EspF Interacts with Nucleation-Promoting Factors To Recruit Junctional Proteins into Pedestals for Pedestal Maturation and Disruption of Paracellular Permeability

Abstract

Many pathogenic bacteria subvert normal host cell processes by delivering effector proteins which mimic eukaryotic functions directly into target cells. EspF is a multifunctional protein injected into host cells by attaching and effacing pathogens, but its mechanism of action is not understood completely. In silico analyses of EspF revealed two key motifs: proline-rich domains and PDZ domain binding motifs. Such functional domains may allow EspF to act as an actin nucleation-promoting factor by mimicking host proteins. In agreement with these predictions, we found that EspF from rabbit enteropathogenic Escherichia coli (E22) participates in the regulation of actin polymerization by binding to a complex of proteins at the tight junctions (TJ). EspF bound to actin and profilin throughout the course of infection. However, after 2 h of infection, EspF also bound to the neural Wiskott-Aldrich syndrome protein and to the Arp2/3, zonula occludens-1 (ZO-1), and ZO-2 proteins. Moreover, EspF caused occludin, claudin, ZO-1, and ZO-2 redistribution and loss of transepithelial electrical resistance, suggesting that actin sequestration by EspF may cause local actin depolymerization leading to EspF-induced TJ disruption. Furthermore, EspF caused recruitment of these TJ proteins into the pedestals. An E22 strain lacking EspF did not cause TJ disruption and pedestals were smaller than those induced by the wild-type strain. Additionally, the pedestals were located mainly in the TJ. The overexpression of EspF caused bigger pedestals located along the length of the cells. Thus, actin sequestration by EspF allows the recruitment of junctional proteins into the pedestals, leading to the maturation of actin pedestals and the disruption of paracellular permeability.

Screening for Host Proteins Interacting with Escherichia Coli O157:H7 EspF using Bimolecular Fluorescence Complementation

“…Moreover, EspF can cause actin sequestration and recruit junctional proteins, such as occludin, claudin, ZO-1 and ZO-2, to the pedestals, inducing the redistribution and loss of transepithelial electrical resistance, disruption of paracellular permeability and depolymerization of actin. Ultimately, this leads to EspF-induced TJ disruption [45]. Our study suggests that EspF may be involved in future science group www.futuremedicine.com the regulation of the actin cytoskeleton and TJs, which confirmed and extended previous findings that indicated the disruption of the TJ barriers might be associated with EspF's regulation of the actin cytoskeleton.…”

“…The YFPn tag may alter the conformation or activity of the labeled protein, having an effect on the binding site on PRR1. Besides, the combination between N-WASP and EspF can be detected by coimmunoprecipitation at 1-1.5-h postinfection, but no interplay was found at 3-h postinfection [45]; this may also be one of the reasons why we did not screen N-WASP. It can be seen that the protein reads were not always proportional to the interaction strength, but it was influenced by the expression level of the protein itself; therefore, we set it as a preliminary criterion for protein interaction, not the gold standard.…”

“…By interacting with numerous proteins such as actin, WASP, GTPase and annexin 1, profilins can regulate actin polymerization or other cellular processes, such as membrane trafficking, smallGTPase signaling and nuclear activities [72]. Profilin has a vital role in the regulation of actin cytoskeleton and by cooperating with actin and EspF, they form a complex which enables EspF to serve as a nucleation-promoting factor [45]. KEGG signaling pathway analysis results indicated that EspF may disrupt TJs through manipulation of the actin cytoskeleton; we hypothesize that PFN4 may play a role in this process by interacting with EspF, and the interplay may affect the polymerization of actin, disrupting the formation of pedestals and leading to the redistribution of TJ proteins.…”

Enterohaemorrhagic Escherichia coli serogroup O111 inhibits NF-κB-dependent innate responses in a manner independent of a type III secreted OspG orthologue

“…Bacterial strains and plasmids used in this study are shown in Table 1. HeLa (ATCC CCL-2) and RK13 actin-GFP cells (Peralta-Ramirez et al, 2008) were maintained in D-MEM (Invitrogen) supplemented with 10 % (v/v) fetal calf serum (FCS, Eurobio) and 80 mg gentamicin ml 21 at 37 uC in 5 % CO 2 . G418 (Invivogen) was added at 400 mg ml 21 to RK13 actin-GFP cells.…”

The Role of Escherichia coli in the Development and Progression of Cancer

“…Additionally, EspF is capable to disrupt the tight junctions proteins in intestinal epithelial cells (67), which could facilitate the release and dissemination of tumor cells, and contributing to metastasis of colon cancer. In addition, EPEC infection increased the expression of macrophage inhibitory citokine 1 (MIC-1), this molecule has been associated with an important increase on survival and spread of colon tumor cells in a GTPase RhoA dependent pathway (68).…”