Melbourne Dental School - Research Publications
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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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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.
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ItemPorphyromonas gingivalis Type IX Secretion Substrates Are Cleaved and Modified by a Sortase-Like Mechanism(PUBLIC LIBRARY SCIENCE, 2015-09)The type IX secretion system (T9SS) of Porphyromonas gingivalis secretes proteins possessing a conserved C-terminal domain (CTD) to the cell surface. The C-terminal signal is essential for these proteins to translocate across the outer membrane via the T9SS. On the surface the CTD of these proteins is cleaved prior to extensive glycosylation. It is believed that the modification on these CTD proteins is anionic lipopolysaccharide (A-LPS), which enables the attachment of CTD proteins to the cell surface. However, the exact site of modification and the mechanism of attachment of CTD proteins to the cell surface are unknown. In this study we characterized two wbaP (PG1964) mutants that did not synthesise A-LPS and accumulated CTD proteins in the clarified culture fluid (CCF). The CTDs of the CTD proteins in the CCF were cleaved suggesting normal secretion, however, the CTD proteins were not glycosylated. Mass spectrometric analysis of CTD proteins purified from the CCF of the wbaP mutants revealed the presence of various peptide/amino acid modifications from the growth medium at the C-terminus of the mature CTD proteins. This suggested that modification occurs at the C-terminus of T9SS substrates in the wild type P. gingivalis. This was confirmed by analysis of CTD proteins from wild type, where a 648 Da linker was identified to be attached at the C-terminus of mature CTD proteins. Importantly, treatment with proteinase K released the 648 Da linker from the CTD proteins demonstrating a peptide bond between the C-terminus and the modification. Together, this is suggestive of a mechanism similar to sortase A for the cleavage and modification/attachment of CTD proteins in P. gingivalis. PG0026 has been recognized as the CTD signal peptidase and is now proposed to be the sortase-like protein in P. gingivalis. To our knowledge, this is the first biochemical evidence suggesting a sortase-like mechanism in Gram-negative bacteria.