Research Article: Crk Adaptors Negatively Regulate Actin Polymerization in Pedestals Formed by Enteropathogenic Escherichia coli (EPEC) by Binding to Tir Effector

Date Published: March 27, 2014

Publisher: Public Library of Science

Author(s): Elvira Nieto-Pelegrin, Eugenia Meiler, José Manuel Martín-Villa, María Benito-León, Narcisa Martinez-Quiles, Guy Tran Van Nhieu.


Infections by enteropathogenic Escherichia coli (EPEC) cause diarrhea linked to high infant mortality in developing countries. EPEC adheres to epithelial cells and induces the formation of actin pedestals. Actin polymerization is driven fundamentally through signaling mediated by Tir bacterial effector protein, which inserts in the plasma membrane of the infected cell. Tir binds Nck adaptor proteins, which in turn recruit and activate N-WASP, a ubiquitous member of the Wiskott-Aldrich syndrome family of proteins. N-WASP activates the Arp2/3 complex to promote actin polymerization. Other proteins aside from components of the Tir-Nck-N-WASP pathway are recruited to the pedestals but their functions are unknown. Here we investigate the function of two alternatively spliced isoforms of Crk adaptors (CrkI/II) and the paralog protein CrkL during pedestal formation by EPEC. We found that the Crk isoforms act as redundant inhibitors of pedestal formation. The SH2 domain of CrkII and CrkL binds to phosphorylated tyrosine 474 of Tir and competes with Nck to bind Tir, preventing its recruitment to pedestals and thereby inhibiting actin polymerization. EPEC infection induces phosphorylation of the major regulatory tyrosine in CrkII and CrkL, possibly preventing the SH2 domain of these proteins from interacting with Tir. Phosphorylated CrkII and CrkL proteins localize specifically to the plasma membrane in contact with EPEC. Our study uncovers a novel role for Crk adaptors at pedestals, opening a new perspective in how these oncoproteins regulate actin polymerization.

Partial Text

Enteropathogenic Escherichia coli (EPEC) causes infant diarrhea worldwide and is a leading cause of death in developing countries. EPEC adheres to intestinal epithelial cells, causing local disappearance of microvilli and altering cell permeability, giving rise to what are classically known as attaching and effacing (A/E) lesions [1]. At A/E lesions, EPEC attaches to host cells and induces the formation of actin-rich structures called pedestals. Although the ultimate function of pedestals is not completely understood, disrupting genes critical for pedestal formation diminishes colonization and subsequent disease in humans [2] and animal models [3]. Pedestals may facilitate EPEC growth and residence inside the intestine by allowing the bacteria to remain attached to the epithelium during peristalsis and host responses to infection [4].

Numerous pathogens have evolved mechanisms to subvert host control of actin polymerization for their own benefit. EPEC manipulates the host actin cytoskeleton from outside the cell, making it a powerful model system to study eukaryotic phosphotyrosine signaling events in response to external stimuli [40]. Several host cell proteins implicated in cytoskeletal remodeling, including Crk adaptor proteins, are recruited to the site of EPEC attachment [12]. In the present study we aimed to investigate a possible role of Crk adaptors in pedestal formation by EPEC.




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