title: Enteropathogenic Escherichia coli outer membrane proteins induce changes in cadherin junctions of Caco-2 cells through activation of PKCalpha.
abstract: Enteropathogenic Escherichia coli (EPEC) is a Gram-negative bacterial pathogen that adheres to human intestinal epithelial cells, resulting in watery, persistent diarrhoea. Despite the advances made in understanding EPEC-host cell interactions, the molecular mechanisms underlying watery diarrhoea have not been understood fully. Loss of transepithelial resistance and increased monolayer permeability by disruption of tight junctions has been implicated in this process. Apart from disruption of tight junctions, an important factor known to regulate monolayer permeability is E-cadherin and its interaction with beta-catenin, both of which constitute the adherens junctions. Our previous studies using HEp-2 cells demonstrated the morphological and cytoskeletal changes caused by cell-free outer membrane preparations (OMPs) of EPEC. In this study, we have shown that EPEC and its OMP induce significant changes in the adherens junctions of Caco-2 monolayers. We also observed significant phosphorylation of protein kinase Calpha (PKCalpha) in cells treated with either whole EPEC or its OMP. Immunoprecipitation of cell lysates with anti-E-cadherin and probing with phospho-PKCalpha monoclonal antibodies and anti-beta-catenins revealed that in these cells, phosphorylated PKCalpha is associated with cadherins, leading to the dissociation of the cadherin/beta-catenin complex. Immunofluorescence showed beta-catenins dissociated from the membrane-bound cadherins and redistributed into the cytoplasm. Expression of dominant negative PKCalpha reversed these effects caused by either whole EPEC or its OMP and also reduced the associated increase in monolayer permeability. It is possible that this mechanism may complement the earlier known pathways for loss of barrier function involving myosin light chain kinase activation and also may play a role in causing host cell death by apoptosis.
authors.affiliation: Centre for Biotechnology, Anna University, 600 025, Chennai, India.
authors.forename: Vasantha
authors.initials: V
authors.lastname: Malladi
authors.forename: Balakrishna
authors.initials: B
authors.lastname: Shankar
authors.forename: Peter H
authors.initials: PH
authors.lastname: Williams
authors.forename: Arun
authors.initials: A
authors.lastname: Balakrishnan
mesh_terms: D022005:Adherens Junctions / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000494:pharmacology; D001692:Biological Transport; D018938:Caco-2 Cells; D015820:Cadherins / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004403:Dysentery / Q000382:microbiology; D004789:Enzyme Activation; D004927:Escherichia coli Infections / Q000382:microbiology; D019453:Escherichia coli O157 / Q000737:chemistry / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D010539:Permeability / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D015534:Trans-Activators / Q000378:metabolism; D051176:beta Catenin
chemical_list: D001425:Bacterial Outer Membrane Proteins; C495270:CTNNB1 protein, human; D015820:Cadherins; D003598:Cytoskeletal Proteins; D029968:Escherichia coli Proteins; C502403:FAT1 protein, human; D007527:Isoenzymes; D015534:Trans-Activators; D051176:beta Catenin; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha