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.
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.
| Kroh, Heather K; Chandrasekaran, Ramyavardhanee; Zhang, Zhifen et al. (2018) A neutralizing antibody that blocks delivery of the enzymatic cargo of Clostridium difficile toxin TcdB into host cells. J Biol Chem 293:941-952 |
| 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 |
| Chandrasekaran, Ramyavardhanee; Lacy, D Borden (2017) The role of toxins in Clostridium difficile infection. FEMS Microbiol Rev 41:723-750 |
| Gupta, Pulkit; Zhang, Zhifen; Sugiman-Marangos, Seiji N et al. (2017) Functional defects in Clostridium difficile TcdB toxin uptake identify CSPG4 receptor-binding determinants. J Biol Chem 292:17290-17301 |
| 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 |
| 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 |
| Smits, Wiep Klaas; Lyras, Dena; Lacy, D Borden et al. (2016) Clostridium difficile infection. Nat Rev Dis Primers 2:16020 |
| 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: |
| Chumbler, Nicole M; Rutherford, Stacey A; Zhang, Zhifen et al. (2016) Crystal structure of Clostridium difficile toxin A. Nat Microbiol 1:15002 |
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