Clostridium perfringens type B, C and D isolates have significant medical, veterinary and biodefense importance. Many highly lethal toxins (such as beta toxin and epsilon toxin, a class B select toxin) produced by type B-D isolates are encoded by large plasmids. The long-term goal of this project is to understand the contributions of these large toxin-encoding plasmids and their encoded toxins to the pathogenicity of type B-D isolates in order to improve the design of vaccines/therapeutics against natural or bioterrorism-related human or animal infections. This work will also lead to development of subtyping assays for molecular epidemiologic or forensic investigations of natural or bioterrorism disease outbreaks involving type B-D isolates. It also has significant implications for understanding the virulence evolution of the major clostridial enteropathogens. To accomplish these goals, the following specific aims will be pursued, i) to determine which known toxins contribute to the pathogenicity of type B-D isolates, Aim A will continue constructing single and multiple toxin null mutants in type B-D backgrounds, using our recently-developed, highly efficient intron mutagenesis approaches;ii) Aim B will compare the pathogenicity of those toxin mutants versus their parent type B-D isolates using our recently optimized animal models that evaluate specific disease aspects, including enteric pathogenicity (using rabbit or goat ileal loops) or lethality (using mouse i.d. or gastric challenge models);when mutants show attenuated pathogenicity, they will be complemented to confirm the attenuation specifically resulted from inactivation of the implicated toxin gene, iii) since we have shown the epsilon toxin-encoding plasmid of type D isolates is conjugative, Aim C will examine whether the toxin plasmids of type B, C and E isolates are also conjugative (using mixed mating approaches), whether type B-D conjugative toxin plasmid transfer can occur in the intestines where this transfer may contribute to pathogenesis, whether C. perfringens can conjugatively exchange toxin plasmids or toxin genes with Clostridium difficile, another major clostridial enteropathogen, and whether certain C. perfringens toxin plasmids are incompatible with one another;and, finally, iv) Aim D will evaluate the genotypic diversity of toxin plasmids in type B and C isolates using pulsed- field gel/Southern blot and plasmid diversity in type B-D isolates using microarray approaches.

Public Health Relevance

Clostridium perfringens type B-D isolates have medical, veterinary, and biodefense importance because they produce a number of highly potent toxins such as epsilon toxin, a class B select toxin. To obtain critical information for developing improved vaccines or therapeutics against natural or bioterrorism-induced type B-D infections, this project will evaluate the contribution of individual known toxins to pathogenesis. In addition, since many toxins of type B-D isolates are encoded by large plasmids, we will study the diversity of the type B-D toxin plasmids in order to develop assays for epidemiologic or forensic purposes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI056177-08
Application #
8107668
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Ranallo, Ryan
Project Start
2003-07-15
Project End
2014-06-30
Budget Start
2011-07-01
Budget End
2012-06-30
Support Year
8
Fiscal Year
2011
Total Cost
$416,537
Indirect Cost
Name
University of Pittsburgh
Department
Genetics
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Garcia, J P; Giannitti, F; Finnie, J W et al. (2015) Comparative neuropathology of ovine enterotoxemia produced by Clostridium perfringens type D wild-type strain CN1020 and its genetically modified derivatives. Vet Pathol 52:465-75
Ma, Menglin; Li, Jihong; McClane, Bruce A (2015) Structure-function analysis of peptide signaling in the Clostridium perfringens Agr-like quorum sensing system. J Bacteriol 197:1807-18
Li, Jihong; Freedman, John C; McClane, Bruce A (2015) NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718. J Bacteriol 197:3339-53
Chen, Jianming; McClane, Bruce A (2015) Characterization of Clostridium perfringens TpeL toxin gene carriage, production, cytotoxic contributions, and trypsin sensitivity. Infect Immun 83:2369-81
Uzal, Francisco A; McClane, Bruce A; Cheung, Jackie K et al. (2015) Animal models to study the pathogenesis of human and animal Clostridium perfringens infections. Vet Microbiol 179:23-33
Freedman, John C; Theoret, James R; Wisniewski, Jessica A et al. (2015) Clostridium perfringens type A-E toxin plasmids. Res Microbiol 166:264-79
Theoret, James R; Uzal, Francisco A; McClane, Bruce A (2015) Identification and characterization of Clostridium perfringens beta toxin variants with differing trypsin sensitivity and in vitro cytotoxicity activity. Infect Immun 83:1477-86
Freedman, John C; Li, Jihong; Uzal, Francisco A et al. (2014) Proteolytic processing and activation of Clostridium perfringens epsilon toxin by caprine small intestinal contents. MBio 5:e01994-14
Uzal, Francisco A; Freedman, John C; Shrestha, Archana et al. (2014) Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease. Future Microbiol 9:361-77
Chen, Jianming; Ma, Menglin; Uzal, Francisco A et al. (2014) Host cell-induced signaling causes Clostridium perfringens to upregulate production of toxins important for intestinal infections. Gut Microbes 5:96-107

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