The Experts below are selected from a list of 3879 Experts worldwide ranked by ideXlab platform
Alison D Obrien - One of the best experts on this subject based on the ideXlab platform.
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the established Intimin receptor tir and the putative eucaryotic Intimin receptors nucleolin and β1 integrin localize at or near the site of enterohemorrhagic escherichia coli o157 h7 adherence to enterocytes in vivo
Infection and Immunity, 2006Co-Authors: James F Sinclair, Evelyn A Deannystrom, Alison D ObrienAbstract:For enterohemorrhagic Escherichia coli (EHEC) O157:H7 to adhere tightly to the intestinal epithelium and produce attach and efface (A/E) lesions, the organism must express the adhesin Intimin and insert the bacterially encoded translocated Intimin receptor Tir into the plasma membrane of the host enterocyte. Additionally, some reports based on tissue culture experiments indicate that Intimin has affinity for the eucaryotic proteins nucleolin and beta1 integrin. To address the potential biological relevance of these eucaryotic proteins in the infection process in vivo, we sought to compare the proximity of Tir, nucleolin, and beta1 integrin to regions of EHEC O157:H7 attachment in intestinal sections from three different inoculated animals: piglets, neonatal calves, and mice. Piglets and neonatal calves were chosen because Intimin-mediated adherence of EHEC O157:H7 and subsequent A/E lesion formation occur at high levels in these animals. Mice were selected because of their ease of manipulation but only after we first demonstrated that in competition with the normal mouse gut flora, an EHEC O157:H7 strain with a nonpolar deletion in the Intimin gene was cleared faster than strains that produced wild-type or hybrid Intimin. In all three animal species, we noted immunostained Tir beneath and stained nucleolin closely associated with adherent bacteria in intestinal sections. We also observed immunostained beta1 integrin clustered at locations of bacterial adherence in porcine and bovine tissue. These findings indicate that nucleolin and beta1 integrin are present on the luminal surface of intestinal epithelia and are potentially accessible as receptors for Intimin during EHEC O157:H7 infection.
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antibody against the carboxyl terminus of Intimin α reduces enteropathogenic escherichia coli adherence to tissue culture cells and subsequent induction of actin polymerization
Infection and Immunity, 2005Co-Authors: Humberto M Carvalho, Louise D Teel, John F Kokaikun, Alison D ObrienAbstract:The C-terminal third of Intimin binds to its translocated receptor (Tir) to promote attaching and effacing lesion formation during infection with enteropathogenic Escherichia coli (EPEC). We observed that the adherence of EPEC strains to HEp-2 cells was reduced and that actin polymerization was blocked by antibody raised against the C-terminal third of Intimin α.
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Intimin types α β and γ bind to nucleolin with equivalent affinity but lower avidity than to the translocated Intimin receptor
Journal of Biological Chemistry, 2004Co-Authors: James F Sinclair, Alison D ObrienAbstract:Abstract The outer membrane adhesins of enteropathogenic Escherichia coli, Citrobacter rodentium, and enterohemorrhagic E. coli (EHEC) O157:H7 that mediate attach and efface intestinal lesions are classified as Intimin α, β, and γ, respectively. Each of these Intimin types binds to its cognate, bacterially encoded receptor (called Tir for translocated Intimin receptor) to promote tight adherence of the organism to the host-cell plasma membrane. We previously reported that γ Intimin of EHEC O157:H7 also bound to a eucaryotic receptor that we determined was nucleolin. The objective of this study was to investigate in vitro and in vivo the interactions of Intimins α, β, and γ with nucleolin in the presence of Tir from EHEC O157:H7. Protein binding experiments demonstrated that Intimin of types α, β, and γ bound nucleolin with similar affinity. Moreover, all three Intimin types co-localized with regions of nucleolin expressed on the surface of HEp-2 cells. When Intimin α, β, or γ bound to Tir in vitro, the Intimin interaction with nucleolin was blocked. Both Tir and nucleolin accumulated beneath Intimin-presenting bacteria that had attached to the surface of HEp-2 cells. Taken together, these findings suggest that nucleolin is involved in bacterial adherence promoted by all Intimin types and that Tir and nucleolin compete for Intimin during adherence.
