Search Results

Now showing 1 - 10 of 51
  • Publication
    Site-directed mutagenesis of intimin alpha modulates intimin-mediated tissue tropism and host specificity
    (2001-04-01) Simmons, Cameron
    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.
  • Publication
    Immunomodulation using bacterialeEnterotoxins
    (2001-03-01) Simmons, Cameron
    Immunologic unresponsiveness (tolerance) is a key feature of the mucosal immune system, and deliberate vaccination by a mucosal route can effectively induce immune suppression. However, some bacterial-derived proteins, e.g. cholera toxin and the heat labile toxin of Escherichia coli, are immunogenic and immunomodulatory at mucosal surfaces and can effectively adjuvant immune responses to codelivered bystander antigens. This review summarizes some of the structural and biological characteristics of these toxins and provides examples of how these properties have been exploited for tolerance induction and mucosal vaccine development.
  • Publication
    Salmonella enterica serovar Typhimurium interaction with dendritic cells: impact of the sifA gene
    (2004-11-01) Simmons, Cameron
    Salmonella enterica serovar Typhimurium (S. Typhimurium) and several mutant derivatives were able to enter efficiently murine bone marrow-derived dendritic cells using mechanisms predominantly independent of the Salmonella pathogenicity island 1 type III secretion system. The levels of intracellular bacteria did not increase significantly over many hours after invasion. Using fluid endocytic tracers and other markers, S. Typhimurium-containing vacuoles (SCVs) were physically distinguishable from early endocytic compartments. Fifty to eighty per cent of SCVs harbouring wild-type S. Typhimurium or aroA, invH and ssaV mutant derivatives were associated with late endosome markers. In contrast, S. Typhimurium sifA was shown to escape the SCVs into the cytosol of infected dendritic cells. S. Typhimurium aroC sifA was more efficient than S. Typhimurium aroC in delivering a eukaryotic promoter-driven green fluorescent protein reporter gene for expression in dendritic cells. In contrast, S. Typhimurium aroC sifA did not detectably increase the efficiency of MHC class I presentation of the model antigen ovalbumin to T cells compared to a similar aroC derivative. Mice infected with the S. Typhimurium aroC sifA expressing ovalbumin did not develop detectably enhanced levels of cytotoxic T cell or interferon-gamma production compared to S. Typhimurium aroC derivatives.
  • Publication
    Mutagenesis of conserved tryptophan residues within the receptor-binding domain of intimin: influence on binding activity and virulence
    (2002-03-01) Simmons, Cameron
    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.
  • Publication
    Host defences to Citrobacter rodentium
    (2003-04-01) Simmons, Cameron
    Citrobacter rodentium is a natural non-invasive bacterial pathogen which infects the distal colon of mice. It uses the same molecular mechanisms of type III secretion as human enteropathogenic and enterohemorrhagic Escherichia coli to colonise the epithelial cells of the gut and is therefore an ideal model to study host-bacterial pathogen interactions in vivo. Infection elicits mucosal inflammation with similarities to inflammatory bowel disease, and so it is a readily accessible model to investigate the relationship between inflammation and anti-bacterial immunity in the gut.
  • Publication
    The immune responses to bacterial antigens encountered in vivo at mucosal surfaces
    (2000-05-29) Simmons, Cameron
    Mammals have evolved a sophisticated immune system for handling antigens encountered at their mucosal surfaces. The way in which mucosally delivered antigens are handled influences our ability to design effective mucosal vaccines. Live attenuated derivatives of pathogens are one route towards the development of mucosal vaccines. However, some molecules, described as mucosal immunogens, are inherently immunogenic at mucosal surfaces. Studies on mucosal immunogens may facilitate the identification of common characteristics that contribute to mucosal immunogenicity and aid the development of novel, non-living mucosal vaccines and immunostimulators.
  • Publication
    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
    (2002-11-01) Simmons, Cameron
    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.
