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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ItemComplementation in trans of Porphyromonas gingivalis Lipopolysaccharide Biosynthetic Mutants Demonstrates Lipopolysaccharide Exchange(American Society for Microbiology, 2021-04-21)Porphyromonas gingivalis, a bacterial pathogen contributing to human periodontitis, exports and anchors cargo proteins to its surface, enabling the production of black pigmentation using a type IX secretion system (T9SS) and conjugation to anionic lipopolysaccharide (A-LPS). To determine whether T9SS components need to be assembled in situ for correct secretion and A-LPS modification of cargo proteins, combinations of nonpigmented mutants lacking A-LPS or a T9SS component were mixed to investigate in trans complementation. Reacquisition of pigmentation occurred only between an A-LPS mutant and a T9SS mutant, which coincided with A-LPS modification of cargo proteins detected by Western blotting and coimmunoprecipitation/quantitative mass spectrometry. Complementation also occurred using an A-LPS mutant mixed with outer membrane vesicles (OMVs) or purified A-LPS. Fluorescence experiments demonstrated that OMVs can fuse with and transfer lipid to P. gingivalis, leading to the conclusion that complementation of T9SS function occurred through A-LPS transfer between cells. None of the two-strain crosses involving only the five T9SS OM component mutants produced black pigmentation, implying that the OM proteins cannot be transferred in a manner that restores function and surface pigmentation, and hence, a more ordered temporal in situ assembly of T9SS components may be required. Our results show that LPS can be transferred between cells or between cells and OMVs to complement deficiencies in LPS biosynthesis and hemin-related pigmentation to reveal a potentially new mechanism by which the oral microbial community is modulated to produce clinical consequences in the human host. IMPORTANCE: Porphyromonas gingivalis is a keystone pathogen contributing to periodontitis in humans, leading to tooth loss. The oral microbiota is essential in this pathogenic process and changes from predominantly Gram-positive (health) to predominantly Gram-negative (disease) species. P. gingivalis uses its type IX secretion system (T9SS) to secrete and conjugate virulence proteins to anionic lipopolysaccharide (A-LPS). This study investigated whether components of this secretion system could be complemented and found that it was possible for A-LPS biosynthetic mutants to be complemented in trans both by strains that had the A-LPS on the cell surface and by exogenous sources of A-LPS. This is the first known example of LPS exchange in a human bacterial pathogen which causes disease through complex microbiota-host interactions.
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ItemBreastmilk influences development and composition of the oral microbiome(TAYLOR & FRANCIS LTD, 2022-12-31)BACKGROUND: Human microbiomes assemble in an ordered, reproducible manner yet there is limited information about early colonisation and development of bacterial communities that constitute the oral microbiome. AIM: The aim of this study was to determine the effect of exposure to breastmilk on assembly of the infant oral microbiome during the first 20 months of life. METHODS: The oral microbiomes of 39 infants, 13 who were never breastfed and 26 who were breastfed for more than 10 months, from the longitudinal VicGeneration birth cohort study, were determined at four ages. In total, 519 bacterial taxa were identified and quantified in saliva by sequencing the V4 region of the bacterial 16S rRNA genes. RESULTS: There were significant differences in the development of the oral microbiomes of never breastfed and breastfed infants. Bacterial diversity was significantly higher in never breastfed infants at 2 months, due largely to an increased abundance of Veillonella and species from the Bacteroidetes phylum compared with breastfed infants. CONCLUSION: These differences likely reflect breastmilk playing a prebiotic role in selection of early-colonising, health-associated oral bacteria, such as the Streptococcus mitis group. The microbiomes of both groups became more heterogenous following the introduction of solid foods.
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ItemTemporal development of the infant oral microbiome(TAYLOR & FRANCIS LTD, 2022-11-02)The human oral microbiome is becoming recognized as playing roles in health and disease well beyond the oral cavity over the lifetime of the individual. The oral microbiome is hypothesized to result from specific colonization events followed by a reproducible and ordered development of complex bacterial communities. Colonization events, proliferation, succession and subsequent community development are dependent on a range of host and environmental factors, most notably the neonate diet. It is now becoming apparent that early childhood and prenatal influences can have long term effects on the development of human oral microbiomes. In this review, the temporal development of the infant human oral microbiome is examined, with the effects of prenatal and postnatal influences and the roles of specific bacteria. Dietary and environmental factors, especially breastfeeding, have a significant influence on the development of the infant oral microbiome. The evidence available regarding the roles and functions of early colonizing bacteria is still limited, and gaps in knowledge where further research is needed to elucidate these specific roles in relation to health and disease still exist.
