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Gad Frankel

Publications and source records attributed to Gad Frankel.

72 records · Page 4Linked to original sources
PubMed2003

Yeast two-hybrid system survey of interactions between LEE-encoded proteins of enteropathogenic Escherichia coli.

Many Gram-negative pathogens employ a specific secretion pathway, termed type III secretion, to deliver virulence effector proteins directly to the membranes and cytosol of host eukaryotic cells. Subsequent functions of many effector proteins delivered in this manner result in subversion of host-signalling pathways to facilitate bacterial entry, survival and dissemination to neighbouring cells and tissues. Whereas the secreted components of type III secretion systems (TTSSs) from different pathogens are structurally and functionally diverse, the structural components and the secretion apparatus itself are largely conserved. TTSSs are large macromolecular assemblies built through interactions between protein components of hundreds of individual subunits. The goal of this project was to screen, using the standard yeast two-hybrid system, pair-wise interactions between components of the enteropathogenic Escherichia coli TTSS. To this end 37 of the 41 genes encoded by the LEE pathogenicity island were cloned into both yeast two-hybrid system vectors and all possible permutations of interacting protein pairs were screened for. This paper reports the identification of 22 novel interactions, including interactions between inner-membrane structural TTSS proteins; between the type III secreted translocator protein EspD and structural TTSS proteins; between established and putative chaperones and their cognate secreted proteins; and between proteins of undefined function.

Bacterial Proteins↗
PubMed2003

CesAB is an enteropathogenic Escherichia coli chaperone for the type-III translocator proteins EspA and EspB.

Enteropathogenic Escherichia coli (EPEC) are extracellular pathogens that colonize mucosal surfaces of the intestine via formation of attaching and effacing (A/E) lesions. The genes responsible for induction of the A/E lesions are located on a pathogenicity island, termed the locus of enterocyte effacement (LEE), which encodes the adhesin intimin and the type III secretion system needle complex, translocator and effector proteins. One of the major EPEC translocator proteins, EspA, forms a filamentous conduit along which secreted proteins travel before they arrive at the translocation pore in the plasma membrane of the host cell, which is composed of EspB and EspD. Prior to secretion, many type III proteins, including translocators, are maintained in the bacterial cytoplasm by association with a specific chaperone. In EPEC, chaperones have been identified for the effector proteins Tir, Map and EspF, and the translocator proteins EspD and EspB. In this study, CesAB (Orf3 of the LEE) was identified as a chaperone for EspA and EspB. Specific CesAB-EspA and CesAB-EspB protein interactions are demonstrated. CesAB was essential for stability of EspA within the bacterial cell prior to secretion. Furthermore, a cesAB mutant failed to secrete EspA, as well as EspB, to assemble EspA filaments, to induce A/E lesion following infection of HEp-2 cells and to adhere to, or cause haemolysis of, erythrocytes.

Bacterial Adhesion↗
PubMed2003

CesD2 of enteropathogenic Escherichia coli is a second chaperone for the type III secretion translocator protein EspD.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli are extracellular pathogens that employ a type III secretion system to export translocator and effector proteins, proteins which facilitates colonization of the mucosal surface of the intestine via formation of attaching and effacing (A/E) lesions. The genes encoding the proteins for A/E lesion formation are located on a pathogenicity island, termed the locus of enterocyte effacement (LEE), which contains eae encoding intimin as well as the type III secretion system and effector genes. Many type III secreted proteins are stabilized and maintained in a secretion-competent conformation in the bacterial cytosol by specific chaperone proteins. Three type III chaperones have been described thus far within the EPEC LEE region: CesD, for the translocator proteins EspB and EspD; CesT, for the effector proteins Tir and Map; and CesF, for EspF. In this study we report the characterization of CesD2 (previously Orf27), a second LEE-encoded chaperone for EspD. We show specific CesD2-EspD protein interaction which appears to be necessary for proper EspD secretion in vitro and pathogenesis in vivo as demonstrated in the A/E-lesion-forming mouse pathogen Citrobacter rodentium.

