Joseph A. Sorg

Bile Salts and Glycine as Cogerminants for Clostridium difficile Spores

Spore formation by Clostridium difficile is a significant obstacle to overcoming hospital-acquired C. difficile-associated disease. Spores are resistant to heat, radiation, chemicals, and antibiotics, making a contaminated environment difficult to clean. To cause disease, however, spores must germinate and grow out as vegetative cells. The germination of C. difficile spores has not been examined in detail. In an effort to understand the germination of C. difficile spores, we characterized the response of C. difficile spores to bile. We found that cholate derivatives and the amino acid glycine act as cogerminants. Deoxycholate, a metabolite of cholate produced by the normal intestinal flora, also induced germination of C. difficile spores but prevented the growth of vegetative C. difficile. A model of resistance to C. difficile colonization mediated by the normal bacterial flora is proposed.

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 spore biology: sporulation, germination, and spore structural proteins

Clostridium difficile is a Gram-positive, spore-forming obligate anaerobe and a major nosocomial pathogen of worldwide concern. Owing to its strict anaerobic requirements, the infectious and transmissible morphotype is the dormant spore. In susceptible patients, C. difficile spores germinate in the colon to form the vegetative cells that initiate Clostridium difficile infections (CDI). During CDI, C. difficile induces a sporulation pathway that produces more spores; these spores are responsible for the persistence of C. difficile in patients and horizontal transmission between hospitalized patients. Although important to the C. difficile lifecycle, the C. difficile spore proteome is poorly conserved when compared to members of the Bacillus genus. Further, recent studies have revealed significant differences between C. difficile and Bacillus subtilis at the level of sporulation, germination, and spore coat and exosporium morphogenesis. In this review, the regulation of the sporulation and germination pathways and the morphogenesis of the spore coat and exosporium will be discussed.

Metabolism of Bile Salts in Mice Influences Spore Germination in Clostridium difficile

Clostridium difficile, a spore-forming bacterium, causes antibiotic-associated diarrhea. In order to produce toxins and cause disease, C. difficile spores must germinate and grow out as vegetative cells in the host. Although a few compounds capable of germinating C. difficile spores in vitro have been identified, the in vivo signal(s) to which the spores respond were not previously known. Examination of intestinal and cecal extracts from untreated and antibiotic-treated mice revealed that extracts from the antibiotic-treated mice can stimulate colony formation from spores to greater levels. Treatment of these extracts with cholestyramine, a bile salt binding resin, severely decreased the ability of the extracts to stimulate colony formation from spores. This result, along with the facts that the germination factor is small, heat-stable, and water-soluble, support the idea that bile salts stimulate germination of C. difficile spores in vivo. All extracts able to stimulate high level of colony formation from spores had a higher proportion of primary to secondary bile salts than extracts that could not. In addition, cecal flora from antibiotic-treated mice was less able to modify the germinant taurocholate relative to flora from untreated mice, indicating that the population of bile salt modifying bacteria differed between the two groups. Taken together, these data suggest that an in vivo-produced compound, likely bile salts, stimulates colony formation from C. difficile spores and that levels of this compound are influenced by the commensal gastrointestinal flora.

Bile acid recognition by the Clostridium difficile germinant receptor, CspC, is important for establishing infection.

Clostridium difficile spores must germinate in vivo to become actively growing bacteria in order to produce the toxins that are necessary for disease. C. difficile spores germinate in vitro in response to certain bile acids and glycine. In other sporulating bacteria, proteins embedded within the inner membrane of the spore sense the presence of germinants and trigger the release of Ca++-dipicolinic acid (Ca++-DPA) from the spore core and subsequent hydrolysis of the spore cortex, a specialized peptidoglycan. Based upon homology searches of known germinant receptors from other spore-forming bacteria, C. difficile likely uses unique mechanisms to recognize germinants. Here, we identify the germination-specific protease, CspC, as the C. difficile bile acid germinant receptor and show that bile acid-mediated germination is important for establishing C. difficile disease in the hamster model of infection. These results highlight the importance of bile acids in triggering in vivo germination and provide the first description of a C. difficile spore germinant receptor. Blocking the interaction of bile acids with the C. difficile spore may represent an attractive target for novel therapeutics.

Chenodeoxycholate Is an Inhibitor of Clostridium difficile Spore Germination

Some cholate derivatives that are normal components of bile can act with glycine to induce the germination of Clostridium difficile spores, but at least one bile component, chenodeoxycholate, does not induce germination. Here we show that chenodeoxycholate inhibits the germination of C. difficile spores in response to cholate and taurocholate.

