Biophysical analysis of the EPEC translocated intimin receptor-binding domain

Enteropathogenic Escherichia coli (EPEC) are Gram (-) bacteria responsible for widespread illness in the form of diarrhea. EPEC cells attach to the intestinal epithelium using a Type III secretion system common to many Gram (-) bacteria. The translocated intimin receptor (TIR) is the first protein secreted through the EPEC secretion complex, and is absolutely required for pathogenesis. It inserts into the intestinal epithelium, serving as an anchor responsible for the attachment of EPEC to the host epithelial cell. Intimin is a transmembrane protein displayed on the EPEC cell surface with an extracellular domain that binds TIR. Observation of a TIR-TIR dimer in the X-ray co-crystal structure of the extracellular domains of intimin and TIR raised the question of how these protein domains interact and function in solution. Herein we report that the extracellular domain of TIR exists in a folded and active monomeric state in solution, as confirmed by analytical ultracentrifugation, analytical size-exclusion HPLC, isothermal titration calorimetry, and surface plasmon resonance.     

Tails of two Tirs: actin pedestal formation by enteropathogenic E. coli and enterohemorrhagic E. coli O157:H7

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli O157:H7 (EHEC) form characteristic lesions on infected mammalian cells called actin pedestals. Each of these two pathogens injects its own translocated intimin receptor (Tir) molecule into the plasma membranes of host cells. Interaction of translocated Tir with the bacterial outer membrane protein intimin is required to trigger the assembly of actin into focused pedestals beneath bound bacteria. Despite similarities between the Tir molecules and the host components that associate with pedestals, recent work indicates that EPEC and EHEC Tir are not functionally interchangeable. For EPEC, Tir-mediated binding of Nck, a host adaptor protein implicated in actin signaling, is both necessary and sufficient to initiate actin assembly. In contrast, for EHEC, pedestals are formed independently of Nck, and require translocation of bacterial factors in addition to Tir to trigger actin signaling.     

A novel category of enteropathogenic Escherichia coli simultaneously utilizes the Nck and TccP pathways to induce actin remodelling

Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) induce drastic reorganization of the microfilament cytoskeleton. EHEC and EPEC translocate Tir (translocated intimin receptor) which, once inserted into the host plasma membrane, binds the bacterial outer membrane adhesin intimin. Tir(EPEC) then becomes tyrosine phosphorylated facilitating the recruitment and site-specific binding of the eukaryotic adaptor Nck, which in turn binds and activates the Wiskott-Aldrich syndrome protein (N-WASP), leading to actin-related protein 2/3 (Arp2/3) complex-mediated actin polymerization. In contrast, Tir(EHEC) has no Nck binding site; instead, EHEC utilizes the translocated effector TccP (Tir-cytoskeleton coupling protein) to bind and activate N-WASP. Here we report a novel class of EPEC that translocates both TccP and Tir(EPEC)-like effector molecules. Consistent with these characteristics, we show that both the Tir-Nck and Tir:TccP actin remodelling pathways function simultaneously during infection, making this a novel and versatile EPEC category.     

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

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.     

A tyrosine-phosphorylated 12-amino-acid sequence of enteropathogenic Escherichia coli Tir binds the host adaptor protein Nck and is required for Nck localization to actin pedestals

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) each promote the reorganization of actin into filamentous pedestal structures beneath attached bacteria during colonization of the intestinal epithelium. Central to this process is the translocation of the protein Tir (translocated intimin receptor) into the plasma membrane of host cells, where it interacts with the bacterial outer membrane protein intimin and triggers cellular signalling events that lead to actin rearrangement. Actin signalling by EPEC Tir requires a tyrosine residue, Y474, which is phosphorylated in the host cell. In contrast, EHEC Tir lacks this residue and generates pedestals independently of tyrosine phosphorylation. Consistent with this difference, recent work indicates that EHEC Tir cannot functionally replace EPEC Tir. To identify the role that tyrosine phosphorylation of EPEC Tir plays in actin signalling, we generated chimeric EHEC/EPEC Tir proteins and identified a 12-residue sequence of EPEC Tir containing Y474 that confers actin-signalling capabilities to EHEC Tir when the chimera is expressed in EPEC. Nck, a mammalian adaptor protein that has been implicated in the initiation of actin signalling, binds to this sequence in a Y474 phosphorylation-dependent manner and is recruited to the pedestals of EPEC, but not of EHEC.     

