Enteropathogenic Escherichia coli dephosphorylates and dissociates occludin from intestinal epithelial tight junctions.
Enteropathogenic Escherichia coli (EPEC) increases tight junction permeability in part by phosphorylating the 20 kDa myosin light chain (MLC20) that induces cytoskeletal contraction. The impact of this enteric pathogen on specific tight junction (TJ) proteins has not been investigated. We examined the effect of EPEC infection on occludin localization and phosphorylation in intestinal epithelial cells. After infection by EPEC, a progressive shift of occludin from a primarily TJ-associated domain to an intracellular compartment occurred, as demonstrated by immunofluorescent staining. A reverse in the ratio of phosphorylated to dephosphorylated occludin accompanied this morphological change. Eradication of EPEC with gentamicin resulted in the normalization of occludin localization and phosphorylation. The serine/threonine phosphatase inhibitor, calyculin A, prevented these events. The EPEC-associated decrease in transepithelial electrical resistance, a measure of TJ barrier function, returned to baseline after gentamicin treatment. Non-pathogenic E. coli, K-12, did not induce these changes. Transformation of K-12 with the pathogenicity island of EPEC, however, conferred the phenotype of wild-type EPEC. Deletion of specific EPEC genes encoding proteins involved in EPEC type III secretion markedly attenuated these effects. These findings suggest that EPEC-induced alterations in occludin contribute to the pathophysiology associated with this infection.
Simonovic
I
I
Department of Medicine, University of Illinois and West Side Department of Veterans Administration Medical Center, Chicago, IL 60612, USA.
Rosenberg
J
J
Koutsouris
A
A
Hecht
G
G
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000472:pathogenicity*; D005455:Fluorescent Antibody Technique; D017353:Gene Deletion; D005839:Gentamicins / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism* / Q000382:microbiology; D008387:Marine Toxins; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism*; D062793:Occludin; D010080:Oxazoles / Q000494:pharmacology; D010766:Phosphorylation; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D014169:Transformation, Bacterial; D014407:Tumor Cells, Cultured; D014774:Virulence
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D005839:Gentamicins; D008387:Marine Toxins; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010080:Oxazoles; C059041:calyculin A