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Christopher G Dowson

Publications and source records attributed to Christopher G Dowson.

15 recordsLinked to original sources
PubMed2025

Transcriptomic and enzymological evidence for plastid peptidoglycan synthesis in the gymnosperm Picea abies.

It is understood that a cyanobacterium was the progenitor of plastids and that the biosynthesis of cell wall peptidoglycan was lost during chloroplast evolution. However, accumulated data, especially from the moss Physcomitrium patens, suggest that peptidoglycan remains essential for plastid division in some land plants. A fundamental set of peptidoglycan biosynthesis (Mur) genes has been identified in the genomes of these land plants, while many angiosperms no longer encode some core Mur genes, including a bifunctional penicillin-binding protein (PBP). Ten incomplete Mur genes were previously identified in the genome of the gymnosperm Picea abies but these could be pseudogenes or encode proteins that have been repurposed. For instance, mutant albino maize and Arabidopsis seedlings possess a defective UDP-N-acetylmuramoyl-l-alanyl-d-glutamate--2,6-diaminopimelate ligase (MurE), an intact MurE ligase being essential for peptidoglycan synthesis. In this study, we isolated a full set of cDNAs for peptidoglycan biosynthesis from P. abies. GFP fusion proteins with either P. abies (Pa)MurE or PaPBP were detected in chloroplasts. Cross-species complementation assays with PaMurE in Arabidopsis albino MurE mutants and Physcomitrium MurE chloroplast division mutants showed that the gymnosperm MurE completely rescued both mutant phenotypes. Enzymatic assay of recombinant PaMurE proteins revealed they catalyze the same reaction performed by their bacterial MurE homologs. Moreover, the expression of the PaPbp cDNA partially rescued the giant chloroplast phenotype in the moss Pbp knockout line. These results are consistent with the operation of a functional Mur gene set in the Norway spruce genome.

Peptidoglycan↗
PubMed2006

Proinflammatory activation of Toll-like receptor-2 during exposure of penicillin-resistant Streptococcus pneumoniae to beta-lactam antibiotics.

OBJECTIVES: Disease caused by penicillin-resistant Streptococcus pneumoniae (PRSP) is associated with more suppurative complications than disease caused by penicillin-susceptible S. pneumoniae (PSSP). Exposure of S. pneumoniae to beta-lactam antibiotics enhances the proinflammatory activation of human cells by pneumococci via Toll-like receptor-2 (TLR2). To test the hypothesis that penicillin resistance influences cellular TLR2 activation by beta-lactam-exposed pneumococci, we compared TLR2 induction by PSSP (MIC 0.06 mg/L) and a high-level PRSP clinical isolate (159; MIC 16 mg/L) following exposure to penicillin and cefotaxime. METHODS: Both organisms were treated with penicillin or cefotaxime at and around the MIC. TLR2 signalling was measured as relative IL-8 promoter activation in transfected HeLa cells. RESULTS: On exposure to penicillin, log-phase PSSP and PRSP induced TLR2-proinflammatory activation at levels significantly higher than unexposed bacteria, and maximal in each case at the MIC. Transformants containing low-affinity penicillin-binding proteins (PBP) 2x, 1a and 2b exhibited stepwise resistance to cefotaxime and penicillin. TLR2 activation following penicillin treatment was dependent on an abnormal cell wall (PBP1a and 2x) and autolysis (PBP2b). High affinity PBP2x was required for this effect to be observed in log-phase pneumococci exposed to cefotaxime at the MIC. Cefotaxime-mediated TLR2 activation was not observed in lag-phase transformants exposed to sub-lethal concentrations. CONCLUSIONS: These data show that PRSP have similar TLR2-proinflammatory effects to PSSP when exposed to beta-lactam antibiotics but the antibiotic concentration relative to the MIC is critical. This has implications for treatment of pneumococcal disease when tissue concentrations of antibiotic are close to the MIC.

Anti-Bacterial Agents↗
PubMed2006

Fluorescent reagents for in vitro studies of lipid-linked steps of bacterial peptidoglycan biosynthesis: derivatives of UDPMurNAc-pentapeptide containing d-cysteine at position 4 or 5.

