Source: OREGON STATE UNIVERSITY submitted to NRP
THE MOLECULAR BASIS FOR CLOSTRIDIUM PERFRINGENS SPORE HEAT RESISTANCE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0193208
Grant No.
2002-35201-12643
Cumulative Award Amt.
(N/A)
Proposal No.
2005-04775
Multistate No.
(N/A)
Project Start Date
Sep 1, 2002
Project End Date
Aug 31, 2007
Grant Year
2005
Program Code
[32.0]- (N/A)
Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331
Performing Department
COLLEGE OF VETERINARY MEDICINE
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.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71240101100100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
The central goal of the project is to determine the molecular basis for the differences in heat resistance between spores of C. perfringens isolates carrying chromosomal versus plasmid enterotoxin (cpe) genes. 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. To investigate this hypothesis, three specific aims will be conducted. Aim #1 will evaluate whether any difference exist between SASP genetics of C. perfringens chromosomal versus plasmid cpe isolates. Fulfilling this aim will answer the following questions, i) are all three ssp genes present in all cpe-positive C. pefringens isolates? ii) does the ssp RFLP (Restriction Fragment Length Polymorphism) pattern of chromosomal cpe isolates differ from that of plasmid cpe isolates? and finally, iii) are the ssp ORF sequences highly conserved in C. perfringens isolates carrying either chromosomal or plasmid cpe genes? Aim#2 will compare the level of SASP production by C. perfringens isolates carrying chromosomal versus plasmid cpe genes. We will first i) prepare the antibodies against C. perfringens a/b-type SASPs and then ii) perform quantitative Western blotting analyses of SASPs produced by chromosomal versus plasmid cpe isolates using C. perfringens a/b-type SASP antibodies. The final aim will examine the role of SASPs in protecting C. perfringens spores from heat damage by constructing the ssp knock-out mutants and comparing the heat sensitivities of spores of mutants with the heat sensitivities of spores of their wild-type parents.
Project Methods
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.

Progress 09/01/02 to 08/31/07

Outputs
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. 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).

Impacts
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.

Publications

  • Raju D., P. Setlow, and M. R. Sarker. 2007. Antisense RNA-Mediated Decreased Synthesis of Small, Acid-Soluble Spore Proteins Leads to Decreased Resistance of Clostridium perfringens Spores to Moist Heat and UV Radiation. Appl. Env. Microbiol. 73: 2048-2053.
  • Hwang, H. J., J. C. Lee, Y. Yammoto, M. R. Sarker, T. Tsuchiya, K. Oguma. 2007. Identification of structural genes of Clostrdium botulinum type C neurotoxin-converting phage particles. FEMS Microbiol. Lett. 270: 82-89.
  • Raju D., and M. R. Sarker. 2007. Production of small, acid-soluble spore proteins in Clostridium perfringens nonfoodborne gastrointestinal disease isolates. Can. J. Microbiol. 53: 514-518.
  • Huang, I., D. Raju, D. Paredes-Sabja, and M. R. Sarker. 2007. Clostridium perfringens: Sporulation, Spore Resistance and Germination. Bangladesh J. Microbiol. 24: 1-8.
  • Paredes-Sabja, D., M. Gonzalez, M. R. Sarker and J. A. Torres. 2007. Combined effects of hydrostatic pressure, temperature and pH on the inactivation of spores of Clostridium perfringens type A and Clostridium sporogenes in buffer solutions. J. Food Sci. 72: M202-M206.
  • Leyva-Illades, J. F., B. Setlow, M. R. Sarker, and P. Setlow. 2007. Effect of a small, acid-soluble spore protein from Clostridium perfringens on the resistance properties of Bacillus subtilis spores. J. Bacteriol. 189: 7927-7931.
  • Mendez, M., I. Huang, K. Ohtani, R. R. Grau, T. Shimizu, and M. R. Sarker 2007. Catabolite repression of Clostridium perfringens gliding motility. J. Bacteriol.190: 48-60.
  • Paredes-Sabja, D., J. A. Torres, P. Setlow, and M. R. Sarker. 2007. Clostridium perfringens spore germination: characterization of germinants and their receptors. J. Bacteriol. (In Press).
  • Paredes-Sabja, D., D. Raju, J. A. Torres, and M. R. Sarker. 2007. Role of Small, Acid-soluble Spore Proteins in the Resistance of Clostridium perfringens Spores to Chemicals. Intl. J. Food Microbiol. (In Press).
  • Raju D., P. Setlow, and M. R. Sarker. 2007. Antisense RNA-Mediated Decreased Synthesis of Small, Acid-Soluble Spore Proteins Leads to Decreased Resistance of Clostridium perfringens Spores to Moist Heat and UV Radiation. In "Proceedings of the 107th Annual General Meeting of the American Society for Microbiology", Toronto, Ontario, Canada, May 21-25, 2007.


Progress 01/01/06 to 12/31/06

Outputs
Previous work has suggested that a group of alpha/beta-type small, acid-soluble spore proteins (SASP) is involved in the resistance of Clostridium perfringens spores to moist-heat. However, this suggestion is based on the analysis of C. perfringens spores lacking only one of the three genes encoding alpha/beta-type SASP in this organism. We have now used antisense RNA (asRNA) to decrease levels of alpha/beta-type SASP in C. perfringens spores by 90%. These spores had significantly reduced resistance to both moist heat and UV radiation, although not to dry heat. These results clearly demonstrate the important role for alpha/beta-type SASP in the resistance of C. perfringens spores.

Impacts
C. perfringens type A food poisoning currently ranks as the third most commonly reported food-boren diseases in the USA. This single food poisoning affects more than 250,000 humans annually, and result in economical losses of over $120 million in the USA. 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. These studies will contribute to the development of strategies to improve the health of humans and drive down the annual economic losses.

Publications

  • Philippe, V. A., M. B. Mendez, I. Huang, L. M. Orsaria, M. R. Sarker and R. R. Grau. 2006. Inorganic Phosphate Induces Spore Morphogenesis and Enterotoxin Production in the Intestinal Pathogen Clostridium perfringens. Infect. Immun.74: 3651-3656.
  • Raju D., M. Waters, P. Setlow, and M. R. Sarker. 2006. Investigating the Role of Small, Acid-Soluble Spore Proteins in the Resistance of Clostridium perfringens Spores to Heat. BMC Microbiol. 6:50.
  • Huang, I., and M. R. Sarker. 2006. Complementation of Clostridium perfringens spo0A Mutant with Wild-type spo0A Gene from other Clostridium species. Appl. Env. Microbiol.72: 6388-6393.
  • Huang, I., M. Mendez, R. Grau, and M. R. Sarker. 2006. Spo0A is Essential for Biofilm Formation and Swarming Motility in the Anaerobic Pathogen Clostridium perfringens. In "Proceedings of the Fifth International Conference on Molecular biology and Pathogenesis of Clostridia", Nottingham, UK., June 21-25, 2006.
  • Huang, I., and M. R. Sarker. 2006. Complementation of Clostridium perfringens spo0A Mutant with Wild-type spo0A from other Clostridium Species. In "Proceedings of the Fifth International Conference on Molecular biology and Pathogenesis of Clostridia", Nottingham, UK., June 21-25, 2006.
  • Sarker, M. R., I. Huang, R. Grau and D. Raju. 2006. Clostridium perfringens Sporulation. In "Proceedings of the Fifth International Conference on Molecular biology and Pathogenesis of Clostridia", Nottingham, UK., June 21-25, 2006.