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plant cell based Intimin vaccine given orally to mice primed with Intimin reduces time of escherichia coli o157 h7 shedding in feces
Infection and Immunity, 2004Co-Authors: Nicole A Judge, Hugh S Mason, Alison D ObrienAbstract:Intimin is the primary adhesin of Escherichia coli O157:H7, the most common infectious cause of bloody diarrhea in the United States and the leading cause of acute kidney failure in children who develop hemolytic uremic syndrome. Cattle are the primary reservoir of E. coli O157:H7. Indeed, most cases of E. coli O157:H7 infection in the United States occur after ingestion of contaminated undercooked hamburger or produce that had contact with bovine manure. Because Intimin is required for persistent colonization of neonatal calves and adult cattle, we hypothesized that an Intimin-based vaccination strategy in calves would reduce colonization of cattle with E. coli O157:H7. To test this concept in a small-animal model, we developed transgenic tobacco plant cells that express the carboxy-terminal host cell-binding domain of E. coli O157:H7 Intimin. Mice were either immunized intraperitoneally with Intimin expressed from the plant cells, fed transgenic plant cells, or both. Here we show that these mice generated an Intimin-specific mucosal immune response when primed parenterally and then boosted orally and also exhibited a reduced duration of E. coli O157:H7 fecal shedding after challenge.
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antibodies to Intimin and escherichia coli secreted proteins a and b in patients with enterohemorrhagic escherichia coli infections
Pediatric Nephrology, 2002Co-Authors: Diana Karpman, Alison D Obrien, Lisa J Gansheroff, Zivile Bekassy, Annchristine Sjogren, Maria S Dubois, Mohamed A Karmali, Mariola Mascarenhas, Karen G Jarvis, Gerald S ArbusAbstract:Enterohemorrhagic Escherichia coli produce an attaching and effacing lesion upon adhering to the intestinal epithelium. Bacterial factors involved in this histopathology include the Intimin adhesin and E. coli secreted proteins (Esps) A and B. In this study we investigated the serum antibody responses to recombinant E. coli O157:H7 Intimin, EspA, and EspB by immunoblotting. Canadian patients with O157:H7 infection (n=10), Swedish patients with O157:H7 (n=21), non-O157 (n=18), or infection from which the serotype was not available (n=3), and asymptomatic household members (n=25) were studied and compared with Canadian (n=20) and Swedish controls (n=52). In Canadian patients, IgG antibodies to Intimin, EspA, and EspB were analyzed, in Swedish patients and their household members IgA, IgG, and IgM antibodies to EspA and EspB were studied. Patients and household members mounted an antibody response to the antigens. Significantly more patients developed an acute response to EspB compared with controls (P<0.01 Canadian patients, P<0.0001 Swedish patients). EspB IgA, IgG, and IgM had a specificity of 100%, 86%, and 86%, positive predictive value of 100%, 83%, and 81%, and sensitivity of 57%, 69%, and 63%, respectively, and appear to be an appropriate assay for the detection of EHEC infection. In cases of hemolytic uremic syndrome or hemorrhagic colitis this assay may be useful when a fecal strain has not been isolated, or in epidemics of non-O157 infection.
Gad Frankel - One of the best experts on this subject based on the ideXlab platform.
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a nanobody targeting the translocated Intimin receptor inhibits the attachment of enterohemorrhagic e coli to human colonic mucosa
PLOS Pathogens, 2019Co-Authors: Gad Frankel, Stephanie Schuller, David Ruanogallego, Daniel Yara, Lorenza Di Ianni, Luis Angel FernandezAbstract:Enterohemorrhagic E. coli (EHEC) is a human intestinal pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. No vaccines or specific therapies are currently available to prevent or treat these infections. EHEC tightly attaches to the intestinal epithelium by injecting the Intimin receptor Tir into the host cell via a type III secretion system (T3SS). In this project, we identified a camelid single domain antibody (nanobody), named TD4, that recognizes a conserved Tir epitope overlapping the binding site of its natural ligand Intimin with high affinity and stability. We show that TD4 inhibits attachment of EHEC to cultured human HeLa cells by preventing Tir clustering by Intimin, activation of downstream actin polymerization and pedestal formation. Furthermore, we demonstrate that TD4 significantly reduces EHEC adherence to human colonic mucosa in in vitro organ cultures. Altogether, these results suggest that nanobody-based therapies hold potential in the development of much needed treatment and prevention strategies against EHEC infection.
