A Novel Small Acid Soluble Protein Variant Is Important for Spore Resistance of Most Clostridium perfringens Food Poisoning Isolates

Clostridium perfringens is a major cause of food poisoning (FP) in developed countries. C. perfringens isolates usually induce the gastrointestinal symptoms of this FP by producing an enterotoxin that is encoded by a chromosomal (cpe) gene. Those typical FP strains also produce spores that are extremely resistant to food preservation approaches such as heating and chemical preservatives. This resistance favors their survival and subsequent germination in improperly cooked, prepared, or stored foods. The current study identified a novel α/β-type small acid soluble protein, now named Ssp4, and showed that sporulating cultures of FP isolates producing resistant spores consistently express a variant Ssp4 with an Asp substitution at residue 36. In contrast, Gly was detected at Ssp4 residue 36 in C. perfringens strains producing sensitive spores. Studies with isogenic mutants and complementing strains demonstrated the importance of the Asp 36 Ssp4 variant for the exceptional heat and sodium nitrite resistance of spores made by most FP strains carrying a chromosomal cpe gene. Electrophoretic mobility shift assays and DNA binding studies showed that Ssp4 variants with an Asp at residue 36 bind more efficiently and tightly to DNA than do Ssp4 variants with Gly at residue 36. Besides suggesting one possible mechanistic explanation for the highly resistant spore phenotype of most FP strains carrying a chromosomal cpe gene, these findings may facilitate eventual development of targeted strategies to increase killing of the resistant spores in foods. They also provide the first indication that SASP variants can be important contributors to intra-species (and perhaps inter-species) variations in bacterial spore resistance phenotypes. Finally, Ssp4 may contribute to spore resistance properties throughout the genus Clostridium since ssp4 genes also exist in the genomes of other clostridial species.
Author Summary Spores made by pathogenic Bacillus and Clostridium spp. contribute to disease transmission. Clostridium perfringens food poisoning (FP) isolates typically produce spores with exceptional resistance to heat and sodium nitrite. This spore resistance probably facilitates FP strain survival in improperly cooked/held foods, contributing to C. perfringens FP outbreaks, which rank among the most common food-borne diseases in developed countries. Currently, the mechanistic basis of the resistant spore phenotype of FP strains is unknown. Here, we report the identification of a novel small acid soluble protein, named Ssp4, and show that sporulating cultures of FP strains producing resistant spores express an Ssp4 variant with Asp at residue 36, while sporulating cultures of C. perfringens strains producing sensitive spores express an Ssp4 with Gly at residue 36. We now demonstrate that i) the Ssp4 Asp variant is required for extreme spore resistance of FP strains and ii) this protein may help protect FP strains via tighter DNA binding than the Ssp4 Gly variant. Our study provides important insights into the transmission of a common FP agent and may suggest strategies to interfere with resistant spores of FP strains. These findings may also have relevance for other pathogenic Clostridium spp. carrying an ssp4 gene.