Enteropathogenic Escherichia coli EspG disrupts microtubules and in conjunction with Orf3 enhances perturbation of the tight junction barrier

Abstract

EspG, a secreted effector of enteropathogenic Escherichia coli (EPEC), as well as its homologue Orf3, has been shown to disrupt microtubules (MTs) in fibroblasts and non-polarized epithelial cells. The roles of MTs and the effects of MT disruption in these cell types differ significantly. The aim of this study was to investigate the effects of EspG on polarized, host target intestinal epithelial cells. Immunofluorescent labelling of tubulin showed that EPEC caused progressive fragmentation and loss of the MT network in cells harbouring attached organisms. Immunoblots of proteins extracted from EPEC-infected cells showed a corresponding loss of alpha-tubulin. Type III secretion system (TTSS)-deficient strains had no effect on MT suggesting TTSS dependence. Mutation of espG, but not espF or map, ablated EPEC's effects on MTs for up to 6 h. Ectopic expression of EspG in HeLa cells caused MT disruption. While deletion of espG alone had no effect on the EPEC-induced decrease in transepithelial electrical resistance (TER), mutation of both espG and orf3 significantly delayed the kinetics of this response. Complementation of the double mutant with espG alone restored the kinetics of TER drop to that of wild type. Herein, we describe a previously unrecognized phenotype for the EPEC effectors EspG and Orf3.

The Bacterial Virulence Factor Lymphostatin Compromises Intestinal Epithelial Barrier Function by Modulating Rho GTPases

“…36 Other molecules that perturb barrier function independent of espF include mitochondria-associated protein 37 and espG in conjunction with orf3. 38 Our findings demonstrate that infection of human intestinal epithelial cells with C. rodentium results in disruption of AJC structure and barrier function. These findings are consistent with a recently published report demonstrating that infection of murine intestinal epithelial cells with C. rodentium resulted in diminished barrier function associated with disruption of TJ expression of claudins-4 and -5.…”

Enteropathogenic Escherichia coli , Shigella flexneri , and Listeria monocytogenes Recruit a Junctional Protein, Zonula Occludens-1, to Actin Tails and Pedestals

“…Amieva and colleagues have reported that Helicobacter pylori causes a similar recruitment of ZO-1 to the bacterial attachment sites in Madin-Darby canine kidney (MDCK) cell monolayers and proposed that the recruitment is linked to a dysfunctional paracellular seal (1). Because EPEC has been reported to affect the integrity of the epithelial barrier (11,32,34,36,37,43,54), we examined if the EPEC-induced recruitment of ZO-1 is also linked with the disruption of the barrier. A polarized Caco/B7 monolayer was infected with wild-type EPEC and the mutants, and the TER across the monolayer, which is a hallmark of the integrity of the epithelial barrier, was measured.…”

Novel fold of VirA, a type III secretion system effector protein from Shigella flexneri

“…It has been postulated that all members of the EspG family exhibit cysteine protease-like activity (Tomson et al 2005;Smollett et al 2006;Yoshida et al 2006). In a similar assay, we investigated the ability of VirA to degrade a/b-tubulin.…”