UDPMurNAc-L-Ala-gamma-D-Glu-X-D-Ala-DAla (X = L-Lys or m-DAP) is the cytoplasmic precursor for the lipid-linked cycle of bacterial peptidoglycan biosynthesis, consisting of at least four enzymatic reactions, which are targets for antibacterial agents. Fluorescent derivatives of the UDPMurNAc-pentapeptide labelled at the 3rd, 4th, and 5th position of the peptide chain were prepared chemoenzymatically, in order to study the reactions catalysed by enzymes in this cycle. Derivatives labelled on the epsilon-amino group of the 3rd amino acid (N-dansyl, N-fluorescamine and N-phthalaldehyde) were prepared by chemical modification. Two methods were developed for preparation of analogues of UDPMurNAc-pentapeptide containing D-cysteine at position 4 or 5: either by MurF-catalysed ligation of the UDPMurNAc-tripeptide to synthetic D-Ala-D-Cys or D-Cys-D-Ala dipeptides; or by enzymatic synthesis of D-Ala-D-Cys by ligase VanD. D-Cys-containing UDPMurNAc-pentapeptides were labelled with pyrene maleimide, to give 4-pyrene and 5-pyrene labelled derivatives. The fluorescent UDPMurNAc-pentapeptides were processed as substrates by Escherichia coli MraY or E. coli membranes, giving 1.5-150-fold changes in fluorescence upon transformation to lipid intermediate I. Subsequent processing to lipid intermediate II gave rise only to small changes in fluorescence. Pyrene-labelled lipid intermediates I and II can be generated using Micrococcus flavus membranes, enabling the study of the later lipid-linked steps.

Bacterial Proteins↗
PubMed2006

Incremental increase in fitness cost with increased beta -lactam resistance in pneumococci evaluated by competition in an infant rat nasal colonization model.

BACKGROUND: We evaluated the impact of resistant penicillin-binding protein (PBP) allele acquisition on the ability of penicillin-resistant (PEN-R) pneumococcal strains to compete with penicillin-susceptible (PEN-S) ancestors for upper-respiratory-tract (URT) colonization. METHODS: PEN-S serotype 2, 6B, and 9V strains were transformed into derivatives expressing an increasing number of PEN-R PBP forms (2X, 2X-1A, and 2X-1A-2B for serotype 2 and 2X, 2X-2B, and 2X-2B-1A for 6B and 9V). Infant rats were inoculated intranasally with a mix of a PEN-R and PEN-S strains. For consecutive days, samples were collected for assessment of the ratio of PEN-S to PEN-R cells colonizing the URT. The selective index (SI), defined as the change in the natural logarithm of the ratio of PEN-S to PEN-R strains from the inoculum to the nasal-wash samples, quantified differences in fitness. RESULTS: SIs significantly > 0 (indicating a cost of resistant allele acquisition) were observed 4-5 days after colonization in all but serotype 6B pbp2x transfomants. Additional replacements with low-affinity forms of pbp2b and pbp1a genes reduced further ability to compete in all strains. CONCLUSIONS: The cost of penicillin-resistance acquisition for the Streptococcus pneumoniae strain competing with its susceptible ancestor to colonize the URT increases with the number of resistant pbp alleles acquired.

Amino Acid Sequence↗
PubMed2006

Distribution and genetic diversity of the ABC transporter lipoproteins PiuA and PiaA within Streptococcus pneumoniae and related streptococci.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. The existence of approximately 90 antigenically distinct capsular serotypes has greatly complicated the development of an effective pneumococcal vaccine. Virulence-associated proteins common and conserved among all capsular types now represent the best strategy to combat pneumococcal infections. PiuA and PiaA are the lipoprotein components of two pneumococcal iron ABC transporters and are required for full virulence in mouse models of infection. Here we describe a study of the distribution and genetic diversity of PiuA and PiaA within typical and atypical S. pneumoniae, Streptococcus oralis, and Streptococcus mitis strains. The genes encoding both PiuA and PiaA were present in all typical pneumococci tested, (covering 20 and 27 serotypes, respectively). The piuA gene was highly conserved within the typical pneumococci (0.3% nucleotide divergence), but was also present in "atypical" pneumococci and the closely related species S. mitis and S. oralis, showing up to 10.4% nucleotide divergence and 7.5% amino acid divergence from the typical pneumococcal alleles. Conversely, the piaA gene was found to be specific to typical pneumococci, 100% conserved, and absent from the oral streptococci, including isolates of S. mitis known to possess pneumolysin and autolysin. These are desirable qualities for a vaccine candidate and as a diagnostic tool for S. pneumoniae.

ATP-Binding Cassette Transporters↗
PubMed2005

PiuA and PiaA, iron uptake lipoproteins of Streptococcus pneumoniae, elicit serotype independent antibody responses following human pneumococcal septicaemia.

Streptococcus pneumoniae causes considerable morbidity and mortality worldwide. The need for a cheap and effective pneumococcal vaccine has necessitated the evaluation of common virulence-associated proteins as potential vaccine antigens. PiuA and PiaA are the lipoprotein components of two pneumococcal iron ABC transporters. Here, we show that patients with culture confirmed pneumococcal septicaemia have elevated levels of antibody to PiuA and PiaA in convalescent-phase, compared with acute-phase serum. Additionally, sera from septicaemic patients infected with 13 pneumococcal strains covering eight different serotypes, cross-reacted with recombinant PiuA-His(6) and PiaA-His(6) from a single pneumococcal strain, indicating that this immune response is serotype independent. Anti-PiuA and anti-PiaA antibodies were also found in healthy seven-month-old infants, indicating that they are immunogenic at a very early age.

ATP-Binding Cassette Transporters↗
PubMed2005

NanA, a neuraminidase from Streptococcus pneumoniae, shows high levels of sequence diversity, at least in part through recombination with Streptococcus oralis.

Streptococcus pneumoniae, an important human pathogen, contains at least two genes, nanA and nanB, that express sialidase activity. NanA is a virulence determinant of pneumococci which is important in animal models of colonization and middle ear infections. The gene encoding NanA was detected in all 106 pneumococcal strains screened that represented 59 restriction profiles. Sequencing confirmed a high level of diversity, up to 17.2% at the nucleotide level and 14.8% at the amino acid level. NanA diversity is due to a number of mechanisms including insertions, point mutations, and recombination generating mosaic genes. The level of nucleotide divergence for each recombinant block is greater than 30% and much higher than the 20% identified within mosaic pbp genes, suggesting that a high selective pressure exists for these alterations. These data indicate that at least one of the four recombinant blocks identified originated from a Streptococcus oralis isolate, demonstrating for the first time that protein virulence determinants of pneumococci have, as identified previously for genes encoding penicillin binding proteins, evolved by recombination with oral streptococci. No amino acid alterations were identified within the aspartic boxes or predicted active site, suggesting that sequence variation may be important in evading the adaptive immune response. Furthermore, this suggests that nanA is an important target of the immune system in the interaction between the pneumococcus and host.

Amino Acid Sequence↗
PubMed2004

Expression, purification, crystallization and preliminary characterization of uridine 5'-diphospho-N-acetylmuramoyl L-alanyl-D-glutamate:lysine ligase (MurE) from Streptococcus pneumoniae 110K/70.

An ORF designated sp1530 (murE) in the Streptococcus pneumoniae TIGR4 genome sequence, identified as uridine 5'-diphospho-N-acetylmuramoyl-L-alanyl-D-glutamate:L-lysine ligase (MurE; EC 6.3.2.7), was cloned into the high-expression plasmid pET21b and overexpressed in Escherichia coli BL21 (DE3) Star. The enzyme was purified in three steps to 99% purity. Crystals were obtained by the hanging-drop vapour-diffusion method at 291 K from solutions containing 25%(w/v) polyethylene glycol 2000 monomethylether, 0.2 M potassium thiocyanate, 0.1 M MES pH 6.5 in the presence of uridine 5'-diphospho-N-acetylmuramoyl alanyl glutamate (UDP-MurNAc-L-Ala-D-Glu) with and without 5'-adenylyl imidophosphate (AMP-PNP), a non-hydrolysable analogue of ATP. Diffraction data to 1.5 and 2.7 A, respectively, were collected at the European Synchrotron Radiation Facility (ESRF). Crystals grown in the presence of two ligands belong to space group P1, with unit-cell parameters a = 68.4, b = 71.4, c = 74.8 A, alpha = 73.4, beta = 80.5, gamma = 72.3 degrees. Crystals grown in the presence of UDP-MurNAc-L-Ala-D-Glu alone belong to space group P2(1), with unit-cell parameters a = 71.1, b = 129.4, c = 74.6 A, beta = 106.3 degrees.

