Melbourne Dental School - Research Publications
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ItemCharacterization of hemin-binding protein 35 (HBP35) in Porphyromonas gingivalis: its cellular distribution, thioredoxin activity and role in heme utilization(BIOMED CENTRAL LTD, 2010-05-25)BACKGROUND: The periodontal pathogen Porphyromonas gingivalis is an obligate anaerobe that requires heme for growth. To understand its heme acquisition mechanism, we focused on a hemin-binding protein (HBP35 protein), possessing one thioredoxin-like motif and a conserved C-terminal domain, which are proposed to be involved in redox regulation and cell surface attachment, respectively. RESULTS: We observed that the hbp35 gene was transcribed as a 1.1-kb mRNA with subsequent translation resulting in three proteins with molecular masses of 40, 29 and 27 kDa in the cytoplasm, and one modified form of the 40-kDa protein on the cell surface. A recombinant 40-kDa HBP35 exhibited thioredoxin activity in vitro and mutation of the two putative active site cysteine residues abolished this activity. Both recombinant 40- and 27-kDa proteins had the ability to bind hemin, and growth of an hbp35 deletion mutant was substantially retarded under hemin-depleted conditions compared with growth of the wild type under the same conditions. CONCLUSION: P. gingivalis HBP35 exhibits thioredoxin and hemin-binding activities and is essential for growth in hemin-depleted conditions suggesting that the protein plays a significant role in hemin acquisition.
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ItemMechanistic Target of Rapamycin (Mtor) Is Essential for Murine Embryonic Heart Development and Growth(PUBLIC LIBRARY SCIENCE, 2013-01-14)Chronic periodontitis has a polymicrobial biofilm aetiology and interactions between key bacterial species are strongly implicated as contributing to disease progression. Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia have all been implicated as playing roles in disease progression. P. gingivalis cell-surface-located protease/adhesins, the gingipains, have been suggested to be involved in its interactions with several other bacterial species. The aims of this study were to determine polymicrobial biofilm formation by P. gingivalis, T. denticola and T. forsythia, as well as the role of P. gingivalis gingipains in biofilm formation by using a gingipain null triple mutant. To determine homotypic and polymicrobial biofilm formation a flow cell system was employed and the biofilms imaged and quantified by fluorescent in situ hybridization using DNA species-specific probes and confocal scanning laser microscopy imaging. Of the three species, only P. gingivalis and T. denticola formed mature, homotypic biofilms, and a strong synergy was observed between P. gingivalis and T. denticola in polymicrobial biofilm formation. This synergy was demonstrated by significant increases in biovolume, average biofilm thickness and maximum biofilm thickness of both species. In addition there was a morphological change of T. denticola in polymicrobial biofilms when compared with homotypic biofilms, suggesting reduced motility in homotypic biofilms. P. gingivalis gingipains were shown to play an essential role in synergistic polymicrobial biofilm formation with T. denticola.
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ItemPorphyromonas gingivalis Peptidylarginine Deiminase, a Key Contributor in the Pathogenesis of Experimental Periodontal Disease and Experimental Arthritis(PUBLIC LIBRARY SCIENCE, 2014-06-24)OBJECTIVES: To investigate the suggested role of Porphyromonas gingivalis peptidylarginine deiminase (PAD) in the relationship between the aetiology of periodontal disease and experimentally induced arthritis and the possible association between these two conditions. METHODS: A genetically modified PAD-deficient strain of P. gingivalis W50 was produced. The effect of this strain, compared to the wild type, in an established murine model for experimental periodontitis and experimental arthritis was assessed. Experimental periodontitis was induced following oral inoculation with the PAD-deficient and wild type strains of P. gingivalis. Experimental arthritis was induced via the collagen antibody induction process and was monitored by assessment of paw swelling and micro-CT analysis of the radio-carpal joints. Experimental periodontitis was monitored by micro CT scans of the mandible and histological assessment of the periodontal tissues around the mandibular molars. Serum levels of anti-citrullinated protein antibodies (ACPA) and P. gingivalis were assessed by ELISA. RESULTS: The development of experimental periodontitis was significantly reduced in the presence of the PAD-deficient P. gingivalis strain. When experimental arthritis was induced in the presence of the PAD-deficient strain there was less paw swelling, less erosive bone damage to the joints and reduced serum ACPA levels when compared to the wild type P. gingivalis inoculated group. CONCLUSION: This study has demonstrated that a PAD-deficient strain of P. gingivalis was associated with significantly reduced periodontal inflammation. In addition the extent of experimental arthritis was significantly reduced in animals exposed to prior induction of periodontal disease through oral inoculation of the PAD-deficient strain versus the wild type. This adds further evidence to the potential role for P. gingivalis and its PAD in the pathogenesis of periodontitis and exacerbation of arthritis. Further studies are now needed to elucidate the mechanisms which drive these processes.