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ItemType B CTD Proteins Secreted by the Type IX Secretion System Associate with PorP-like Proteins for Cell Surface Anchorage(MDPI, 2022-05)The Bacteroidetes type IX secretion system (T9SS) consists of at least 20 components that translocate proteins with type A or type B C-terminal domain (CTD) signals across the outer membrane (OM). While type A CTD proteins are anchored to the cell surface via covalent linkage to the anionic lipopolysaccharide, it is still unclear how type B CTD proteins are anchored to the cell surface. Moreover, very little is known about the PorE and PorP components of the T9SS. In this study, for the first time, we identified a complex comprising the OM β-barrel protein PorP, the OM-associated periplasmic protein PorE and the type B CTD protein PG1035. Cross-linking studies supported direct interactions between PorE-PorP and PorP-PG1035. Furthermore, we show that the formation of the PorE-PorP-PG1035 complex was independent of PorU and PorV. Additionally, the Flavobacterium johnsoniae PorP-like protein, SprF, was found bound to the major gliding motility adhesin, SprB, which is also a type B CTD protein. Together, these results suggest that type B-CTD proteins may anchor to the cell surface by binding to their respective PorP-like proteins.
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ItemProtein Interactome Analysis of the Type IX Secretion System Identifies PorW as the Missing Link between the PorK/N Ring Complex and the Sov Translocon(AMER SOC MICROBIOLOGY, 2022-02)The type IX secretion system (T9SS) transports cargo proteins through the outer membrane of Bacteroidetes and attaches them to the cell surface for functions including pathogenesis, gliding motility, and degradation of carbon sources. The T9SS comprises at least 20 different proteins and includes several modules: the trans-envelope core module comprising the PorL/M motor and the PorK/N ring, the outer membrane Sov translocon, and the cell attachment complex. However, the spatial organization of these modules is unknown. We have characterized the protein interactome of the Sov translocon in Porphyromonas gingivalis and identified Sov-PorV-PorA as well as Sov-PorW-PorN-PorK to be novel networks. PorW also interacted with PGN_1783 (PorD), which was required for maximum secretion efficiency. The identification of PorW as the missing link completes a continuous interaction network from the PorL/M motor to the Sov translocon, providing a pathway for cargo delivery and energy transduction from the inner membrane to the secretion pore. IMPORTANCE The T9SS is a newly identified protein secretion system of the Fibrobacteres-Chlorobi-Bacteroidetes superphylum used by pathogens associated with diseases of humans, fish, and poultry for the secretion and cell surface attachment of virulence factors. The T9SS comprises three known modules: (i) the trans-envelope core module comprising the PorL/M motor and the PorK/N ring, (ii) the outer membrane Sov translocon, and (iii) the cell surface attachment complex. The spatial organization and interaction of these modules have been a mystery. Here, we describe the protein interactome of the Sov translocon in the human pathogen Porphyromonas gingivalis and have identified PorW as the missing link which bridges PorN with Sov and so completes a continuous interaction network from the PorL/M motor to the Sov translocon, providing, for the first time, a pathway for cargo delivery and energy transduction from the inner membrane to the secretion pore.
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ItemMicrobiome profiles of non-responding and responding paired periodontitis sites within the same participants following non-surgical treatment(TAYLOR & FRANCIS LTD, 2022-12-31)AIM: Periodontitis is a site-specific, chronic disease treated by non-surgical debridement of subgingival plaque. We aimed to determine the microbiome of sites that did not respond to this treatment (NR) compared with paired good responding (GR) sites before and after treatment. MATERIALS AND METHODS: In a longitudinal cohort study, clinical parameters of disease and biological samples were taken prior to and 3 months after treatment. Twelve NR sites from six participants were paired with GR sites within the same participant. Subgingival plaque samples were subjected to bacterial community analysis using 16S rRNA gene sequencing. RESULTS: There were no significant differences in clinical parameters and microbial communities at baseline between GR and NR sites. Bacterial communities in deep pockets were dominated by a small number of species, notably Porphyromonas gingivalis and Treponema denticola. In NR sites three months after treatment there was no significant change in bacterial composition whilst there was a collapse in the abundance of pathobionts in GR sites. CONCLUSION: NR sites were not identifiable prior to treatment by clinical or microbiological parameters. Treatment failed to disrupt pathogenic bacterial community in NR sites. Targeted suppression of particular species should be considered to initiate community collapse and aid disease resolution.
