Enteropathogenic Escherichia coli infection leads to appearance of aberrant tight junctions strands in the lateral membrane of intestinal epithelial cells

Infection of intestinal epithelial cells with enteropathogenic Escherichia coli (EPEC) disrupts tight junction (TJ) architecture and barrier function. The aim of this study was to determine the impact of EPEC on TJ protein interactions and localization. Human intestinal epithelial cells (T84) were infected for 1, 3 or 6 h with EPEC. To probe the TJ protein–protein interactions, co‐immunoprecipitations were performed. The associations between ZO‐1, occludin and claudin‐1 progressively decreased after infection. Corresponding morphological changes were analysed by immunofluorescence confocal microscopy. Tight junction proteins progressively lost their apically restricted localization. Freeze‐fracture electron microscopy revealed the appearance of aberrant strands throughout the lateral membrane that contained claudin‐1 and occludin as determined by immunogold labelling. These structural alterations were accompanied by a loss of barrier function. Mutation of the gene encoding EspF, important in the disruption of TJs by EPEC, prevented the disruption of TJs. Tight junction structure normalized following eradication of EPEC with gentamicin and overnight recovery. This is the first demonstration that a microbial pathogen can cause aberrant TJ strands in the lateral membrane of host cells. We speculate that the disruption of integral and cytoplasmic TJ protein interactions following EPEC infection allows TJ strands to form or diffuse into the lateral plasma membrane.

[1]  Torsten Schöneberg,et al.  Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells , 2002, Journal of Cell Science.

[2]  S. Lees-Miller,et al.  Intestinal infection with Giardia spp. reduces epithelial barrier function in a myosin light chain kinase-dependent fashion. , 2002, Gastroenterology.

[3]  S. Tsukita,et al.  Claudin-based barrier in simple and stratified cellular sheets. , 2002, Current opinion in cell biology.

[4]  C. V. Van Itallie,et al.  Claudins create charge-selective channels in the paracellular pathway between epithelial cells. , 2002, American journal of physiology. Cell physiology.

[5]  T. Sakaguchi,et al.  Shigella flexneri regulates tight junction‐associated proteins in human intestinal epithelial cells , 2002, Cellular microbiology.

[6]  T. Inai,et al.  Formation of tight junction strands by expression of claudin-1 mutants in their ZO-1 binding site in MDCK cells , 2002, Histochemistry and Cell Biology.

[7]  L. Shoshani,et al.  Biogenesis of Epithelial Polarity and Tight Junctions , 2001 .

[8]  G. Meer,et al.  Tight Junctions and Cell Surface Lipid Polarity , 2001 .

[9]  Shoichiro Tsukita,et al.  Multifunctional strands in tight junctions , 2001, Nature Reviews Molecular Cell Biology.

[10]  P. Verkade,et al.  Clostridium difficile Toxins Disrupt Epithelial Barrier Function by Altering Membrane Microdomain Localization of Tight Junction Proteins , 2001, Infection and Immunity.

[11]  J. Nougayrède,et al.  Translocated EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function. , 2001, The Journal of clinical investigation.

[12]  E. Schneeberger,et al.  Inducible expression of claudin-1-myc but not occludin-VSV-G results in aberrant tight junction strand formation in MDCK cells. , 2000, Journal of cell science.

[13]  J. Rosenberg,et al.  Enteropathogenic Escherichia coli dephosphorylates and dissociates occludin from intestinal epithelial tight junctions , 2000, Cellular microbiology.

[14]  S. Tsukita,et al.  Clostridium perfringens enterotoxin binds to the second extracellular loop of claudin‐3, a tight junction integral membrane protein , 2000, FEBS letters.

[15]  C. V. Van Itallie,et al.  CaCo-2 Cells Treated with Clostridium perfringensEnterotoxin Form Multiple Large Complex Species, One of Which Contains the Tight Junction Protein Occludin* , 2000, The Journal of Biological Chemistry.

[16]  S. Tsukita,et al.  Pores in the Wall , 2000, The Journal of cell biology.

[17]  B. P. McNamara,et al.  Enteropathogenic E. coli secreted protein F is required for alteration of host intestinal epithelial barrier function , 2000 .

[18]  K. Magnusson,et al.  Distinct effects of Vibrio cholerae haemagglutinin/protease on the structure and localization of the tight junction‐associated proteins occludin and ZO‐1 , 2000, Cellular microbiology.

[19]  Yusuke Nakamura,et al.  Localization of membrane-associated guanylate kinase (MAGI)-1/BAI-associated protein (BAP) 1 at tight junctions of epithelial cells , 1999, Oncogene.

[20]  M. Itoh,et al.  Direct Binding of Three Tight Junction-Associated Maguks, Zo-1, Zo-2, and Zo-3, with the Cooh Termini of Claudins , 1999, The Journal of cell biology.

[21]  Erika S Wittchen,et al.  Protein Interactions at the Tight Junction , 1999, The Journal of Biological Chemistry.

[22]  S. Tsukita,et al.  Manner of Interaction of Heterogeneous Claudin Species within and between Tight Junction Strands , 1999, The Journal of cell biology.

[23]  G. Hecht,et al.  Myosin regulation of NKCC1: effects on cAMP-mediated Cl- secretion in intestinal epithelia. , 1999, American journal of physiology. Cell physiology.

[24]  B. Kachar,et al.  Redistribution and Phosphorylation of Occludin During Opening and Resealing of Tight Junctions in Cultured Epithelial Cells , 1999, The Journal of Membrane Biology.

[25]  S. Nigam,et al.  Role of tyrosine phosphorylation in the reassembly of occludin and other tight junction proteins. , 1999, American journal of physiology. Renal physiology.

[26]  G. Hecht,et al.  Enteropathogenic E. coli attenuates secretagogue-induced net intestinal ion transport but not Cl- secretion. , 1999, American journal of physiology. Gastrointestinal and liver physiology.

[27]  K. Fujimoto,et al.  Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[28]  C. Sears,et al.  Bacteroides fragilis enterotoxin cleaves the zonula adherens protein, E-cadherin. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[29]  K. Fujimoto,et al.  A Single Gene Product, Claudin-1 or -2, Reconstitutes Tight Junction Strands and Recruits Occludin in Fibroblasts , 1998, The Journal of cell biology.

[30]  K. Kemphues,et al.  An Atypical PKC Directly Associates and Colocalizes at the Epithelial Tight Junction with ASIP, a Mammalian Homologue of Caenorhabditis elegans Polarity Protein PAR-3 , 1998, The Journal of cell biology.

[31]  B. P. McNamara,et al.  A novel proline-rich protein, EspF, is secreted from enteropathogenic Escherichia coli via the type III export pathway. , 1998, FEMS microbiology letters.

[32]  L. Kelland.  Signal transduction pathways. , 1998, IDrugs : the investigational drugs journal.

[33]  Kazushi Fujimoto,et al.  Claudin-1 and -2: Novel Integral Membrane Proteins Localizing at Tight Junctions with No Sequence Similarity to Occludin , 1998, The Journal of cell biology.

[34]  L. Gu,et al.  ZO-3, a Novel Member of the MAGUK Protein Family Found at the Tight Junction, Interacts with ZO-1 and Occludin , 1998, The Journal of cell biology.

[35]  James P. Nataro,et al.  Diarrheagenic Escherichia coli , 1998, Clinical Microbiology Reviews.

[36]  D. Philpott,et al.  Signal Transduction Pathways Involved in Enterohemorrhagic Escherichia coli-Induced Alterations in T84 Epithelial Permeability , 1998, Infection and Immunity.

[37]  T. Chakraborty,et al.  EspE, a novel secreted protein of attaching and effacing bacteria, is directly translocated into infected host cells, where it appears as a tyrosine‐phosphorylated 90 kDa protein , 1998, Molecular microbiology.

[38]  S. Savkovic,et al.  Enteropathogenic Escherichia coli-induced myosin light chain phosphorylation alters intestinal epithelial permeability. , 1997, Gastroenterology.

[39]  V. Wong.  Phosphorylation of occludin correlates with occludin localization and function at the tight junction. , 1997, American journal of physiology. Cell physiology.

[40]  B. Finlay,et al.  Enteropathogenic E. coli (EPEC) Transfers Its Receptor for Intimate Adherence into Mammalian Cells , 1997, Cell.

[41]  J. Katahira,et al.  Clostridium perfringens Enterotoxin Utilizes Two Structurally Related Membrane Proteins as Functional Receptors in Vivo * , 1997, The Journal of Biological Chemistry.

[42]  S. Savkovic,et al.  Activation of NF-κB in intestinal epithelial cells by enteropathogenic Escherichia coli. , 1997, American journal of physiology. Cell physiology.

[43]  D. Clark,et al.  Bacteroides fragilis toxin exhibits polar activity on monolayers of human intestinal epithelial cells (T84 cells) in vitro , 1997, Infection and immunity.