Animals↗
PubMed2004

Distribution, genetic diversity, and variable expression of the gene encoding hyaluronate lyase within the Streptococcus suis population.

Although Streptococcus suis is an economically important pathogen of pigs and an occasional cause of zoonotic infections of humans knowledge of crucial virulence factors, and as a consequence targets for therapeutic or prophylactic intervention, remains limited. Here we describe a detailed study of the distribution, diversity, and in vitro expression of hyaluronate lyase, a protein implicated as a virulence factor of many mucosal pathogens. The gene encoding hyaluronate lyase, hyl, was present in all 309 bona fide S. suis isolates examined representing diverse serotypes, geographic sources, and clinical backgrounds. Examination of the genetic diversity of hyl by RFLP and sequence analysis indicated a pattern of diversity shared by many gram-positive surface proteins with a variable 5' region encoding the most distal cell surface-exposed regions of the protein and a much more conserved 3' region encoding domains more closely associated with the bacterial cell. Variation occurs by several mechanisms, including the accumulation of point mutations and deletion and insertion events, and there is clear evidence that genetic recombination has contributed to molecular variation in this gene. Despite the ubiquitous presence of hyl, the corresponding enzyme activity was detected in fewer than 30% of the 309 isolates. In several cases this lack of activity correlates with the presence of mutations (either sequence duplications or point mutations) within hyl that result in a truncated polypeptide. There is a striking absence of hyaluronate lyase activity in a large majority of isolates from classic S. suis invasive disease, indicating that this protein is probably not a crucial virulence factor, although activity is present in significantly higher numbers of isolates associated with pneumonia.

Animals↗
PubMed2004

Development of a multilocus sequence typing scheme for the opportunistic pathogen Pseudomonas aeruginosa.

A multilocus sequence typing (MLST) scheme has been developed for Pseudomonas aeruginosa which provides molecular typing data that are highly discriminatory and electronically portable between laboratories. MLST data confirm the data from previous studies that suggest that P. aeruginosa is best described as nonclonal but as having an epidemic population. The index of association was 0.17, indicating a freely recombining population; however, there was evidence of clusters of closely related strains or clonal complexes among the members of this population. It is apparent that the sequence types (STs) from single isolates, representing each of the present epidemic clones in the United Kingdom from Liverpool, Manchester, and the West Midlands, are not closely related to each other. This suggests distinct evolutionary origins for each of these epidemic clones in the United Kingdom. Furthermore, these clones are distinct from European clone C. Comparison of the results of MLST with those of toxA typing and serotyping revealed that strains with identical STs may possess different toxA types and diverse serotypes. Given that recombination is important in the population of P. aeruginosa, the lack of a linkage between toxA type and serotype is not surprising and reveals the strength of the MLST approach for obtaining a better understanding of the epidemiology of P. aeruginosa.

Alleles↗
PubMed2003

Spontaneous sequence duplications within capsule genes cap8E and tts control phase variation in Streptococcus pneumoniae serotypes 8 and 37.

Capsule phase variants were isolated from serotype 8 and serotype 37 pneumococcal sorbarods. Sequence duplications within the essential capsule genes - cap8E (type 8) and tts (type 37) - were found to introduce frameshifts and generate acapsular phenotypes. Capsular revertants possessed wild-type cap8E and tts genes, indicating the precise excision of these duplications. Reversion frequencies (OFF-ON) fit a linear relationship between log(frequency of reversion) and log(length of duplication), previously found for serotype three pneumococci [Waite, R. D., Struthers, J. K. & Dowson, C. G. (2001). Mol Microbiol 42, 1223-1232]. This study provides evidence that capsule phase variation can occur in pneumococcal serotypes with either simple (one to three genes) or complex capsule-encoding loci (12 genes). Given the key role of CapE (the first monosaccharide transferase) in other clinically important pneumococci, such as serotypes 14 and 19F with complex capsular loci, the observed duplication within cap8E suggests that capsule phase variation could be controlled by tandem sequence duplication in capE homologues in other pneumococcal serotypes that construct their capsules through polymerization of lipid-linked intermediates.

Bacterial Proteins↗
PubMed2003

Multilocus sequence typing for comparison of veterinary and human isolates of Campylobacter jejuni.

