Melbourne Dental School - Research Publications
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ItemNo Preview AvailableProduction and properties of adhesin-free gingipain proteinase RgpA(NATURE PORTFOLIO, 2023-07-04)The Arg-specific gingipains of Porphyromonas gingivalis RgpA and RgpB have 97% identical sequences in their catalytic domains yet their propeptides are only 76% identical. RgpA isolates as a proteinase-adhesin complex (HRgpA) which hinders direct kinetic comparison of RgpAcat as a monomer with monomeric RgpB. We tested modifications of rgpA identifying a variant that enabled us to isolate histidine-tagged monomeric RgpA (rRgpAH). Kinetic comparisons between rRgpAH and RgpB used benzoyl-L-Arg-4-nitroanilide with and without cysteine and glycylglycine acceptor molecules. With no glycylglycine, values of Km, Vmax, kcat and kcat/Km for each enzyme were similar, but with glycylglycine Km decreased, Vmax increased and kcat increased ~ twofold for RgpB but ~ sixfold for rRgpAH. The kcat/Km for rRgpAH was unchanged whereas that of RgpB more than halved. Recombinant RgpA propeptide inhibited rRgpAH and RgpB with Ki 13 nM and 15 nM Ki respectively slightly more effectively than RgpB propeptide which inhibited rRgpAH and RgpB with Ki 22 nM and 29 nM respectively (p < 0.0001); a result that may be attributable to the divergent propeptide sequences. Overall, the data for rRgpAH reflected observations previously made by others using HRgpA, indicating rRgpAH fidelity and confirming the first production and isolation of functional affinity tagged RgpA.
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ItemPorphyromonas gingivalis laboratory strains and clinical isolates exhibit different distribution of cell surface and secreted gingipains(TAYLOR & FRANCIS LTD, 2021-01-01)Background: The cell-surface cysteine proteinases RgpA, RgpB (Arg-gingipain), and Kgp (Lys-gingipain) are major virulence factors of P. gingivalis, a keystone pathogen in the development of destructive periodontal disease. The gingipains function as proteinases and transpeptidases utilising small peptides such as glycylglycine as acceptor molecules. However, the characteristics of the gingipains from most P. gingivalis strains have not been determined. Methods: We determined the phenotypes of a panel of P. gingivalis laboratory strains and global clinical isolates with respect to growth on blood agar plus whole-cell and vesicle-free culture supernatant (VFSN) Arg- and Lys-specific proteinase activities. Results: The P. gingivalis isolates exhibited different growth characteristics and hydrolysis of haemoglobin in solid media. Whole-cell Arg-gingipain Vmax varied 5.8-fold and the whole cell Lys-gingipain Vmax varied 2.1-fold across the strains. Furthermore, the P. gingivalis strains showed more than 107-fold variance in soluble Arg-gingipain activity in VFSN and more than 371-fold variance in soluble Lys-gingipain activity in VFSN. Glycylglycine and cysteine stimulated Arg- and Lys-specific cleavage activities of all strains. The stimulation by cysteine was in addition to its redox effect consistent with both glycylglycine and cysteine promoting transpeptidation. Conclusion: The global P. gingivalis clinical isolates exhibit different Arg- and Lys‑gingipain activities with substantial variability in the level of soluble proteinases released into the environment.
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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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ItemA therapeutic Porphyromonas gingivalis gingipain vaccine induces neutralising IgG1 antibodies that protect against experimental periodontitis(SPRINGERNATURE, 2016-12-01)Porphyromonas gingivalis infected mice with an established P. gingivalis-specific inflammatory immune response were protected from developing alveolar bone resorption by therapeutic vaccination with a chimera (KAS2-A1) immunogen targeting the major virulence factors of the bacterium, the gingipain proteinases. Protection was characterised by an antigen-specific IgG1 isotype antibody and Th2 cell response. Adoptive transfer of KAS2-A1-specific IgG1 or IgG2 expressing B cells confirmed that IgG1-mediated protection. Furthermore, parenteral or intraoral administration of KAS2-A1-specific polyclonal antibodies protected against the development of P. gingivalis-induced bone resorption. The KAS2-A1-specific antibodies neutralised the gingipains by inhibiting: proteolytic activity, binding to host cells/proteins and co-aggregation with other periodontal bacteria. Combining key gingipain sequences into a chimera vaccine produced an effective therapeutic intervention that protected against P. gingivalis-induced periodontitis.
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ItemCasein Phosphopeptide-Amorphous Calcium Phosphate Nanocomplexes: A Structural Model(AMER CHEMICAL SOC, 2016-08-09)Tryptic digestion of the calcium-sensitive caseins yields casein phosphopeptides (CPP) that contain clusters of phosphorylated seryl residues. The CPP stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the CPP with calcium phosphate using a variety of nuclear magnetic resonance (NMR) techniques. Translational diffusion measurements indicated that the β-CN(1-25)-ACP nanocomplex has a hydrodynamic radius of 1.526 ± 0.044 nm at pH 6.0, which increases to 1.923 ± 0.082 nm at pH 9.0. (1)H NMR spectra were well resolved, and (3)JH(N)-H(α) measurements ranged from a low of 5.5 Hz to a high of 8.1 Hz. Total correlation spectroscopy and nuclear Overhauser effect spectroscopy spectra were acquired and sequentially assigned. Experiments described in this paper have allowed the development of a structural model of the β-CN(1-25)-amorphous calcium phosphate nanocomplex.