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functional studies of Intimin in vivo and ex vivo implications for host specificity and tissue tropism
Microbiology, 2007Co-Authors: Rosanna Mundy, Alan D. Phillips, Stephanie Schuller, Francis Girard, John M Fairbrother, Gad FrankelAbstract:Intimin is an outer-membrane adhesin that is essential for colonization of the host gastrointestinal tract by attaching and effacing pathogens including enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR). The N-terminus of Intimin from the different strains is highly conserved while the C-terminus, which harnesses the active receptor-binding site, shows sequence and antigenic polymorphism. This diversity was used to define a number of distinct Intimin types, the most common of which are α, β and γ. Intimin binds the type III secretion system effector protein Tir. However, a large body of evidence suggests that Intimin also binds a host-cell-encoded receptor(s) (Hir), and interaction of different Intimin types with Hir contributes to tissue and host specificity. The aims of this study were to compare the activity of the major Intimin types (α, β and γ) in vivo and ex vivo, using the CR mouse model and in vitro organ culture (IVOC), and to determine their exchangeability. The results confirm that Intimin γ is not functional in the CR mouse model. In the pig, Intimin β can substitute for EPEC Intimin α but when placed in an EHEC O157 : H7 background it does not produce an Intimin α-like tropism, although some adhesion to the small and large intestine was observed. In contrast, in human IVOC, Intimin β in an EHEC background produces small intestinal colonization in a similar manner to Intimin α.
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enteropathogenic escherichia coli epec adhesion to intestinal epithelial cells role of bundle forming pili bfp espa filaments and Intimin
Microbiology, 2004Co-Authors: Jennifer Cleary, Gad Frankel, Liching Lai, Robert K Shaw, Anna Straatmaniwanowska, Michael S Donnenberg, Stuart KnuttonAbstract:Enteropathogenic Escherichia coli (EPEC), an important paediatric diarrhoeal pathogen, employs multiple adhesins to colonize the small bowel and produces characteristic ‘attaching and effacing’ (A/E) lesions on small intestinal enterocytes. EPEC adhesins that have been associated with A/E adhesion and intestinal colonization include bundle-forming pili (BFP), EspA filaments and Intimin. BFP are involved in bacteria–bacteria interaction and microcolony formation but their role in cell adhesion remains unclear; EspA filaments are components of the EPEC type III secretion system but since they interact directly with host cells they may also function as adhesins; Intimin is the well characterized intimate EPEC adhesin which binds the translocated Intimin receptor, Tir. However, other uncharacterized host cell receptors have been implicated in Intimin-mediated adhesion. In this study, the role of BFP, EspA filaments and Intimin in EPEC adhesion to intestinal brush border cells was assessed by observing adhesion of wild-type EPEC strain E2348/69 and a set of isogenic single, double and triple mutants in bfpA, espA and eae (Intimin gene) to differentiated human intestinal Caco-2 cells. E2348/69 (bfpA + espA + eae +) adhered rapidly (<10 min) to the brush border of Caco-2 cells and subsequently produced microcolonies and typical A/E lesions. Non-intimate brush border adhesion of double mutant strain UMD880 (bfpA + espA − eae −) also occurred rapidly, whereas adhesion of strain UMD886 (bfpA − espA + eae −) occurred later in the infection (>1 h) and with much lower efficiency; confocal microscopy indicated BFP and EspA-mediated adhesion, respectively. Strain UMD883 (bfpA − espA − eae +), which is unable to translocate Tir, was non-adherent although this strain was able to form intimate attachment and A/E lesions when co-cultured with strain CVD206 (bfpA + espA + eae −) which supplied Tir to the membrane. Single mutant strains CVD206 (bfpA + espA + eae −) and UMD872 (bfpA + espA − eae +) showed adherence characteristics of strain UMD880 (bfpA + espA − eae −), whilst triple mutant strain UMD888 (bfpA − espA − eae −) was totally non-adherent. These results support an adhesive role for BFP and EspA in initial brush border cell attachment, and in typical EPEC which express both BFP and EspA filaments suggest a predominant role for BFP; EspA filaments, however, could serve as initial attachment factors in atypical EPEC which lacks BFP. The study found no evidence for an independent host cell Intimin receptor or for other adhesive factors able to support bacterial adherence.