Amino Acid Sequence↗
PubMed2003

Polymorphisms within EspA filaments of enteropathogenic and enterohemorrhagic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) possess a filamentous type III secretion system (TTSS) employed to deliver effector proteins into host cells. EspA is a type III secreted protein which forms the filamentous extension to the TTSS and which interacts with host cells during early stages of attaching and effacing (A/E) lesion formation. By immunofluorescence, a polyclonal antibody previously raised to EspA from EPEC strain E2348/69 (O127:H6) stained approximately 12-nm-diameter EspA filaments produced by this strain but did not stain similar filaments produced by EHEC serotype O157:H7. Similarly, an antibody that we subsequently raised to EHEC strain 85-170 (O157:H7) EspA stained approximately 12-nm-diameter EspA filaments produced by strain 85-170 but did not stain E2348/69 EspA filaments. Given such heterogeneity between EPEC and EHEC EspA filaments, we examined polymorphisms of functional EspA filaments among different EPEC and EHEC serotypes. With use of the EPEC EspA antiserum, EspA filaments were observed only with EPEC serotypes O127:H6 and O55:H6, serotypes which encode an identical EspA protein. When stained with the EHEC EspA antiserum, EspA filaments were detected only on EHEC strains belonging to serotype O157:H7; the EHEC antiserum did, however, stain EspA filaments produced by the closely related EPEC serotype O55:H7 but not filaments of any other EPEC serotype tested. Such polymorphisms among functional EspA filaments of EPEC and EHEC would be expected to have important implications for the development of broad-range EspA-based vaccines.

Animals↗
PubMed2003

Binding of intimin from enteropathogenic Escherichia coli to lymphocytes and its functional consequences.

Intimin-conjugated fluorescent beads bind to spleen CD4 T cells and Peyer's patch, mesenteric lymph node, and cecal follicle lymphocytes, with less binding to lamina propria T cells and intraepithelial lymphocytes. Intimin costimulates proliferation of spleen CD4 T cells and cells from organized lymphoid tissues but does not costimulate cells from the lamina propria of normal or inflamed colon.

Adhesins, Bacterial↗
PubMed2003

Central role for B lymphocytes and CD4+ T cells in immunity to infection by the attaching and effacing pathogen Citrobacter rodentium.

Citrobacter rodentium, an attaching-effacing bacterial pathogen, establishes an acute infection of the murine colonic epithelium and induces a mild colitis in immunocompetent mice. This study describes the role of T-cell subsets and B lymphocytes in immunity to C. rodentium. C57Bl/6 mice orally infected with C. rodentium resolved infection within 3 to 4 weeks. Conversely, systemic and colonic tissues of RAG1(-/-) mice orally infected with C. rodentium contained high and sustained pathogen loads, and in the colon this resulted in a severe colitis. C57Bl/6 mice depleted of CD4(+) T cells, but not CD8(+) T cells, were highly susceptible to infection and also developed severe colitis. Mice depleted of CD4(+) T cells also had diminished immunoglobulin G (IgG) and IgA antibody responses to two C. rodentium virulence-associated determinants, i.e., EspA and intimin, despite having a massively increased pathogen burden. Mice with an intact T-cell compartment, but lacking B cells ( micro MT mice), were highly susceptible to C. rodentium infection. Systemic immunity, but not mucosal immunity, could be restored by adoptive transfer of convalescent immune sera to infected micro MT mice. Adoptive transfer of immune B cells, but not naïve B cells, provided highly variable immunity to recipient micro MT mice. The results suggest that B-cell-mediated immune responses are central to resolution of a C. rodentium infection but that the mechanism through which this occurs requires further investigation. These data are relevant to understanding immunity to enteric attaching and effacing bacterial pathogens of humans.

Adoptive Transfer↗
PubMed2003

Distribution of the saa gene in strains of Shiga toxin-producing Escherichia coli of human and bovine origins.

Certain strains of Shiga toxin-producing Escherichia coli (STEC) which do not have the locus of enterocyte effacement pathogenicity island carry the STEC autoagglutinating adhesin (saa) gene. The distribution of the saa gene in STEC isolates from patients with hemolytic-uremic syndrome (HUS), patients with less severe diarrheal disease, asymptomatic individuals, and healthy cattle was examined. saa-positive strains were detected more frequently (P < 0.001) in STEC strains from bovines (32 of 56 strains) than in those from humans (8 of 91 strains). No significant association (P = 0.135) was found between the saa gene and STEC isolated from patients with HUS (6 of 46 strains) or diarrhea (2 of 29 strains) and from healthy controls (0 of 16 strains).

Adhesins, Bacterial↗
PubMed2002

Impaired resistance and enhanced pathology during infection with a noninvasive, attaching-effacing enteric bacterial pathogen, Citrobacter rodentium, in mice lacking IL-12 or IFN-gamma.