Progress 01/01/05 to 12/31/05

Outputs
Previous studies have indicated that Clostridium perfringens isolates are strongly associated with food poisoning (FP) because these isolates have the ability to form heat-resistant spores, which regulates the synthesis of enterotoxin (CPE) as well as facilitates the survival of C. perfringens isolates in incompletely-cooked, or inadequately-warmed, foods. To investigate the molecular basis for C. perfringens spore formation and its role in heat resistance, we performed research in the following areas: A) Comparison of the Heat Resistance of Wild-type, cpe Knock-out and cpe Plasmid-cured Clostridium perfringens Type A: Recent studies suggested that the specific association between chromosomal cpe isolates and FP is attributable, at least in part, to the chromosomal cpe isolates being considerably more heat resistant than plasmid cpe isolates, which favors their survival in incompletely cooked or inadequately warmed foods. The basis for the differences of heat resistance between FP versus non-foof-borne gastrointestinal disease (NFBGID) isolates remains unknown. However, at least two possible explanations for this phenomenon can be envisioned. First, since a previous study revealed differences in heat resistance between CPE-producing versus non CPE-producing FP isolates, it is possible that CPE expression is involved in mediating high heat resistance to C. perfringens. In contrast, CPE-producing NFBGID isolates exhibit less heat resistance than that of FP isolates because these isolates carry a large 75-kb cpe plasmid that might encode gene(s) conferring heat sensitivity. To evaluate these two hypothesis, in this study we cured the cpe plasmid from a NFBGID isolate and compared the heat resistance of wild-type, cpe knock-out and cpe plasmid-cured C. perfringens type A. Our results demonstrated that i) wild-type cpe has no role in mediating high heat resistance to C. perfringens, and ii) cpe plasmid does not carry gene(s) that might confer heat sensitivity to NFBGID isolates B) Investigating the Role of Small Acid-soluble Spore Proteins (SASPs) in C. perfringens Spore Heat Resistance: It has been reported that C. perfringens alpha/beta SASPs are encoded by only three ssp genes (ssp1, ssp2 and ssp3). Our PCR and Nucleotide sequencing analyses showed that all three ssp ORF sequences are highly conserved in our surveyed chromosomal and plasmid cpe isolates. To evaluate the contribution of SASPs to the heat resistance of spores, we constructed an isogenic ssp3 knock-out mutant of a C. perfringens type A chromosomal cpe isolate. The spores of the ssp3 mutant were found to exhibit a slightly lower decimal reduction value than exhibited by the spores of wild type strain. Ongoing double and triple ssp knock-out studies should fully address the contributions of SASPs to C. perfringens spore heat resistance.

Impacts
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 USA. This single food poisoning affects more than 250,000 humans annually, and result in economical losses of over $120 million in the USA. 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 sporulation and its role in heat resistance. These studies will contribute to the development of strategies to improve the health of humans and drive down the annual economic loss.

Publications

  • Fisher, D. J., K. Miyamoto, B. Harrison, M. R, Sarker, and B. A. McClane. 2005. Association of Beta2 Toxin Production with Clostridium perfringens Type A Human Gastrointestinal Disease Isolates Carrying a Plasmid Enterotoxin Gene. Molec. Microbiol. 65: 747-762.
  • Waters, M., D. Raju, H. S. Garmory, M. R. Popoff, and M. R. Sarker. 2005. Regulated Expression of Beta2-toxin gene (cpb2) in Clostridium perfringens Isolates Associated with Horse Gastrointestinal Diseases. J. Clin. Microbiol. 43: 4002-4009.
  • Raju, D. and M. R. Sarker. 2005. Comparison of the Levels of Heat Resistance of Wild-type, cpe Knock-out and cpe Plasmid-cured Clostridium perfringens Type A Strains. Appl. Env. Microbiol. 71: 7618-7620.
  • Harrison, B., D. Raju, H. S. Garmory, M. M. Brett, R. W. Titball and M. R. Sarker. 2005. Molecular Characterization of Clostridium perfringens Isolates from Humans with Infectious Intestinal Diseases: Evidence for Transcriptional Regulation of Beta2-toxin Gene. Appl. Env. Microbiol. In Press.
  • Raju, D., I. Huang, M. Waters, and M. R. Sarker. 2005. Transcriptional Analysis of the spo0VD-sigG Locus in Enterotoxigenic Clostridium perfringens Type A and Characterization of a pilT Knock-out Mutant. Abstract in "Proceedings of the 105th Annual General Meeting of the American Society for Microbiology", Atlanta, GA., June 5-9, 2005.
  • Huang, I., M. Waters, and M. R. Sarker, 2005. Complementation Studies on Clostridium perfringens spo0A Mutant using spo0A from other Clostridium Species. Abstract in "Proceedings of the 105th Annual General Meeting of the American Society for Microbiology", Atlanta, GA., June 5-9, 2005.
  • Sarker, M. R., M. Waters, H. S. Garmory, M. R. Popoff and D. Raju. 2005. Regulated Expression of the Beta2-Toxin gene (cpb2) in Clostridium perfringens Type A Isolates from Horses with Gastrointestinal Diseases. Abstract in "Proceedings of the 105th Annual General Meeting of the American Society for Microbiology", Atlanta, GA., June 5-9, 2005.