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subtyping of virulence genes in verocytotoxin producing escherichia coli vtec other than serogroup o157 associated with disease in the united kingdom
Journal of Medical Microbiology, 2003Co-Authors: Claire Jenkins, Gad Frankel, G A Willshaw, J Evans, T Cheasty, H Chart, D J Shaw, G Dougan, H R SmithAbstract:Verocytotoxin-producing Escherichia coli (VTEC) causes a wide spectrum of disease in humans, from mild diarrhoea to haemolytic uraemic syndrome (HUS). The verocytotoxin (vtx) and Intimin (eae) genes of VTEC strains, other than those of serogroup O157, were subtyped to identify common properties that may be associated with increased pathogenicity. Strains were isolated from patients with HUS, those with diarrhoea or from asymptomatic individuals. Strains of VTEC that carried vtx 2 gene subtypes vtx 2 and vtx 2c were most commonly associated with HUS, whereas strains from patients with less severe disease and from the healthy control group were more likely to have vtx 1c or vtx 2d genes. The eae gene was detected more frequently in strains isolated from HUS patients than in those associated with cases of diarrhoea; β-Intimin was the most common Intimin subtype in strains isolated from both groups of patients. None of the strains from the healthy control group carried the eae gene.
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subtyping Intimin genes from enteropathogenic escherichia coli associated with outbreaks and sporadic cases in the united kingdom and eire
Molecular and Cellular Probes, 2003Co-Authors: Claire Jenkins, Gad Frankel, G A Willshaw, T Cheasty, G Dougan, Andrew J Lawson, P Wright, H R SmithAbstract:Abstract PCR-RFLP methods for subtyping the Intimin gene from strains of typical and atypical enteropathogenic Escherichia coli (EPEC) and Verocytotoxin-producing E. coli (VTEC) were compared. A novel Hha I PCR-RFLP method was developed that was rapid, easy to use and amplified an 1852 bp fragment of the Intimin gene from all isolates examined. This method was used to investigate the Intimin sub-types of EPEC strains associated with 14 outbreaks of diarrhoeal disease between 1967 and 2001, and 20 sporadic cases between January and December 2000, in the UK and Eire. In this study, genes encoding α, β, γ, δ and ζ-Intimin were detected in the EPEC strains associated with outbreaks and β, γ, ϵ, θ and ζ-Intimin genes were identified in isolates from sporadic cases. The β-Intimin gene was the most frequently detected sub-type in both the outbreak and sporadic strains.
Gordon Dougan - One of the best experts on this subject based on the ideXlab platform.
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Site-directed mutagenesis of Intimin α modulates Intimin-mediated tissue tropism and host specificity
Molecular microbiology, 2001Co-Authors: Stephen Reece, Alan D. Phillips, Robert J. Fitzhenry, Cameron P. Simmons, Stephen Matthews, Gordon DouganAbstract:Summary 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 (a, b, g, d and 1) 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 twohybrid 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 Int280a/whole Intimin a )i n Intimin a 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.
