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EPEC Perturbation of Crb3 and Pals1 Localization Precedes Tight Junction Disruption
Author(s) -
Tapia Rocio,
Kralicek Sarah,
Hecht Gail
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.1043.10
Subject(s) - cell polarity , epithelial polarity , microbiology and biotechnology , tight junction , cytoplasm , chemistry , biology , biophysics , membrane , cell , biochemistry
Enteropathogenic Escherichia coli (EPEC) alters both the architecture and barrier function of intestinal tight junctions (TJ). EPEC also perturbs the apical‐basal polarity of intestinal cells, demonstrated by redistribution of the basolateral proteins Na + /K + ATPase and β1‐integrin to the apical membrane domain and the formation of aberrant TJ strands throughout the lateral membrane. TJ are crucial for the establishment and maintenance of epithelial apical‐basal polarity. The apical Crumbs (Crb) complex (Crb3/Pals1/Patj) is essential for epithelial cell polarization and TJ assembly. We hypothesized that EPEC disrupts intestinal barrier function via perturbation of Crb polarity complex. The aim of this study was to determine the impact of EPEC on Crb polarity complex in vivo and in vitro . Our results indicate that EPEC redistributes Crb3 and Pals1, but not Patj, from the membrane to the cytoplasm of colonic epithelial cells. Depletion of espF , but not map , ablates the cytoplasmic accumulation of Crb3. Complementation of espF (Δ espF /p espF ) restores the wild‐type EPEC phenotype of Crb3 internalization. Depletion of both espF and map , attenuates the redistribution of Pals1, while complemented strains (Δ espF /p espF and Δ map /p map ) reverted this phenotype. Immunofluoresce intensity of murine colonic tissues showed that EPEC significantly reduces the level of membrane‐associated Crb3 and Pals1 (−36.0±4.0% and −38.0±3.0%; p<0.001; n=3, respectively) and increases cytoplasmic accumulation (82.0±5.0% and 106.0±6.0%; p<0.001; n=3, respectively) compared to uninfected mice. To determine the temporal sequence of Crb3 and TJ disruption, SKCO‐15 monolayers were infected and Crb3 and occludin localization and transepithelial resistance (TER) were analyzed over time. Crb3 internalization occurred as early as 1 hr yet occludin was not disrupted until 3 hr post‐infection. Decrease in TER occurred as early as ‐‐2hr (−28.0±7.0% vs +11.0±6.0% change from baseline, respectively; n=3, ***p<0.0001) and continued to decline over the course of infection dropping 41.0±8.0% from baseline vs 14±9.0% increase of controls from baseline at 3 hr (n=3, ***p<0.0001). These results show that EPEC‐induced perturbation of Crb3 and Pals1 precedes alteration of TJ structure and function and may contribute to TJ disruption.