Point mutants of EHEC intimin that diminish Tir recognition and actin pedestal formation highlight a putative Tir binding pocket

Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worchester, MA, USA.
Molecular Microbiology (Impact Factor: 4.42). 10/2002; 45(6):1557-73. DOI: 10.1046/j.1365-2958.2002.03137.x
Source: PubMed


Attachment to host cells by enterohaemorrhagic Escherichia coli (EHEC) is associated with the formation of a highly organized cytoskeletal structure containing filamentous actin, termed an attaching and effacing (AE) lesion. Intimin, an outer membrane protein of EHEC, is required for the formation of AE lesions, as is Tir, a bacterial protein that is translocated into the host cell to function as a receptor for intimin. We established a yeast two-hybrid assay for intimin-Tir interaction and, after random mutagenesis, isolated 24 point mutants in intimin, which disrupted Tir recognition in this system. Analysis of 11 point mutants revealed a correlation between recognition of recombinant Tir and the ability to trigger AE lesions. Many of the mutations fell within a 50-residue region near the C-terminus of intimin. Alanine-scanning mutagenesis of this region revealed four residues (Ser890, Thr909, Asn916 and Asn927) that are critical for Tir recognition. Mapping the sequences of EHEC intimin and Tir onto the crystal structure of the intimin-Tir complex of enteropathogenic E. coli predicts that each of these four intimin residues lies at the intimin-Tir interface and contributes to a pocket that interacts with Ile298 of EHEC Tir. Thus, this genetic approach to intimin function both identified residues critical for Tir binding and demonstrated a correlation between the ability to bind Tir and the ability to trigger actin focusing.

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Available from: Moses M Prabu, Oct 08, 2014
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    • "Yeast Two-Hybrid Analyses The two-hybrid expression vectors pGAD424 and pBTM116 as well as reporter strain L40 were used to define the interaction between IRTKS, IRSp53, EspF U , and EHEC Tir as previously described (Cheng et al., 2008; Liu et al., 2002). ONPG assays were performed as previously described (Garmendia et al., 2004; Cheng et al., 2008). "
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    ABSTRACT: Intrinsically disordered protein (IDP)-mediated interactions are often characterized by low affinity but high specificity. These traits are essential in signaling and regulation that require reversibility. Enterohaemorrhagic Escherichia coli (EHEC) exploit this situation by commandeering host cytoskeletal signaling to stimulate actin assembly beneath bound bacteria, generating "pedestals" that promote intestinal colonization. EHEC translocates two proteins, EspF(U) and Tir, which form a complex with the host protein IRTKS. The interaction of this complex with N-WASP triggers localized actin polymerization. We show that EspF(U) is an IDP that contains a transiently α-helical N-terminus and dynamic C-terminus. Our structure shows that single EspF(U) repeat forms a high-affinity trimolecular complex with N-WASP and IRTKS. We demonstrate that bacterial and cellular ligands interact with IRTKS SH3 in a similar fashion, but the bacterial protein has evolved to outcompete cellular targets by utilizing a tryptophan switch that offers superior binding affinity enabling EHEC-induced pedestal formation.
    Structure 08/2012; 20(10):1692-703. DOI:10.1016/j.str.2012.07.015 · 5.62 Impact Factor
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    • "We next characterized the ability of C. rodentiumΔeae expressing EHEC intimin to attach to host cells that express high levels of Tir on their surface, which more sensitively assesses Tir–intimin interactions compared to conventional infection assays (Liu et al., 2002). Pre-infection (i.e., " priming " ) of cells with an EPECΔeae mutant permits efficient delivery of Tir to the eukaryotic cell (Rosenshine et al., 1996; Liu et al., 1999). "
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    ABSTRACT: Upon binding to intestinal epithelial cells, enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and Citrobacter rodentium trigger formation of actin pedestals beneath bound bacteria. Pedestal formation has been associated with enhanced colonization, and requires intimin, an adhesin that binds to the bacterial effector Tir, which is translocated to the host cell membrane and promotes bacterial adherence and pedestal formation. Intimin has been suggested to also promote cell adhesion by binding one or more host receptors, and allelic differences in intimin have been associated with differences in tissue and host specificity. We assessed the function of EHEC, EPEC, or C. rodentium intimin, or a set of intimin derivatives with varying Tir-binding abilities in animal models of infection. We found that EPEC and EHEC intimin were functionally indistinguishable during infection of gnotobiotic piglets by EHEC, and that EPEC, EHEC, and C. rodentium intimin were functionally indistinguishable during infection of C57BL/6 mice by C. rodentium. A derivative of EHEC intimin that bound Tir but did not promote robust pedestal formation on cultured cells was unable to promote C. rodentium colonization of conventional mice, indicating that the ability to trigger actin assembly, not simply to bind Tir, is required for intimin-mediated intestinal colonization. Interestingly, streptomycin pre-treatment of mice eliminated the requirement for Tir but not intimin during colonization, and intimin derivatives that were defective in Tir-binding still promoted colonization of these mice. These results indicate that EPEC, EHEC, and C. rodentium are functionally interchangeable during infection of gnotobiotic piglets or conventional C57BL/6 mice, and that whereas the ability to trigger Tir-mediated pedestal formation is essential for colonization of conventional mice, intimin provides a Tir-independent activity during colonization of streptomycin pre-treated mice.
    Frontiers in Microbiology 01/2012; 3:11. DOI:10.3389/fmicb.2012.00011 · 3.99 Impact Factor
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    • "After 3 h incubation at 37°C, the monolayers were washed with PBS and further incubated for 3 h after addition of fresh RHFM. The cells were washed, fixed with 2.5% para-formaldehyde, stained for bacteria with DAPI and filamentous actin with TRITC-conjugated phalloidin (Sigma; Knutton et al., 1989; Liu et al., 1999a, 2002). To quantify pedestal formation, bacteria-associated with actin pedestals was determined as a function of 100 HEp-2 cells examined. "
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    ABSTRACT: Upon intestinal colonization, enterohemorrhagic Escherichia coli (EHEC) induces epithelial cells to generate actin "pedestals" beneath bound bacteria, lesions that promote colonization. To induce pedestals, EHEC utilizes a type III secretion system to translocate into the mammalian cell bacterial effectors such as translocated intimin receptor (Tir), which localizes in the mammalian cell membrane and functions as a receptor for the bacterial outer membrane protein intimin. Whereas EHEC triggers efficient pedestal formation during mammalian infection, EHEC cultured in vitro induces pedestals on cell monolayers with relatively low efficiency. To determine whether growth within the mammalian host enhances EHEC pedestal formation, we compared in vitro-cultivated bacteria with EHEC directly isolated from infected piglets. Mammalian adaptation by EHEC was associated with a dramatic increase in the efficiency of cell attachment and pedestal formation. The amounts of intimin and Tir were significantly higher in host-adapted than in in vitro-cultivated bacteria, but increasing intimin or Tir expression, or artificially increasing the level of bacterial attachment to mammalian cells, did not enhance pedestal formation by in vitro-cultivated EHEC. Instead, a functional assay suggested that host-adapted EHEC translocate Tir much more efficiently than does in vitro-cultivated bacteria. These data suggest that adaptation of EHEC to the mammalian intestine enhances bacterial cell attachment, expression of intimin and Tir, and translocation of effectors that promote actin signaling.
    Frontiers in Microbiology 11/2011; 2:226. DOI:10.3389/fmicb.2011.00226 · 3.99 Impact Factor
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