The Molecular Basis for Clostridium Perfringens Spore Heat Resistance

NON-TECHNICAL SUMMARY: Clostridium perfringens type A food poisoning is caused by C. perfringens type A isolates carrying their enterotoxin gene (cpe) on the chromosome, while non-food-borne human gastrointestinal diseases are caused by isolates carrying cpe on extra-chromosomal DNA, i.e., plasmids. Our recent study suggested that the chromosomal cpe isolates are strongly associated with food poisoning because their cells and spores possess greater heat resistance than the cells and spores of plasmid cpe isolates. The molecular basis for this difference in heat resistance between chromosomal versus plasmid cpe isolates remains unclear. We hypothesize that the differences in heat resistance between spores of chromosomal versus plasmid cpe isolates may be largely attributable to the spores of chromosomal cpe isolates producing more small, acid-soluble spore proteins (SASPs) than the spores of plasmid cpe isolates. Our proposed project will investigate this hypothesis by comparing the genetics and expression of SASPs produced by C. perfringens isolates carrying chromosomal versus plasmid cpe genes. Determining the molecular basis for the differences in heat resistance between spores of C. perfringens isolates carrying chromosomal versus plasmid cpe genes holds profound implications for our understanding of the strong relationship between chromosomal cpe isolates and C. perfringens type A food poisoning. These studies may improve our abilities to prevent C. perfringens type A food poisoning which currently ranks as the third most commonly reported food-borne disease in the United States. <P>

APPROACH: Polymerase Chain Reaction (PCR) and Southern hybridization analyses will be performed to determine whether all three C. perfringens ssp genes are present in all cpe-positive C. perfringens isolates. Molecular cloning and sequencing of the ssp genes will be conducted to find out whether ssp gene sequences are highly conserved in C. perfringens isolates carrying either chromosomal or plasmid cpe genes. Quantitative Western blotting analysis will be performed to compare the production of SASPs by chromosomal versus plasmid cpe isolates. To examine the role of SASPs in protecting C. perfringens spores from heat damage, ssp knock-out mutants will be constructed by allelic exchange using our recently described double-antibiotic selection strategy. Briefly, entire ssp ORFs will be deleted and replaced with the chrolamphenicol resistance gene (catP). A suicidal mutator plasmid will be constructed by re-cloning the inactivated ssp-containing fragment into a plasmid carrying no origin of replication for clostridia. This mutator plasmid will be transformed into the cpe-positive C. perfringens strains by electroporation and the ssp double cross-over mutant will be selected by growing on antibiotic-containing plates. Once ssp knock-out mutants are available, the heat sensitivities of spores of ssp mutants will be compared with the heat sensitivities of spores of their wild type parents. Briefly, C. perfringens cultures will grown in Duncan-Strong (DS) medium for sporulation and determine the total viable spores per ml of DS culture at the start of heating. The DS culture will then be heated at either 90*C or 100*C for selected time periods, ranging from 1 min to 6 h, depending on the individual isolates and the temperature being used. At each time point, the viable spores present per ml of heated DS culture will be determined. These data will be graphed to determine D values (the decimal reduction value, i.e., the duration of time that a culture had to be kept at a given temperature to obtain a 90% reduction in viable cell numbers) for spores of each isolate tested.

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PROGRESS: 2002/09 TO 2007/08<BR>
OUTPUTS: Enterotoxin-producing C. perfringens isolates have been associated with C. perfringens type A food poisoning, which currently ranks as the third most commonly reported foodborne illness in the U.S. This single food poisoning affects more than 250,000 humans annually, and result in economical losses of over $120 million in the U.S. C. perfringens type A food poisoning is acquired when people consume a food (typically a beef or poultry product) contaminated with large numbers of vegetative cells of enterotoxigenic C. perfringens type A isolates. Recent studies suggested that the C. perfringens isolates are strongly associated with food poisoning because these isolates have the ability to form heat-resistant spores, which should enhance their survival in incompletely cooked or inadequately warmed foods. The central goal of our research is to determine the molecular basis for C. perfringens spore heat resistance. The significant outputs of our studies are: spore heat resistance is critical for C. perfringens to cause foodborne illness; thoroughly cooking food is the best approach to prevent and control C. perfringens type A foodborne illness; rapid cooling of cooked foods and then storing and serving these foods at nonpermissive conditions for vegetative growth of C. perfringens also a step for preventing C. perfringens type A foodborne illness. <BR>PARTICIPANTS: Pricipal investigator: Mahfuzur R. Sarker; Researchers: Nahid Sarker, Amanda Savoie, Mike Waters, Ben Harrison, Deepa Raju, I-Hsiu Huang and Daniel Paredes-Sabja; Collaborators: J. A. Torres (Department of Food Science and Technology, Oregon State University) and P. Setlow (Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center).
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IMPACT: 2002/09 TO 2007/08<BR>
Previous studies have shown that alpha/beta-type small, acid-soluble proteins (SASP) play a major role in the resistance of C. perfringens spores to moist heat, UV radiation and some chemicals. Additional major factors in B. subtilis spore resistance are the spore's core water content and cortex peptidoglycan (PG) structure, with the latter properties modulated by the spm and dacB gene products and the sporulation temperature. In our work we have shown that the spm and dacB genes are expressed only during C. perfringens sporulation and have examined the effects of spm and dacB mutations and sporulation temperature on spore core water content and spore resistance to moist heat, UV radiation and a number of chemicals. The results of these analyses indicate that for C. perfringens: (i) core water content and probably cortex PG structure has little if any role in spore resistance to UV and formaldehyde, presumably because these spore's DNA is saturated with alpha/beta-type SASP; (ii) spore resistance to moist heat and nitrous acid is determined to a large extent by core water content and probably cortex structure; (iii) core water content and cortex PG cross-linking play little or no role in spore resistance to hydrogen peroxide; (iv) spore core water content decreases with higher sporulation temperatures resulting in more moist heat resistant spores; and (v) factors in addition to SpmAB, DacB and sporulation temperature play roles in determining spore core water content and thus spore resistance to moist heat. These studies will contribute to the development of strategies to improve the health of humans and drive down the annual economic losses.