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

Abstract

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.

Actin Pedestal Formation by Enterohemorrhagic Escherichia coli Enhances Bacterial Host Cell Attachment and Concomitant Type III Translocation

“…In contrast, priming of monolayers with EHEC ⌬tir-cesT-eae/pTir resulted in high-level binding by GFP-expressing E. coli K-12/ pIntimin with an average of 48 bound bacteria per high-power field. As previously observed (36,41,47), bound bacteria also triggered the formation of actin pedestals ( Fig. 2A, middle inset).…”

Detection and Characterization of Verocytotoxin-Producing Escherichia coli by Automated 5′ Nuclease PCR Assay

“…The sequences of the probes and primers for the subtype-specific PCR assays were in the extracellular C-terminal region of intimin, as large variations between intimin subtypes exist in this region. This is also the Tir-binding region of intimin; however, it has recently been shown that only a few amino acids are likely to be critical for Tir binding, and these residues are conserved among the different intimin types (26). Also, the intimin-binding area of Tir, the central portion of Tir, is conserved (23); and likewise, it has been predicted that only a few conserved residues are critical for binding (26).…”

“…This is also the Tir-binding region of intimin; however, it has recently been shown that only a few amino acids are likely to be critical for Tir binding, and these residues are conserved among the different intimin types (26). Also, the intimin-binding area of Tir, the central portion of Tir, is conserved (23); and likewise, it has been predicted that only a few conserved residues are critical for binding (26). Our primers and probes for Tir are placed in the variable regions outside the central intimin-binding area.…”

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

“…Screening of the phage-displayed SH3 domain library was performed as described previously (23). Yeast 2-hybrid assays were used to assess interaction among IRTKS, IRSp53, EspF U, and EHEC Tir as previously described (36). Interaction between IRTKS and recombinant EHEC Tir was assessed in GST pull-down assays.…”