Melbourne Dental School - Research Publications
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ItemComparative transcriptomic analysis of Porphyromonas gingivalis biofilm and planktonic cells(BMC, 2009-01-29)BACKGROUND: Porphyromonas gingivalis in subgingival dental plaque, as part of a mature biofilm, has been strongly implicated in the onset and progression of chronic periodontitis. In this study using DNA microarray we compared the global gene expression of a P. gingivalis biofilm with that of its planktonic counterpart grown in the same continuous culture. RESULTS: Approximately 18% (377 genes, at 1.5 fold or more, P-value < 0.01) of the P. gingivalis genome was differentially expressed when the bacterium was grown as a biofilm. Genes that were down-regulated in biofilm cells, relative to planktonic cells, included those involved in cell envelope biogenesis, DNA replication, energy production and biosynthesis of cofactors, prosthetic groups and carriers. A number of genes encoding transport and binding proteins were up-regulated in P. gingivalis biofilm cells. Several genes predicted to encode proteins involved in signal transduction and transcriptional regulation were differentially regulated and may be important in the regulation of biofilm growth. CONCLUSION: This study analyzing global gene expression provides insight into the adaptive response of P. gingivalis to biofilm growth, in particular showing a down regulation of genes involved in growth and metabolic activity.
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ItemHemoglobin hydrolysis and heme acquisition by Porphyromonas gingivalis(BLACKWELL MUNKSGAARD, 2004-02)Porphyromonas gingivalis has been implicated in the progression of chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. This bacterium is a gram-negative, black-pigmented, asaccharolytic anaerobe that relies on the fermentation of amino acids for the production of metabolic energy. The Arg- and Lys-specific extracellular cysteine proteinases of P. gingivalis, RgpA, RgpB and Kgp have been implicated as major virulence factors. In this study we investigated the hydrolysis of human hemoglobin by whole cells of P. gingivalis W50 and the mutants W501 (RgpA-), W50AB (RgpA-RgpB-) and W50ABK (RgpA-RgpB-Kgp-) under strictly anaerobic conditions in a physiological buffer (pH 7.5) using mass spectrometric analysis. Incubation of P. gingivalis W50 with hemoglobin over a period of 30 min resulted in the detection of 20 hemoglobin peptides, all with C-terminal Arg or Lys residues. The majority of the hemoglobin alpha- and beta-chain sequences were recovered as peptides except for two similar regions of the C-terminal half of each chain, alpha(92-127) and beta(83-120). The residues of the unrecovered sequences form part of the interface between the alpha- and beta-chains and an exposed surface area of the hemoglobin tetramer that may be involved in binding to P. gingivalis. P. gingivalis W501 (RgpA-) produced similar peptides to those seen in the wild-type. All identified peptides from the hydrolysis of hemoglobin by the P. gingivalis W50AB (RgpA-RgpB-) mutant were the result of cleavage at Lys. The triple mutant W50ABK was unable to hydrolyze hemoglobin under the assay conditions used, suggesting that on whole cells the major cell surface activity responsible for hydrolysis of hemoglobin is from the RgpA/B and Kgp proteinases. However, the triple proteinase mutant W50ABK grew as well as the wild-type in a medium containing hemoglobin as the only iron source, indicating that the RgpA/B and Kgp proteinases are not essential for iron assimilation from hemoglobin by P. gingivalis.
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ItemCharacterization and expression of a novel Porphyromonas gingivalis outer membrane protein, Omp28(WILEY, 2002-06)We report the characterization of a Porphyromonas gingivalis gene, designated omp28, encoding a protein that we have previously purified and characterized as a 28-kDa outer membrane protein. The deduced amino acid sequence of the omp28 open reading frame displayed an outer membrane leader sequence and lipoprotein attachment site but did not exhibit any significant overall sequence identity with protein sequences in the databases. A small stretch of amino acids (19 residues) exhibits 50% sequence identity with a segment of a fimbrial protein from Dichelobacter nodosus involved in adhesion, suggesting that Omp28 may be a surface adhesin/receptor of P. gingivalis. Using the pET-24 vector we expressed recombinant Omp28 (rOmp28) in Escherichia coli. Western blot analyses of purified rOmp28 with rabbit antisera to a P. gingivalis outer membrane preparation, protective rat anti-whole P. gingivalis antisera and pooled human sera from chronic periodontitis patients showed that the recombinant was recognized by all antisera. Further, anti-rOmp28 antisera exhibited strong reactivity with a panel of four laboratory strains and 10 clinical isolates of P. gingivalis from the United States, Sudan, Romania and Norway. These results suggest that Omp28 is expressed by a wide distribution of P. gingivalis strains.
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ItemMajor outer membrane proteins and proteolytic processing of RgpA and Kgp of Porphyromonas gingivalis W50(PORTLAND PRESS, 2002-04-01)