Enterohaemorrhagic Escherichia coli Tir requires a C-terminal 12-residue peptide to initiate EspFU-mediated actin assembly and harbours N-terminal sequences that influence pedestal length

Abstract

SummaryEnterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) both utilize type III secretion systems that translocate the effector protein Tir into the plasma membrane of mammalian cells in order to stimulate localized actin assembly into 'pedestals'. The Tir molecule that EPEC delivers is phosphorylated within its C-terminus on tyrosine-474, and a clustered 12-residue phosphopeptide encompassing this residue initiates an efficient signalling cascade that triggers actin polymerization. In addition to Y474, tyrosine-454 of EPEC Tir is phosphorylated, although inefficiently, and promotes actin polymerization at low levels. In contrast to EPEC Tir, EHEC Tir lacks Y474 and triggers pedestal formation in a phosphotyrosine-independent manner by interacting with an additional effector protein, EspF U . To identify EHEC Tir sequences that regulate localized actin assembly, we circumvented the strict requirements for type III translocation and directly expressed Tir derivatives in mammalian cells by transfection. Infection of Tir-expressing cells with a Tir-deficient EHEC strain demonstrated that ectopically expressed Tir localizes to the plasma membrane, is modified by mammalian serine-threonine kinases and is fully functional for actin pedestal formation. Removal of portions of the cytoplasmic N-terminus of Tir resulted in the generation of abnormally long pedestals, indicating that this region of EHEC Tir influences pedestal length. In the presence of the entire N-terminal domain, a 12-residue peptide from the C-terminus of EHEC Tir is both necessary and sufficient to recruit EspF U and initiate actin pedestal formation. This peptide encompasses the portion of EHEC Tir analogous to the EPEC Tir-Y454 region and is present within the Tir molecules of all pedestal-forming bacteria, suggesting that this sequence harbours a conserved signalling function.

Cortactin Recruitment by Enterohemorrhagic Escherichia coli O157:H7 during Infection In Vitro and Ex Vivo

“…Tir Y458 has been implicated in actin polymerization and the recruitment of TccP (3,4). In order to determine if Tir Y458 plays a role in the recruitment of cortactin to sites of bacterial attachment, we infected Swiss 3T3 fibroblasts with wild-type EHEC, EHEC⌬tir, and EHEC⌬tir complemented with plasmids encoding either wild-type Tir (pTir WT /pICC421) or Tir Y458A (pTir Y458A /pICC422).…”

“…Although robust pedestal formation in vitro requires TccP, an EHEC O157:H7 tccP mutant can trigger inefficient actin polymerization on cultured monolayers (4), suggesting that EHEC triggers multiple pathways of actin assembly in host cells. Consistent with this, EHEC O157:H7 tccP mutants can induce A/E lesions on ex vivo human intestinal organ cultures (13) and in animal models (22,26).…”

Insulin receptor tyrosine kinase substrate links theE. coliO157:H7 actin assembly effectors Tir and EspFUduring pedestal formation

“…Culture of bacteria before infection of mammalian cells, and culture and transfection of HeLa cells, were performed as previously described (14,15). Transfection of mammalian cells for ectopic expression of proteins, and infection of mammalian cells with bacteria were performed as previously described (15,37).…”

“…The C-terminal cytoplasmic domain of EHEC Tir harbors an Asn-Pro-Tyr 458 (NPY 458 ) sequence that is essential for actin signaling (12)(13)(14). In addition, EHEC translocates into host cells a second effector, EspF U (aka TccP) that acts in concert with Tir to promote pedestal formation (15,16).…”

Modelling of Infection by EnteropathogenicEscherichia coliStrains in Lineages 2 and 4 Ex Vivo and In Vivo by UsingCitrobacter rodentiumExpressing TccP

“…Instead, EHEC O157:H7 employs the T3SS effector TccP (also known as EspF U ) (3,13), which mimics Nck in terms of linking Tir to the N-WASP actin polymerization machinery. Recruitment of TccP to Tir is dependent on the conserved NPY Tir motif (1,4).…”