title: High temporal resolution of glucosyltransferase dependent and independent effects of Clostridium difficile toxins across multiple cell types.
abstract: BACKGROUND
  Clostridium difficile toxins A and B (TcdA and TcdB), considered to be essential for C. difficile infection, affect the morphology of several cell types with different potencies and timing. However, morphological changes over various time scales are poorly characterized. The toxins' glucosyltransferase domains are critical to their deleterious effects, and cell responses to glucosyltransferase-independent activities are incompletely understood. By tracking morphological changes of multiple cell types to C. difficile toxins with high temporal resolution, cellular responses to TcdA, TcdB, and a glucosyltransferase-deficient TcdB (gdTcdB) are elucidated.
  
  
  RESULTS
  Human umbilical vein endothelial cells, J774 macrophage-like cells, and four epithelial cell lines (HCT8, T84, CHO, and immortalized mouse cecal epithelial cells) were treated with TcdA, TcdB, gdTcdB. Impedance across cell cultures was measured to track changes in cell morphology. Metrics from impedance data, developed to quantify rapid and long-lasting responses, produced standard curves with wide dynamic ranges that defined cell line sensitivities. Except for T84 cells, all cell lines were most sensitive to TcdB. J774 macrophages stretched and increased in size in response to TcdA and TcdB but not gdTcdB. High concentrations of TcdB and gdTcdB (>10 ng/ml) greatly reduced macrophage viability. In HCT8 cells, gdTcdB did not induce a rapid cytopathic effect, yet it delayed TcdA and TcdB's rapid effects. gdTcdB did not clearly delay TcdA or TcdB's toxin-induced effects on macrophages.
  
  
  CONCLUSIONS
  Epithelial and endothelial cells have similar responses to toxins yet differ in timing and degree. Relative potencies of TcdA and TcdB in mouse epithelial cells in vitro do not correlate with potencies in vivo. TcdB requires glucosyltransferase activity to cause macrophages to spread, but cell death from high TcdB concentrations is glucosyltransferase-independent. Competition experiments with gdTcdB in epithelial cells confirm common TcdA and TcdB mechanisms, yet different responses of macrophages to TcdA and TcdB suggest different, additional mechanisms or targets in these cells. This first-time, precise quantification of the response of multiple cell lines to TcdA and TcdB provides a comparative framework for delineating the roles of different cell types and toxin-host interactions.
authors: Department of Biomedical Engineering, University of Virginia, PO Box 800759, Charlottesville, VA, 22908, USA. kd3jd@virginia.edu.
authors: Kevin M
authors: KM
authors: D'Auria
authors: Department of Biomedical Engineering, University of Virginia, PO Box 800759, Charlottesville, VA, 22908, USA. mjb8vv@virginia.edu.|Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, PO Box 801340, Charlottesville, VA, 22908, USA. mjb8vv@virginia.edu.
authors: Meghan J
authors: MJ
authors: Bloom
authors: Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, PO Box 801340, Charlottesville, VA, 22908, USA. yesenia.reyes@utdallas.edu.
authors: Yesenia
authors: Y
authors: Reyes
authors: Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, PO Box 801340, Charlottesville, VA, 22908, USA. mrc6r@virginia.edu.
authors: Mary C
authors: MC
authors: Gray
authors: Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, PO Box 801340, Charlottesville, VA, 22908, USA. edward.j.van.opstal@vanderbilt.edu.|Current address: Vanderbilt University School of Medicine, 340 Light Hall, Nashville, TN, 27232, USA. edward.j.van.opstal@vanderbilt.edu.
authors: Edward J
authors: EJ
authors: van Opstal
authors: Department of Biomedical Engineering, University of Virginia, PO Box 800759, Charlottesville, VA, 22908, USA. papin@virginia.edu.
authors: Jason A
authors: JA
authors: Papin
authors: Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, PO Box 801340, Charlottesville, VA, 22908, USA. eh2v@virginia.edu.
authors: Erik L
authors: EL
authors: Hewlett
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D016360:Clostridioides difficile / Q000378:metabolism*; D055724:Electrophysiological Phenomena / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects* / Q000502:physiology; D004768:Enterotoxins / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects* / Q000502:physiology; D005964:Glucosyltransferases / Q000378:metabolism*; D006801:Humans; D008264:Macrophages / Q000187:drug effects* / Q000502:physiology; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D005964:Glucosyltransferases
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