Clostridium difficile is a gram-positive, spore-forming anaerobe that infects the colon, causing a range of human disease including diarrhea, pseudomembranous colitis, and toxic megacolon. The incidence, severity, and costs associated with C. difficile infection (CDI) are increasing, making C. difficile a significant public health concern. The principle virulence factors in C. difficile pathogenesis are TcdA and TcdB, two large homologous toxins capable of entering and modifying multiple targets within eukaryotic host cells. This proposal is designed to follow up two important discoveries made in the previous funding cycle, a crystal structure of TcdA and the cellular receptor for TcdB.
In Aim 1, we will use the structure of the TcdA delivery domain as a framework for understanding toxin pore formation within the host endosomal membrane. A hybrid approach of electron microscopy, crosslinking, and fluorescence intensity measurements will generate a structural view of the toxin pore.
In Aim 2, we will define the interaction of TcdB with its receptor, PVRL3, using a hybrid of electron microscopy, mutagenesis, quantitative binding studies, and X-ray crystallography.
In Aim 3, we will evaluate the relevance of the TcdB-PVRL3 interaction in mouse models of intoxication and infection. Results from these aims will provide a molecular understanding of the events that allow toxins access to the host cell and a framework for therapeutic intervention.

Public Health Relevance

Clostridium difficile is a toxin-producing bacterium that is a frequent cause of hospital-acquired and antibiotic-associated diarrhea. The incidence, severity, and costs associated with C. difficile infection (CDI) are increasing, making C. difficile a significant public health concern. The goal of the proposed project is to delineate the structural and molecular mechanisms by which the two primary toxins, TcdA and TcdB, gain access to host cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI095755-10
Application #
9688159
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Ranallo, Ryan
Project Start
2011-05-15
Project End
2021-04-30
Budget Start
2019-05-01
Budget End
2020-04-30
Support Year
10
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Type
DUNS #
079917897
City
Nashville
State
TN
Country
United States
Zip Code
37232
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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
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Chumbler, Nicole M; Rutherford, Stacey A; Zhang, Zhifen et al. (2016) Crystal structure of Clostridium difficile toxin A. Nat Microbiol 1:15002
Craven, Ryan; Lacy, D Borden (2016) Clostridium sordellii Lethal-Toxin Autoprocessing and Membrane Localization Activities Drive GTPase Glucosylation Profiles in Endothelial Cells. mSphere 1:

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