Biomedical subjects

Mark J Walker

Publications and source records attributed to Mark J Walker.

27 records · Page 2Linked to original sources

PubMed2003

An aromatic amino acid auxotrophic mutant of Bordetella bronchiseptica is attenuated and immunogenic in a mouse model of infection.

We have constructed an aromatic amino acid auxotrophic mutant of Bordetella bronchiseptica, harbouring mutations in aroA and trpE to investigate the use of such a strain as a live-attenuated vaccine. B. bronchiseptica aroA trpE was unable to grow in minimal medium without aromatic supplementation. Compared to the parental wild-type strain, the mutant displayed significantly reduced abilities to invade and survive within the mouse macrophage-like cell line J774A.1 in vitro and in the murine respiratory tract following experimental intranasal infection. Mice vaccinated with B. bronchiseptica aroA trpE displayed significant dose-dependent increases in B. bronchiseptica-specific antibody responses, and exhibited increases in the number of B. bronchiseptica-reactive spleen cells in lymphoproliferation assays. Immunised animals were protected against lung colonisation after challenge with the wild-type parental strain. With such a broad host range displayed by B. bronchiseptica, the attenuated strain constructed in this study may not only be used for the prevention of B. bronchiseptica-associated disease, but also for the potential delivery of heterologous antigen.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed2003

Distribution of intimin subtypes among Escherichia coli isolates from ruminant and human sources.

The intimin gene eae, located within the locus of enterocyte effacement pathogenicity island, distinguishes enteropathogenic Escherichia coli (EPEC) and some Shiga toxin-producing E. coli (STEC) strains from all other pathotypes of diarrheagenic E. coli. EPEC is a leading cause of infantile diarrhea in developing countries, and intimin-positive STEC isolates are typically associated with life-threatening diseases such as hemolytic-uremic syndrome and hemorrhagic colitis. Here we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that reliably differentiates all 11 known intimin types (alpha1, alpha2, beta, gamma, kappa, epsilon, eta, iota, lambda, theta, and zeta) and three new intimin genes that show less than 95% nucleotide sequence identity with existing intimin types. We designated these new intimin genes Int- micro, Int-nu, and Int-xi. The PCR-RFLP assay was used to screen 213 eae-positive E. coli isolates derived from ovine, bovine, and human sources comprising 60 serotypes. Of these, 82 were STEC isolates, 89 were stx-negative (stx(-)) and ehxA-positive (ehxA(+)) isolates, and 42 were stx(-) and ehxA-negative isolates. Int-beta, the most commonly identified eae subtype (82 of 213 [38.5%] isolates), was associated with 21 serotypes, followed by Int-zeta (39 of 213 [18.3%] isolates; 11 serotypes), Int-theta (25 of 213 [11.7%] isolates; 15 serotypes), Int-gamma (19 of 213 [8.9%] isolates; 9 serotypes), and Int-epsilon (21 of 213 [9.9%] isolates; 5 serotypes). Intimin subtypes alpha1, alpha2, kappa, lambda, xi, micro, nu, and iota were infrequently identified; and Int-eta was not detected. Phylogenetic analyses with the Phylip package of programs clustered the intimin subtypes into nine distinct families (alpha, beta-xi, gamma, kappa, epsilon-eta-nu, iota- micro, lambda, theta, and zeta). Our data confirm that ruminants are an important source of serologically and genetically diverse intimin-containing E. coli strains.

Animals↗

PubMed2003

Evolution of sfbI encoding streptococcal fibronectin-binding protein I: horizontal genetic transfer and gene mosaic structure.

Streptococcal fibronectin-binding protein is an important virulence factor involved in colonization and invasion of epithelial cells and tissues by Streptococcus pyogenes. In order to investigate the mechanisms involved in the evolution of sfbI, the sfbI genes from 54 strains were sequenced. Thirty-four distinct alleles were identified. Three principal mechanisms appear to have been involved in the evolution of sfbI. The amino-terminal aromatic amino acid-rich domain is the most variable region and is apparently generated by intergenic recombination of horizontally acquired DNA cassettes, resulting in a genetic mosaic in this region. Two distinct and divergent sequence types that shared only 61 to 70% identity were identified in the central proline-rich region, while variation at the 3' end of the gene is due to deletion or duplication of defined repeat units. Potential antigenic and functional variabilities in SfbI imply significant selective pressure in vivo with direct implications for the microbial pathogenesis of S. pyogenes.