  • Publication
    Modulation of dendritic cell endocytosis and antigen processing pathways by Escherichia coli heat-labile enterotoxin and mutant derivatives
    (2003-03-28) Simmons, Cameron
    Escherichia coli heat-labile enterotoxin (LT) is known to be a potent adjuvant of both the mucosal and systemic immune systems but the mechanism of action leading to adjuvant activity remains incompletely understood. This study investigates the action of LT and LT mutants with impaired enzymatic activity, on the function of dendritic cells. Wild-type LT and LTR72, which retains some ADP ribosyltransferase activity, induced a selective increase in cell surface expression of B7.1, and a selective decrease of CD40 expression on mouse bone marrow derived dendritic cells. LTK63 and LT-B had no obvious effect on the expression of these antigens on similar dendritic cells. LT-treated dendritic cells also showed a profoundly impaired ability to present protein antigen (ovalbumin) to cognate T cells, although this effect was not observed with non-toxic LT mutants. LT and LTR72-treated cells showed a slower rate of receptor-mediated endocytosis as measured by flow cytometric analysis of uptake of fluorescently labelled dextran. Furthermore, confocal microscopy showed changes in the intracellular distribution of endocytosed molecules, and of the class II containing acidic antigen processing compartments. This response of dendritic cells to toxin is likely to play an important role in determining the adjuvant activity of these molecules.
  • Publication
    Refocusing of B-cell responses following a single amino acid substitution in an antigen
    (2001-06-01) Simmons, Cameron
    Intranasal immunization of BALB/c strain mice was carried out using baculovirus-derived human chorionic gonadotrophin (hCG) beta-chain, together with Escherichia coli heat-labile enterotoxin. Gonadotrophin-reactive immunoglobulin A (IgA) was induced in a remote mucosal site, the lung, in addition to a systemic IgG response. The extensive sequence homology with luteinizing hormone (LH) results in the production of LH cross-reactive antibodies when holo-hCG is used as an immunogen. In contrast to wild-type hCGbeta, a mutated hCGbeta-chain containing an arginine to glutamic acid substitution at position 68 did not induce the production of antibodies which cross-react with LH. Furthermore, the epitopes utilized in the B-cell response to the mutated hCGbeta shifted away from the immunodominant region of the parent wild-type molecule towards epitopes within the normally weakly immunogenic C terminus. This shift in epitope usage was also seen following intramuscular immunization of rabbits. Thus, a single amino acid change, which does not disrupt the overall structure of the molecule, refocuses the immune response away from a disadvantageous cross-reactive epitope region and towards a normally weakly immunogenic but antigen-unique area. Similar mutational strategies for epitope-refocusing may be applicable to other vaccine candidate molecules.
  • Publication
    Identification of a novel type IV pilus gene cluster required for gastrointestinal colonization of Citrobacter rodentium
    (2003-05-01) Simmons, Cameron
    Citrobacter rodentium is used as an in vivo model system for clinically significant enteric pathogens such as enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens all colonize the lumen side of the host gastrointestinal tract via attaching and effacing (A/E) lesion formation. In order to identify genes required for the colonization of A/E-forming pathogens, a library of signature-tagged transposon mutants of C. rodentium was constructed and screened in mice. Of the 576 mutants tested, 14 were attenuated in their ability to colonize the descending colon. Of these, eight mapped to the locus of enterocyte effacement (LEE), which is required for the formation of A/E lesions, underlying the importance of this mechanism for pathogenesis. Another mutant, P5H2, was found to have a transposon insertion in an open reading frame that has strong similarity to type IV pilus nucleotide-binding proteins. The region flanking the transposon insertion was sequenced, identifying a cluster of 12 genes that encode the first described pilus of C. rodentium (named colonization factor Citrobacter, CFC). The proteins encoded by cfc genes have identity to proteins of the type IV COF pilus of enterotoxigenic E. coli (ETEC), the toxin co-regulated pilus of Vibrio cholerae and the bundle-forming pilus of EPEC. A non-polar mutation in cfcI, complementation of this strain with wild-type cfcI and complementation of strain P5H2 with wild-type cfcH confirmed that these genes are required for colonization of the gastrointestinal tract by C. rodentium. Thus, CFC provides a convenient model to study type IV pilus-mediated pathogen-host interactions under physiological conditions in the natural colonic environment.