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Mark J Walker

Publications and source records attributed to Mark J Walker.

At least 19 recordsLinked to original sources
PubMed2006

Comparative analysis of virulence genes, genetic diversity, and phylogeny of commensal and enterotoxigenic Escherichia coli isolates from weaned pigs.

If the acquisition of virulence genes (VGs) for pathogenicity were not solely acquired through horizontal gene transfers of pathogenicity islands, transposons, and phages, then clonal clusters of enterotoxigenic Escherichia coli (ETEC) would contain few or even none of the VGs found in strains responsible for extraintestinal infections. To evaluate this possibility, 47 postweaning diarrhea (PWD) ETEC strains from different geographical origins and 158 commensal E. coli isolates from the gastrointestinal tracts of eight group-housed healthy pigs were screened for 36 extraintestinal and 18 enteric VGs using multiplex PCR assays. Of 36 extraintestinal VGs, only 8 were detected (fimH, traT, fyuA, hlyA, kpsMtII, k5, iha, and ompT) in the ETEC collection. Among these, hlyA (alpha-hemolysin) and iha (nonhemagglutinating adhesin) occurred significantly more frequently among the ETEC isolates than in the commensal isolates. Clustering analysis based on the VG profiles separated commensal and ETEC isolates and even differentiated serogroup O141 from O149. On the other hand, pulsed-field gel electrophoresis (PFGE) successfully clustered ETEC isolates according to both serotype and geographical origin. In contrast, the commensal isolates were heterogeneous with respect to both serotype and DNA fingerprint. This study has validated the use of VG profiling to examine pathogenic relationships between porcine ETEC isolates. The clonal relationships of these isolates can be further clarified by PFGE fingerprinting. The presence of extraintestinal VGs in porcine ETEC confirmed the hypothesis that individual virulence gene acquisitions can occur concurrently against a background of horizontal gene transfers of pathogenicity islands. Over time, this could enable specific clonotypes to respond to host selection pressure and to evolve into new strains with increased virulence.

Animals↗
PubMed2006

The plasminogen-binding group A streptococcal M protein-related protein Prp binds plasminogen via arginine and histidine residues.

The migration of the human pathogen Streptococcus pyogenes (group A streptococcus) from localized to deep tissue sites may result in severe invasive disease, and sequestration of the host zymogen plasminogen appears crucial for virulence. Here, we describe a novel plasminogen-binding M protein, the plasminogen-binding group A streptococcal M protein (PAM)-related protein (Prp). Prp is phylogenetically distinct from previously described plasminogen-binding M proteins of group A, C, and G streptococci. While competition experiments indicate that Prp binds plasminogen with a lower affinity than PAM (50% effective concentration = 0.34 microM), Prp nonetheless binds plasminogen with high affinity and at physiologically relevant concentrations of plasminogen (K(d) = 7.8 nM). Site-directed mutagenesis of the putative plasminogen binding site indicates that unlike the majority of plasminogen receptors, Prp does not interact with plasminogen exclusively via lysine residues. Mutagenesis to alanine of lysine residues Lys(96) and Lys(101) reduced but did not abrogate plasminogen binding by Prp. Plasminogen binding was abolished only with the additional mutagenesis of Arg(107) and His(108) to alanine. Furthermore, mutagenesis of Arg(107) and His(108) abolished plasminogen binding by Prp despite the presence of Lys(96) and Lys(101) in the binding site. Thus, binding to plasminogen via arginine and histidine residues appears to be a conserved mechanism among plasminogen-binding M proteins.

Amino Acid Sequence↗
PubMed2006

Serum opacity factor promotes group A streptococcal epithelial cell invasion and virulence.

