing rapidly (14, 39). Thus, there is an urgent need to find alternative therapies for CDI.C. difficile infection likely is initiated by infection with the spore form of C. difficile (12). C. difficile elicits disease through the actions of two secreted toxins, TcdA and TcdB (48). TcdB was recently shown to be critical for pathogenesis in an animal model of disease (18). Since the toxins are produced by vegetative cells, not by spores (17), germination and outgrowth are prerequisites for pathogenesis.Spore germination is triggered by the interaction of small molecules, called germinants, with receptors within the spore inner membrane. These germinants vary by bacterial species and can include ions, amino acids, sugars, nucleotides, surfactants, or combinations thereof (43). The recognition of germinants triggers irreversible germination, leading to Ca²⁺-dipicolinic acid release, the uptake of water, the degradation of the cortex, and, eventually, the outgrowth of the vegetative bacterium (43). The germination receptors that C. difficile uses to sense the environment have not been identified. Based on homology searches, C. difficile germination receptors must be very different from known germination receptors (42), but they appear to be proteinaceous (13).Taurocholate, a primary bile acid, has been used for approximately 30 years by researchers and clinical microbiologists to increase colony formation by C. difficile spores from patient and environmental samples (3, 49, 51, 52). This suggested that C. difficile spores interact with bile acids along the gastrointestinal (GI) tract and that spores use a host-derived signal to initiate germination.The liver synthesizes the two major primary bile acids, cholate and chenodeoxycholate (40). These compounds are modified by conjugation with either taurine (to give taurocholate or taurochenodeoxycholate) or glycine (producing glycocholate or glycochenodeoxycholate). Upon secretion into the digestive tract, bile aids in the absorption of fat and cholesterol; much of the secreted bile is actively absorbed and recycled back to the liver for reutilization (40). Though efficient, enterohepatic recirculation is not complete; bile enters the cecum of the large intestine at a concentration of approximately 2 mM (30).In the cecum, bile is modified by the normal, benign colonic flora. First, bile salt hydrolases found on the surfaces of many bacterial species remove the conjugated amino acid, producing the deconjugated primary bile acids cholate and chenodeoxycholate (40). These deconjugated primary bile acids are further metabolized by only a few species of intestinal bacteria, including Clostridium scindens. C. scindens actively transports unconjugated primary bile acids into the cytoplasm, where they are 7α-dehydroxylated, converting cholate to deoxycholate and chenodeoxycholate to lithocholate (21, 40). The disruption of the colonic flora by antibiotic treatment abolishes 7α-dehydroxylation activity (41).Building upon the work on Wilson and others (51, 52), we demonstrated that taurocholate and glycine, acting together, trigger the loss of the birefringence of C. difficile spores (45). All cholate derivatives (taurocholate, glycocholate, cholate, and deoxycholate) stimulate the germination of C. difficile spores (45). Recently it was shown that taurocholate binding is prerequisite to glycine binding (37). At physiologically relevant concentrations, chenodeoxycholate inhibits taurocholate-mediated germination (46) and also inhibits C. difficile vegetative growth, as does deoxycholate (45). In fact, C. difficile spores use the relative concentrations of the various bile acids as cues for germination within the host (10).Since chenodeoxycholate is absorbed by the colonic epithelium and metabolized to lithocholate by the colonic flora (25, 40), the use of chenodeoxycholate as a therapy against C. difficile disease might be hindered by its absorption and conversion to lithocholate.Here, we further characterize the interaction of C. difficile spores with various bile acids and demonstrate that chenodeoxycholate is a competitive inhibitor of taurocholate-mediated germination. Further, we identify chemical analogs of chenodeoxycholate that are more potent inhibitors of germination and that resist 7α-dehydroxylation by the colonic flora, potentially increasing their stability and effectiveness as inhibitors of C. difficile spore germination in the colonic environment.