Tir phosphorylation and Nck/N-WASP recruitment by enteropathogenic and enterohaemorrhagic Escherichia coli during ex vivo colonization of human intestinal mucosa is different to cell culture models

Tir, the translocated intimin receptor of enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) and Citrobacter rodentium, is translocated into the host cell by a filamentous type III secretion system. Epithelial cell culture has demonstrated that Tir tyrosine phosphorylation is necessary for attaching effacing (A/E) lesion formation by EPEC and C. rodentium, but is not required by EHEC O157:H7. Recent in vivo work on C. rodentium has reported that Tir translocation, but not its phosphorylation, is necessary for colonization of the mouse colon. In this study we investigated the involvement of Tir and its tyrosine phosphorylation in EPEC and EHEC human intestinal colonization, N-WASP accumulation and F-actin recruitment using in vitro organ culture (IVOC). We showed that both EPEC and EHEC Tir are translocated into human intestinal epithelium during IVOC and that Tir is necessary for ex vivo intestinal colonization by both EPEC and EHEC. EPEC, but not EHEC, Tir is tyrosine phosphorylated but Tir phosphorylation-deficient mutants still colonize intestinal explants. While EPEC Tir recruits the host adaptor protein Nck to initiate N-WASP-Arp2/3-mediated actin polymerization, Tir derivatives deficient in tyrosine phosphorylation recruit N-WASP independently of Nck indicating the presence of a tyrosine phosphorylation-independent mechanism of A/E lesion formation and actin recruitment ex vivo by EPEC in man.     

Enterohaemorrhagic and enteropathogenic Escherichia coli Tir proteins trigger a common Nck-independent actin assembly pathway

The Tir proteins of enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC respectively) are each translocated into the host plasma membrane where they promote F-actin pedestals in epithelial cells beneath adherent bacteria, but the two proteins act by different means. The canonical EPEC Tir becomes phosphorylated on tyrosine residue 474 (Y474) to recruit the host adaptor protein Nck, and also stimulates an inefficient, Nck-independent pathway utilizing tyrosine residue 454 (Y454). In contrast, the canonical EHEC Tir lacks Y474 and instead utilizes residues 452-463 to recruit EspF(U), an EHEC-specific effector that stimulates robust Nck-independent actin assembly. EHEC Tir Y458 and EPEC Tir Y454 are both part of an asparagine-proline-tyrosine (NPY) sequence. We report that each of the EHEC Tir NPY residues is required for EspF(U) recruitment and pedestal formation, and each of the EPEC Tir NPY residues is critical for inefficient, Nck-independent pedestal formation. Introduction of EspF(U) into EPEC dramatically enhanced Nck-independent actin assembly by EPEC Tir in a manner dependent on NPY(454). These results suggest that EPEC and EHEC Tir trigger a common Nck-independent actin assembly pathway and are both derived from an ancestral Tir molecule that utilized NPY to stimulate low-level pedestal formation.     