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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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ItemProspects for treatment of Porphyromonas gingivalis-mediated disease - immune-based therapy(CO-ACTION PUBLISHING, 2015)Chronic periodontitis is an inflammatory disease of the supporting tissues of the teeth associated with a polymicrobial biofilm (subgingival plaque) accreted to the tooth which results in destruction of the tooth's supporting tissues. A characteristic feature of the disease-associated plaque is the emergence of proteolytic species. One of these species, Porphyromonas gingivalis has recently been described as a keystone pathogen as it dysregulates the host immune response to favour the polymicrobial biofilm disrupting homeostasis to cause dysbiosis and disease. The level of P. gingivalis in subgingival plaque above threshold levels (~10% of total bacterial cell load) has been demonstrated to predict imminent clinical attachment loss (disease progression) in humans. Porphyromonas gingivalis is found as microcolonies in the superficial layers of subgingival plaque adjacent to the periodontal pocket epithelium which helps explain the strong association with underlying tissue inflammation and disease at relatively low proportions (10%) of the total bacterial cell load of the plaque. The mouse periodontitis model has been used to show that inflammation is essential to allow establishment of P. gingivalis at the levels in plaque (10% or greater of total bacterial cell load) necessary to produce dysbiosis and disease. The extracellular proteinases "gingipains" (RgpA/B and Kgp) of P. gingivalis have been implicated as major virulence factors that are critical for dysbiosis and disease. This has resulted in the strategy of targeting the gingipains by vaccination. We have produced a recombinant immunogen which induces an immune response in mice that neutralises the proteolytic and host/bacterial binding functions of the gingipains. Using this immunogen as a therapeutic vaccine in mice already infected with P. gingivalis, we have shown that inflammation and alveolar bone loss can be substantially reduced. The protection was characterised by a predominant Th2 cytokine and antibody (IgG1) response and shown to be mediated by the gingipain neutralising antibodies using adoptive transfer and systemic/topical passive antibody experiments. Vaccination may be a useful adjunct to scaling and root planing in the treatment of P. gingivalis-mediated chronic periodontitis.
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ItemNo Preview AvailableBacterial Fluorescent-dextran Diffusion Assay(Bio-Protocol, LLC, 2014)
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ItemFluoride content of tank water in Australia(WILEY-BLACKWELL, 2014-06)BACKGROUND: The aims of this study were to: (1) analyse the fluoride content of tank water; (2) determine whether the method of water collection or storage influenced fluoride content; and (3) survey participant attitudes towards water fluoridation. METHODS: Plastic tubes and a questionnaire were distributed through dentists to households with water tanks in Victoria. A midstream tank water sample was collected and fluoride analysed in triplicate using ion chromatography RESULTS: All samples (n = 123) contained negligible amounts of fluoride, with a mean fluoride concentration of <0.01 ppm (range: <0.01-0.18 ppm). No statistically significant association was found between fluoride content and variables investigated such as tank material, tank age, roof material and gutter material. Most people did not know whether their tank water contained fluoride and 40.8% preferred to have access to fluoridated water. The majority thought fluoride was safe and more than half of the respondents supported fluoridation. Fluoride content of tank water was well below the optimal levels for caries prevention. CONCLUSIONS: People who rely solely on tank water for drinking may require additional exposure to fluoride for optimal caries prevention.
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ItemOxantel Disrupts Polymicrobial Biofilm Development of Periodontal Pathogens(AMER SOC MICROBIOLOGY, 2014-01)Bacterial pathogens commonly associated with chronic periodontitis are the spirochete Treponema denticola and the Gram-negative, proteolytic species Porphyromonas gingivalis and Tannerella forsythia. These species rely on complex anaerobic respiration of amino acids, and the anthelmintic drug oxantel has been shown to inhibit fumarate reductase (Frd) activity in some pathogenic bacteria and inhibit P. gingivalis homotypic biofilm formation. Here, we demonstrate that oxantel inhibited P. gingivalis Frd activity with a 50% inhibitory concentration (IC50) of 2.2 μM and planktonic growth of T. forsythia with a MIC of 295 μM, but it had no effect on the growth of T. denticola. Oxantel treatment caused the downregulation of six P. gingivalis gene products and the upregulation of 22 gene products. All of these genes are part of a regulon controlled by heme availability. There was no large-scale change in the expression of genes encoding metabolic enzymes, indicating that P. gingivalis may be unable to overcome Frd inhibition. Oxantel disrupted the development of polymicrobial biofilms composed of P. gingivalis, T. forsythia, and T. denticola in a concentration-dependent manner. In these biofilms, all three species were inhibited to a similar degree, demonstrating the synergistic nature of biofilm formation by these species and the dependence of T. denticola on the other two species. In a murine alveolar bone loss model of periodontitis oxantel addition to the drinking water of P. gingivalis-infected mice reduced bone loss to the same level as the uninfected control.
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ItemNo Preview AvailableProspects for treatment of Porphyromonas gingivalis-mediated disease – immune-based therapy(Informa UK Limited, 2015-01)
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ItemPorphyromonas gingivalis-derived RgpA-Kgp Complex Activates the Macrophage Urokinase Plasminogen Activator System IMPLICATIONS FOR PERIODONTITIS(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2015-06-26)Urokinase plasminogen activator (uPA) converts plasminogen to plasmin, resulting in a proteolytic cascade that has been implicated in tissue destruction during inflammation. Periodontitis is a highly prevalent chronic inflammatory disease characterized by destruction of the tissue and bone that support the teeth. We demonstrate that stimulation of macrophages with the arginine- and lysine-specific cysteine protease complex (RgpA-Kgp complex), produced by the keystone pathogen Porphyromonas gingivalis, dramatically increased their ability to degrade matrix in a uPA-dependent manner. We show that the RgpA-Kgp complex cleaves the inactive zymogens, pro-uPA (at consensus sites Lys(158)-Ile(159) and Lys(135)-Lys(136)) and plasminogen, yielding active uPA and plasmin, respectively. These findings are consistent with activation of the uPA proteolytic cascade by P. gingivalis being required for the pathogen to induce alveolar bone loss in a model of periodontitis and reveal a new host-pathogen interaction in which P. gingivalis activates a critical host proteolytic pathway to promote tissue destruction and pathogen virulence.
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