title: Aerolysin from Aeromonas hydrophila perturbs tight junction integrity and cell lesion repair in intestinal epithelial HT-29/B6 cells.
abstract: BACKGROUND
  Aeromonads cause a variety of infections, including gastroenteritis, sepsis, and wound necrosis. Pathogenesis of Aeromonas hydrophila and its hemolysin has been characterized, but the mechanism of the epithelial barrier dysfunction is currently poorly understood.
  
  
  METHODS
  Human colon epithelial monolayers HT-29/B6 were apically inoculated with clinical isolates of A. hydrophila or with the secreted pore-forming toxin aerolysin. Epithelial resistance and permeability for several markers were determined in Ussing chambers, using 2-path impedance spectroscopy. The subcellular distribution of tight junction (TJ) and cytoskeleton proteins was analyzed by Western blotting and confocal laser-scanning microscopy.
  
  
  RESULTS
  A. hydrophila infection induces chloride secretion with a small decrease in transcellular resistance. However, the major effect of A. hydrophila, mediated by its toxin aerolysin, was a sustained reduction of paracellular resistance by retracting sealing TJ proteins from the TJ strands. Aerolysin-treated monolayers showed increased paracellular permeability to FITC-dextran-4000 (0.104 ± 0.014 vs 0.047 ± 0.001 10(-6) cm/s in control; P < .05). Moreover, restitution of epithelial lesions was impaired. The effects were myosin light chain kinase (MLCK) dependent and mediated by intracellular Ca(2+) signaling.
  
  
  CONCLUSIONS
  During Aeromonas infection, pore formation by aerolysin impairs epithelial integrity by promoting TJ protein redistribution and consequently affecting wound closure. Thus, Aeromonas-induced diarrhea is mediated by 2 mechanisms, transcellular secretion and paracellular leak flux.
authors.affiliation: Department of Gastroenterology, Infectious Diseases, and Rheumotology, Charité Berlin, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Bücker
authors.forename: Susanne M
authors.initials: SM
authors.lastname: Krug
authors.forename: Rita
authors.initials: R
authors.lastname: Rosenthal
authors.forename: Dorothee
authors.initials: D
authors.lastname: Günzel
authors.forename: Anja
authors.initials: A
authors.lastname: Fromm
authors.forename: Martin
authors.initials: M
authors.lastname: Zeitz
authors.forename: Trinad
authors.initials: T
authors.lastname: Chakraborty
authors.forename: Michael
authors.initials: M
authors.lastname: Fromm
authors.forename: Hans-Jörg
authors.initials: HJ
authors.lastname: Epple
authors.forename: Jörg-Dieter
authors.initials: JD
authors.lastname: Schulzke
mesh_terms: D016980:Aeromonas hydrophila / Q000378:metabolism / Q000502:physiology*; D001427:Bacterial Toxins; D015153:Blotting, Western; D002118:Calcium / Q000502:physiology; D002463:Cell Membrane Permeability / Q000502:physiology*; D003967:Diarrhea / Q000382:microbiology* / Q000503:physiopathology; D058266:Dielectric Spectroscopy; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology / Q000503:physiopathology*; D019073:HT29 Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000382:microbiology* / Q000503:physiopathology; D008856:Microscopy, Fluorescence; D009219:Myosin-Light-Chain Kinase / Q000502:physiology; D052899:Pore Forming Cytotoxic Proteins / Q000502:physiology*; D019108:Tight Junctions / Q000502:physiology*
chemical_list: D001427:Bacterial Toxins; D052899:Pore Forming Cytotoxic Proteins; C032676:aerolysin; D009219:Myosin-Light-Chain Kinase; D002118:Calcium