Adhesins, Bacterial↗

PubMed2003

stx1c Is the most common Shiga toxin 1 subtype among Shiga toxin-producing Escherichia coli isolates from sheep but not among isolates from cattle.

Unlike Shiga toxin 2 (stx(2)) genes, most nucleotide sequences of Shiga toxin 1 (stx(1)) genes from Shiga toxin-producing Escherichia coli (STEC), Shigella dysenteriae, and several bacteriophages (H19B, 933J, and H30) are highly conserved. Consequently, there has been little incentive to investigate variants of stx(1) among STEC isolates derived from human or animal sources. However stx(1OX3), originally identified in an OX3:H8 isolate from a healthy sheep in Germany, differs from other stx(1) subtypes by 43 nucleotides, resulting in changes to 12 amino acid residues, and has been renamed stx(1c). In this study we describe the development of a PCR-restriction fragment length polymorphism (RFLP) assay that distinguishes stx(1c) from other stx(1) subtypes. The PCR-RFLP assay was used to study 378 stx(1)-containing STEC isolates. Of these, 207 were isolated from sheep, 104 from cattle, 45 from humans, 11 from meat, 5 from swine, 5 from unknown sources, and 1 from a cattle water trough. Three hundred fifty-five of the 378 isolates (93.9%) also possessed at least one other associated virulence gene (ehxA, eaeA, and/or stx(2)); the combination stx(1), stx(2), and ehxA was the most common (175 of 355 [49.3%]), and 90 of 355 (25.4%) isolates possessed eaeA. One hundred thirty-six of 207 (65.7%) ovine isolates possessed stx(1c) alone and belonged to 41 serotypes. Seventy-one of 136 (52.2%) comprised the common ovine serotypes O5:H(-), O128:H2, and O123:H(-). Fifty-two of 207 isolates (25.1%) possessed an stx(1) subtype; 27 (51.9%) of these belonged to serotype O91:H(-). Nineteen of 207 isolates (9.2%) contained both stx(1c) and stx(1) subtypes, and 14 belonged to serotype O75:H8. In marked contrast, 97 of 104 (93.3%) bovine isolates comprising 44 serotypes possessed an stx(1) subtype, 6 isolates possessed stx(1c), and the remaining isolate possessed both stx(1c) and stx(1) subtypes. Ten of 11 (91%) isolates cultured from meat in New Zealand possessed stx(1c) (serotypes O5:H(-), O75:H8/H40, O81:H26, O88:H25, O104:H(-)/H7, O123:H(-)/H10, and O128:H2); most of these serotypes are commonly recovered from the feces of healthy sheep. Serotypes containing stx(1) recovered from cattle rarely were the same as those isolated from sheep. Although an stx(1c) subtype was never associated with the typical enterohemorrhagic E. coli serogroups O26, O103, O111, O113, and O157, 13 human isolates possessed stx(1c). Of these, six isolates with serotype O128:H2 (from patients with diarrhea), four O5:H(-) isolates (from patients with hemolytic-uremic syndrome), and three isolates with serotypes O123:H(-) (diarrhea), OX3:H8 (hemolytic-uremic syndrome), and O81:H6 (unknown health status) represent serotypes that are commonly isolated from sheep.

Adhesins, Bacterial↗

PubMed2003

Bovine non-O157 Shiga toxin 2-containing Escherichia coli isolates commonly possess stx2-EDL933 and/or stx2vhb subtypes.

stx(2) genes from 138 Shiga toxin-producing Escherichia coli (STEC) isolates, of which 127 were of bovine origin (58 serotypes) and 11 of human origin (one serotype; O113:H21), were subtyped. The bovine STEC isolates from Australian cattle carried ehxA and/or eaeA and predominantly possessed stx(2-EDL933) (103 of 127; 81.1%) either in combination with stx(2vhb) (32 of 127; 25.2%) or on its own (52 of 127; 40.4%). Of 22 (90.9%) bovine isolates of serotype O113:H21, a serotype increasingly recovered from patients with hemolytic uremic syndrome (HUS) or hemorrhagic colitis, 20 contained both stx(2-EDL933) and stx(2vhb); 2 isolates contained stx(2vhb) only. Although 7 of 11 (63.6%) human O113:H21 isolates associated with diarrhea possessed stx(2-EDL933), the remaining 4 isolates possessed a combination of stx(2-EDL933) and stx(2vhb). Three of the four were from separate sporadic cases of HUS, and one was from an unknown source.