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ItemTaxonomy of Oral Bacteria(ELSEVIER ACADEMIC PRESS INC, 2018-01-01)The oral cavity is a collection of diverse microenvironments, each inhabited by a community of microorganisms, the majority of which are bacteria and their phages. Given the appropriate conditions, some of these bacteria can cause destruction of the teeth or their supporting hard and soft tissues. For over 300 years microbiologists have been characterising these microbial communities, in both oral health and disease. In this chapter, we take the reader on a journey through time as we discuss the various methods that have been utilised in the characterisation of the bacteria calling the oral cavity home, and how the use of these methods has informed our understanding of oral bacterial communities and the diversity of their members.
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ItemThe prebiotic effect of CPP-ACP sugar-free chewing gum(ELSEVIER SCI LTD, 2019-12)OBJECTIVES: To determine if chewing gum containing casein phosphopeptide stabilised amorphous calcium phosphate (CPP-ACP) promoted an increase in the abundance of Streptococcus sanguinis and other species associated with dental health in supragingival plaque in a clinical study. MATERIALS AND METHODS: Nineteen participants were recruited for a three-leg cross-over, randomised, controlled clinical trial. Participants chewed a sugar-free gum with or without CPP-ACP six times daily for 20 min over two weeks. The study also involved no gum chewing (no gum) for the same two week period. Participants were randomly assigned to one of the test gums or no gum for each intervention period. Participants abstained from oral hygiene and had washout periods of two weeks between intervention periods. After each intervention period, supragingival plaque was collected and analysed for bacterial composition by sequencing the V4 variable region of the 16S rRNA gene. Data were analysed using a linear mixed model. RESULTS: The CPP-ACP gum intervention produced a significant (p < 0.01) increase in the proportions of S. sanguinis (112%), as well as the commensal species Rothia dentocariosa (127%), Corynebacterium durum (80%) and Streptococcus mitis (55%) when compared with the no gum intervention. All the species that were promoted by the CPP-ACP gum are known to possess one or both of the alkali-producing enzymes arginine deiminase and nitrate reductase. CONCLUSION: This clinical study demonstrated that chewing a sugar-free gum containing CPP-ACP promoted prebiosis by significantly increasing the proportion of S. sanguinis and other health-associated bacterial species in supragingival plaque. CLINICAL SIGNIFICANCE: Regular chewing of CPP-ACP sugar-free gum increases the proportions of health-associated commensal species in supragingival plaque to promote prebiosis and oral homeostasis.
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ItemNo Preview AvailableIn situ structure and organisation of the type IX secretion system( 2020-05-14)Abstract The Bacteroidetes type IX secretion system (T9SS) consists of at least 19 components that translocate proteins with a type A or type B C-terminal domain (CTD) signal across the outer membrane. The overall organisation and architecture of this system including how the secretion pore (Sov) interacts with the other components is unknown. We used cryo-electron tomography to obtain the first images of the T9SS including PorK/N rings inside intact Porphyromonas gingivalis cells. Using proteomics, we identified a novel complex between Sov, PorV and PorA and showed that Sov interacts with the PorK/N rings via PorW and a new component PGN_1783. A separate complex comprising the outer membrane β-barrel protein PorP, PorE, and the type B CTD protein PG1035 was also identified. Similarly, the Flavobacterium johnsoniae PorP-like protein, SprF was found bound to the major gliding motility adhesin, SprB. Based on these data, we propose cell surface anchorage for type B CTD proteins to PorP-like proteins and a unique model where the PorK/N rings function as an outer membrane barrier to maintain the close proximity of the translocon to the shuttle and attachment complexes inside the rings, ensuring the harmonized secretion and cell surface attachment of the T9SS substrates.