Serum opacity factor (SOF) is a bifunctional cell surface protein expressed by 40-50% of group A streptococcal (GAS) strains comprised of a C-terminal domain that binds fibronectin and an N-terminal domain that mediates opacification of mammalian sera. The sof gene was recently discovered to be cotranscribed in a two-gene operon with a gene encoding another fibronectin-binding protein, sfbX. We compared the ability of a SOF(+) wild-type serotype M49 GAS strain and isogenic mutants lacking SOF or SfbX to invade cultured HEp-2 human pharyngeal epithelial cells. Elimination of SOF led to a significant decrease in HEp-2 intracellular invasion while loss of SfbX had minimal effect. The hypoinvasive phenotype of the SOF(-) mutant could be restored upon complementation with the sof gene on a plasmid vector, and heterologous expression of sof49 in M1 GAS or Lactococcus lactis conferred marked increases in HEp-2 cell invasion. Studies using a mutant sof49 gene lacking the fibronectin-binding domain indicated that the N-terminal opacification domain of SOF contributes to HEp-2 invasion independent of the C-terminal fibronectin binding domain, findings corroborated by observations that a purified SOF N-terminal peptide could promote latex bead adherence to HEp-2 cells and inhibit GAS invasion of HEp-2 cells in a dose-dependent manner. Finally, the first in vivo studies to employ a single gene allelic replacement mutant of SOF demonstrate that this protein contributes to GAS virulence in a murine model of necrotizing skin infection.

Animals↗
PubMed2006

The maintenance of high affinity plasminogen binding by group A streptococcal plasminogen-binding M-like protein is mediated by arginine and histidine residues within the a1 and a2 repeat domains.

Subversion of the plasminogen activation system is implicated in the virulence of group A streptococci (GAS). GAS displays receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M-like protein (PAM). The plasminogen binding domain of PAM is highly variable, and this variation has been linked to host selective immune pressure. Site-directed mutagenesis of full-length PAM protein from an invasive GAS isolate was undertaken to assess the contribution of residues in the a1 and a2 repeat domains to plasminogen binding function. Mutagenesis to alanine of key plasminogen binding lysine residues in the a1 and a2 repeats (Lys98 and Lys111) did not abrogate plasminogen binding by PAM nor did additional mutagenesis of Arg101 and His102 and Glu104, which have previously been implicated in plasminogen binding. Plasminogen binding was only abolished with the additional mutagenesis of Arg114 and His115 to alanine. Furthermore, mutagenesis of both arginine (Arg101 and Arg114) and histidine (His102 and His115) residues abolished interaction with plasminogen despite the presence of Lys98 and Lys111 in the binding repeats. This study shows for the first time that residues Arg101, Arg114, His102, and His115 in both the a1 and a2 repeat domains of PAM can mediate high affinity plasminogen binding. These data suggest that highly conserved arginine and histidine residues may compensate for variation elsewhere in the a1 and a2 plasminogen binding repeats, and may explain the maintenance of high affinity plasminogen binding by naturally occurring variants of PAM.

Amino Acid Sequence↗
PubMed2006

Trigger for group A streptococcal M1T1 invasive disease.

The globally disseminated Streptococcus pyogenes M1T1 clone causes a number of highly invasive human diseases. The transition from local to systemic infection occurs by an unknown mechanism; however invasive M1T1 clinical isolates are known to express significantly less cysteine protease SpeB than M1T1 isolates from local infections. Here, we show that in comparison to the M1T1 strain 5448, the isogenic mutant delta speB accumulated 75-fold more human plasmin activity on the bacterial surface following incubation in human plasma. Human plasminogen was an absolute requirement for M1T1 strain 5448 virulence following subcutaneous (s.c.) infection of humanized plasminogen transgenic mice. S. pyogenes M1T1 isolates from the blood of infected humanized plasminogen transgenic mice expressed reduced levels of SpeB in comparison with the parental 5448 used as inoculum. We propose that the human plasminogen system plays a critical role in group A streptococcal M1T1 systemic disease initiation. SpeB is required for S. pyogenes M1T1 survival at the site of local infection, however, SpeB also disrupts the interaction of S. pyogenes M1T1 with the human plasminogen activation system. Loss of SpeB activity in a subpopulation of S. pyogenes M1T1 at the site of infection results in accumulation of surface plasmin activity thus triggering systemic spread.