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Intimin specific immune responses prevent bacterial colonization by the attaching effacing pathogen citrobacter rodentium
Infection and Immunity, 2001Co-Authors: Marjan Ghaemmaghami, Gad Frankel, Cameron P. Simmons, Sarah Daniell, Mariagrazia Pizza, David J M Lewis, Gordon DouganAbstract:The formation of attaching and effacing (A/E) lesions on gut enterocytes is central to the pathogenesis of enterohemorrhagic (EHEC) Escherichia coli, enteropathogenic E. coli (EPEC), and the rodent pathogen Citrobacter rodentium. Genes encoding A/E lesion formation map to a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Here we show that the LEE-encoded proteins EspA, EspB, Tir, and Intimin are the targets of long-lived humoral immune responses in C. rodentium-infected mice. Mice infected with C. rodentium developed robust acquired immunity and were resistant to reinfection with wild-type C. rodentium or a C. rodentium derivative, DBS255(pCVD438), which expressed Intimin derived from EPEC strain E2348/69. The receptor-binding domain of Intimin polypeptides is located within the carboxy-terminal 280 amino acids (Int280). Mucosal and systemic vaccination regimens using enterotoxin-based adjuvants were employed to elicit immune responses to recombinant Int280α from EPEC strain E2348/69. Mice vaccinated subcutaneously with Int280α, in the absence of adjuvant, were significantly more resistant to oral challenge with DBS255(pCVD438) but not with wild-type C. rodentium. This type-specific immunity could not be overcome by employing an exposed, highly conserved domain of Intimin (Int388–667) as a vaccine. These results show that anti-Intimin immune responses can modulate the outcome of a C. rodentium infection and support the use of Intimin as a component of a type-specific EPEC or EHEC vaccine.
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Intimin and the host cell is it bound to end in tir s
Trends in Microbiology, 2001Co-Authors: Gad Frankel, Gordon Dougan, Alan D. Phillips, Stuart Knutton, Luiz Rachid Trabulsi, Stephen MatthewsAbstract:Intimate bacterial adhesion to the intestinal epithelium is a pathogenic mechanism shared by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli. Two bacterial protein partners involved in this intimate association have been identified, Intimin and Tir. Some key remaining questions include whether Intimin specifically interacts with one or more host-cell-encoded molecules and whether these contacts are a prerequisite for the subsequent intimate Intimin-Tir association. Recent data support the hypothesis that the formation of a stable Intimin-Tir relationship is the consequence of Intimin protein interactions involving both host and bacterial components.
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Expression of Intimin gamma from enterohemorrhagic Escherichia coli in Citrobacter rodentium.
Infection and Immunity, 2000Co-Authors: Elizabeth L Hartland, Thomas T. Macdonald, Gordon Dougan, Nathalie S. Gonçalves, Veronika Huter, Lisa M. Higgins, Alan D. Phillips, Gad FrankelAbstract:The carboxy-terminal 280 amino acids (Int280) of the bacterial adhesion molecule Intimin include the receptor-binding domain. At least five different types of Int280, designated alpha, beta, gamma, delta, and epsilon, have been described based on sequence variation in this region. Importantly, the Intimin types are associated with different evolutionary branches and contribute to distinct tissue tropism of Intimin-positive bacterial pathogens. In this study we engineered a strain of Citrobacter rodentium, which normally displays Intimin beta, to express Intimin gamma from enterohemorrhagic Escherichia coli. We show that Intimin gamma binds to the translocated Intimin receptor (Tir) from C. rodentium and has the ability to produce attaching and effacing lesions on HEp-2 cells. However, C. rodentium expressing Intimin gamma could not colonize orally infected mice or induce mouse colonic hyperplasia. These results suggest that Intimin may contribute to host specificity, possibly through its interaction with a receptor on the host cell surface.