Mice infected with Citrobacter rodentium represent an excellent model in which to examine immune defenses against an attaching-effacing enteric bacterial pathogen. Colonic tissue from mice infected with C. rodentium harbors increased transcripts for IL-12 and IFN-gamma and displays mucosal pathology compared with uninfected controls. In this study, the role of IL-12 and IFN-gamma in host defense and mucosal injury during C. rodentium infection was examined using gene knockout mice. IL-12p40(-/-) and IFN-gamma(-/-) mice were significantly more susceptible to mucosal and gut-derived systemic C. rodentium infection. In particular, a proportion of IL-12p40(-/-) mice died during infection. Analysis of the gut mucosa of IL-12p40(-/-) mice revealed an influx of CD4(+) T cells and a local IFN-gamma response. Infected IL-12p40(-/-) and IFN-gamma(-/-) mice also mounted anti-Citrobacter serum and gut-associated IgA responses and strongly expressed inducible NO synthase (iNOS) in mucosal tissue, despite diminished serum nitrite/nitrate levels. However, iNOS does not detectably contribute to host defense against C. rodentium, as iNOS(-/-) mice were not more susceptible to infection. However, C57BL/6 mice infected with C. rodentium up-regulated expression of the mouse beta-defensin (mBD)-1 and mBD-3 in colonic tissue. In contrast, expression of mBD-3, but not mBD-1, was significantly attenuated during infection of IL-12- and IFN-gamma-deficient mice, suggesting mBD-3 may contribute to host defense. These studies are among the first to examine mechanisms of host resistance to an attaching-effacing pathogen and show an important role for IL-12 and IFN-gamma in limiting bacterial infection of the colonic epithelium.

Administration, Oral↗
PubMed2002

Induction of cell death in T lymphocytes by invasin via beta1-integrin.

Ligand binding to beta1-integrins exerts multiple effects on cells of the immune system including adhesion, spreading, haptotaxis and costimulation of T cells activated by anti-CD3. Here we show that a high-affinity ligand for beta1-integrins, the invasin (Inv) protein of Yersinia pseudotuberculosis, can induce cell death in T lymphocytes via a rapid process. Partially purified native Inv protein and an Inv fusion protein caused apoptotic/necrotic caspase-independent cell death in T lymphocytes as determined by phosphatidylserine exposure on the cell surface, uptake of propidium iodide, labeling of DNA strand breaks and presence of DNA ladder. Inv-induced cell death was mediated via beta1-integrins as indicated by the fact that Inv bound to the beta1-integrin subunit (CD29), that anti-beta(1)-integrin antibodies blocked Inv-induced cell death and that Inv-induced cell death was absent in two beta1-integrin- cell lines produced by different procedures. Killing via beta1-integrins represents a novel pathway for cell death in T lymphocytes.

Adhesins, Bacterial↗
PubMed2002

Tyrosine residues at the immunoglobulin-C-type lectin inter-domain boundary of intimin are not involved in Tir-binding but implicated in colonisation of the host.

Intimin is an outer membrane adhesion molecule involved in bacterial adhesion to intestinal epithelium by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli and Citrobacter rodentium. Intimin binds to the translocated intimin receptor, Tir, which is delivered to the plasma membrane of the host cell by a type III protein translocation system. Intimin is also implicated in binding to a host cell-encoded intimin receptor (Hir). The receptor-binding activity of intimin resides within the carboxy terminus 280 amino acids (Int280) of the polypeptide. Structural analysis of this region revealed two immunoglobulin-like domains, the second of which forms a number of contacts with the distal C-type lectin-like module. Specific orientation differences at this inter-domain boundary, which consists of several tyrosine residues, were detected between the crystal and solution structures. In this study, we determined the influence of site-directed mutagenesis of each of four tyrosine residues on intimin-Tir interactions and on intimin-mediated intimate attachment. The mutant intimins were also studied using a variety of in vitro and in vivo infection models. The results show that three of the four Tyr, although not essential for A/E lesion formation in vitro, are required for efficient colonisation of the mouse host following oral challenge.

Adhesins, Bacterial↗
PubMed2002

Functional analysis of the enteropathogenic Escherichia coli type III secretion system chaperone CesT identifies domains that mediate substrate interactions.

In many Gram-negative bacteria, a key indicator of pathogenic potential is the possession of a specialized type III secretion system, which is utilized to deliver virulence effector proteins directly into the host cell cytosol. Many of the proteins secreted from such systems require small cytosolic chaperones to maintain the secreted substrates in a secretion-competent state. One such protein, CesT, serves a chaperone function for the enteropathogenic Escherichia coli (EPEC) translocated intimin receptor (Tir) protein, which confers upon EPEC the ability to alter host cell morphology following intimate bacterial attachment. Using a combination of complementary biochemical approaches, functional domains of CesT that mediate intermolecular interactions, involved in both chaperone-chaperone and chaperone-substrate associations, were determined. The CesT N-terminal is implicated in chaperone dimerization, whereas the amphipathic alpha-helical region of the C-terminal, is intimately involved in substrate binding. By functional complementation of chaperone domains using the Salmonella SicA chaperone to generate chaperone chimeras, we show that CesT-Tir interaction proceeds by a mechanism potentially common to other type III secretion system chaperones.

Amino Acid Sequence↗
PubMed2002

Coiled-coil proteins associated with type III secretion systems: a versatile domain revisited.

The pathogenic potential of many Gram-negative bacteria is indicated by the possession of a specialized type III secretion system that is used to deliver virulence effector proteins directly into the cellular environment of the eukaryotic host. Extracellular assemblies of secreted proteins contrive a physical link between the pathogen and host cytosol and enable the translocated effectors to bypass the bacterial and host membranes in a single step. Subsequent interactions of some effector proteins with host cytoskeletal and signalling proteins result in modulation of the cytoskeletal architecture of the aggressed cell and facilitate entry, survival and dissemination of the pathogen. Although the secreted components of type III secretion systems are diverse, many are predicted to share a common coiled-coil structural feature. Coiled-coils are ubiquitous and highly versatile assembly motifs found in a wide range of structural and regulatory proteins. The prevalence of these domains in secreted virulence effector proteins suggests a fundamental contribution to multiple aspects of their function, and evidence accumulating from functional studies suggests an intrinsic involvement of coiled-coils in subunit assembly, translocation and flexible interactions with multiple bacterial and host proteins. The known functional flexibility that coiled-coil domains confer upon proteins provides insights into some of the pathogenic mechanisms used during interaction with the host.

Bacteria↗
PubMed2002

Mutagenesis of conserved tryptophan residues within the receptor-binding domain of intimin: influence on binding activity and virulence.

Intimate bacterial adhesion to intestinal epithelium is a pathogenic mechanism shared by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli and Citrobacter rodentium. The proteins directly involved in this process are the outer-membrane adhesion molecule intimin and the translocated intimin receptor, Tir. The receptor-binding activity of intimin resides within the carboxy terminus 280 aa (Int280) of the polypeptide. Four tryptophan residues, W117/776, W136/795, W222/881 and W240/899, are conserved within different Int280 molecules that otherwise show considerable sequence variation. In this study the influence of site-directed mutagenesis of each of the four tryptophan residues on intimin-Tir interactions and on intimin-mediated intimate attachment was determined. The mutant intimins were also studied using a variety of in vitro and in vivo infection models. The results show that all the substitutions modulated intimin activity, although some mutations had more profound effects than others.

Adhesins, Bacterial↗
PubMed2002

Enteropathogenic Escherichia coli translocate Tir and form an intimin-Tir intimate attachment to red blood cell membranes.

Type III secretion allows bacteria to inject effector proteins into host cells. In enteropathogenic Escherichia coli (EPEC) the type III secreted protein, Tir, is translocated to the host-cell plasma membrane where it functions as a receptor for the bacterial adhesin intimin, leading to intimate bacterial attachment and "attaching and effacing" (A/E) lesion formation. To study EPEC type III secretion the interaction of EPEC with monolayers of red blood cells (RBCs) has been exploited and in a recent study [Shaw, R. K., Daniell, S., Ebel, F., Frankel, G. & Knutton, S. (2001 ). Cell Microbiol 3, 213-222] it was shown that EPEC induced haemolysis of RBCs and translocation of EspD, a putative pore-forming type III secreted protein in the RBC membrane. Here it is demonstrated that EPEC are able to translocate and correctly insert Tir into the RBC membrane and produce an intimin-Tir intimate bacterial attachment, identical to that seen in A/E lesions. Following translocation Tir did not undergo any change in apparent molecular mass or become tyrosine-phosphorylated and there was no focusing of RBC cytoskeletal actin beneath intimately adherent bacteria, and no pedestal formation. This study, employing an RBC model of infection, has demonstrated that Tir translocation can be separated from host-cell-mediated Tir modifications; the data show that the EPEC type III protein translocation apparatus is sufficient to deliver and correctly insert Tir into host-cell membranes independent of eukaryotic cell functions.

Adhesins, Bacterial↗
PubMed2002

Intimin, tir, and shiga toxin 1 do not influence enteropathogenic responses to shiga toxin-producing Escherichia coli in bovine ligated intestinal loops.