Progress 01/01/04 to 12/31/04

Outputs
To investigate the molecular basis for C. perfringens spore heat resistance, we compared the genetics and expression of small acid soluble proteins (SASPs) produced by C. perfringens isolates carrying chromosomal versus extrachromosomal cpe gene. The rationale for studying SASPs is that since Bacillus subtilis SASPs can protect their spores from heat damage, we hypothesized that C. perfringens SASPs may play a similar role in spore heat resistance. It has been reported that C. perfringens alpha/beta-type SASPs are encoded by only three ssp genes (ssp1, ssp2 and ssp3) and comprise a protein family of Mol. wt. 6-7 kDa. Our PCR analyses, using three ssp-specific primers on DNA isolated from 5 chromosomal and 5 plasmid cpe isolates, demonstrated that all three ssp genes are present in all of our surveyed C. perfringens isolates. Nucleotide sequencing analyses of the PCR amplified ssp genes showed that all three ssp ORF sequences are highly conserved in our surveyed C. perfringens chromosomal and plasmid cpe isolates. These results suggest that all three ssp genes are present in most, or all, cpe-positive C. perfringens type A. Western blot analysis of SASPs produced our surveyed C. perfringens isolates, using antibodies against B. subtilis SASPs, demonstrated the expression of SASPs in both chromosomal and plasmid cpe isolates. These results suggested that no obvious differences exist between the genetics and expression of SASP in C. perfringens chromosomal versus plasmid cpe isolates. In order to evaluate the contribution of SASPs to the heat resistance of C. perfringens spores, in our current study we constructed an isogenic ssp3 knock-out mutant of a C. perfringens type A chromosomal cpe isolate. When the heat sensitivities of spores produced by ssp3 knock-out mutant was compared with that of spores of the wild type strain, spores of the ssp3 mutant were found to exhibit a slightly lower decimal reduction value than exhibited by the spores of wild type strain. However, this effect could be restored by complementing the ssp3 mutant with a recombinant plasmid carrying the wild type ssp3. Collectively, these results suggest that SASPs may play a role in the resistance of C. perfringens spores to heat. Ongoing double (ssp1 and ssp3) and triple (ssp1, ssp2 and ssp3) ssp knock-out studies should fully address the contributions of SASPs to C. perfringens spore heat resistance.

Impacts
For over 30 years, 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 USA. This single food poisoning affects over 600,000 Americans per year and results in annual economic losses of over $120 million dollars. 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. These studies will provide important insights into the pathogenesis of enterotoxigenic C. perfringens isolates, and may improve our abilities to prevent C. perfringens type A food poisoning.

Publications

  • Huang, I., M. Waters, R. R. Grau, and M. R. Sarker. 2004. Disruption of the Gene (spo0A) Encoding Sporulation Transcription Factor Blocks Endospore Formation and Enterotoxin Production in Enterotoxigenic Clostridium perfringens. FEMS Microbiol. Lett. 233: 233-240.
  • Raju, D., M. Waters, P. Setlow, and M. R. Sarker, 2004. Role of small acid-soluble spore proteins (SASPs) in Clostridium perfringens spore heat resistance. Abstract in "Proceedings of the 104th Annual General Meeting of the American Society for Microbiology", New Oleans, LA., May 23-27, 2004.
  • Huang, I., M. Waters, R. R. Grau, and M. R. Sarker. 2004. Disruption of the Gene (spo0A) Encoding Sporulation Transcription Factor Blocks Endospore Formation and Enterotoxin Production in Enterotoxigenic Clostridium perfringens Type A. Abstract in "Proceedings of the 104th Annual General Meeting of the American Society for Microbiology", New Oleans, LA., May 23-27, 2004.


