title: Nck adaptors, besides promoting N-WASP mediated actin-nucleation activity at pedestals, influence the cellular levels of enteropathogenic Escherichia coli Tir effector.
abstract: Enteropathogenic Escherichia coli (EPEC) binding to human intestinal cells triggers the formation of disease-associated actin rich structures called pedestals. The latter process requires the delivery, via a Type 3 secretion system, of the translocated Intimin receptor (Tir) protein into the host plasma membrane where binding of a host kinase-modified form to the bacterial surface protein Intimin triggers pedestal formation. Tir-Intimin interaction recruits the Nck adaptor to a Tir tyrosine phosphorylated residue where it activates neural Wiskott-Aldrich syndrome protein (N-WASP); initiating the major pathway to actin polymerization mediated by the actin-related protein (Arp) 2/3 complex. Previous studies with Nck-deficient mouse embryonic fibroblasts (MEFs) identified a key role for Nck in pedestal formation, presumed to reflect a lack of N-WASP activation. Here, we show the defect relates to reduced amounts of Tir within Nck-deficient cells. Indeed, Tir delivery and, thus, pedestal formation defects were much greater for MEFs than HeLa (human epithelial) cells. Crucially, the levels of two other effectors (EspB/EspF) within Nck-deficient MEFs were not reduced unlike that of Map (Mitochondrial associated protein) which, like Tir, requires CesT chaperone function for efficient delivery. Interestingly, drugs blocking various host protein degradation pathways failed to increase Tir cellular levels unlike an inhibitor of deacetylase activity (Trichostatin A; TSA). Treatments with TSA resulted in significant recovery of Tir levels, potentiation of actin polymerization and improvement in bacterial attachment to cells. Our findings have important implications for the current model of Tir-mediated actin polymerization and opens new lines of research in this area.
authors.affiliation: a Department of Microbiology ; School of Pharmacy; Complutense University; Madrid, Spain.
authors.forename: Elvira
authors.initials: E
authors.lastname: Nieto-Pelegrin
authors.forename: Brendan
authors.initials: B
authors.lastname: Kenny
authors.forename: Narcisa
authors.initials: N
authors.lastname: Martinez-Quiles
mesh_terms: D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D018829:Adhesins, Bacterial / Q000378:metabolism; D000818:Animals; D001422:Bacterial Adhesion; D054308:Enteropathogenic Escherichia coli / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism / Q000382:microbiology; D055786:Gene Knockout Techniques; D006367:HeLa Cells; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D051379:Mice; D015513:Oncogene Proteins / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D011956:Receptors, Cell Surface / Q000378:metabolism*; D051316:Wiskott-Aldrich Syndrome Protein / Q000378:metabolism*
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D018829:Adhesins, Bacterial; D029968:Escherichia coli Proteins; D006877:Hydroxamic Acids; C078332:Nck protein; D015513:Oncogene Proteins; D011956:Receptors, Cell Surface; C109287:Tir protein, E coli; D051316:Wiskott-Aldrich Syndrome Protein; C073819:eaeA protein, E coli; C012589:trichostatin A
keywords: A/E, Attaching and effacing; Ab, Polyclonal antibody; Actin; BFP, Bundle-forming pili; Cmp, Chloramphenicol; CrkII, CT10 regulator of kinase; CrkL, Crk-like; Ctr., Control; EHEC, Enterohaemorrhagic Escherichia coli; EPEC; EPEC, Enteropathogenic Escherichia coli; Esp, EPEC-secreted proteins; FBS, Fetal bovine serum; GBD, GTPase-binding domain; HDAC, Histone deacetylases; HeLa, Human cervical epithelial cancer cell line; IRSp53, Insulin receptor tyrosine kinase substrate p53; IRTKS, Insulin receptor tyrosine kinase substrate; LEE, Locus of enterocyte effacement; MEFs, Mouse embryonic fibroblasts; MOI, Multiplicity of infection; Map, Mitochondrial associated protein; MoAb, Monoclonal antibody; N-WASP; N-WASP, Neural Wiskott–Aldrich syndrome protein; NF-kB, Nuclear factor kB; NPF, Nucleation promoting factor; Nck; Nck, Non-catalytic tyrosine kinase; Nle, Non-LEE effectors; PRD, Proline-rich domain; SH3, Src homology 3; T3SS, Type 3 secretion system; TNF- α, Tumor necrosis factor-α; TSA; TSA, Trichostatin A; Tir; Tir, Translocated Intimin receptor; WB, Western Blot; WT, Wild type.; bacterial adhesion; pedestals