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Title page for ETD etd-11092017-150619

Type of Document Dissertation
Author Chandrasekaran, Ramyavardhanee
URN etd-11092017-150619
Title Mechanism of Clostridium difficile Toxin A Entry into Host Cells
Degree PhD
Department Microbiology and Immunology
Advisory Committee
Advisor Name Title
Eric P. Skaar Committee Chair
Anne K. Kenworthy Committee Member
Christopher R. Aiken Committee Member
D. Borden Lacy Committee Member
Matthew J. Tyska Committee Member
  • genetic screen
  • receptor-binding
  • endocytosis
  • toxins
  • Clostridium difficile
Date of Defense 2017-11-07
Availability restricted
Clostridium difficile infection affects a significant number of hospitalized patients in the United States. Two homologous exotoxins, TcdA and TcdB, are the major virulence factors in C. difficile pathogenesis. The toxins are glucosyltransferases that inactivate Rho family-GTPases to disrupt host cellular function and cause fluid secretion, inflammation, and cell death. Toxicity depends on receptor binding and subsequent endocytosis. While the receptors and endocytic pathway utilized by TcdB for host cell intoxication are known, the mechanism of TcdA uptake remained unclear. Utilizing a combination of RNAi-based knockdown, pharmacological inhibition, and cell imaging approaches, we show that TcdA and TcdB utilize distinct endocytic mechanisms to intoxicate host cells. Unlike TcdB, which enters by the clathrin pathway, TcdA uptake and cellular intoxication occurs by a novel clathrin- and caveolae-independent mechanism that requires PACSIN2 and dynamin. Furthermore, our recent efforts to characterize the mechanism of neutralization of PA50, a humanized monoclonal anti-TcdA antibody, by X-ray crystallography, electron microscopy, and cell functional assays have identified regions within the C-terminus of TcdA that may promote toxin binding to the host cell surface. Current efforts are focused on identifying receptor molecules and additional host factors critical for TcdA entry and intoxication.

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