Crystal structure of EHEC intimin: insights into the complementarity between EPEC and EHEC

Enterohaemorrhagic E. coli (EHEC) O157:H7 is a primary food-borne bacterial pathogen capable of causing life-threatening human infections which poses a serious challenge to public health worldwide. Intimin, the bacterial outer-membrane protein, plays a key role in the initiating process of EHEC infection. This activity is dependent upon translocation of the intimin receptor (Tir), the intimin binding partner of the bacteria-encoded host cell surface protein. Intimin has attracted considerable attention due to its potential function as an antibacterial drug target. Here, we report the crystal structure of the Tir-binding domain of intimin (Int188) from E. coli O157:H7 at 2.8 Å resolution, together with a mutant (IntN916Y) at 2.6 Å. We also built the structural model of EHEC intimin-Tir complex and analyzed the key binding residues. It suggested that the binding pattern of intimin and Tir between EHEC and Enteropathogenic E. coli (EPEC) adopt a similar mode and they can complement with each other. Detailed structural comparison indicates that there are four major points of structural variations between EHEC and EPEC intimins: one in Domain I (Ig-like domain), the other three located in Domain II (C-type lectin-like domain). These variations result in different binding affinities. These findings provide structural insight into the binding pattern of intimin to Tir and the molecular mechanism of EHEC O157: H7.     

The Tir-binding region of enterohaemorrhagic Escherichia coli intimin is sufficient to trigger actin condensation after bacterial-induced host cell signalling

Enterohaemorrhagic Escherichia coli (EHEC) has emerged as an important agent of diarrhoeal disease. Attachment to host cells, an essential step during intestinal colonization by EHEC, is associated with the formation of a highly organized cytoskeletal structure containing filamentous actin, termed an attaching and effacing (A/E) lesion, directly beneath bound bacteria. The outer membrane protein intimin is required for the formation of this structure, as is Tir, a bacterial protein that is translocated into the host cell and is thought to function as a receptor for intimin. To understand intimin function better, we fused EHEC intimin to a homologous protein, Yersinia pseudotuberculosis invasin, or to maltose-binding protein. The N-terminal 539 amino acids of intimin were sufficient to promote outer membrane localization of the C-terminus of invasin and, conversely, the N-terminal 489 amino acids of invasin were sufficient to promote the localization of the C-terminus of intimin. The C-terminal 181 residues of intimin were sufficient to bind mammalian cells that had been preinfected with an enteropathogenic E. coli strain that expresses Tir but not intimin. Binding of intimin derivatives to preinfected cells correlated with binding to recombinant Tir protein. Finally, the 181-residue minimal Tir-binding region of intimin, when purified and immobilized on latex beads, was sufficient to trigger A/E lesions on preinfected mammalian cells.     

Identification of the intimin-binding domain of Tir of enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) attaches intimately to mammalian cells via a bacterial outer membrane adhesion molecule, intimin, and its receptor in the host cell membrane, Tir. Tir is a bacterial protein translocated into the host cell membrane and tyrosine phosphorylated after insertion. Tir–intimin binding induces organized actin polymerization beneath the adherent bacteria, resulting in the formation of pedestal-like structures. A series of Tir deletion derivatives were constructed to analyse which Tir domains are involved in intimin binding. We have localized the intimin-binding domain (IBD) of Tir using a yeast two-hybrid system and a gel-overlay approach to a region of 109 amino acids that is predicted to be exposed on the surface of the plasma membrane. A truncated Tir protein lacking this domain was translocated to the host cell membrane and tyrosine phosphorylated, but failed to bind intimin or to induce either actin polymerization or Tir accumulation beneath the bacteria. These results indicate that only a small region of Tir is needed to bind intimin and support the predicted topology for Tir, with both N- and C-terminal regions in the mammalian cell cytosol. They also confirm that Tir–intimin interactions are needed for cytoskeletal organization. We have also identified N-terminal regions involved in Tir stability and Tir secretion to the media.     

