Abstract
Enteropathogenic Escherichia coli (EPEC) uses a type three secretion system to inject effector proteins into host intestinal epithelial cells, causing diarrhea. EPEC induces the formation of pedestals underlying attached bacteria, disrupts tight junction (TJ) structure and function, and alters apico-basal polarity by redistributing the polarity proteins Crb3 and Pals1, although the mechanisms are unknown. Here we investigate the temporal relationship of PAR polarity complex and TJ disruption following EPEC infection. EPEC recruits active aPKCζ, a PAR polarity protein, to actin within pedestals and at the plasma membrane prior to disrupting TJ. The EPEC effector EspF binds the endocytic protein sorting nexin 9 (SNX9). This interaction impacts actin pedestal organization, recruitment of active aPKCζ to actin at cell–cell borders, endocytosis of JAM-A S285 and occludin, and TJ barrier function. Collectively, data presented herein support the hypothesis that EPEC-induced perturbation of TJ is a downstream effect of disruption of the PAR complex and that EspF binding to SNX9 contributes to this phenotype. aPKCζ phosphorylates polarity and TJ proteins and participates in actin dynamics. Therefore, the early recruitment of aPKCζ to EPEC pedestals and increased interaction with actin at the membrane may destabilize polarity complexes ultimately resulting in perturbation of TJ.
Highlights
Enteropathogenic Escherichia coli (EPEC) delivers bacterial effector proteins into host intestinal epithelial cells (IECs) through a type III secretion system (TTSS), inducing actin pedestal formation, attaching and effacing lesions, and physiological changes in IECs that contribute to diarrhea [1]
The present study provides evidence that EPEC perturbs PAR polarity complex integrity and induces the recruitment of aPKCζ to actin pedestals almost immediately following EPEC attachment
The sorting nexin 9 (SNX9)-binding domain of EspF is important for the recruitment and organization of p-aPKCζ–T560 organization within EPEC pedestals, and the endocytosis of tight junctions (TJ) proteins
Summary
Enteropathogenic Escherichia coli (EPEC) delivers bacterial effector proteins into host intestinal epithelial cells (IECs) through a type III secretion system (TTSS), inducing actin pedestal formation, attaching and effacing lesions, and physiological changes in IECs that contribute to diarrhea [1]. TJ are localized at the most apical region of the lateral membrane and constitute a paracellular diffusion barrier modulating the flow of ions and solutes. These structures consist of integral membrane proteins (claudin family, occludin, tricellulin, MarvelD3, and JAM-A) that interact with adhesion molecules of adjacent cells and with intracellular domains that associate with cytoplasmic adaptor proteins (MAGUK family, cingulin, paracingulin, MAGI-1-3, and MUPP-1) [4,5]. Apico-basal polarity contributes to cell morphology, directional vesicle transportation, ion and solute transport, and specific localization of proteins and lipids to different membrane domains [6,7]
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