The Clostridium perfringens epsilon toxin, a Category B Select Agent, is responsible for a severe, often fatal enterotoxemia characterized by cardiac, pulmonary, kidney, and brain edema. The mechanism by which epsilon toxin acts is incompletely understood. However, it is believed that toxin monomers bind to specific receptors on sensitive cells, assemble into oligomeric complexes, and form pores in the cell membrane. We hypothesize that inhibitors can be prepared that block one or more of these key steps in the process by which the Clostridium perfringens epsilon toxin mediates cytotoxic effects.
The specific aims of this proposal are designed to develop inhibitors of epsilon toxin activity by (1) identifying dominant-negative mutants and (2) identifying small molecule inhibitors.
In aim 1, amino acid deletions, insertions, and substitutions will be introduced into the gene encoding epsilon toxin, with special emphasis placed on a region of the protein believed to insert into the target cell membrane. Recombinant mutant proteins will be examined for both a lack of cytotoxicity and for the ability to inhibit the cytotoxic activity of wild-type epsilon toxin. Mutants identified in this aim will increase our understanding of the structure-function relationships that govern epsilon toxin activity, will provide insight into the nature of dominant-negative mutant toxins, and identify new therapeutic candidates for countering exposure to epsilon toxin.
In aim 2, a high-throughput screen will be used to identify small molecules that inhibit the cytotoxic activity of epsilon toxin. In addition to identifying novel inhibitors, this aim will validate high-throughput assays that may be used in future studies to test additional compounds for the ability to either inhibit toxin activity or mitigate the effects of the toxin. The inhibitors identified in this R21 Exploratory/Developmental proposal will provide the foundation for future studies aimed at optimizing the inhibitory activities, examining the mechanisms of inhibition, and testing the effectiveness of the inhibitors using established animal models. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI065435-02
Application #
7244032
Study Section
Special Emphasis Panel (ZRG1-IDM-A (90))
Program Officer
Van de Verg, Lillian L
Project Start
2006-06-01
Project End
2008-05-31
Budget Start
2007-06-01
Budget End
2008-05-31
Support Year
2
Fiscal Year
2007
Total Cost
$149,008
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
Ivie, Susan E; Fennessey, Christine M; Sheng, Jinsong et al. (2011) Gene-trap mutagenesis identifies mammalian genes contributing to intoxication by Clostridium perfringens ?-toxin. PLoS One 6:e17787
Lewis, Michelle; Weaver, Charles David; McClain, Mark S (2010) Identification of Small Molecule Inhibitors of Clostridium perfringens ?-Toxin Cytotoxicity Using a Cell-Based High-Throughput Screen. Toxins (Basel) 2:1825-1847
Pelish, Teal M; McClain, Mark S (2009) Dominant-negative inhibitors of the Clostridium perfringens epsilon-toxin. J Biol Chem 284:29446-53
McClain, Mark S; Cover, Timothy L (2007) Functional analysis of neutralizing antibodies against Clostridium perfringens epsilon-toxin. Infect Immun 75:1785-93