Laboratory Maintenance of Clostridium difficile

Clostridium difficile is a Gram‐positive, spore‐forming, anaerobe and is the leading cause of antibiotic‐associated diarrhea, pseudomembranous colitis, and toxic megacolon. Essential to the lifestyle of C. difficile is the ability to form a metabolically dormant spore, germinate, and grow out upon appropriate signals and elicit disease with the secretion of two toxins. To aid in the study of this organism, this unit describes the growth and maintenance of C. difficile. Included are methods to isolate C. difficile from environmental samples, grow in laboratory medium, and produce and purify spores. Curr. Protoc. Microbiol. 12:9A.1.1‐9A.1.10.

Secretion signal recognition by YscN, the Yersinia type III secretion ATPase

Yersinia type III machines secrete protein substrates across the bacterial envelope. Secretion signals of some substrates have been identified; however, the mechanisms whereby these signals interact with type III machines are not known. Here we show that fusion of YopR, an early secretion substrate, to the N terminus of dihydrofolate reductase (DHFR) or other tightly folded proteins generates impassable hybrids that cannot travel the type III pathway. YopR hybrids capture YscN, the ATPase that provides energy for type III transport reactions, in the bacterial cytoplasm. Eleven N-terminal residues function as the YopR secretion signal, which is required for both binding to YscN and blocking the type III pathway. When expressed during type III machine assembly, YopR-DHFR blocks all secretion. Delayed expression of YopR-DHFR, when yersiniae have already engaged the type III pathway, blocks secretion of early (YscP) but not of late (effector Yops) substrates. These observations support a model whereby type III machines are programmed to secrete a sequence of proteins that can be disrupted when an impassable early substrate interacts with the YscN ATPase and blocks the transport of late substrates.

Binding of SycH Chaperone to YscM1 and YscM2 Activates Effector yop Expression in Yersinia enterocolitica

Yersinia enterocolitica transports YscM1 and YscM2 via the type III pathway, a mechanism that is required for the establishment of bacterial infections. Prior to host cell contact, YscM1 and YscM2 exert posttranscriptional regulation to inhibit expression of effector yop genes, which encode virulence factors that travel the type III pathway into the cytoplasm of macrophages. Relief from repression has been predicted to occur via the type III secretion of YscM1 and YscM2 into the extracellular medium, resulting in the depletion of regulatory molecules from the bacterial cytoplasm. Using digitonin fractionation and fluorescence microscopy of FlAsH-labeled polypeptides in Yersinia-infected cells, we have localized YscM1 and YscM2 within the host cell cytoplasm. Type III injection of YscM1 and YscM2 required the SycH chaperone. Expression of C-terminal fusions of YscM1 and YscM2 to the neomycin phosphotransferase reporter revealed sequences required for regulatory activity and for secretion in the absence of SycH. Coexpression of SycH and glutathione S-transferase (GST)-YscM1 or GST-YscM2, hybrid GST variants that cannot be transported by the type III apparatus, also relieved repression of Yop synthesis. GST-SycH bound to YscM1 and YscM2 and activated effector yop expression without initiation of the bound regulatory molecules into the type III pathway. Further, regulation of yop expression by YscM1, YscM2, and SycH is shown to act independently of factors that regulate secretion, and gel filtration chromotography revealed populations of YscM1 and YscM2 that are not bound to SycH under conditions where Yop synthesis is repressed. Taken together, these results suggest that YscM1- and YscM2-mediated repression may be relieved through binding to the cytoplasmic chaperone SycH prior to their type III injection into host cells.

Substrate recognition of type III secretion machines –testing the RNA signal hypothesis

Secretion by the type III pathway of Gram‐negative microbes transports polypeptides into the extracellular medium or into the cytoplasm of host cells during infection. In pathogenic Yersinia spp., type III machines recognize 14 different Yop protein substrates via discrete signals genetically encoded in 7–15 codons at the 5′ portion of yop genes. Although the signals necessary and sufficient for substrate recognition of Yop proteins have been mapped, a clear mechanism on how proteins are recognized by the machinery and then initiated into the transport pathway has not yet emerged. As synonymous substitutions, mutations that alter mRNA sequence but not codon specificity, affect the function of some secretion signals, recent work with several different microbes tested the hypothesis of an RNA‐encoded secretion signal for polypeptides that travel the type III pathway. This review summarizes experimental observations and mechanistic models for substrate recognition in this field.