title: Clostridium difficile Toxin A Undergoes Clathrin-Independent, PACSIN2-Dependent Endocytosis.
abstract: 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. TcdB has been shown to enter cells by clathrin-dependent endocytosis, but the mechanism of TcdA uptake is still unclear. Here, we utilize a combination of RNAi-based knockdown, pharmacological inhibition, and cell imaging approaches to investigate the endocytic mechanism(s) that contribute to TcdA uptake and subsequent cytopathic and cytotoxic effects. We show that TcdA uptake and cellular intoxication is dynamin-dependent but does not involve clathrin- or caveolae-mediated endocytosis. Confocal microscopy using fluorescently labeled TcdA shows significant colocalization of the toxin with PACSIN2-positive structures in cells during entry. Disruption of PACSIN2 function by RNAi-based knockdown approaches inhibits TcdA uptake and toxin-induced downstream effects in cells indicating that TcdA entry is PACSIN2-dependent. We conclude that TcdA and TcdB utilize distinct endocytic mechanisms to intoxicate host cells.
authors.affiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, United States of America.
authors.forename: Ramyavardhanee
authors.identifier: http://orcid.org/0000-0002-6620-0422
authors.initials: R
authors.lastname: Chandrasekaran
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, United States of America.|Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, United States of America.|Epithelial Biology Program, Vanderbilt University School of Medicine, Nashville, TN, United States of America.
authors.forename: Anne K
authors.initials: AK
authors.lastname: Kenworthy
authors.affiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, United States of America.|Epithelial Biology Program, Vanderbilt University School of Medicine, Nashville, TN, United States of America.|The Veterans Affairs Tennessee Valley Healthcare System, Nashville, TN, United States of America.
authors.forename: D Borden
authors.identifier: http://orcid.org/0000-0003-2273-8121
authors.initials: DB
authors.lastname: Lacy
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D015153:Blotting, Western; D018938:Caco-2 Cells; D002966:Clathrin; D016360:Clostridioides difficile; D003015:Clostridium Infections / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D004768:Enterotoxins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D051379:Mice; D018613:Microscopy, Confocal; D021381:Protein Transport / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D037521:Virulence Factors / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D001427:Bacterial Toxins; D002966:Clathrin; D004768:Enterotoxins; C120714:PACSIN2 protein, human; D037521:Virulence Factors; C041348:tcdA protein, Clostridium difficile