Nck adaptors, besides promoting N-WASP mediated actin-nucleation activity at pedestals, influence the cellular levels of enteropathogenicEscherichia coliTir effector

Abstract

Enteropathogenic Escherichia coli (EPEC) binding to human intestinal cells triggers the formation of diseaseassociated 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 kinasemodified 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.

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

“…Enrichment analysis showed that the target proteins were enriched in five main types of MF: ribosome structure (p = 0.012), GTPase activity (p = 0.013), protein binding (p = 0.030), RNA binding (p = 0.038) and RNA splicing activity (p = 0.048; Table 3). Studies have shown that EspF has the ability to target mitochondria and nucleoli [6,16], and interacts with N-WASP, SNX9 and Abcf2 [8,[17][18], leading to microvilli disappearance, cytoskeleton rearrangement, actin aggregation, mitochondrial dysfunction and apoptosis in intestinal epithelial cells, nucleolysis and other functions [11,39]. EspF with GTPase activity combined with splicing RNA and other functions in the host cell remains unknown research territory, deserving of further investigation.…”

“…We performed Individual Gateway recombination reactions (in 96-well plates) for all 18,000 open-reading frames (ORFs) from the hORFeome v7.1 library [26] with a mixture of pBabe-CMV-YFPc-DEST-puro (pB-CMV-NCpuro) and pBabe-CMV-DEST-YFPc-puro (pB-CMV-CC-puro; 1:1; Invitrogen, CA, USA) to generate ORFs tagged with YFPc at either the N or C terminus, obtaining a library of YFPc-prey pool plasmids.…”

“…Such structural features enable EspF to target the mitochondria, resulting in disruption of the mitochondrial membrane potential, release of cytochrome c into the cytoplasm, cleavage of caspases 9 and 3; all of these events are associated with mitochondrial apoptotic signaling pathways [15,16]. However, the SH3 and CRIB domains enable EspF to interact with the SNX9 protein and the neuronal Wiskott-Aldrich syndrome (N-WASP) protein directly [17,18]. Moreover, VK Viswanathan et al have shown that EspF can interact with the host intermediate filament protein cytokeratin 18 (CK18) in a complex with adaptor protein 14-3-3, which can alter the architecture of the intermediate filament network in EPEC-infected cells [19].…”

Enteropathogenic Escherichia coli (EPEC) Recruitment of PAR Polarity Protein Atypical PKCζ to Pedestals and Cell–Cell Contacts Precedes Disruption of Tight Junctions in Intestinal Epithelial Cells

“…aPKC activation during endothelial morphogenesis is determined by the adaptor protein Nck [76]. After Nck is recruited to actin pedestals [77,78], it could serve as a hub for aPKCζ pedestal activity. Therefore, one could speculate that the early recruitment of aPKCζ to pedestals redirects its kinase activity away from TJ and polarity complexes, thus disrupting apico-basal polarity and barrier function.…”

The NCK and ABI adaptor genes in catfish and their involvement in ESC disease response

“…NCK is essential for the actin-based motility of vaccinia virus (Frischknecht et al, 1999). NCK has also been implicated in the initiation of actin signaling, binding to a tyrosine-phosphorylated 12 amino acid sequences of enteropathogenic Escherichia coli (EPEC) translocated intimin receptor (TIR) in a Y474 phosphorylation-dependent manner (Nieto-Pelegrin et al, 2014). Recently, NCK1 was identified to be a candidate gene for enteric septicemia of catfish (ESC) disease resistance of catfish, and was speculated to play similar roles during ESC and EPEC pathogenicity (Zhou et al, 2016).…”