Multilocus sequence typing (MLST) has been applied to 266 Campylobacter jejuni isolates, mainly from veterinary sources, including cattle, sheep, poultry, pigs, pets, and the environment, as well as isolates from human cases of campylobacteriosis. The populations of veterinary and human isolates overlap, suggesting that most veterinary sources should be considered reservoirs of pathogenic campylobacters. There were some associations between source and sequence type complex, indicating that host or source adaptation may exist. The pig isolates formed a distinct group by MLST and may well represent a potential pig-adapted clone of C. jejuni. A subset (n = 82) of isolates was reanalyzed with a second MLST scheme which provided a unique set of isolates that had been analyzed at a total of 12 loci. The distribution of isolates among the complexes in each of the two schemes was similar but not identical. In addition to isolates from human outbreaks, one group of isolates that were not epidemiologically linked was also identical at all 12 loci. This group of isolates is believed to represent another stable strain of C. jejuni.

Abattoirs↗
PubMed2002

Development of a multilocus sequence typing scheme for the pig pathogen Streptococcus suis: identification of virulent clones and potential capsular serotype exchange.

Streptococcus suis is an important pathogen of pigs and occasionally causes serious human disease. However, little is known about the S. suis population structure, the clonal relationships between strains, the potential of particular clones to cause disease, and the relevance of serotype as a marker for epidemiology. Here we describe a multilocus sequence typing (MLST) scheme for S. suis developed in order to begin to address these issues. Seven housekeeping gene fragments from each of 294 S. suis isolates obtained from various S. suis diseases and from asymptomatic carriage representing 28 serotypes and nine distinct countries of origin were sequenced. Between 32 and 46 alleles per locus were identified, giving the ability to distinguish >1.6 x 10(11) sequence types (STs). However only 92 STs were identified in this study. Of the 92 STs 18 contained multiple isolates, the most common of which, ST1, was identified on 141 occasions from six countries. Assignment of the STs to lineages resulted in 37 being identified as unique and unrelated STs while the remaining 55 were assigned to 10 complexes. ST complexes ST1, ST27, and ST87 dominate the population; while the ST1 complex was strongly associated with isolates from septicemia, meningitis, and arthritis, the ST87 and ST27 complexes were found to contain significantly higher numbers of lung isolates. In agreement with the observed distribution of disease-causing isolates of S. suis, most isolates previously characterized as of high virulence in porcine infection models belong to ST1, while isolates belonging to other STs appear to be less virulent in general. Finally nine STs were found to contain isolates of multiple serotypes, and many isolates belonging to the same serotypes were found to have very disparate genetic backgrounds. As well as highlighting that the serotype can often be a poor indicator of genetic relatedness between S. suis isolates, these findings suggest that capsular genes may be moving horizontally through the S. suis population.

Animals↗
PubMed1999

Population biology of Streptococcus pneumoniae isolated from oropharyngeal carriage and invasive disease.

The population structure of Streptococcus pneumoniae in a sample of 134 carried antibiotic-susceptible isolates, and 53 resistant and susceptible invasive isolates, was examined using a DNA-based version of multilocus enzyme electrophoresis: multilocus restriction typing (MLRT). This involved RFLP analysis of PCR products generated from nine loci of housekeeping genes located around the pneumococcal chromosome. The combination of alleles at each of the nine loci gave an allelic profile or restriction type (RT). All carried (throat or nasopharyngeal) isolates from children or adults in Oxford and Manchester, UK, and from an HIV-seropositive cohort in Nairobi, Kenya, showed an epidemic population structure. Twelve carried clonal groups, each with different serotypes, were identified at both locations within the UK. Almost all of the carried clones examined (16/17) were found to possess identical RTs or sequence types (STs) to invasive isolates, indicating that frequently carried clones are also associated with cases of invasive disease. As expected from previous studies, the population of 53 invasive, mainly penicillin-resistant, isolates was also found to be at linkage equilibrium. Serotype switching was identified among 14% of RTs that possessed two or more members, or 5.7% of individual isolates within these RTs. In support of a population structure in which there is frequent recombination, there is also clear evidence that the trpA/B locus within pneumococci has evolved by horizontal gene transfer. A non-serotypable isolate from an HIV-seropositive patient in Kenya was clearly genetically distinct from other strains studied, with unique alleles at eight out of nine loci examined. However, it was initially identified as a pneumococcus by a 16S RNA gene probe (Gen-Probe), optochin susceptibility and the presence of pneumolysin and autolysin.

Adolescent↗