Melbourne Dental School - Research Publications
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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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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.
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ItemThe Porphyromonas gingivalis Ferric Uptake Regulator Orthologue Binds Hemin and Regulates Hemin-Responsive Biofilm Development(PUBLIC LIBRARY SCIENCE, 2014-11-06)Porphyromonas gingivalis is a Gram-negative pathogen associated with the biofilm-mediated disease chronic periodontitis. P. gingivalis biofilm formation is dependent on environmental heme for which P. gingivalis has an obligate requirement as it is unable to synthesize protoporphyrin IX de novo, hence P. gingivalis transports iron and heme liberated from the human host. Homeostasis of a variety of transition metal ions is often mediated in Gram-negative bacteria at the transcriptional level by members of the Ferric Uptake Regulator (Fur) superfamily. P. gingivalis has a single predicted Fur superfamily orthologue which we have designated Har (heme associated regulator). Recombinant Har formed dimers in the presence of Zn2+ and bound one hemin molecule per monomer with high affinity (Kd of 0.23 µM). The binding of hemin resulted in conformational changes of Zn(II)Har and residue 97Cys was involved in hemin binding as part of a predicted -97C-98P-99L- hemin binding motif. The expression of 35 genes was down-regulated and 9 up-regulated in a Har mutant (ECR455) relative to wild-type. Twenty six of the down-regulated genes were previously found to be up-regulated in P. gingivalis grown as a biofilm and 11 were up-regulated under hemin limitation. A truncated Zn(II)Har bound the promoter region of dnaA (PGN_0001), one of the up-regulated genes in the ECR455 mutant. This binding decreased as hemin concentration increased which was consistent with gene expression being regulated by hemin availability. ECR455 formed significantly less biofilm than the wild-type and unlike wild-type biofilm formation was independent of hemin availability. P. gingivalis possesses a hemin-binding Fur orthologue that regulates hemin-dependent biofilm formation.
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ItemPorphyromonas gingivalis and Treponema denticola Exhibit Metabolic Symbioses(PUBLIC LIBRARY SCIENCE, 2014-03)Porphyromonas gingivalis and Treponema denticola are strongly associated with chronic periodontitis. These bacteria have been co-localized in subgingival plaque and demonstrated to exhibit symbiosis in growth in vitro and synergistic virulence upon co-infection in animal models of disease. Here we show that during continuous co-culture a P. gingivalis:T. denticola cell ratio of 6∶1 was maintained with a respective increase of 54% and 30% in cell numbers when compared with mono-culture. Co-culture caused significant changes in global gene expression in both species with altered expression of 184 T. denticola and 134 P. gingivalis genes. P. gingivalis genes encoding a predicted thiamine biosynthesis pathway were up-regulated whilst genes involved in fatty acid biosynthesis were down-regulated. T. denticola genes encoding virulence factors including dentilisin and glycine catabolic pathways were significantly up-regulated during co-culture. Metabolic labeling using 13C-glycine showed that T. denticola rapidly metabolized this amino acid resulting in the production of acetate and lactate. P. gingivalis may be an important source of free glycine for T. denticola as mono-cultures of P. gingivalis and T. denticola were found to produce and consume free glycine, respectively; free glycine production by P. gingivalis was stimulated by T. denticola conditioned medium and glycine supplementation of T. denticola medium increased final cell density 1.7-fold. Collectively these data show P. gingivalis and T. denticola respond metabolically to the presence of each other with T. denticola displaying responses that help explain enhanced virulence of co-infections.