SleC Is Essential for Germination of Clostridium difficile Spores in Nutrient-Rich Medium Supplemented with the Bile Salt Taurocholate

Clostridium difficile is the major cause of infectious diarrhea and a major burden to health care services. The ability of this organism to form endospores plays a pivotal role in infection and disease transmission. Spores are highly resistant to many forms of disinfection and thus are able to persist on hospital surfaces and disseminate infection. In order to cause disease, the spores must germinate and the organism must grow vegetatively. Spore germination in Bacillus is well understood, and genes important for this process have recently been identified in Clostridium perfringens; however, little is known about C. difficile. Apparent homologues of the spore cortex lytic enzyme genes cwlJ and sleB (Bacillus subtilis) and sleC (C. perfringens) are present in the C. difficile genome, and we describe inactivation of these homologues in C. difficile 630Δerm and a B1/NAP1/027 clinical isolate. Spores of a sleC mutant were unable to form colonies when germination was induced with taurocholate, although decoated sleC spores formed the same number of heat-resistant colonies as the parental control, even in the absence of germinants. This suggests that sleC is absolutely required for conversion of spores to vegetative cells, in contrast to CD3563 (a cwlJ/sleB homologue), inactivation of which had no effect on germination and outgrowth of C. difficile spores under the same conditions. The B1/NAP1/027 strain {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 was found to sporulate more slowly and produce fewer spores than 630Δerm. Furthermore, fewer {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 spores germinated, indicating that there are differences in both sporulation and germination between these epidemic and nonepidemic C. difficile isolates.The Gram-positive anaerobe Clostridium difficile causes diarrheal diseases ranging from asymptomatic carriage to a fulminant, relapsing, and potentially fatal colitis (8, 30). This organism is resistant to various broad-spectrum antibiotics and capitalizes on disruption of the normal intestinal flora to colonize and cause disease symptoms through the action of toxins A and B (16, 40). While these toxins are the principal virulence factors, the ability of the organism to produce endospores is necessary for disease transmission.Clostridial spores are extremely resistant to all kinds of chemical and physical agents and provide the mechanism by which C. difficile can evade the potentially fatal consequences of exposure to heat, oxygen, alcohol, and certain disinfectants (35). Thus, the spores shed in fecal matter are very difficult to eradicate and can persist on contaminated surfaces in health care facilities for extended periods of time (35). This leads to infection or reinfection of cohabitating individuals through inadvertent ingestion of infected material (10, 32). Once in the anaerobic environment of the gut, spores presumably germinate to form toxin-producing vegetative cells and, in susceptible individuals, diarrheal disease.Spore germination is defined as the events that result in the irreversible loss of spore characteristics. However, current mechanistic knowledge of the germination process is based principally on data derived from studying Bacillus subtilis. Little is known about spore germination in clostridia and, in particular, in C. difficile. Germination is initiated when the bacterial spore senses specific effectors, termed germinants. These effectors can include nutrients, cationic surfactants, peptidoglycan, and a 1:1 chelate of pyridine-2,6-dicarboxylic acid (dipicolinic acid) and Ca²⁺ (CaDPA) (23, 34, 36). Spores of B. subtilis can germinate in response to nutrients through the participation of three sensory receptors located in the spore inner membrane, GerA, GerB, and GerK (23). After activation, the events include the release of monovalent cations (H⁺, K⁺, and Na⁺) and CaDPA (accounting for approximately 10% of the spore dry weight) (36). The third major step in germination involves hydrolysis of the spore peptidoglycan (PG) cortex. It is during this hydrolysis that the previously low water content of the spore is restored to the water content of a normal vegetative cell and the core is able to expand, which in turn allows enzyme activity, metabolism, and spore outgrowth (36).CwlJ and SleB are two specific spore cortex-lytic enzymes (SCLEs) involved in Bacillus cortex hydrolysis, which break down PG containing muramic-δ-lactam (28). SleB has been shown to localize in both the inner and outer layers of B. subtilis spores through interaction of the enzyme peptidoglycan-binding motif and the δ-lactam structure of the cortex (7, 19) and in association with YpeB, which is required for sleB expression during sporulation (4, 7). SleB is a lytic transglycosylase muramidase, but so far its mode of activation is unknown (21). CwlJ is localized to the spore coat during sporulation (3) and is required for CaDPA-induced germination in B. subtilis. Activation can be due to either CaDPA released from the spore core at the onset of germination or exogenous CaDPA (22). Neither enzyme is individually essential for complete cortex hydrolysis during nutrient germination, although inactivation of both cwlJ and sleB in B. subtilis results in a spore unable to complete this process (15). The role of SleL has recently been studied in Bacillus anthracis. Mutants unable to produce this enzyme are still able to germinate, but the process is retarded (18).The SCLEs of Clostridium are less well studied than those of Bacillus. The SCLEs SleC (20) and SleM (6) have been identified in Clostridium perfringens, and a recent study demonstrated that SleC is required during germination for complete cortex hydrolysis (26). Although SleM can degrade spore cortex peptidoglycan and inactivation of both sleC and sleM decreased the ability of spores to germinate more than inactivation of sleC alone did, SleM was not essential (26). It has also been shown that the germination-specific serine protease CspB is essential for cortex hydrolysis and converts the inactive pro-SleC found in dormant spores to an active enzyme (24). So far, there has been no detailed study of any gene responsible for spore germination in C. difficile, although genes showing homology to cwlJ and sleB of B. subtilis (CD3563) and sleC of C. perfringens (CD0551) have now been identified in the C. difficile 630 genome (33).With germinant receptors in C. difficile yet to be identified, the mechanism by which the spores sense a suitable environment for germination is unclear. Recent studies have suggested that this process may involve the interaction of C. difficile with bile. Taurocholate has been shown to enhance recovery of C. difficile spores in nutrient-rich medium (42), and it has been proposed that glycine and taurocholate act as cogerminants (38), while chenodeoxycholate inhibits C. difficile spore germination (39).The emergence of C. difficile B1/NAP1/027 strains has increased the burden on health care services worldwide. Such strains have been shown to produce higher levels of toxin in the laboratory than many other types of strains (41), although the mechanism behind this production is not fully understood. However, while the observed higher levels of toxin production is doubtless important, perhaps the recent attention given to B1/NAP1/027 strains has focused too much on toxins. As spores represent the infectious stage of C. difficile, processes such as spore germination may also contribute to the greater virulence of these strains. In this study we evaluated the sporulation and germination efficiencies of an “epidemic” B1/NAP1/027 C. difficile strain ({"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291, isolated from the Stoke Mandeville outbreak in 2004 and 2005) and the “nonepidemic” strain 630Δerm (14). We then constructed strains with mutations in CD3563 (a cwlJ/sleB homologue) and a sleC homologue to analyze the role of these genes in the germination of C. difficile spores.