Molecular basis for Clostridium perfringens spore resistance to heat and other environmental stress factors

Clostridium perfringens type A isolates producing enterotoxin (CPE) are an important cause of both food poisoning (FP) and non food borne gastrointestinal diseases (NFBGID) in both humans and animals. C. perfringens type A food poisoning is caused by isolates carrying the CPE encoding gene (cpe) on the chromosome while the NFBGID isolates carry cpe on a large plasmid. Recent studies indicate that the association between chromosomal cpe isolates and food poisoning is, in part, because spores of these isolates exhibit heat resistance which enhances their survival in incompletely cooked or inadequately warmed foods. The molecular basis for resistance of spores to heat and other environmental stress factors remains unknown. Since in Bacillus subtilis, small acid soluble spore proteins (SASPs) have been shown to play a major role in spore resistance to heat and other environmental factors, in the current study, we hypothesized that alpha/beta-type SASPs in C. perfringens play a similar role. Results of this study showed that the SASP encoding genes, ssp1, 2 and 3 are present and expressed in food poisoning isolates of C. perfringens. Consistent with the results in B. subtilis , alpha/beta-type SASPs in C. perfringens protect the spores by binding to spore DNA. A single C. perfringens ssp3 knock out mutant showed a slightly decreased spore heat resistance compared to wild type. Since there is no genetic tool available to make a triple ssp (ssp1, 2 and 3) knock out mutant in C. perfringens, the antisense RNA strategy was used to down regulate the production of alpha/beta-type SASPs in a food poisoning isolate. The strains with lower alpha/beta-type SASP production had lower resistance to moist heat and UV radiation. These results support our hypothesis that C. perfringens alpha/beta-type SASPs play a major role in spore resistance to heat and other environmental factors.; The second hypothesis in this study was that the NFBGID isolates carrying cpe on a plasmid exhibit lower heat resistance than the FP isolates because these isolates produce less alpha/beta-type SASPs. PCR and nucleotide sequencing analysis demonstrated that all three ssp genes are present in NFBGID isolates. Quantitative analysis of SASPs production demonstrated that NFBGID isolates produce similar levels of SASPs as the FP isolates. Further studies showed that the cpe plasmid does not encode any factors responsible for heat sensitivity in NFBGID isolates. The cpe plasmid cured strain of these isolates showed a similar heat resistance to that of the wild type strain carrying the cpe plasmid. However, it was observed that the dry heat resistance of the plasmid cpe isolates was similar to that of the chromosomal cpe isolates indicating that there might be additional factors (eg: spore water content) involved in spore resistance to dry heat. Future studies on identifying the genes encoding factors that affect spore water content should help in understanding the mechanism of resistance to dry heat.