Philip N Ward

Oxford University Hospitals NHS Trust, Oxford, England, United Kingdom

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Publications (28)85.35 Total impact

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    ABSTRACT: The regulation and control of gene expression in response to differing environmental stimuli is crucial for successful pathogen adaptation and persistence. The regulatory gene vru of Streptococcus uberis encodes a stand-alone response regulator with similarity to the Mga of group A Streptococcus. Mga controls expression of a number of important virulence determinants. Experimental intramammary challenge of dairy cattle with a mutant of S. uberis carrying an inactivating lesion in vru showed reduced ability to colonize the mammary gland and an inability to induce clinical signs of mastitis compared with the wild-type strain. Analysis of transcriptional differences of gene expression in the mutant, determined by microarray analysis, identified a number of coding sequences with altered expression in the absence of Vru. These consisted of known and putative virulence determinants, including Lbp (Sub0145), SclB (Sub1095), PauA (Sub1785) and hasA (Sub1696).
    Microbiology 03/2012; 158(Pt 6):1581-92. · 3.06 Impact Factor
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    ABSTRACT: The bovine pathogen Streptococcus uberis was assessed for biofilm growth. The transition from planktonic to biofilm growth in strain 0140J correlated with an upregulation of several gene products that have been shown to be important for pathogenesis, including a glutamine ABC transporter (SUB1152) and a lactoferrin binding protein (gene lbp; protein SUB0145).
    Applied and Environmental Microbiology 11/2010; 77(1):382-4. · 3.95 Impact Factor
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    ABSTRACT: Streptococcus uberis, strain 0140J, contains a single copy sortase A (srtA), encoding a transamidase capable of covalently anchoring specific proteins to peptidoglycan. Unlike the wild-type, an isogenic mutant carrying an inactivating ISS1 insertion within srtA was only able to infect the bovine mammary gland in a transient fashion. For the first 24 h post challenge, the srtA mutant colonised at a similar rate and number to the wild type strain, but unlike the wild type did not subsequently colonise in higher numbers. Similar levels of host cell infiltration were detected in response to infection with both strains, but only in those mammary quarters infected with the wild type strain were clinical signs of disease evident. Mutants that failed to express individual sortase substrate proteins (sub0135, sub0145, sub0207, sub0241, sub0826, sub0888, sub1095, sub1154, sub1370, and sub1730) were isolated and their virulence determined in the same challenge model. This revealed that mutants lacking sub0145, sub1095 and sub1154 were attenuated in cattle. These data demonstrate that a number of sortase anchored proteins each play a distinct, non-redundant and important role in pathogenesis of S. uberis infection within the lactating bovine mammary gland.
    Veterinary Research 09/2010; 41(5):63. · 3.43 Impact Factor
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    ABSTRACT: Sortase (a transamidase) has been shown to be responsible for the covalent attachment of proteins to the bacterial cell wall. Anchoring is effected on secreted proteins containing a specific cell wall motif toward their C-terminus; that for sortase A (SrtA) in Gram-positive bacteria often incorporates the sequence LPXTG. Such surface proteins are often characterized as virulence determinants and play important roles during the establishment and persistence of infection. Intramammary infection with Streptococcus uberis is a common cause of bovine mastitis, which impacts on animal health and welfare and the economics of milk production. Comparison of stringently produced cell wall fractions from S. uberis and an isogenic mutant strain lacking SrtA permitted identification of 9 proteins likely to be covalently anchored at the cell surface. Analysis of these sequences implied the presence of two anchoring motifs for S. uberis, the classical LPXTG motif and an additional LPXXXD motif.