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ItemPorphyromonas gingivalis Uses Specific Domain Rearrangements and Allelic Exchange to Generate Diversity in Surface Virulence Factors(FRONTIERS MEDIA SA, 2017-01-26)Porphyromonas gingivalis is a keystone pathogen of chronic periodontitis. The virulence of P. gingivalis is reported to be strain related and there are currently a number of strain typing schemes based on variation in capsular polysaccharide, the major and minor fimbriae and adhesin domains of Lys-gingipain (Kgp), amongst other surface proteins. P. gingivalis can exchange chromosomal DNA between strains by natural competence and conjugation. The aim of this study was to determine the genetic variability of P. gingivalis strains sourced from international locations over a 25-year period and to determine if variability in surface virulence factors has a phylogenetic basis. Whole genome sequencing was performed on 13 strains and comparison made to 10 previously sequenced strains. A single nucleotide polymorphism-based phylogenetic analysis demonstrated a shallow tri-lobed phylogeny. There was a high level of reticulation in the phylogenetic network, demonstrating extensive horizontal gene transfer between the strains. Two highly conserved variants of the catalytic domain of the major virulence factor the Kgp proteinase (KgpcatI and KgpcatII) were found. There were three variants of the fourth Kgp C-terminal cleaved adhesin domain. Specific variants of the cell surface proteins FimA, FimCDE, MfaI, RagAB, Tpr, and PrtT were also identified. The occurrence of all these variants in the P. gingivalis strains formed a mosaic that was not related to the SNP-based phylogeny. In conclusion P. gingivalis uses domain rearrangements and genetic exchange to generate diversity in specific surface virulence factors.
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ItemPropeptide-Mediated Inhibition of Cognate Gingipain Proteinases(PUBLIC LIBRARY SCIENCE, 2013-06-10)Porphyromonas gingivalis is a major pathogen associated with chronic periodontitis. The organism's cell-surface cysteine proteinases, the Arg-specific proteinases (RgpA, RgpB) and the Lys-specific proteinase (Kgp), which are known as gingipains have been implicated as major virulence factors. All three gingipain precursors contain a propeptide of around 200 amino acids in length that is removed during maturation. The aim of this study was to characterize the inhibitory potential of the Kgp and RgpB propeptides against the mature cognate enzymes. Mature Kgp was obtained from P. gingivalis mutant ECR368, which produces a recombinant Kgp with an ABM1 motif deleted from the catalytic domain (rKgp) that enables the otherwise membrane bound enzyme to dissociate from adhesins and be released. Mature RgpB was obtained from P. gingivalis HG66. Recombinant propeptides of Kgp and RgpB were produced in Escherichia coli and purified using nickel-affinity chromatography. The Kgp and RgpB propeptides displayed non-competitive inhibition kinetics with K(i) values of 2.04 µM and 12 nM, respectively. Both propeptides exhibited selectivity towards their cognate proteinase. The specificity of both propeptides was demonstrated by their inability to inhibit caspase-3, a closely related cysteine protease, and papain that also has a relatively long propeptide. Both propeptides at 100 mg/L caused a 50% reduction of P. gingivalis growth in a protein-based medium. In summary, this study demonstrates that gingipain propeptides are capable of inhibiting their mature cognate proteinases.
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ItemBioinformatic investigation of the cost management strategies of five oral microbes(WILEY, 2015-04)Some amino acids are more energetically costly to synthesize de novo, therefore many microbes have evolved to regulate the metabolic expenditure of the cell and reduce the energy burden of extracellular unrecyclable proteins. Several oral bacterial species take up amino acids and peptides obtained from proteolysis of host proteins and hence do not rely only on de novo synthesis. The aim of this study was to investigate if five oral bacterial species implement cost management strategies to reduce the energy burden of extracellular unrecyclable proteins. Since the relative de novo amino acid synthesis costs are proportional to the masses of the amino acids, the energy costs of producing proteins were assessed by calculating the mean amino acid mass for each protein. For Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Prevotella intermedia and Streptococcus sanguinis, the outer membrane/extracellular proteins are made up of a much larger percentage of lower average mass amino acids whereas cytoplasmic proteins are made up of a larger proportion of higher average mass amino acid residues. These results are consistent with the five oral bacterial species employing energy-saving mechanisms in the production of extracellular unrecyclable proteins. Interestingly, the P. gingivalis and S. sanguinis genomes exhibited significantly lower predicted mean amino acid masses compared with those of the genomes of the other three species, suggesting that this may provide them with an energy advantage with respect to protein biosynthetic cost.
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ItemThe Interactions of CPP-ACP with Saliva(MDPI, 2016-06)The repair of early dental caries lesions has been demonstrated by the application of the remineralisation technology based on casein phosphopeptide-stabilised amorphous calcium phosphate complexes (CPP-ACP). These complexes consist of an amorphous calcium phosphate mineral phase stabilised and encapsulated by the self-assembly of milk-derived phosphopeptides. During topical application of CPP-ACP complexes in the oral cavity, the CPP encounters the enamel pellicle consisting of salivary proteins and peptides. However the interactions of the CPP with the enamel salivary pellicle are not known. The studies presented here reveal that the predominant peptides of CPP-ACP complexes do interact with specific salivary proteins and peptides of the enamel pellicle, and provide a mechanism by which the CPP-ACP complexes are localised at the tooth surface to promote remineralisation.
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ItemA bio-informatics study of the c25 cysteine protease family(Scientific Research Publishing, Inc., 2012)The oral pathogen Porphyromonas gingivalis is recognized as one of the major aetiological agents of chronic periodontitis. The gingipains, which are the principal virulence factors of P. gingivalis, are multi-domain proteins containing an N-terminal C25 cysteine protease domain. We have conducted a bio-informatics study of the C25 cysteine protease domains and have identified related domains in over two thousand proteins from 739 organisms in 35 distinct phyla. Proteins having significant similarity to the gingipain C25 cysteine protease domain are also found in Gram +ve bacteria, Archaea, algae, higher fungi, and a wide variety of Eukaryotic species.