Animals↗
PubMed2006

Inhibition of indoleamine 2,3 dioxygenase activity by H2O2.

Indoleamine 2,3-dioxygenase is the first and rate limiting enzyme of the kynurenine pathway of tryptophan metabolism, has potent effects on cell proliferation and mediates antimicrobial, antitumorogenic, and immunosuppressive effects. As a potent cytotoxic effector, the mechanisms of indoleamine 2,3-dioxygenase inhibition deserve greater attention. The work presented here represents the first systematic study exploring the mechanisms by which low levels of hydrogen peroxide (10-100 microM) inhibit indoleamine 2,3-dioxygenase in vitro. Following brief peroxide exposure both enzyme inhibition and structural changes were observed. Loss of catalysis was accompanied by oxidation of several cysteine residues to sulfinic and sulfonic acids, observed by electrospray and MALDI mass spectrometry. Enzyme activity could in part be preserved in the presence of sulfhydryl containing compounds, particularly DTT and methionine. However, these structural alterations did not prevent substrate (l-tryptophan) binding. Some enzyme activity could be recovered in the presence of thioredoxin, indicating that the inhibitory effect of H(2)O(2) is at least partially reversible in vitro. We present evidence that cysteine oxidation represents one mechanism of indoleamine 2,3-dioxygenase inhibition.

Dose-Response Relationship, Drug↗
PubMed2006

Domains of group A streptococcal M protein that confer resistance to phagocytosis, opsonization and protection: implications for vaccine development.

Streptococcus pyogenes (group A streptococcus) colonizes skin and throat tissues resulting in a range of benign and serious human diseases. Opsonization and phagocytosis are important defence mechanisms employed by the host to destroy group A streptococci. Antisera against the cell-surface M protein, of which over 150 different types have been identified, are opsonic and contribute to disease protection. In this issue of Molecular Microbiology, Sandin and colleagues have comprehensively analysed the regions of M5 protein that contribute to phagocytosis resistance and opsonization. Human plasma proteins bound to M5 protein B- and C-repeats were shown to block opsonization, an observation that needs to be carefully considered for the development of M protein-derived vaccines. While safe and efficacious human group A streptococcal vaccines are not commercially available, candidate M protein-derived vaccines have shown promise in murine vaccine models and a recent phase 1 human clinical trial.

Animals↗
PubMed2006

P159 is a proteolytically processed, surface adhesin of Mycoplasma hyopneumoniae: defined domains of P159 bind heparin and promote adherence to eukaryote cells.

Mycoplasma hyopneumoniae, the causative agent of porcine enzootic pneumonia, colonizes the respiratory cilia of affected swine causing significant economic losses to swine production worldwide. Heparin is known to inhibit adherence of M. hyopneumoniae to porcine respiratory epithelial cilia. M. hyopneumoniae cells bind heparin but the identity of the heparin-binding proteins is limited. Proteomic analysis of M. hyopneumoniae lysates identified 27 kDa (P27), 110 kDa (P110) and 52 kDa (P52) proteins representing different regions of a 159 kDa (P159) protein derived from mhp494. These cleavage fragments were surface located and present at all growth stages. Following purification of four recombinant proteins spanning P159 (F1P159, F2P159, F3P159 and F4P159), only F3P159 and F4P159 bound heparin in a dose-dependent manner (K(d) values 142.37 +/- 22.01 nM; 75.37 +/- 7.34 nM respectively). Scanning electron microscopic studies showed M. hyopneumoniae bound intimately to porcine kidney epithelial-like cells (PK15 cells) but these processes were inhibited by excess heparin and F4P159. Similarly, latex beads coated with F2P159 and F4P159 adhered to and entered PK15 cells, but heparin, F2P159 and F4P159 was inhibitory. These findings indicate that P159 is a post-translationally cleaved, glycosaminoglycan-binding adhesin of M. hyopneumoniae.

