Clostridium difficile infection (CDI) is an important source of morbidity and mortality among U.S. Military Veterans. The primary virulence factors are TcdA and TcdB, toxins that induce diarrhea, inflammation, and significant damage within the colon. This proposal is designed around the hypothesis that inhibition of toxin activity represents a therapeutic approach that can impact clinical treatment and outcome for individuals suffering from CDI. TcdA and TcdB are homologous glucosyltransferases that modify and inactivate Rho family GTPases. The glucosyltransferase activity of the toxins has been linked to a `cytopathic' disruption of the acti cytoskeleton and is important for systemic toxicity in a mouse intoxication model. The X-ray crystal structures of small molecule glucosyltransferase inhibitors bound to the TcdA and TcdB glucosyltransferase domains (Aim 1) will provide a foundation for structure-guided design of molecules with enhanced potency. In addition to the cytopathic effects, TcdB is a potent cytotoxin that causes necrotic damage in cells and tissue. The cytotoxicity results from TcdB-induced assembly of the epithelial cell NADPH oxidase (NOX) complex. The assembly results in the production of reactive oxygen species (ROS), which cause necrosis. Preliminary data indicate that a Nox1 knockout mouse is protected from CDI tissue damage and underscores the hypothesis that inhibition of the NOX1 pathway will protect against the colonic tissue damage observed in severe cases of CDI. A high-throughput screen has led to the identification of 176 small molecules that inhibit TcdB-induced necrosis. Experiments in Aim 2 will categorize these compounds according to their mechanism of action and result in the identification of lead compounds for further analysis.
In Aim 3, the efficacy of N- acetylcysteine, an FDA-approved antioxidant, along with lead compounds from Aims 1 and 2 will be evaluated in a mouse model of CDI. Spore challenge with an epidemic M7404 strain and a panel of variants with defined mutations in one or both toxins will permit dissection of the specific effect each compound has on TcdA- and TcdB-mediated events. These are the key studies needed to advance small molecule inhibitors of the C. difficile toxins into clinical practice.

Public Health Relevance

Clostridium difficile is the leading cause of nosocomial diarrhea in the United States and an important source of morbidity and mortality among U.S. Military Veterans. C. difficile infection (CDI) can result in a spectrum of diseases that range from mild diarrhea to pseudomembranous colitis and can include life- threatening complications such as perforation of the colon, toxic megacolon, and sepsis. Problems with current treatment strategies include issues with relapse, recurrence, and instances where antibiotics fail to alter the disease process (no response). CDI pathology is associated with the action of two toxins, TcdA and TcdB. The toxins and the pathways they use to cause disease, therefore, represent attractive targets for the development of novel therapeutics that can be used either to prevent relapse and recurrence or to prevent the life-threatening aspects of existing infections.

Agency
National Institute of Health (NIH)
Institute
Veterans Affairs (VA)
Type
Non-HHS Research Projects (I01)
Project #
5I01BX002943-04
Application #
9605227
Study Section
Infectious Diseases B (INFB)
Project Start
2015-10-01
Project End
2019-09-30
Budget Start
2018-10-01
Budget End
2019-09-30
Support Year
4
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Veterans Health Administration
Department
Type
DUNS #
156385783
City
Nashville
State
TN
Country
United States
Zip Code
37212
Kroh, Heather K; Chandrasekaran, Ramyavardhanee; Rosenthal, Kim et al. (2017) Use of a neutralizing antibody helps identify structural features critical for binding of Clostridium difficile toxin TcdA to the host cell surface. J Biol Chem 292:14401-14412
Alvin, Joseph W; Lacy, D Borden (2017) Clostridium difficile toxin glucosyltransferase domains in complex with a non-hydrolyzable UDP-glucose analogue. J Struct Biol 198:203-209
Chumbler, Nicole M; Rutherford, Stacey A; Zhang, Zhifen et al. (2016) Crystal structure of Clostridium difficile toxin A. Nat Microbiol 1:15002
Chumbler, Nicole M; Farrow, Melissa A; Lapierre, Lynne A et al. (2016) Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms. Infect Immun 84:2871-7
Chandrasekaran, Ramyavardhanee; Kenworthy, Anne K; Lacy, D Borden (2016) Clostridium difficile Toxin A Undergoes Clathrin-Independent, PACSIN2-Dependent Endocytosis. PLoS Pathog 12:e1006070
Chumbler, Nicole M; Rutherford, Stacey A; Zhang, Zhifen et al. (2016) Crystal structure of Clostridium difficile toxin A. Nat Microbiol 1: