Enteropathogenic (EPEC) binding to human intestinal cells triggers the formation of disease-associated actin rich structures called pedestals. major pathway to actin polymerization mediated by the actin-related protein (Arp) 2/3 complex. Previous studies ETC-1002 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 ETC-1002 have important implications for the current model of Tir-mediated actin polymerization and opens new lines of research in this area. (EPEC) is one of the leading causes of infantile diarrhea worldwide especially in developing countries. EPEC is a non-invasive bacterium that colonizes the intestinal epithelium through the formation of characteristic attaching and effacing (A/E) lesions. These lesions are characterized by a localized loss of epithelium microvilli close adherence of the bacteria to the host cell membrane and the generation of filamentous actin-rich structures beneath these bacteria called pedestals.2 Although they have been described more than two decades ago the biological purpose of pedestals is not completely understood. Importantly the disruption of genes critical for the formation of these structures has been shown to diminish colonization and subsequent ETC-1002 disease in humans3 and in experimental animals.4 The capacity to generate actin pedestals depends on the translocation of bacterial effector proteins into host cells via a type 3 secretion system (T3SS). During the first steps of infection EPEC adheres non-intimately to the host epithelium in discrete microcolonies whose formation ETC-1002 is mediated by the type 4 pili termed bundle-forming pili (BFP) owing to their capacity to laterally aggregate into long braided structures.5 Microcolony formation enhances EPEC attachment to host cells and facilitates the injection of effectors via T3SS.6 7 The attached EPEC delivers the translocated Intimin receptor (Tir) which drives the major pathway responsible for regulating actin polymerization. Other translocated effectors include Mitochondrial associated protein (Map) and EPEC-secreted proteins (Esp) H F G and Z that are encoded within a pathogenicity island termed the locus of enterocyte effacement (LEE).8 Upon injection into the cell cytoplasm Tir is inserted into the plasma membrane in a hairpin-loop conformation exposing ETC-1002 an extracellular loop which interacts ETC-1002 with the bacterial surface protein Intimin.9 This binding facilitates extremely tight attachment10 and results in the clustering of Tir in the plasma membrane that contributes to the downstream signaling events leading to the formation of actin-rich pedestals11 in a manner that depends on Tir tyrosine phosphorylation.9 Tir is phosphorylated by various host tyrosine kinases12 13 at tyrosine 474 (Y474)14 within the C-terminal cytoplasmic domain thereby recruiting the host cell adaptor proteins non-catalytic tyrosine kinase (Nck) 1 and 2 (collectively referred as Nck). Nck in turns recruits the neural Wiskott-Aldrich syndrome protein (N-WASP) 15 a member of the WAS family of proteins that promote actin polymerization Rabbit polyclonal to AKR1A1. by binding and activating the actin related protein (Arp) 2/3 complex.16 17 N-WASP presents a closed inactive conformation mainly due to intramolecular autoinhibitory interactions that involve the C-terminal acidic domain and the GTPase-binding domain (GBD).18 19 N-WASP requires the interaction with other proteins through its GBD or proline-rich domain (PRD) and possibly post-translational modifications to be fully active. Thus Nck binds directly to the numerous proline motifs in the PRD of N-WASP through its Src homology 3 (SH3) domains and activates N-WASP by destabilizing the inhibitory interactions.20 Although it is not clear whether N-WASP is recruited to Tir via direct.