Shiga toxin-producing Escherchia coli (STEC) comprises a group of attaching and effacing (A/E) enteric pathogens of animals and humans. Natural and experimental infection of calves with STEC may result in acute enteritis or subclinical infection, depending on serotype- and host-specific factors. To quantify intestinal secretory and inflammatory responses to STEC in the bovine intestine, serotypes that are associated with human disease (O103:H2 and O157:H7) were introduced into ligated mid-ileal loops in gnotobiotic and conventional calves, and fluid accumulation and recruitment of radiolabeled neutrophils were measured after 12 h. STEC serotype O103:H2, but not serotype O157:H7, elicited strong enteropathogenic responses. To determine if the inflammatory response to STEC O103:H2 in calves requires Shiga toxin 1 or intimate bacterial attachment to the intestinal epithelium, defined mutations were made in the stx1, eae, and tir genes. Our data indicate that some STEC induce intestinal inflammatory responses in calves by a mechanism that is independent of A/E-lesion formation, intimin, or Shiga toxin 1. This may have implications for strategies to reduce STEC carriage in cattle.

Adhesins, Bacterial↗
PubMed2002

Efa1 influences colonization of the bovine intestine by shiga toxin-producing Escherichia coli serotypes O5 and O111.

Shiga toxin-producing Escherichia coli (STEC) comprises a broad group of bacteria, some of which cause attaching and effacing (AE) lesions and enteritis in animals and humans. Non-O157 STEC serotypes contain a gene (efa1) that mediates attachment to cultured epithelial cells. An almost-identical gene in enteropathogenic E. coli (lifA) encodes lymphostatin, which inhibits the proliferation of mitogen-activated lymphocytes and the synthesis of proinflammatory cytokines. We have investigated the role of the efa1 gene in colonization of 4- and 11-day-old conventional calves by STEC serotypes O5 and O111. Our findings show that Efa1 is required for efficient colonization of the bovine intestinal tract by STEC, since efa1 deletion and insertion mutants were shed in the feces in significantly lower numbers. In addition, efa1 mutations dramatically reduced the number of bacteria associated with the intestinal epithelium. Expression and secretion of locus for enterocyte effacement-encoded type III secreted proteins that are required for adhesion and AE-lesion formation were impaired by mutation of efa1 in STEC but not by mutation of lifA in enteropathogenic E. coli. However, STEC efa1 mutants retain the ability to nucleate filamentous actin under sites of bacterial attachment to cultured eukaryotic cells. Efa1 is only the second STEC factor shown to influence carriage of the bacteria in the bovine intestine. Our data may have implications for strategies to reduce the prevalence of STEC in cattle.

Animals↗
PubMed2000

Intimin from enteropathogenic Escherichia coli mediates remodelling of the eukaryotic cell surface.

Adhesion to cultured epithelial cells by enteropathogenic Escherichia coli (EPEC) is associated with extensive rearrangement of the host cell cytoskeleton. Evidence has been presented that EPEC adhesion is associated with activation of signal transduction pathways leading to production of a characteristic histopathological feature known as the attaching and effacing (A/E) lesion. A/E lesion formation requires intimin, an EPEC adhesion molecule and several EPEC secreted proteins (EspA, B, D and Tir) involved in cell signalling and protein translocation. In this study it is shown that HEp-2 cells respond during the early stages of infection with two wild-type EPEC strains (B171 and E2348/69) by producing microvillus-like processes (MLP) at the site of initial bacterial adherence. Intimin appears to play a key role in MLP elongation. At later stages of infection with these wild-type EPEC strains, when A/E lesions have formed, the MLP were reduced in number and length to appear as at time zero, and the cell surface in the vicinity of bacterial clusters appeared unaffected. In contrast, infection with EspA- or EspB-negative, but intimin-positive, EPEC strains (UMD872 and UMD864, respectively) resulted in enhanced MLP proliferation and formation of 'cage-like' structures engulfing the bacteria. Inoculating HEp-2 cells with intimin-coated latex spheres induced similar 'cage-like' structures. Caco-2 cells did not show intimin-induced microvillus elongation in response to EPEC infection, although microvillus effacement and reduction in number occurred. Similar phenomena appeared on B171 and E2348/69 infection of paediatric intestine using in vitro organ culture, i.e. elongated microvilli were seen in association with small colonies and at the periphery of large localized colonies, along with evidence of microvillus breakdown and debris in the colony centre. These results show that intimin activates signal transduction pathways involved in the remodelling of the eukaryotic cell surface, probably via binding to a receptor encoded by the host cell.

Adhesins, Bacterial↗