Progress 01/01/03 to 12/31/03

Outputs
To investigate the molecular basis for C. perfringens spore heat resistance, we compared the genetics and expression of small acid-soluble spore proteins (SASPs) produced by C. perfringens isolates carrying chromosomal versus extrachromosomal cpe gene. The rationale for studying SASPs is that since Bacillus subtilis SASPs can protect their spores from heat damage, we hypothesized that C. perfringens SASPs may play a similar role in spore heat resistance. It has been reported that C. perfringens alpha/beta SASPs are encoded by only three ssp genes (ssp1, ssp2 and ssp3) and comprise a protein family of Mol. wt. 6-7 kDa. Our PCR analyses, using three ssp-specific primers on DNA isolated from 5 chromosomal and 5 plasmid cpe isolates, demonstrated that all three ssp genes are present in all of our surveyed C. perfringens isolates. Nucleotide sequencing analyses of the PCR amplified ssp genes showed that all three ssp ORF sequences are highly conserved in our surveyed C. perfringens chromosomal and plasmid cpe isolates. These results suggest that all three ssp genes are present in most, or all, cpe-positive C. perfringens type A. Western blot analysis of SASPs produced by our surveyed C. perfringens isolates, using antibodies against B. subtilis SASPs, demonstrated the expression of SASPs in both chromosomal and plasmid cpe isolates. These results suggested that no obvious differences exist between the genetics and expression of SASPs in C. perfringens chromosomal versus plasmid cpe isolates. In order to evaluate the contribution of SASPs to the heat resistance of C. perfringens spores, in our current study we constructed an isogenic ssp3 knock-out mutant of a C. perfringens type A chromosomal cpe isolate. When the heat sensitivities of spores produced by ssp3 knock-out mutant was compared with that of spores of the wild type strain, spores of the ssp3 mutant were found to exhibit a slightly lower decimal reduction value at 100 degree celcius than exhibited by the spores of wild type strain. Ongoing double (ssp1 and ssp3) and triple (ssp1, ssp2 and ssp3) ssp knock-out studies should fully address the contributions of SASPs to C. perfringens spore heat resistance.

Impacts
For over 30 years, 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 USA. This single food poisoning affects over 600,000 Americans per year and results in annual economic losses of over $120 million dollars. 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. These studies will provide important insights into the pathogenesis of enterotoxigenic C. perfringens isolates, and may improve our abilities to prevent C. perfringens type A food poisoning.

Publications

  • Waters, M., Savoie, A., Garmory, H., Bueschel, D., Popoff, M. R., Songer, J. G., Titball, R. W., McClane, B. A., and Sarker, M. R. 2003. Genotyping an Phenotyping of Beta2-Toxigenic Clostridium perfringens Fecal Isolates Associated with Gastrointestinal Diseases in Piglets. J. Clin. Microbiol. 41: 3584-3591.
  • Raju, D., Waters, M., Setlow, P., and Sarker, M. R. 2003. Evaluation of the role of small acid-soluble spore proteins (SASPs) in Clostridium perfringens spore heat resistance. Abstract in "Proceedings of the Fourth International Conference on Molecular biology and Pathogenesis of Clostridia", Woods Hole, MA., April 26-30, 2003.
  • Huang, I., Waters, M., Grau, R. R., and Sarker, M. R. 2003. Characterization of spo0A homologue in enterotoxigenic Clostridium perfringens type A. Abstract in "Proceedings of the Fourth International Conference on Molecular biology and Pathogenesis of Clostridia", Woods Hole, MA., April 26-30, 2003.
  • Raju, D., Waters, M., Setlow, P., and Sarker, M. R. 2004. Role of small acid-soluble spore proteins (SASPs) in Clostridium perfringens spore heat resistance. Abstract in "Proceedings of the 104th Annual General Meeting of the American Society for Microbiology", New Oleans, LA., May 23-27, 2004.
  • Huang, I., Waters, M., Grau, R. R., and Sarker, M. R. 2004. Disruption of the Gene (spo0A) Encoding Sporulation Transcription Factor Blocks Endospore Formation and Enterotoxin Production in Enterotoxigenic Clostridium perfringens Type A. Abstract in "Proceedings of the 104th Annual General Meeting of the American Society for Microbiology", New Oleans, LA., May 23-27, 2004.