Adhesins, Bacterial↗

PubMed2002

Immunological response mounted by Aboriginal Australians living in the Northern Territory of Australia against Streptococcus pyogenes serum opacity factor.

Streptococcus pyogenes (Group A streptococcus) interacts with host fibronectin via a number of distinct surface components. The streptococcal serum opacity factor (SOF) is a cell-surface protein of S. pyogenes which causes opalescence of human serum and mediates bacterial binding to fibronectin. In this study, hexahistidyl-tagged fusion proteins encompassing full-length SOF, and domains of SOF encompassing opacity factor activity and fibronectin-binding regions, were used in the characterization of the Aboriginal immune response to SOF. Anti-SOF serum IgG responses were found to be significantly higher (P<0.0001) in Aboriginal adults and children when compared to a non-Aboriginal adult group. The Aboriginal immune response against the fibronectin-binding region of SOF was significantly reduced when compared to the response against the whole SOF protein and N-terminal domains examined in this study (P<0.001). This pattern of immune response was also observed in rabbits immunized with recombinant SOF. Comparison of the deduced amino acid sequence of SOF from a number of common Australian isolates with other SOF sequences revealed that the N-terminus of SOF exhibits sequence similarity values ranging from 42.9% to 96.5%. The C-terminus containing the fibronectin-binding domain and membrane-spanning regions was more highly conserved, exhibiting sequence similarity values ranging from 84.6% to 100% within the fibronectin-binding repeats. These data suggest that the immune response against SOF is directed toward the variable N-terminus of the SOF protein. Phylogenetic analysis indicated that the sof genes of S. pyogenes do not exhibit geographical variation.

Adhesins, Bacterial↗

PubMed2002

Molecular analysis of group B protective surface protein, a new cell surface protective antigen of group B streptococci.

Group B streptococci (GBS) express various surface antigens designated c, R, and X antigens. A new R-like surface protein from Streptococcus agalactiae strain Compton R has been identified by using a polyclonal antiserum raised against the R protein fraction of this strain to screen a lambda Zap library. DNA sequence analysis of positive clones allowed the prediction of the primary structure of a 105-kDa protein designated BPS protein (group B protective surface protein) that exhibited typical features of streptococcal surface proteins such as a signal sequence and a membrane anchor region but did not show significant similarity with other known sequences. Immunogold electron microscopy using a BPS-specific antiserum confirmed the surface location of BPS protein on S. agalactiae strain Compton R. Anti-BPS antibodies did not cross-react with R1 and R4 proteins expressed by two variant type III GBS strains but reacted with the parental streptococcal strain in Western blot and immunoprecipitation analyses. Separate R3 and BPS immunoprecipitation bands were observed when a cell extract of strain Compton R was tested with an antiserum against Compton R previously cross-absorbed to remove R4 antibodies. Immunization of mice with recombinant BPS protein by the subcutaneous route produced an efficient antigen-specific response, and immunized animals survived challenge with a lethal dose of a virulent strain. Therefore, BPS protein represents a new R-like protective antigen of GBS.

Amino Acid Sequence↗

PubMed1999

Contribution of adjuvant to adaptive immune responses in mice against Actinobacillus pleuropneumoniae.

