Melbourne Dental School - Research Publications
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ItemRemineralization and fluoride uptake of white spot lesions under dental varnishes(WILEY, 2020-12)INTRODUCTION: The aim of this study was to evaluate white spot lesion (WSL) remineralization and fluoride uptake by the application of fluoride varnishes directly onto artificial WSLs in vitro. METHODS: MI varnish containing casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and 2.26% fluoride and Duraphat varnish containing 2.26% fluoride (no added calcium) were compared with a placebo varnish (no added calcium or fluoride). Two WSLs were prepared in enamel slabs and varnish applied to cover one of the two lesions. Each slab was immersed in artificial saliva for 14 days at 37°C. Mineral content was determined using transverse microradiography and fluoride uptake using electron probe microanalysis. The data were statistically analysed using a linear mixed model. RESULTS: Both MI and Duraphat varnishes significantly remineralized the covered and uncovered WSLs when compared with the placebo varnish (P < 0.001). The WSLs covered with varnish showed greater remineralization than those uncovered. MI varnish produced the highest level of remineralization and significantly greater fluoride uptake (0.44 ± 0.08 wt%) compared with Duraphat (0.24 ± 0.03 wt%) and the placebo varnish (0.06 ± 0.05 wt%). CONCLUSION: Varnish containing fluoride and CPP-ACP was superior to varnish containing fluoride alone in promoting WSL remineralization and fluoride uptake.
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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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ItemSelf-assembly of dental surface nanofilaments and remineralisation by SnF2 and CPP-ACP nanocomplexes(NATURE PUBLISHING GROUP, 2019-02-04)Dental caries, erosion and hypersensitivity are major public health problems. SnF2 is used widely in oral care products to help prevent/treat these conditions. Casein phosphopeptide-stabilised amorphous calcium phosphate nanocomplexes (CPP-ACP) are a biomimetic nanotechnology of salivary phosphopeptide-ACP complexes that deliver bioavailable calcium and phosphate ions to promote dental remineralisation (repair). We show here using in vitro studies and a double-blind, randomised controlled, cross-over design in situ clinical trial that SnF2 and CPP-ACP interact to form a nanofilament coating on the tooth surface and that together they are superior in their ability to promote dental remineralisation. Sn(II) by cross-linking the CPP-ACP helps to stabilise the complexes which improves delivery to the tooth surface and enhances binding and ion incorporation into tooth mineral. The combination of SnF2 and CPP-ACP in oral care products may significantly improve their efficacy in prevention/treatment of dental caries/erosion and hypersensitivity.
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ItemThe Role of Treponema denticola Motility in Synergistic Biofilm Formation With Porphyromonas gingivalis(FRONTIERS MEDIA SA, 2019-12-18)Chronic periodontitis has a polymicrobial biofilm etiology and interactions between key oral bacterial species, such as Porphyromonas gingivalis and Treponema denticola contribute to disease progression. P. gingivalis and T. denticola are co-localized in subgingival plaque and have been previously shown to exhibit strong synergy in growth, biofilm formation and virulence in an animal model of disease. The motility of T. denticola, although not considered as a classic virulence factor, may be involved in synergistic biofilm development between P. gingivalis and T. denticola. We determined the role of T. denticola motility in polymicrobial biofilm development using an optimized transformation protocol to produce two T. denticola mutants targeting the motility machinery. These deletion mutants were non-motile and lacked the gene encoding the flagellar hook protein of the periplasmic flagella (ΔflgE) or a component of the stator motor that drives the flagella (ΔmotB). The specificity of these gene deletions was determined by whole genome sequencing. Quantitative proteomic analyses of mutant strains revealed that the specific inactivation of the motility-associated gene, motB, had effects beyond motility. There were 64 and 326 proteins that changed in abundance in the ΔflgE and ΔmotB mutants, respectively. In the ΔflgE mutant, motility-associated proteins showed the most significant change in abundance confirming the phenotype change for the mutant was related to motility. However, the inactivation of motB as well as stopping motility also upregulated cellular stress responses in the mutant indicating pleiotropic effects of the mutation. T. denticola wild-type and P. gingivalis displayed synergistic biofilm development with a 2-fold higher biomass of the dual-species biofilms than the sum of the monospecies biofilms. Inactivation of T. denticola flgE and motB reduced this synergy. A 5-fold reduction in dual-species biofilm biomass was found with the motility-specific ΔflgE mutant suggesting that T. denticola periplasmic flagella are essential in synergistic biofilm formation with P. gingivalis.
