Characterization of Clostridium perfringens Spores That Lack SpoVA Proteins and Dipicolinic Acid

Abstract

Spores of Clostridium perfringens possess high heat resistance, and when these spores germinate and return to active growth, they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of Ca 2؉ and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little, if any, Ca 2؉ and DPA, and their core water content was approximately twofold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as DPA-less B. subtilis spores do. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of L-asparagine and KCl (AK), KCl alone, or a 1:1 chelate of Ca 2؉ and DPA (Ca-DPA). However, the viability of C. perfringens spoVA spores was 20-fold lower than the viability of wild-type spores. Decoated wild-type and spoVA spores exhibited little, if any, germination with AK, KCl, or exogenous Ca-DPA, and their colony-forming efficiency was 10 3 -to 10 4 -fold lower than that of intact spores. However, lysozyme treatment rescued these decoated spores. Although the levels of DNAprotective ␣/␤-type, small, acid-soluble spore proteins in spoVA spores were similar to those in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid, and UV radiation than wild-type spores did. In sum, these results suggest the following. (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation. (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination. (iii) A low spore core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation, and chemicals.Clostridium perfringens food poisoning is caused mainly by enterotoxigenic type A isolates that have the ability to form metabolically dormant spores that are extremely resistant to heat, radiation, and toxic chemicals (45,49,50,55). These highly resistant spores can survive traditional methods for cooking meat and poultry products as well as other processing treatments used in the food industry. The surviving spores can then go through germination and outgrowth, generating the vegetative cells that cause disease (30).Spores of Bacillus species also have the extreme resistance of C. perfringens spores. The factors involved in Bacillus subtilis spore resistance include the following: (i) relatively impermeable spore inner membrane; (ii) low water content of the spore core; (iii) high levels of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) in the spore core and the type and amount of cations chelated by DPA; and (iv) the saturation of spore DNA with ␣/␤-type small, acid-s...

Germination of spores of Clostridium difficile strains, including isolates from a hospital outbreak of Clostridium difficile-associated disease (CDAD)

“…One possible explanation is that K + ions and P i directly activate a CLE and/or Ca-DPA release. Ongoing work is thus oriented towards investigating the roles played by a number of proteins, including CLEs that may be involved in cortex PG hydrolysis (Foster & Johnstone, 1987;Ishikawa et al, 1998;Makino et al, 1994;Miyata et al, 1995Miyata et al, , 1997Shimamoto et al, 2001) and SpoVA proteins implicated in Ca-DPA release (Paredes-Sabja et al, 2008b), in the germination of C. difficile spores with K + and P i . The resolution of the paradox noted above as well as a more thorough understanding of the germination of C. difficile spores may well have applications in the areas of public health and food safety.…”

GerO, a Putative Na + /H + -K + Antiporter, Is Essential for Normal Germination of Spores of the Pathogenic Bacterium Clostridium perfringens

“…The forward and reverse primers (the sequences of all primers used in the present study are given in Table S2 in the supplemental material) had SalI and PstI cleavage sites, respectively, at the 5Ј ends. These PCR fragments were digested with SalI and PstI and cloned between the SalI and PstI sites in plasmid pMRS127 in E. coli DH5␣, the host for all plasmid construction, as described previously (20,25) to create gerO-and gerQ-gusA fusions, giving plasmids pDP81 and pDP82 (see Table S1 in the supplemental material). These plasmids were introduced by electroporation (4) into C. perfringens SM101, and erythromycin-resistant (Em r ) transformants were selected.…”

“…Spores of C. perfringens isolates were prepared and purified as described previously (20,21). Briefly, C. perfringens sporulating cultures were prepared by inoculating 0.2 ml of an overnight culture grown at 37°C in fluid thioglycolate broth (Difco) into 10 ml of DS sporulation medium (5), followed by incubation for 24 h at 37°C, and the presence of spores was confirmed by phase-contrast microscopy.…”

“…With the exception of dodecylamine germination, spore suspensions in water were heat activated (80°C, 10 min) prior to germination, cooled in water at ambient temperature for 5 min, and incubated at 40°C for 10 min prior to the addition of buffer and germinants as described previously (20,21). Except for germination with dodecylamine (see below), spore germination was routinely measured by monitoring the OD 600 of spore cultures (Smartspec 3000 spectrophotometer; Bio-Rad Laboratories, Hercules, CA), which falls ϳ60% upon complete spore germination, and levels of germination were confirmed by phase-contrast microscopy.…”

Host serum factor triggers germination of Clostridium perfringens spores lacking the cortex hydrolysis machinery

“…Spore coats were extracted as previously described (Paredes-Sabja et al, 2008a). Briefly, spore suspensions at an OD 600 of~20 were incubated in 1 ml 50 mM Tris/HCl (pH 8.0), 8 M urea, 1 % (w/v) SDS, 50 mM DTT for 90 min at 37 uC, and washed five times with 150 mM NaCl and twice with distilled water.…”