The N-terminus of enteropathogenic Escherichia coli (EPEC) Tir mediates transport across bacterial and eukaryotic cell membranes

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to translocate into host cells several effector molecules that are required for virulence. One of these, the translocated intimin receptor, Tir, inserts into the host cell cytoplasmic membrane, where it functions as the receptor for intimin, an outer membrane adhesin expressed by EPEC. A chaperone for Tir, called CesT, is required for stability of Tir in the EPEC cytoplasm. In this study, the cyaA gene reporter system was used to identify domains in Tir that mediate secretion into the culture supernatant and translocation into host cells. A Tir-CyaA fusion containing the first 15 N-terminal residues of Tir was secreted and translocated into HeLa cells by a deltatirdeltacesT mutant; however, maximal secretion and translocation was observed with the first 26 N-terminal residues of Tir. Fusions containing progressively larger N-terminal sequences of Tir were also efficiently secreted and translocated into HeLa cells by the deltatirdeltacesT strain. However, in a deltatir mutant that expresses CesT, Tir26-CyaA and an additional fusion containing the first 69 N-terminal residues of Tir were not secreted or translocated, but fusions containing larger N-terminal Tir sequences were secreted and translocated by the deltatir mutant. Wild-type EPEC secreted and translocated the Tir15-CyaA fusion, whereas longer fusions, such as Tir26-CyaA and Tir69-CyaA, were translocated to higher levels, similar to what was observed with the deltatirdeltacesT mutant. A Tir-CyaA fusion containing the CesT binding domain was translocated into HeLa cells more rapidly in the presence of CesT compared with translocation in the absence of CesT. Collectively, these results suggest that an N-terminal domain of 26 amino acids functions as a CesT-independent signal that is capable of delivering Tir into both the culture supernatant and the cytosol of host cells. Furthermore, in addition to its role in the stability of Tir, CesT may function in translocation by mediating rapid delivery of Tir into host cells.     

Seric and secretory antibodies reactive to α, β and γ intimins of Escherichia coli in healthy Brazilian adults

Intimin is essential for attaching and effacing lesions by pathogens such as enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC), and the antigenic polymorphism of intimin determines distinct subtypes. Our aim was to investigate the presence of immunoglobulin G (IgG) and IgA antibodies reactive to α, β and γ intimins in serum and colostrum from healthy Brazilian adults. We found seric IgG and secretory IgA antibodies reactive to conserved and variable regions of α, β and γ intimins and a positive correlation between the concentrations of these antibodies in both serum and colostrum that suggested cross reactivity among anti-intimin antibodies, as was confirmed by immunoblotting and absorption. The concentrations of anti-conserved region antibodies were higher than those of variable region antibodies. The presence of antibodies reactive to EHEC antigens could result from contact with EPEC or with other bacteria of the environment even though this bacterium is not frequent in Brazil, and suggests possible protection against EHEC.     

Identification of enteropathogenic and enterohaemorrhagic Escherichia coli strains by immunoserological detection of intimin

Aims: To evaluate the sensitivity and specificity of polyclonal and monoclonal antibodies (Mabs) against intimin in the detection of enteropathogenic and enterohaemorrhagic Escherichia coli isolates using immunoblotting. Methods and Results: Polyclonal and Mabs against the intimin‐conserved region were raised, and their reactivities were compared in enteropathogenic E. coli (EPEC) and enterohaemorrhagic E. coli (EHEC) isolates using immunoblotting analysis. In comparison with rat antiserum, rabbit anti‐intimin IgG‐enriched fraction had a stronger recognition pattern to a wide spectrum of intimin types in different EPEC and EHEC serotypes. On the other hand, murine monoclonal IgG2b specific to intimin, with dissociation constant of 1·3 × 10^?8 mol l^?1, failed in the detection of some of these isolates. Conclusion: All employed antibodies showed 100% specificity, not reacting with any of the eae‐negative isolates. The sensitivity range was according to the employed antisera, and 97% for rabbit anti‐intimin IgG‐enriched fraction, followed by 92% and 78% sensitivity with rat antisera and Mab. Significance and Impact of the Study: The rabbit anti‐intimin IgG‐enriched fraction in immunoblotting analysis is a useful tool for EPEC and EHEC diagnoses.     