Adhesins, Bacterial↗
PubMed2006

Two domains within the Mycoplasma hyopneumoniae cilium adhesin bind heparin.

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, a chronic and economically significant respiratory disease that affects swine production worldwide. M. hyopneumoniae adheres to and adversely affects the function of ciliated epithelial cells of the respiratory tract, and the cilium adhesin (Mhp183, P97) is intricately but not exclusively involved in this process. Although binding of pathogenic bacteria to glycosaminoglycans is a recognized step in pathogenesis, knowledge of glycosaminoglycan-binding proteins in M. hyopneumoniae is lacking. However, heparin and other sulfated polysaccharides are known to block the binding of M. hyopneumoniae to purified swine respiratory cilia. In this study, four regions within the cilium adhesin were examined for the ability to bind heparin. Cilium adhesin fragments comprising 653 amino acids of the N terminus and 301 amino acids of the C terminus (containing two repeat regions, R1 and R2) were cloned and expressed. These fragments bound heparin in a dose-dependent and saturable manner with physiologically significant binding affinities of 0.27 +/- 0.02 microM and 1.89 +/- 0.33 microM, respectively. Heparin binding of both fragments was strongly inhibited by the sulfated polysaccharides fucoidan and mucin but not by chondroitin sulfate B. When the C-terminal repeat regions R1 and R2 were cloned separately and expressed, heparin-binding activity was lost, suggesting that both regions are required for heparin binding. The ability of the cilium adhesin to bind heparin indicates that this molecule plays a multifunctional role in the adherence of M. hyopneumoniae to host respiratory surfaces and therefore has important implications with respect to the pathogenesis of this organism.

Adhesins, Bacterial↗
PubMed2005

Divergence in the plasminogen-binding group a streptococcal M protein family: functional conservation of binding site and potential role for immune selection of variants.

Group A streptococci (GAS) display receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M protein (PAM). Characterization of PAM genes from 12 GAS isolates showed significant variation within the plasminogen-binding repeat motifs (a1/a2) of this protein. To determine the impact of sequence variation on protein function, recombinant proteins representing five naturally occurring variants of PAM, together with a recombinant M1 protein, were expressed and purified. Equilibrium dissociation constants for the interaction of PAM variants with biotinylated Glu-plasminogen ranged from 1.58 to 4.99 nm. Effective concentrations of prototype PAM required for 50% inhibition of plasminogen binding to immobilized PAM variants ranged from 0.68 to 22.06 nm. These results suggest that although variation in the a1/a2 region of the PAM protein does affect the comparative affinity of PAM variants, the functional capacity to bind plasminogen is conserved. Additionally, a potential role for the a1 region of PAM in eliciting a protective immune response was investigated by using a mouse model for GAS infection. The a1 region of PAM was found to protect immunized mice challenged with a PAM-positive GAS strain. These data suggest a link between selective immune pressure against the plasminogen-binding repeats and the functional conservation of the binding domain in PAM variants.

Amino Acid Motifs↗
PubMed2005

Development of a group-specific PCR combined with ARDRA for the identification of Bacillus species of environmental significance.

A group-specific primer pair was designed to amplify the 16S rRNA gene of representative reference strains from environmentally sourced, mesophilic aerobic spore-forming Bacillus taxa. The PCR generated a 1114 bp amplicon but did not do so with DNA extracted from 16 other Eubacterial species. When amplicons were digested with restriction enzymes AluI or TaqI, different profiles containing between 2 and 5 fragments ranging in size from 76 to 804 base pairs were seen with different Bacillus species. This procedure, known otherwise as amplified ribosomal DNA restriction analysis or ARDRA, produced unique and distinguishable patterns to differentiate between 15 ATCC reference strains (10 Bacillus, 3 Paenibacillus and 2 Brevibacillus member species) as well as 3 misidentified Bacillus probiotic strains in a commercial collection. Our simplified PCR-ARDRA protocol provides a facile method for the identification of most environmentally important species of Bacillus.

Animals↗
PubMed2005

Is plasminogen deployed as a Streptococcus pyogenes virulence factor?