    Journal of Proteome Research 02/2010; 9(2):1088-95. · 5.06 Impact Factor
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    ABSTRACT: Streptococcus uberis, a Gram positive bacterial pathogen responsible for a significant proportion of bovine mastitis in commercial dairy herds, colonises multiple body sites of the cow including the gut, genital tract and mammary gland. Comparative analysis of the complete genome sequence of S. uberis strain 0140J was undertaken to help elucidate the biology of this effective bovine pathogen. The genome revealed 1,825 predicted coding sequences (CDSs) of which 62 were identified as pseudogenes or gene fragments. Comparisons with related pyogenic streptococci identified a conserved core (40%) of orthologous CDSs. Intriguingly, S. uberis 0140J displayed a lower number of mobile genetic elements when compared with other pyogenic streptococci, however bacteriophage-derived islands and a putative genomic island were identified. Comparative genomics analysis revealed most similarity to the genomes of Streptococcus agalactiae and Streptococcus equi subsp. zooepidemicus. In contrast, streptococcal orthologs were not identified for 11% of the CDSs, indicating either unique retention of ancestral sequence, or acquisition of sequence from alternative sources. Functions including transport, catabolism, regulation and CDSs encoding cell envelope proteins were over-represented in this unique gene set; a limited array of putative virulence CDSs were identified. S. uberis utilises nutritional flexibility derived from a diversity of metabolic options to successfully occupy a discrete ecological niche. The features observed in S. uberis are strongly suggestive of an opportunistic pathogen adapted to challenging and changing environmental parameters.
    BMC Genomics 02/2009; 10:54. · 4.40 Impact Factor
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    E L Denham, P N Ward, J A Leigh
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    ABSTRACT: The role of lipoprotein diacylglyceryl transferase (Lgt) and lipoprotein signal peptidase (Lsp) responsible for processing lipoproteins was investigated in Streptococcus uberis, a common cause of bovine mastitis. In the absence of Lgt, three lipoproteins [MtuA (SUB0473), Hap (SUB1625) and an extracellular solute-binding protein (SUB0365)] were detected in extracellular locations. All were shown by Edman degradation analysis to be cleaved on the carboxy side of the LXXC lipobox. Detection of MtuA, a lipoprotein shown previously to be essential for infectivity and virulence, was used as a surrogate lipoprotein marker to locate and assess processing of lipoproteins. The absence of Lgt did not prevent location of MtuA to the cell membrane, its location in the wild-type strain but, in contrast to the situation with wild-type, did result in a widespread location of this protein. In the absence of both Lgt and Lsp, MtuA was similarly released from the bacterial cell. In such strains, however, the cell-associated MtuA represented the full-length gene product, indicating that Lsp was able to cleave non-lipidated (lipo)proteins but was not responsible for their release from this bacterium.
    Microbiology 02/2009; 155(Pt 1):134-41. · 2.85 Impact Factor
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    ABSTRACT: Streptococcus suis is a zoonotic pathogen that infects pigs and can occasionally cause serious infections in humans. S. suis infections occur sporadically in human Europe and North America, but a recent major outbreak has been described in China with high levels of mortality. The mechanisms of S. suis pathogenesis in humans and pigs are poorly understood. The sequencing of whole genomes of S. suis isolates provides opportunities to investigate the genetic basis of infection. Here we describe whole genome sequences of three S. suis strains from the same lineage: one from European pigs, and two from human cases from China and Vietnam. Comparative genomic analysis was used to investigate the variability of these strains. S. suis is phylogenetically distinct from other Streptococcus species for which genome sequences are currently available. Accordingly, approximately 40% of the approximately 2 Mb genome is unique in comparison to other Streptococcus species. Finer genomic comparisons within the species showed a high level of sequence conservation; virtually all of the genome is common to the S. suis strains. The only exceptions are three approximately 90 kb regions, present in the two isolates from humans, composed of integrative conjugative elements and transposons. Carried in these regions are coding sequences associated with drug resistance. In addition, small-scale sequence variation has generated pseudogenes in putative virulence and colonization factors. The genomic inventories of genetically related S. suis strains, isolated from distinct hosts and diseases, exhibit high levels of conservation. However, the genomes provide evidence that horizontal gene transfer has contributed to the evolution of drug resistance.
    PLoS ONE 02/2009; 4(7):e6072. · 3.53 Impact Factor
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    E L Denham, P N Ward, J A Leigh
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    ABSTRACT: Lipoprotein signal peptidase (lsp) is responsible for cleaving the signal peptide sequence of lipoproteins in gram-positive bacteria. Investigation of the role of Lsp in Streptococcus uberis, a common cause of bovine mastitis, was undertaken using the lipoprotein MtuA (a protein essential for virulence) as a marker. The S. uberis lsp mutant phenotype displayed novel lipoprotein processing. Not only was full-length (uncleaved) MtuA detected by Western blotting, but during late log phase, a lower-molecular-weight derivative of MtuA was evident. Similar analysis of an S. uberis double mutant containing insertions disrupting both lsp and eep (a homologue of the Enterococcus faecalis "enhanced expression of pheromone" gene) indicated a role for eep in cleavage of lipoproteins in the absence of Lsp. Such a function may indicate a role for eep in maintenance of secretion pathways during disruption of normal lipoprotein processing.
    Journal of bacteriology 08/2008; 190(13):4641-7. · 3.94 Impact Factor
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    ABSTRACT: The characteristics of a streptococcal plasminogen activator (PA) displaying specificity for ruminant plasminogen (Plg) were defined using molecular approaches. The 16-kDa secreted protein PadA was found to be prevalent in Streptococcus dysgalactiae subspecies dysgalactiae isolated from cases of bovine mastitis and septic arthritis in lambs. PadA was able to activate bovine, ovine and caprine Plg, but not human Plg. Amino acid sequence analysis identified a limited level of homology to other streptococcal PAs, including streptokinase; however, PadA was found to align well with and match in size the staphylococcal PA, staphylokinase. Recombinant PadA was used to investigate interaction with bovine Plg, leading to formation of an activator complex that was capable of recruiting and converting further substrate Plg into plasmin. Individual non-overlapping peptides of PadA or bovine microplasminogen were found to block the interaction between PadA and bovine Plg, preventing the formation of the activation complex. Homology modelling based upon structures of staphylokinase complexed with human microplasminogen supported these findings by placing critical residues in close proximity to the plasmin component of the activation complex.
    Journal of Molecular Biology 07/2008; 381(3):734-47. · 3.91 Impact Factor
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    ABSTRACT: Streptococcus (S.) uberis is a common cause of mastitis in cattle. A protein (PauA) secreted by this bacterium is capable of activating plasminogen from sheep and cattle. The PauA first binds to bovine plasminogen (b-plg) to form a PauA-plasminogen complex that subsequently binds to and activates b-plg to form plasmin. We have identified several linear epitopes of PauA that are recognized by murine monoclonal antibodies to PauA. Two of the monoclonal antibodies which neutralized the enzymatic activity of PauA, EC3 and 2.22, recognized common linear peptide sequences with similar charge and spacing patterns. These neutralization epitopes are located in the predicted alpha-domain of the PauA molecule. Further, these same epitopes are in critical structure/function domains identified in other studies. These characterizations may facilitate the design of an efficacious vaccine for streptococcal mastitis in the dairy cow.
    Veterinary Immunology and Immunopathology 05/2005; 104(3-4):155-62. · 1.88 Impact Factor
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    ABSTRACT: The interactions between bovine plasminogen and the streptococcal plasminogen activator PauA that culminate in the generation of plasmin are not fully understood. Formation of an equimolar activation complex comprising PauA and plasminogen by non-proteolytic means is a prerequisite to the recruitment of substrate plasminogen; however the determinants that facilitate these interactions have yet to be defined. A mutagenesis strategy comprising nested deletions and random point substitutions indicated roles for both amino and carboxyl-terminal regions of PauA and identified further essential residues within the alpha domain of the plasminogen activator. A critical region within the alpha domain was identified using non-overlapping PauA peptides to block the interaction between PauA and bovine plasminogen, preventing formation of the activation complex. Homology modelling of the activation complex based upon the known structures of streptokinase complexed with human plasmin supported these findings by placing critical residues in close proximity to the plasmin component of the activation complex.
    Journal of Molecular Biology 10/2004; 342(4):1101-14. · 3.91 Impact Factor
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    ABSTRACT: Despite much success in the control of mastitis in dairy cattle, intramammary infection with Streptococcus uberis remains a threat to herd health. This organism is a frequent cause of mastitis worldwide. Recent advances in the ability to genetically manipulate this bacterium, coupled to the determination of a representative genome sequence have already enabled the investigation of certain aspects of disease pathogenesis. Further use of such technology coupled to reliable models of disease and post-genomic analysis will permit the elucidation of further interactions between pathogen and host. This additional information can be usefully targeted at identification of candidates for inclusion in effective vaccines. This communication reviews the current, reported progress using this technology for S. uberis.
    Veterinary Immunology and Immunopathology 09/2004; 100(3-4):145-9. · 1.88 Impact Factor
  • The Veterinary record 06/2004; 154(21):671-2. · 1.80 Impact Factor
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    Philip N Ward, James A Leigh
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    ABSTRACT: Streptococci produce a diverse range of secreted plasminogen activators capable of converting mammalian plasminogen to plasmin in a species-specific manner. In all examples to date, the host animal's plasminogen and that of a number of additional species have been shown to interact with these molecules leading to the conclusion that the pathogenesis of streptococci is in some way dependent upon activation of host plasminogen. PauA was the first plasminogen activator described from Streptococcus uberis, a pathogen frequently isolated from cases of bovine mastitis. Recently, a second S. uberis plasminogen activator (PauB) was identified from a Danish mastitis isolate. Interestingly, the pauB open reading frame occupied the locus normally filled by pauA. In the present study a genetic screen of streptococcal and field isolates frequently associated with mastitis was undertaken to assess the distribution, chromosomal location and sequence variation of these putative virulence factors. Southern analysis of a diverse panel of streptococci and additional bacterial isolates frequently associated with bovine mastitis was performed using pauA and pauB probes. Sequence variation of PauA was assessed at the protein level following nucleotide sequence analysis of pauA alleles amplified from isolates picked from different geographical locations. We observed plasminogen activators to be universally distributed amongst S. uberis. A pauA allele was identified in all but one strain of S. uberis. This strain had a pauB allele substituted for pauA at the same locus. The remarkably low level of sequence variation demonstrated by PauA was further restricted to a limited number of residues within the molecule. The high prevalence of PauA alleles in field isolates of S.uberis supported the observation that plasminogen activators are likely to confer an advantage with respect to colonization and growth. The findings of the present study support the theory that PauA plays a critical role in the pathogenesis of S. uberis.
    The Indian Journal of Medical Research 05/2004; 119 Suppl:136-40. · 2.06 Impact Factor
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    ABSTRACT: To determine the localization of MtuA, an LraI lipoprotein within Streptococcus uberis and assess whether the protein was able to induce an antibody response capable of growth inhibition. Immunoblots and ELISAs were performed on S. uberis cell fractions to localize the protein. The strongest reactivity was within the membrane-enriched fraction. Electron micrographs also showed labelling consistent with a location within the membrane. Specific antibodies from both rabbits and calves were unable to inhibit the growth of S. uberis in milk. In addition, MtuA was not detectable in a whole-cell ELISA and whole bacterial cells were unable to adsorb specific antibodies from antiserum raised against MtuA. The MtuA protein appears to be located within the cell membrane and is not on the bacterial surface and thus not available for interaction with potentially growth-inhibiting antibodies. Unlike PsaA of S. pneumoniae and MtsA of S. pyogenes, MtuA of S. uberis does not appear to be located at the cell surface. Therefore, in contrast to studies with other similar proteins, MtuA is unlikely to be a good vaccine candidate.
    Journal of Applied Microbiology 02/2004; 97(1):149-57. · 2.20 Impact Factor
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    ABSTRACT: A mutant of Streptococcus uberis carrying a single copy of ISS1 within pauA was unable to activate bovine plasminogen. Contrary to a hypothesis postulated previously, this mutation did not alter the ability of the bacterium to grow in milk or to infect the lactating bovine mammary gland.
    Infection and Immunity 01/2004; 71(12):7193-6. · 4.07 Impact Factor
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    ABSTRACT: A mutant strain of Streptococcus uberis (AJS001) that was unable to grow in bovine milk was isolated following random insertional mutagenesis. The level of growth in milk was restored to that of the parental strain (strain 0140J) following addition of MnSO(4) but not following addition of other metal ions. The mutant contained a single insertion within mtuA, a homologue of mtsA and psaA, which encode metal-binding proteins in Streptococcus pyogenes and Streptococcus pneumoniae, respectively. Strain AJS001 was unable to infect any of eight quarters on four dairy cows following intramammary challenge with 10(5) CFU. Bacteria were never recovered directly from milk of these animals but were detected following enrichment in Todd-Hewitt broth in three of eight milk samples obtained within 24 h of challenge. The animals showed no inflammatory response and no signs of mastitis. Three mammary quarters on two different animals simultaneously challenged with 600 CFU of the parental strain, strain 0140J, became colonized, shed high numbers of S. uberis organisms in milk, displayed a marked inflammatory response to infection, and showed overt signs of mastitis. These data indicate that mtuA was required for efficient uptake of Mn(2+) during growth in bovine milk and infection of the lactating bovine mammary gland.