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structural basis for recognition of the translocated Intimin receptor tir by Intimin from enteropathogenic escherichia coli
The EMBO Journal, 2000Co-Authors: Miranda Batchelor, Gad Frankel, Gordon Dougan, Stephen Reece, Ian F Connerton, Sunil Prasannan, Sarah Daniell, Graham B Bloomberg, Stephen MatthewsAbstract:Intimin is a bacterial adhesion molecule involved in intimate attachment of enteropathogenic and enterohaemorrhagic Escherichia coli to mammalian host cells. Intimin targets the translocated Intimin receptor (Tir), which is exported by the bacteria and integrated into the host cell plasma membrane. In this study we localized the Tir‐binding region of Intimin to the C‐terminal 190 amino acids (Int190). We have also determined the region9s high‐resolution solution structure, which comprises an immunoglobulin domain that is intimately coupled to a novel C‐type lectin domain. This fragment, which is necessary and sufficient for Tir interaction, defines a new super domain in Intimin that exhibits striking structural similarity to the integrin‐binding domain of the Yersinia invasin and C‐type lectin families. The extracellular portion of Intimin comprises an articulated rod of immunoglobulin domains extending from the bacterium surface, conveying a highly accessible ’adhesive tip9 to the target cell. The interpretation of NMR‐titration and mutagenesis data has enabled us to identify, for the first time, the binding site for Tir, which is located at the extremity of the Int190 moiety.
Alan D. Phillips - One of the best experts on this subject based on the ideXlab platform.
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functional studies of Intimin in vivo and ex vivo implications for host specificity and tissue tropism
Microbiology, 2007Co-Authors: Rosanna Mundy, Alan D. Phillips, Stephanie Schuller, Francis Girard, John M Fairbrother, Gad FrankelAbstract:Intimin is an outer-membrane adhesin that is essential for colonization of the host gastrointestinal tract by attaching and effacing pathogens including enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR). The N-terminus of Intimin from the different strains is highly conserved while the C-terminus, which harnesses the active receptor-binding site, shows sequence and antigenic polymorphism. This diversity was used to define a number of distinct Intimin types, the most common of which are α, β and γ. Intimin binds the type III secretion system effector protein Tir. However, a large body of evidence suggests that Intimin also binds a host-cell-encoded receptor(s) (Hir), and interaction of different Intimin types with Hir contributes to tissue and host specificity. The aims of this study were to compare the activity of the major Intimin types (α, β and γ) in vivo and ex vivo, using the CR mouse model and in vitro organ culture (IVOC), and to determine their exchangeability. The results confirm that Intimin γ is not functional in the CR mouse model. In the pig, Intimin β can substitute for EPEC Intimin α but when placed in an EHEC O157 : H7 background it does not produce an Intimin α-like tropism, although some adhesion to the small and large intestine was observed. In contrast, in human IVOC, Intimin β in an EHEC background produces small intestinal colonization in a similar manner to Intimin α.
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Intimin type influences the site of human intestinal mucosal colonisation by enterohaemorrhagic escherichia coli o157 h7
Gut, 2002Co-Authors: Robert J. Fitzhenry, Elizabeth L Hartland, Alan D. Phillips, Stephen Reece, G Dougan, Derek Pickard, Gad FrankelAbstract:Background: Enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli epithelial cell adhesion is characterised by intimate attachment, and attaching and effacing (A/E) lesion formation. This event is mediated in part by Intimin binding to another bacterial protein, Tir (translocated Intimin receptor), which is exported by the bacteria and integrated into the host cell plasma membrane. Importantly, EPEC (O127:H6) and EHEC (O157:H7) express antigenically distinct Intimin types known as Intimin α and γ, respectively. EHEC (O157:H7) colonises human intestinal explants although adhesion is restricted to the follicle associated epithelium of Peyer's patches. This phenotype is also observed with EPEC O127:H6 engineered to express EHEC Intimin γ. Aims: To investigate the influence of Intimin on colonisation of human intestine by E coli O157:H7, and Intimin types on tissue tropism in humans. Methods: Human intestinal in vitro organ culture with wild type and mutant strains of O157:H7 were employed. Results: Introducing a deletion mutation in the eae gene encoding Intimin γ in EHEC (O157:H7) caused the strain (ICC170) to fail to colonise human intestinal explants. However, colonisation of Peyer's patches and A/E lesion formation were restored with Intimin γ expression from a plasmid (ICC170 (pICC55)). In contrast, complementing the mutation with Intimin α resulted in a strain (ICC170 (pCVD438)) capable of colonising and producing A/E lesions on both Peyer's patch and other small intestinal explants. Conclusion: Intimin is necessary for human intestinal mucosal colonisation by E coli O157:H7. Intimin type influences the site of colonisation in a Tir type independent mechanism; Intimin γ appears to restrict colonisation to human follicle associated epithelium.