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ItemMolecular Interactions of Peptide Encapsulated Calcium Phosphate Delivery Vehicle at Enamel Surfaces(Springer, 2018-12-31)Phosphorylated peptides derived from milk caseins, known as casein phosphopeptides (CPP) self-assemble and encapsulate the calcium and phosphate mineral in the form of amorphous calcium phosphate (ACP) thus forming CPP-ACP nanocomplexes that are non-toxic and bio-compatible. The biomedical application is the repair of tooth surfaces (enamel) at early stages of tooth decay. These nanocomplexes release calcium and phosphate ions to rebuild demineralised HA crystals in enamel subsurface lesions. The topical application of CPP-ACP at the tooth surface initiates a series of interactions at the enamel mineral hydroxyapatite surface, and at the enamel salivary pellicle that are not well understood. In this study, we have shown that the β-casein (1-25) peptide binds reversibly to Ca2+, Mg2+, Mn2+, La2+, Ni2+, and Cd2+ metal ions. In contrast, binding to Sn2+, Fe2+, and Fe3+ ions, resulted in ion-induced aggregation. The casein peptides as well as the mineral ions dissociate from the CPP-ACP complexes to adsorb to both the un-coated and saliva-coated mineral surface with the mineralisation increasing monotonically with increasing pH. Furthermore, SEM of the CPP-ACP revealed images of spherical particles surrounded by ACP mineral. In conclusion, the enamel remineralisation process involves an array of interactions between the peptide and mineral ions of the CPP-ACP delivery vehicle and the tooth enamel mineral with its salivary pellicle.
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ItemPG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System(PUBLIC LIBRARY SCIENCE, 2016-10-06)Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a β-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS.
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ItemReversible redox regulation of specificityof Arg-gingipain B in Porphyromonas gingivalis(ELSEVIER SCIENCE BV, 2013-05-02)Arg-gingipain B (RgpB), a major virulence factor secreted by the periodontal pathogen Porphyromonas gingivalis is an Arg-specific cysteine proteinase. By monitoring proteolytic cleavage of a human salivary peptide histatin 5 using MALDI-TOF MS, RgpB purified from P. gingivalis HG66 was found to shift from a dominant Arg-X to dominant Lys-X activity, both in vitro and in vivo, upon reversible cysteine oxidation. Native PAGE analysis revealed the association of novel Lys-X activity with a reversible state change of the oxidized enzyme. The redox-regulated Lys-X activity of RgpB may provide a survival advantage to P. gingivalis against the oxidative host defence.
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ItemTannerella forsythia Outer Membrane Vesicles Are Enriched with Substrates of the Type IX Secretion System and TonB-Dependent Receptors(AMER CHEMICAL SOC, 2015-12)Tannerella forsythia, a Gram-negative oral bacterium closely associated with chronic periodontitis, naturally produces outer membrane vesicles (OMVs). In this study, OMVs were purified by gradient centrifugation, and the proteome was investigated together with cellular fractions using LC-MS/MS analyses of SDS-PAGE fractions, resulting in the identification of 872 proteins including 297 OMV proteins. Comparison of the OMV proteome with the subcellular proteomes led to the localization of 173 proteins to the vesicle membrane and 61 proteins to the vesicle lumen, while 27 substrates of the type IX secretion system were assigned to the vesicle surface. These substrates were generally enriched in OMVs; however, the stoichiometry of the S-layer proteins, TfsA and TfsB, was significantly altered, potentially to accommodate the higher curvature required of the S-layer around OMVs. A vast number of TonB-dependent receptors related to SusC, together with their associated SusD-like lipoproteins, were identified, and these were also relatively enriched in OMVs. In contrast, other lipoproteins were significantly depleted from the OMVs. This study identified the highest number of membrane-associated OMV proteins to date in any bacterium and conclusively demonstrates cargo sorting of particular classes of proteins, which may have significant impact on the virulence of OMVs.
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ItemSpheres of influence: Porphyromonas gingivalis outer membrane vesicles(WILEY, 2016-10)Outer membrane vesicles (OMVs) are asymmetrical single bilayer membranous nanostructures produced by Gram-negative bacteria important for bacterial interaction with the environment. Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces OMVs that act as a virulence factor secretion system contributing to its pathogenicity. Despite their biological importance, the mechanisms of OMV biogenesis have not been fully elucidated. The ~14 times more curvature of the OMV membrane than cell outer membrane (OM) indicates that OMV biogenesis requires energy expenditure for significant curvature of the OMV membrane. In P. gingivalis, we propose that this may be achieved by upregulating the production of certain inner or outer leaflet lipids, which causes localized outward curvature of the OM. This results in selection of anionic lipopolysaccharide (A-LPS) and associated C-terminal domain (CTD) -family proteins on the outer surface due to their ability to accommodate the curvature. Deacylation of A-LPS may further enable increased curvature leading to OMV formation. Porphyromonas gingivalis OMVs that are selectively enriched in CTD-family proteins, largely the gingipains, can support bacterial coaggregation, promote biofilm development and act as an intercessor for the transport of non-motile bacteria by motile bacteria. The P. gingivalis OMVs are also believed to contribute to host interaction and colonization, evasion of immune defense mechanisms, and destruction of periodontal tissues. They may be crucial for both micro- and macronutrient capture, especially heme and probably other assimilable compounds for its own benefit and that of the wider biofilm community.