The authors have previously demonstrated that adjuvant-mediated differences in early cellular responses to antigens significantly affect subsequent adaptive immune responses. To investigate further the contribution of adjuvant to adaptive immune responses, outer-membrane proteins (OMP) purified from the respiratory pathogen Actinobacillus pleuropneumoniae, given either alone (antigen group) or complexed with SAMA4 (vaccine group), were injected intradermally into groups of mice. Controls were given PBS. Inclusion of adjuvant did not significantly alter the kinetics of antibody responses against OMP in serum or respiratory tract washings (RTW) over 21 weeks. Re-exposure to OMP at 21 weeks also induced identical recall responses in both immunized groups. However, differences between the responses of the vaccine and antigen groups were apparent when sera and RTW were reacted against OMP and OMP-derived polysaccharide antigens (ODPA). Serum and RTW reactivity against protein antigens was stronger in the vaccine group than in the antigen group. Serum and RTW from the vaccine group also reacted against a greater number of proteins than did the antigen group. Although serum reactivity against ODPA was equivalent for both groups, RTW from the vaccine group reacted only faintly against ODPA compared with the antigen group. The results suggested that shifting of antibody reactivity away from polysaccharide antigens toward protein antigens was an adjuvant-mediated effect. The rapid death of controls following intranasal inoculation confirmed that protection was ultimately dependent on the presence of specific antibodies in the serum and respiratory tract. However, since both groups responded equally to intranasal infection with A. pleuropneumoniae, as seen by the rapid clearance of bacteria from the lungs, the biological significance of any differences between the groups was unclear. Knowledge of the effects of adjuvants may provide a rational basis for adjuvant selection and the ability to manipulate immunological outcomes more precisely.

Actinobacillus Infections↗

PubMed1998

Reiterated repeat region variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is a highly prevalent pathogen which colonizes the ciliated epithelial lining of the porcine respiratory tract. Expression libraries constructed from genomic DNA of the non-pathogenic strain M. hyopneumoniae J were screened with porcine hyperimmune antiserum against M. hyopneumoniae. One clone expressed a 28 kDa protein which was also reactive with monospecific antiserum raised against a putative M. hyopneumoniae-specific 94 kDa antigen derived from strain J. Trypsin digestion of whole M. hyopneumoniae cells showed the 94 kDa antigen to be surface-accessible. DNA sequence analysis of the gene encoding the 94 kDa antigen revealed greater than 90% homology to two adhesin genes, encoding P97 and Mhp1, cloned from pathogenic strain 232 and strain P5722 of M. hyopneumoniae, respectively. Two regions of repetitive DNA sequence were identified in the gene encoding the 94 kDa antigen. The first encoded the deduced amino acid sequence A(T)-K-P-E(V)-A(T) arranged as nine tandem repeats (RR1). The second region of repetitive DNA sequence encoded the deduced amino acid sequence G-A(E,S)-P-N(S)-Q-G-K-K-A-E arranged as five tandem repeats (RR2). Comparison of the three M. hyopneumoniae adhesin genes revealed that the genes encoding P97 and Mhp1, and the strain J gene encoding the 94 kDa antigen contained 15, 12 and 9 tandem repeats, respectively, in RR1, and 4, 5 and 5 tandem repeats, respectively, in RR2. Southern hybridization analysis of EcoRI-digested genomic DNA probed with an 820 bp fragment spanning RR1 and RR2 identified a strongly hybridizing fragment ranging in size from 2.15 to 2.30 kb among seven geographically diverse strains of M. hyopneumoniae but failed to hybridize with DNA from four strains of Mycoplasma hyorhinis or Mycoplasma flocculare strain Ms42. PCR primers flanking the DNA sequence encoding RR1 and RR2 were used to amplify DNA from the seven strains of M. hyopneumoniae and DNA sequence analysis of the amplification products showed that the number of tandem amino acid repeats in RR1 varied considerably between strains. RR1 from M. hyopneumoniae strains YZ, Beaufort, Sue, OMZ407 and C1735/2 comprised 11, 15, 12, 15 and 8 tandem copies, respectively, of the 5-aa repeat whilst RR2 comprised 4, 3, 4, 3 and 4 tandem copies, respectively, of the 10-aa repeat. Two putative integrin binding sites (L-E-T and R-X-X-X-D) were identified in the 94 kDa ciliary adhesin. Variability in the number of amino acid repeats in RR1 amongst strains of M. hyopneumoniae may influence ciliary binding.

Adhesins, Bacterial↗