Streptococcus pyogenes (group A streptococcus) causes human skin and throat infections as well as highly invasive diseases including necrotizing fasciitis. Group A streptococcal infections and invasive disease have made a resurgence in developed countries during the past two decades. S. pyogenes use multiple pathways for the acquisition and activation of human plasminogen, securing potent proteolytic activity on the bacterial cell surface. Recent experimental evidence using a humanized transgenic mouse model suggests a crucial role for human plasminogen in the dissemination of S. pyogenes in vivo.

Plasminogen↗
PubMed2005

R120G alphaB-crystallin promotes the unfolding of reduced alpha-lactalbumin and is inherently unstable.

alpha-Crystallin is the principal lens protein which, in addition to its structural role, also acts as a molecular chaperone, to prevent aggregation and precipitation of other lens proteins. One of its two subunits, alphaB-crystallin, is also expressed in many nonlenticular tissues, and a natural missense mutation, R120G, has been associated with cataract and desmin-related myopathy, a disorder of skeletal muscles [Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D & Fardeau M (1998) Nat Genet20, 92-95]. In the present study, real-time 1H-NMR spectroscopy showed that the ability of R120G alphaB-crystallin to stabilize the partially folded, molten globule state of alpha-lactalbumin was significantly reduced in comparison with wild-type alphaB-crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced alpha-lactalbumin, but showed limited chaperone activity for other target proteins. Using NMR spectroscopy, gel electrophoresis, and MS, we observed that, unlike the wild-type protein, R120G alphaB-crystallin is intrinsically unstable in solution, with unfolding of the protein over time leading to aggregation and progressive truncation from the C-terminus. Light scattering, MS, and size-exclusion chromatography data indicated that R120G alphaB-crystallin exists as a larger oligomer than wild-type alphaB-crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of alphaB-crystallin causes partial unfolding, increased exposure of hydrophobic regions, and enhances its susceptibility to proteolysis, thus reducing its solubility and promoting its aggregation and complexation with other proteins. These characteristics may explain the involvement of R120G alphaB-crystallin with human disease states.

Animals↗
PubMed2005

Surface analyses and immune reactivities of major cell wall-associated proteins of group a streptococcus.

A proteomic analysis was undertaken to identify cell wall-associated proteins of Streptococcus pyogenes. Seventy-four distinct cell wall-associated proteins were identified, 66 of which were novel. Thirty-three proteins were immunoreactive with pooled S. pyogenes-reactive human antisera. Biotinylation of the GAS cell surface identified 23 cell wall-associated proteins that are surface exposed.

Bacterial Proteins↗
PubMed2004

Plasminogen binding by group A streptococcal isolates from a region of hyperendemicity for streptococcal skin infection and a high incidence of invasive infection.

Reports of resurgence in invasive group A streptococcal (GAS) infections come mainly from affluent populations with infrequent exposure to GAS. In the Northern Territory (NT) of Australia, high incidence of invasive GAS disease is secondary to endemic skin infection, serotype M1 clones are rare in invasive infection, the diversity and level of exposure to GAS strains are high, and no particular strains dominate. Expression of a plasminogen-binding GAS M-like protein (PAM) has been associated with skin infection in isolates elsewhere (D. Bessen, C. M. Sotir, T. M. Readdy, and S. K. Hollingshead, J. Infect. Dis. 173:896-900, 1996), and subversion of the host plasminogen system by GAS is thought to contribute to invasion in animal models. Here, we describe the relationship between plasminogen-binding capacity of GAS isolates, PAM genotype, and invasive capacity in 29 GAS isolates belonging to 25 distinct strains from the NT. In the presence of fibrinogen and streptokinase, invasive isolates bound more plasminogen than isolates from uncomplicated infections (P < or = 0.004). Only PAM-positive isolates bound substantial levels of plasminogen by a fibrinogen-streptokinase-independent pathway (direct binding). Despite considerable amino acid sequence variation within the A1 repeat region of PAM where the plasminogen-binding domain maps, the critical lysine residue was conserved.