[44]  B. Finlay,et al.  Intimin-dependent binding of enteropathogenic Escherichia coli to host cells triggers novel signaling events, including tyrosine phosphorylation of phospholipase C-gamma1 , 1997, Infection and immunity.

[45]  M. Saitou,et al.  Possible Involvement of Phosphorylation of Occludin in Tight Junction Formation , 1997, The Journal of cell biology.

[46]  B. Gumbiner,et al.  A Synthetic Peptide Corresponding to the Extracellular Domain of Occludin Perturbs the Tight Junction Permeability Barrier , 1997, The Journal of cell biology.

[47]  S. Savkovic,et al.  Attachment of a noninvasive enteric pathogen, enteropathogenic Escherichia coli, to cultured human intestinal epithelial monolayers induces transmigration of neutrophils , 1996, Infection and immunity.

[48]  R. D. Lynch,et al.  Occludin is a functional component of the tight junction. , 1996, Journal of cell science.

[49]  M. Balda,et al.  Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical- basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein , 1996, The Journal of cell biology.

[50]  M. Itoh,et al.  Interspecies diversity of the occludin sequence: cDNA cloning of human, mouse, dog, and rat-kangaroo homologues , 1996, The Journal of cell biology.

[51]  D. Philpott,et al.  Infection of T84 cells with enteropathogenic Escherichia coli alters barrier and transport functions. , 1996, The American journal of physiology.

[52]  K. Fujimoto.  Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Application to the immunogold labeling of intercellular junctional complexes. , 1995, Journal of cell science.

[53]  C. V. Van Itallie,et al.  Tight junctions and the molecular basis for regulation of paracellular permeability. , 1995, The American journal of physiology.

[54]  R. Sartor.  Current concepts of the etiology and pathogenesis of ulcerative colitis and Crohn's disease. , 1995, Gastroenterology clinics of North America.

[55]  R. Gilbert,et al.  Involvement of Ras-related Rho proteins in the mechanisms of action of Clostridium difficile toxin A and toxin B , 1995, Infection and immunity.

[56]  K. Aktories,et al.  The low molecular mass GTP-binding protein Rho is affected by toxin A from Clostridium difficile. , 1995, The Journal of clinical investigation.

[57]  T. McDaniel,et al.  A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[58]  J. Alverdy,et al.  Enteropathogenic Escherichia coli adherence to intestinal epithelial monolayers diminishes barrier function. , 1995, The American journal of physiology.

[59]  K. Aktories,et al.  Clostridium difficile toxin B acts on the GTP-binding protein Rho. , 1994, The Journal of biological chemistry.

[60]  M. Itoh,et al.  Occludin: a novel integral membrane protein localizing at tight junctions , 1993, The Journal of cell biology.

[61]  B. Gumbiner.  Breaking through the tight junction barrier , 1993, The Journal of cell biology.

[62]  B. Finlay,et al.  Enteropathogenic Escherichia coli decreases the transepithelial electrical resistance of polarized epithelial monolayers , 1993, Infection and immunity.

[63]  S. Colgan,et al.  A simple approach to measurement of electrical parameters of cultured epithelial monolayers: Use in assessing neutrophil-epithelial interactions , 1992 .

[64]  P. Williams,et al.  Purification of a 20 kDa phosphoprotein from epithelial cells and identification as a myosin light chain Phosphorylation induced by enteropathogenicEscherichia coli and phorbol ester , 1991, FEBS letters.

[65]  P. Williams,et al.  Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli , 1989, Infection and immunity.

[66]  S. Knutton,et al.  Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa , 1987, Infection and immunity.

[67]  J. Siliciano,et al.  Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (zonula occludens) in a variety of epithelia , 1986, The Journal of cell biology.

[68]  L. Staehelin.  Further observations on the fine structure of freeze-cleaved tight junctions. , 1973, Journal of cell science.

[69]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[70]  G. Palade,et al.  JUNCTIONAL COMPLEXES IN VARIOUS EPITHELIA , 1963, The Journal of cell biology.

[71]  P. Nava,et al.  Tight junction proteins. , 2003, Progress in biophysics and molecular biology.

[72]  園田 紀之.  Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands : Evidence for direct involvement of claudins in tight junction barrier , 2002 .

[73]  小林 順一.  Formation of tight junction strands by expression of claudin-1 mutants in their ZO-1 binding site in MDCK cells , 2002 .

[74]  S. Savkovic,et al.  Activation of NFk B in intestinal epithelial cells by enteropathogenic Escherichia coli , 1997 .