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 associated disease are substantial and 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 modifying multiple targets within eukaryotic host cells. The toxins contain four functional domains that are associated with host-receptor binding, delivery across a membrane, autoprocessing, and the enzymatic inactivation of host Rho-proteins. The interaction with receptors, the kinetics of delivery, the ease of autoprocessing, and the specificity for cellular substrates are affected by sequence variation among different toxin isoforms and distinct environmental conditions within the host cell. The central objective of the proposed research program is to elucidate structural and molecular mechanisms of toxin function. We propose a hybrid approach that combines cryo-electron microscopy (cryo-EM), X-ray crystallography, biochemical and cell-based functional studies.
In Aim 1, we will elucidate a structure of the TcdA holotoxin using cryo-EM and probe the multiple conformational stages that are accessed as the toxin is exposed to host environmental cues (low pH, inostol-6-phosphate and reductant).
In Aim 2, we will determine the X-ray crystal structure of the TcdA delivery domain and identify the sequences that are associated with membrane pore formation.
In Aim 3, we will compare the glucosyltransferase properties of TcdA to those of TcdB. A mechanistic understanding of the conserved and divergent features of these two toxins will provide a necessary platform for therapeutic inhibitor design.

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 associated disease are substantial and increasing, making C. difficile a significant public health concern. The goal of the proposed project is to identify the structural and molecular mechanisms by which the two primary toxins, TcdA and TcdB, disrupt host cell function.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI095755-04
Application #
8651869
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Ranallo, Ryan
Project Start
2011-05-15
Project End
2016-04-30
Budget Start
2014-05-01
Budget End
2015-04-30
Support Year
4
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Nashville
State
TN
Country
United States
Zip Code
37212
Chumbler, Nicole M; Rutherford, Stacey A; Zhang, Zhifen et al. (2016) Crystal structure of Clostridium difficile toxin A. Nat Microbiol 1:
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:
Quesada-Gómez, Carlos; López-Ureña, Diana; Chumbler, Nicole et al. (2016) Analysis of TcdB Proteins within the Hypervirulent Clade 2 Reveals an Impact of RhoA Glucosylation on Clostridium difficile Proinflammatory Activities. Infect Immun 84:856-65
LaFrance, Michelle E; Farrow, Melissa A; Chandrasekaran, Ramyavardhanee et al. (2015) Identification of an epithelial cell receptor responsible for Clostridium difficile TcdB-induced cytotoxicity. Proc Natl Acad Sci U S A 112:7073-8
Wang, Xia; Yamamoto, Yusuke; Wilson, Lane H et al. (2015) Cloning and variation of ground state intestinal stem cells. Nature 522:173-8
Zhang, Zhifen; Park, Minyoung; Tam, John et al. (2014) Translocation domain mutations affecting cellular toxicity identify the Clostridium difficile toxin B pore. Proc Natl Acad Sci U S A 111:3721-6
Farrow, Melissa A; Chumbler, Nicole M; Lapierre, Lynne A et al. (2013) Clostridium difficile toxin B-induced necrosis is mediated by the host epithelial cell NADPH oxidase complex. Proc Natl Acad Sci U S A 110:18674-9
Pruitt, Rory N; Chumbler, Nicole M; Rutherford, Stacey A et al. (2012) Structural determinants of Clostridium difficile toxin A glucosyltransferase activity. J Biol Chem 287:8013-20
Chumbler, Nicole M; Farrow, Melissa A; Lapierre, Lynne A et al. (2012) Clostridium difficile Toxin B causes epithelial cell necrosis through an autoprocessing-independent mechanism. PLoS Pathog 8:e1003072

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