Antigens, Bacterial↗
PubMed2003

The pyruvate dehydrogenase complex of Mycoplasma hyopneumoniae contains a novel lipoyl domain arrangement.

The genes encoding the pyruvate dehydrogenase (PDH) complex (pdhA, pdhB, pdhC and pdhD) from Mycoplasma hyopneumoniae have been cloned and sequenced. The genes are arranged into two operons, designated pdhAB and pdhCD, which are not found together in the chromosome. The pdhA, pdhB, pdhC and pdhD genes encode proteins of predicted molecular masses of 44.2 kDa (pyruvate dehydrogenase major subunit; E1alpha), 36.6 kDa (pyruvate dehydrogenase minor subunit; E1beta), 33.1 kDa (dihydrolipoyl acetyltransferase; E2) and 66.3 kDa (dihydrolipoyl dehydrogenase; E3), respectively. Sequence analysis of the pdhCD operon revealed the presence of a lipoyl-binding domain in pdhD but not in pdhC. The lipoyl domain is believed to act as a "swinging arm" that spans the gaps between the catalytic domains of each of the subunits. Portions of the N-terminal regions of pdhA and pdhD were expressed as 6xHis-tag fusion proteins in Escherichia coli and purified by nickel affinity chromatography. The purified proteins were used to raise antibodies in rabbits, and Western blot analysis was performed with the polyclonal rabbit antiserum. Both the pdhA and pdhD genes were expressed among various strains of M. hyopneumoniae as well as the porcine mycoplasmas, Mycoplasma hyorhinis and Mycoplasma flocculare. Southern hybridisation analysis using probes from pdhA and pdhD detected one copy of each gene in the chromosome of M. hyopneumoniae. Since previous studies have shown pyruvate dehydrogenase activity in M. hyopneumoniae [J. Gen. Microbiol. 134 (1988) 791], it appears likely that a functional lipoyl-binding domain in the N terminus of PdhC is not an absolute prerequisite for pyruvate dehydrogenase enzyme activity. We hypothesise that the lipoyl-binding domain of PdhD is performing the enzymatic function normally attributed to the PdhC lipoyl-binding domain in other organisms. Searches of pyruvate dehydrogenase gene sequences derived from other Mycoplasma species showed that a putative lipoyl domain was absent in the pdhC gene from Mycoplasma pulmonis. However, like other bacterial species, pdhC gene sequences from Mycoplasma capricolum, Mycoplasma genitalium and Mycoplasma pneumoniae contain a putative lipoyl domain.

Amino Acid Sequence↗
PubMed2003

Asp274 and his346 are essential for heme binding and catalytic function of human indoleamine 2,3-dioxygenase.

L-Tryptophan is the least abundant essential amino acid in humans. Indoleamine 2,3-dioxgyenase (IDO) is a cytosolic heme protein which, together with the hepatic enzyme tryptophan 2,3-dioxygenase, catalyzes the first and rate-limiting step in the major pathway of tryptophan metabolism, the kynurenine pathway. The physiological role of IDO is not fully understood but is of great interest, because IDO is widely distributed in human tissues, can be up-regulated via cytokines such as interferon-gamma, and can thereby modulate the levels of tryptophan, which is vital for cell growth. To identify which amino acid residues are important in substrate or heme binding in IDO, site-directed mutagenesis of conserved residues in the IDO gene was undertaken. Because it had been proposed that a histidine residue might be the proximal heme ligand in IDO, mutation to alanine of the three highly conserved histidines His16, His303, and His346 was conducted. Of these, only His346 was shown to be essential for heme binding, indicating that this histidine residue may be the proximal ligand and suggesting that neither His303 nor His16 act as the proximal ligand. Site-directed mutagenesis of Asp274 also compromised the ability of IDO to bind heme. This observation indicates that Asp274 may coordinate to heme directly as the distal ligand or is essential in maintaining the conformation of the heme pocket.

Alanine↗