P-037. A Novel Small Acid Soluble Protein Variant Is Important for the Exceptional Spore Resistance of Most Clostridium perfringens Food Poisoning Isolates

B. A. McClane, J. Li;
Univ. of Pittsburgh Sch. of Med., Pittsburgh, PA.

Background: 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 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. Methods: A genome bioinformatics search identified a novel α/β-type small acid soluble protein, now named Ssp4. Sequencing of the FP ORF showed the isolates producing resistant spores were found to consistently express a variant Ssp4 with an Asp substitution at residue 36, as opposed to the Gly present at Ssp4 residue 36 in C. perfringens strains producing sensitive spores. Isogenic Ssp4 mutants were constructed with the targetron system. Purified recombinant Ssp4 was tested for DNA binding by gel shift assay and in vitro binding. Results: Isogenic Ssp4 mutants and complementing strains demonstrated the importance of Ssp4 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 C. perfringens DNA than does a Ssp4 variant with Gly at residue 36. Conclusion: These findings provide the first mechanistic explanation for the highly resistant spore phenotype of most FP strains carrying a chromosomal cpe gene and may facilitate development of targeted strategies to increase killing of these resistant spores in foods. They also induct that SASP variants can be important contributors to intra-species (and perhaps inter-species) variations in bacterial spore resistance phenotypes. Ssp4 may also contribute to spore resistance properties throughout the genus Clostridium since similar ssp4 genes were identified in the genomes of other clostridial species.