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Site-directed mutagenesis of Intimin α modulates Intimin-mediated tissue tropism and host specificity
Molecular microbiology, 2001Co-Authors: Stephen Reece, Alan D. Phillips, Robert J. Fitzhenry, Cameron P. Simmons, Stephen Matthews, Gordon DouganAbstract:Summary 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 (a, b, g, d and 1) 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 twohybrid 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 Int280a/whole Intimin a )i n Intimin a 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.
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Intimin and the host cell is it bound to end in tir s
Trends in Microbiology, 2001Co-Authors: Gad Frankel, Gordon Dougan, Alan D. Phillips, Stuart Knutton, Luiz Rachid Trabulsi, Stephen MatthewsAbstract:Intimate bacterial adhesion to the intestinal epithelium is a pathogenic mechanism shared by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli. Two bacterial protein partners involved in this intimate association have been identified, Intimin and Tir. Some key remaining questions include whether Intimin specifically interacts with one or more host-cell-encoded molecules and whether these contacts are a prerequisite for the subsequent intimate Intimin-Tir association. Recent data support the hypothesis that the formation of a stable Intimin-Tir relationship is the consequence of Intimin protein interactions involving both host and bacterial components.
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Expression of Intimin gamma from enterohemorrhagic Escherichia coli in Citrobacter rodentium.
Infection and Immunity, 2000Co-Authors: Elizabeth L Hartland, Thomas T. Macdonald, Gordon Dougan, Nathalie S. Gonçalves, Veronika Huter, Lisa M. Higgins, Alan D. Phillips, Gad FrankelAbstract:The carboxy-terminal 280 amino acids (Int280) of the bacterial adhesion molecule Intimin include the receptor-binding domain. At least five different types of Int280, designated alpha, beta, gamma, delta, and epsilon, have been described based on sequence variation in this region. Importantly, the Intimin types are associated with different evolutionary branches and contribute to distinct tissue tropism of Intimin-positive bacterial pathogens. In this study we engineered a strain of Citrobacter rodentium, which normally displays Intimin beta, to express Intimin gamma from enterohemorrhagic Escherichia coli. We show that Intimin gamma binds to the translocated Intimin receptor (Tir) from C. rodentium and has the ability to produce attaching and effacing lesions on HEp-2 cells. However, C. rodentium expressing Intimin gamma could not colonize orally infected mice or induce mouse colonic hyperplasia. These results suggest that Intimin may contribute to host specificity, possibly through its interaction with a receptor on the host cell surface.
Martin J Woodward - One of the best experts on this subject based on the ideXlab platform.
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role of Intimin tir interactions and the tir cytoskeleton coupling protein in the colonization of calves and lambs by escherichia coli o157 h7
Infection and Immunity, 2006Co-Authors: Isabella Vlisidou, G Frankel, Francis Dziva, R M La Ragione, Angus Best, Junkal Garmendia, Pippa Hawes, Paul Monaghan, Shaun Cawthraw, Martin J WoodwardAbstract:Intimin facilitates intestinal colonization by enterohemorrhagic Escherichia coli O157:H7; however, the importance of Intimin binding to its translocated receptor (Tir) as opposed to cellular coreceptors is unknown. The Intimin-Tir interaction is needed for optimal actin assembly under adherent bacteria in vitro, a process which requires the Tir-cytoskeleton coupling protein (TccP/EspF(U)) in E. coli O157:H7. Here we report that E. coli O157:H7 tir mutants are at least as attenuated as isogenic eae mutants in calves and lambs, implying that the role of Intimin in the colonization of reservoir hosts can be explained largely by its binding to Tir. Mutation of tccP uncoupled actin assembly from the Intimin-Tir-mediated adherence of E. coli O157:H7 in vitro but did not impair intestinal colonization in calves and lambs, implying that pedestal formation may not be necessary for persistence. However, an E. coli O157:H7 tccP mutant induced typical attaching and effacing lesions in a bovine ligated ileal loop model of infection, suggesting that TccP-independent mechanisms of actin assembly may operate in vivo.