    Infection and Immunity 10/2003; 71(9):4842-9. · 4.07 Impact Factor
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    ABSTRACT: Streptococcus uberis is an increasingly significant cause of intramammary infection in the dairy cow, presently responsible for approximately 33% of all cases of bovine mastitis in the United Kingdom. Following experimentally induced infection of the lactating mammary gland, S. uberis is found predominantly in the luminal areas of secretory alveoli and ductular tissue, indicating that much of the bacterial growth occurs in residual and newly synthesized milk. With the objective of identifying potential virulence determinants in a clinical isolate of S. uberis, we have used representational difference analysis of cDNA to identify genes that show modified expression in milk. We have identified a number of differentially expressed genes that may contribute to the overall pathogenicity of the organism. Of these, a transcript encoding a putative oligopeptide binding protein (OppA) was further characterized. We have found that S. uberis possesses two oppA-like open reading frames, oppA1 and oppA2, which are up-regulated to different degrees following growth in milk. Mutants lacking either oppA1 or oppA2 are viable and have an increased resistance to the toxic peptide derivative aminopterin; however, only mutants lacking oppA1 display a lower rate of growth in milk. In addition, expression of the oppA genes appears to be coordinated by different mechanisms. We conclude that the oppA genes encode oligopeptide binding proteins, possibly displaying different specificities, required for the efficient growth of S. uberis in milk.
    Journal of Bacteriology 10/2003; 185(17):5210-9. · 3.19 Impact Factor
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    ABSTRACT: The frequency at which the genes responsible for capsule biosynthesis occurred in field isolates of Streptococcus uberis was determined. Of the two genotypes detected (hasABC and hasC), the capsular genotype (hasABC) was more common. This genotype was present at a higher frequency in a population isolated from mastitis cases than in a population isolated from cattle bedding. The virulence of a mutant strain of S. uberis (TRF0-6) that lacked the ability to produce a hyaluronic acid capsule due to an insertion within its single copy of hasA (P. N. Ward, T. R. Field, W. G. F. Ditcham, E. Maguin, and J. A. Leigh, Infect. Immun. 69:392-399, 2001) was compared to that of the capsular parental strain (0140J). Strains TRF0-6 and 0140J infected all mammary gland quarters following experimental challenge. The wild type and the mutant induced overt signs of disease in four out of four and in six out of eight mammary gland quarters, respectively. Both the wild type and the hasA mutant were resistant to killing by bovine neutrophils following cultivation in bovine milk. The ability to withstand the bactericidal action of neutrophils following growth in milk was therefore independent of the capsule and coincided with the ability of supernatants from such cultures to prevent the bactericidal action of neutrophils. This investigation revealed that, in the absence of the capsule, S. uberis is able to withstand the bactericidal effect of bovine neutrophils and induce mastitis in dairy cows.
    Infection and Immunity 02/2003; 71(1):132-9. · 4.07 Impact Factor
  • J. A. Leigh, P. N. Ward, T. R. Field
    Research in Veterinary Science 01/2003; 74:26-26. · 1.77 Impact Factor

Publication Stats

393 Citations
85.35 Total Impact Points

Institutions

  • 2008–2012
    • Oxford University Hospitals NHS Trust
      • Nuffield Department of Clinical Laboratory Sciences
      Oxford, England, United Kingdom
    • University of Nottingham
      • School of Veterinary Medicine and Science
      Nottingham, ENG, United Kingdom
  • 2010
    • University of Exeter
      Exeter, England, United Kingdom
  • 2009
    • Wellcome Trust Sanger Institute
      Cambridge, England, United Kingdom
  • 1999–2004
    • Biotechnology and Biological Sciences Research Council
      Swindon, England, United Kingdom