Citrobacter rodentium translocated intimin receptor (Tir) is an essential virulence factor needed for actin condensation,intestinal colonization and colonic hyperplasia in mice

Citrobacter rodentium infection of mice serves as a relevant small animal model to study enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) infections in man. Enteropathogenic E. coli and EHEC translocate Tir into the host cytoplasmic membrane, where it serves as the receptor for the bacterial adhesin intimin and plays a central role in actin condensation beneath the adherent bacterium. In this report, we examined the function of C. rodentium Tir both in vitro and in vivo. Similar to EPEC, C. rodentium Tir is tyrosine phosphorylated and is essential for actin condensation. Citrobacter Tir and EPEC Tir are functionally interchangeable and both require tyrosine phosphorylation to mediate actin rearrangements. In contrast, Citrobacter Tir supports actin nucleation in EHEC independent of tyrosine phosphorylation, while EHEC Tir cannot replace Citrobacter Tir for this function. This indicates that C. rodentium and EPEC use an actin nucleating mechanism different from EHEC. We also found that Tir is expressed and translocated into mouse enterocytes in vivo by C. rodentium during infections. This represents the first direct demonstration of a type III effector translocated in vivo into a natural host by any pathogen. In addition, we showed that Tir, but not its tyrosine phosphorylation, is essential for C. rodentium to colonize the large bowel and induce attaching/effacing (A/E) lesions and colonic hyperplasia in mice, and that both EPEC Tir and EHEC Tir can substitute for Citrobacter Tir for these activities in vivo. These results thus demonstrate that Tir is an essential virulence factor in this infection model. The data also show that the function of Tir tyrosine phosphorylation and its subsequent actin nucleating activity are not essential for C. rodentium colonization of the mouse gut nor for inducing A/E lesions and colonic hyperplasia, thereby uncoupling colonization and disease from actin condensation for this A/E pathogen.     

Site-directed mutagenesis of intimin alpha modulates intimin-mediated tissue tropism and host specificity

The hallmark of enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherchia coli adhesion to host cells is intimate attachment leading to the formation of distinctive 'attaching and effacing' lesions. This event is mediated, in part, by binding of the bacterial adhesion molecule intimin to a second bacterial protein, Tir, delivered by a type III secretion system into the host cell plasma membrane. The receptor-binding activity of intimin is localized to the C-terminal 280 amino acids (Int280) and at least five distinct intimin types (alpha, beta, gamma, delta and epsilon) have been identified thus far. In addition to binding to Tir, intimin can also bind to a component encoded by the host. The consequence of latter intimin-binding activity may determine tissue tropism and host specificity. In this study we selected three amino acids in intimin, which are implicated in Tir binding, for site-directed mutagenesis. We used the yeast two-hybrid system and gel overlays to study intimin-Tir protein interaction. In addition, the biological consequences of the mutagenesis was tested using a number of infection models (cultured epithelial cells, human intestinal explants and a mouse model). We report that while an I237/897A substitution (positions numbered according to Int280alpha/whole intimin alpha) in intimin alpha did not have any affect on its biological activity, a T255/914A substitution attenuated intimin activity in vivo. In contrast, the mutation V252/911A affected tissue targeting in the human intestinal explant model and attenuated the biological activity of intimin in the mouse model. This study provides the first clues of the molecular basis of how intimin mediates tissue tropism and host specificity.     