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Role for Flagella but Not Intimin in the Persistent Infection of the Gastrointestinal Tissues of Specific-Pathogen-Free Chicks by Shiga Toxin-Negative Escherichia coli O157:H7
Infection and Immunity, 2005Co-Authors: Angus I. Best, Roberto M La Ragione, A. Robin Sayers, Martin J WoodwardAbstract:Shiga toxin (Stx)-positive Escherichia coli O157:H7 readily colonize and persist in specific-pathogen-free (SPF) chicks, and we have shown that an Stx-negative E. coli O157:H7 isolate (NCTC12900) readily colonizes SPF chicks for up to 169 days after oral inoculation at 1 day of age. However, the role of Intimin in the persistent colonization of poultry remains unclear. Thus, to investigate the role of Intimin and flagella, which is a known factor in the persistence of non-O157 E. coli in poultry, isogenic single- and double-Intimin and aflagellar mutants were constructed in E. coli O157:H7 isolate NCTC12900. These mutants were used to inoculate (105 CFU) 1-day-old SPF chicks. In general, significant attenuation of the aflagellate and Intimin-aflagellate mutants, but not the Intimin mutant, was noted at similar time points between 22 and 92 days after inoculation. The Intimin-deficient mutant was still being shed at the end of the experiment, which was 211 days after inoculation, 84 days more than the wild type. Shedding of the aflagellar and Intimin-aflagellar mutants ceased 99 and 113 days after inoculation, respectively. Histological analysis of gastrointestinal tissues from inoculated birds gave no evidence for true microcolony formation by NCTC12900 or Intimin and aflagellar mutants to epithelial cells. However, NCTC12900 mutant derivatives associated with the mucosa were observed as individual cells and/or as large aggregates. Association with luminal contents was also noted. These data suggest that O157 organisms do not require Intimin for the persistent colonization of chickens, whereas flagella do play a role in this process.
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Isolation of recombinant antibodies against EspA and Intimin of escherichia coli O157:H7
Journal of Clinical Microbiology, 2004Co-Authors: Sarah A. Kühne, William S. Hawes, Martin J Woodward, Roberto M La Ragione, Garry C. Whitelam, Kevin C. GoughAbstract:Intimin, Tir, and EspA proteins are expressed by attaching-effacing Escherichia coli, which include enteropathogenic and enterohemorrhagic E. coli pathotypes. EspA proteins are part of the type three secretion system needle complex that delivers Tir to the host epithelial cell, while surface arrayed Intimin docks the bacterium to the translocated Tir. This intimate attachment leads to attaching and effacing lesions. Recombinant forms of these effector proteins from enterohemorrhagic E. coli O157:H7 were produced by using E. coli expression vectors. Binding of Intimin and Tir fragments in enzyme-linked immunosorbent assay (ELISAs) demonstrated the interaction of Intimin fragments containing the C-terminal 282 or 188 amino acids to a Tir fragmentcontaining amino acid residues 258 to 361. Recombinant Intimin and EspA proteins were used to elicit immune responses in rabbits and immune phage-display antibody libraries were produced. Screening of these immune libraries by conventional phage-antibody panning and colony filter screening produced a panel of antibodies with specificity for EspA or Intimin. Antibodies recognizing different C-terminal epitopes on Intimin bound specifically to the gamma Intimin of O157:H7 and not to other classes of Intimin. Antibodies recognizing EspAfrom E. coli O157 also recognized the protein from the eae-deficient O157 mutant DM3 and from E. coli O111. Anti-Intimin antibodies were also produced as fusion proteins coupled to the reporter molecule alkaline phosphatase, allowing the one-step detection of Intimin. The isolated recombinant monoclonal antibodies were functional in a range of assay formats, including ELISA, Western blotting, and dot blots, thus demonstrating their diagnostic potential.
