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

Abstract

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 630⌬erm 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 R20291 was found to sporulate more slowly and produce fewer spores than 630⌬erm. Furthermore, fewer R20291 spores germinated, indicating that there are differences in both sporulation and germination between these epidemic and nonepidemic C. difficile isolates.

Characterization of the Dynamic Germination of Individual Clostridium difficile Spores Using Raman Spectroscopy and Differential Interference Contrast Microscopy

“…It was also notable that while germination of C. difficile spores was slowed tremendously in the absence of SleC, a small percentage of the sleC mutant spores released CaDPA but did not exhibit cortex hydrolysis. The latter observation is consistent with the low viability of sleC mutant C. difficile spores and the restoration of significant viability by inclusion of lysozyme in plating medium (30). An important question then concerns the mechanism of the germination of the small percentage of sleC mutant spores that germinated in 10 h, as measured by their release of CaDPA, as this is perhaps analogous in some ways to the spontaneous germination of small percentages of B. subtilis spores that lack all GRs (53).…”

“…In addition to these differences in germinant sensing, the regulation of cortex hydrolysis differs between C. difficile and B. subtilis (15,16,29). C. difficile spores require only a single CLE, SleC (30), which is made as a zymogen that is proteolytically activated by the serine protease CspB, similar to the situation in Clostridium perfringens (31)(32)(33). Thus, the environmental signals that trigger germination, the germinant receptors, and the regulation of cortex hydrolysis exhibit notable differences between C. difficile and B. subtilis.…”

Multiple Factors Modulate Biofilm Formation by the Anaerobic Pathogen Clostridium difficile

“…C. difficile spores were measured as previously described (27). At day 1, 3, and 5 of biofilm formation samples were removed from the anaerobic chamber and heated at 80°C for 25 min to kill the vegetative cells but not the spores.…”

“…Mutations in the genes sleC, spo0A, fliC, and luxS were generated using the insertional inactivation system, ClosTron, as previously described (27)(28)(29)(30). Construction of the fliC and luxS mutants is described in the supplemental material.…”

ClosTron-Mediated Engineering of Clostridium

“…By way of example, it has been used to establish the role of bacterial factors (toxins, flagella and adhesins) in infection, 23,[26][27][28][29] to garner a greater understanding of the role and activities of enzymes in primary metabolism, [30][31][32][33] to begin to elucidate mechanistic details of the developmental process of spore formation/ germination 21,34,35 and provide fundamental information on regulatory processes important in virulence factor and metabolite production. [36][37][38][39] Its availability has provided the research community with a sequence of the re-targeted region used to generate the insertion should be given.…”