Characterization of the eaeA gene from rabbit enteropathogenic Escherichia coli strain RDEC-1 and comparison to other eaeA genes from bacteria that cause attaching-effacing lesions

Abstract A number of enteric pathogens, including enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli , Hafnia alvei , a strain of Citrobacter freundii , and rabbit EPEC strain RDEC-1 cause attaching-effacing (AE) lesions in the gut mucosa. These bacteria have a pathogenicity cassette (locus of enterocyte effacement or LEE) containing the eaeA gene. This gene encodes intimin, an outer membrane protein required for production of AE lesions. RDEC-1, a non-invasive enteropathogen in young rabbits, produces AE lesions morphologically indistinguishable from lesions caused by human AE bacterial strains. The RDEC-1 example of E. coli diarrhea in rabbits is an important model for studying the pathogenesis of AE bacteria in a natural infection and for analyzing specific roles of the components of LEE. In order to better understand the role of intimin in the development of AE lesions, a portion of DNA within RDEC-1 LEE, containing the eaeA gene and an upstream open reading frame (ORF), was sequenced. The RDEC-1 eaeA gene shared 87%, 92%, and 93% DNA sequence identity and > 80% amino acid sequence identity with the eaeA genes of C. freundii biotype 4280, EHEC O157:H7, and EPEC O127:H6, respectively. The carboxy-terminal 280 amino acid residues of intimin has 80%, 56%, and 54% identity with C. freundii , EHEC O157:H7, and EPEC O127:H6 intimins, respectively. The predicted protein encoded by the upstream ORF (156 amino acids) shares 95%, 97%, and 99% amino acid identity with predicted proteins from C. freundii , EHEC O157:H7, and EPEC O127:H6, respectively. The high degree of sequence homology of the ORF and the eaeA gene of RDEC-1 with those of other AE bacteria suggests an evolutionary relationship of LEE and supports and facilitates the use of the RDEC-1 model for studying the role of LEE in pathogenesis.     

Enteropathogenic E. coli Tir binds Nck to initiate actin pedestal formation in host cells

Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that causes infantile diarrhea worldwide. EPEC injects a bacterial protein, translocated intimin receptor (Tir), into the host-cell plasma membrane where it acts as a receptor for the bacterial outer membrane protein, intimin. The interaction of Tir and intimin triggers a marked rearrangement of the host actin cytoskeleton into pedestals beneath adherent bacteria. On delivery into host cells, EPEC Tir is phosphorylated on tyrosine 474 of the intracellular carboxy-terminal domain, an event that is required for pedestal formation. Despite its essential role, the function of Tir tyrosine phosphorylation has not yet been elucidated. Here we show that tyrosine 474 of Tir directly binds the host-cell adaptor protein Nck, and that Nck is required for the recruitment of both neural Wiskott-Aldrich-syndrome protein (N-WASP) and the actin-related protein (Arp)2/3 complex to the EPEC pedestal, directly linking Tir to the cytoskeleton. Cells with null alleles of both mammalian Nck genes are resistant to the effects of EPEC on the actin cytoskeleton. These results implicate Nck adaptors as host-cell determinants of EPEC virulence.     

Molecular evolution and mosaic structure of alpha, beta, and gamma intimins of pathogenic Escherichia coli.

Two types of pathogenic Escherichia coli, enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), cause diarrheal disease by disrupting the intestinal environment through the intimate attachment of the bacteria to the intestinal epithelium. This process is mediated by intimin, an outer membrane protein that is homologous to the invasins of pathogenic Yersinia. The intimin (eae) gene is part of a pathogenicity island, a 35-kb segment of DNA that has been acquired independently in different groups of pathogens. Nucleotide sequences of eae of three EPEC and four EHEC strains representing distinct clonal lineages revealed an exceptionally high level of divergence (15%) in the amino acid sequences of alpha, beta, and gamma intimin molecules, most of which is concentrated in the C-terminal region. The gamma intimin sequences from E. coli strains with serotypes O157:H7, O55:H7, and O157:H- are virtually identical, supporting the hypothesis that these bacteria belong to a single clonal lineage. Sequences of beta intimin of EPEC strains of serotypes O111:H2 and O128:H2 show substantial differences from alpha and gamma intimins, indicating that these strains have evolved independently. Strong nonrandom clustering of polymorphic sites indicates that the intimin genes are mosaics, suggesting that protein divergence has been accelerated by recombination and diversifying selection.