Melbourne Dental School - Theses
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ItemCharacterization of the Porphyromonas gingivalis protein PG1881 and its roles in outer membrane vesicle biogenesis and biofilm formation( 2016)Porphyromonas gingivalis is a non-motile, Gram-negative coccobacillus, oral anaerobe that is implicated in chronic periodontitis, a polymicrobial inflammatory disease resulting in the destruction of the supporting tissues of the teeth. P. gingivalis OMVs are an important vehicle for secretion of virulence factors, nutrient acquisition, biofilm development and pathogenesis. PG1881 was first identified to be preferentially sorted and enriched to the OMV membrane, and thus became the focus of this study. The purpose of this thesis was to characterize PG1881 protein and its potential roles in P. gingivalis virulence particularly in OMV biogenesis and biofilm formation. This study determined that pg1881 was transcribed as part of a four gene operon with pg1878-pg1880 in P. gingivalis strain W50. PG1881 was bioinformatically identified as a Type V pilin capable of forming filament-like structures and is exclusively conserved in limited families of the Bacteroidales order. PG1881 shares a similar overall structure to FimA and Mfa1 and possesses the essential conserved motifs in the N- and C-terminal regions. Similar to FimA and Mfa1, PG1881 undergoes a step-wise maturation process including maturation by proteolytic cleavage by the gingipains. PG1881 polymerisation is mediated by non-covalent interactions likely to be utilized via a donor stand mechanism. Phenotypic characterization of a P. gingivalis W50 mutant strain, ΔPG1881 lacking PG1881 revealed no significant difference in ΔPG1881 growth, whole cell morphology, SDS sensitivity, autoaggregation activity and cellular arginine-specific proteinase activity as compared to the parent strain W50. Although proteomic analyses of ΔPG1881 whole cells revealed minimal altered protein expression, ΔPG1881 displayed a significant decrease in OMV production as compared to W50. ΔPG1881 OMVs showed a significant decrease in the abundance of proteinases (including RgpA), consistent with the significant reduction in ΔPG1881 OMV arginine-specific proteinase activity in comparison to W50 OMVs. ΔPG1881 OMVs were also more enriched in TonB-dependent receptors. ΔPG1881 OMVs contained greater amount of nucleic acids on the surface and in the lumen than W50 OMVs. These results suggest that PG1881 may play a role in the vesiculation mechanism(s) of P. gingivalis given that OMVs originate from the bulging of bacterial OM, thus substantiating the correlation between pilins and vesiculation. PG1881 protein expression was increased in acidic pH conditions suggesting a role of PG1881 in P. gingivalis adaptability in response to pH changes. Using a flow cell model, PG1881 was shown to play a suppressive regulatory role in mono-species biofilm formation. PG1881 expression was also crucial for mutualistic biofilm formation with T. denticola as determined using a static biofilm model. These data suggest that PG1881 may play a role in response to environmental cues and for polymicrobial interaction with other oral bacteria such as T. denticola. Collectively, this study has identified a novel Type V pilin which has been designated as filament-forming protein 1, Ffp1 that affects P. gingivalis vesiculation and biofilm formation and extends the current knowledge concerning P. gingivalis pili. Further investigation may lead to greater insights in host-microbe interactions and the pathogenicity of P. gingvalis in periodontitis disease progression.
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ItemInvestigation of the role of Treponema denticola motility and uncharacterized protein TDE0659 in synergistic biofilm formation with Porphyromonas gingivalis( 2018)Chronic periodontitis has a polymicrobial biofilm etiology and interactions between key oral bacterial species such as Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia contribute to disease progression. P. gingivalis and T. denticola both have a set of virulence factors that are believed to contribute to the initiation and development of disease. It has been previously shown that P. gingivalis and T. denticola 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, is likely to be involved in synergistic biofilm development between P. gingivalis and T. denticola. In order to investigate this, an optimized protocol for the transformation of T. denticola was developed and used to produce a number of T. denticola mutants targeting the motility machinery. The resulting mutants lacked periplasmic flagella (∆flgE) or possessed periplasmic flagella that were either non-functional (∆motA and ∆motB) or non-regulatable (∆cheY). ∆cheY contained a large genomic excision and was thus omitted from the study, however analyses of ∆flgE, ∆motA and ∆motB showed that they were impaired in motility and growth. Quantitative proteomic analyses of mutant strains showed that the inactivation of these motility-associated genes, especially motA and motB, have far reaching effects beyond motility. The inactivation of motA and motB activated a cellular stress response in the mutants and directly or indirectly impacted the growth of the mutants through the change in abundance of a number of proteins. T. denticola motility mutant and WT strains were grown as mono- and dual-species biofilms with P. gingivalis. Results showed that T. denticola motility and/or spiral morphology are required for monospecies biofilm formation and T. denticola periplasmic flagella are essential for synergistic biofilm formation with P. gingivalis. Zones of clearing were observed between T. denticola sibling colonies grown close to one another on agar, similar to the sibling killing phenomenon observed in Paenibacillus dendritiformis. Given that a sibling killing phenomenon could have a considerable effect on biofilm formation, this phenomenon was investigated further in T. denticola. Although T. denticola was found to possess a homologue (TDE0659) of the dendritiformis sibling bacteriocin (DfsB) produced by P. dendritiformis, the protein did not undertake the same function in T. denticola. Construction and analysis of a T. denticola mutant strain lacking TDE0659 showed that the loss of this gene product from T. denticola did not prevent adjacent sibling colonies from forming zones of clearing on agar, further the mutant was able to form monospecies biofilms similar to WT. However, the lack of TDE0659 prevented the incorporation of P. gingivalis in a dual-species static biofilm with T. denticola, indicating that TDE0659 is essential for promoting synergistic biofilm formation with P. gingivalis. Together, the findings from this study will further our understanding about how P. gingivalis and T. denticola interact with one another and proliferate during disease.
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ItemBiochemical characterisation of outer membrane vesicles from periodontal pathogens and their role in innate immunity( 2017)This project characterises and assesses the immunological effects of outer membrane vesicles (OMVs) produced by major periodontal pathogens Porphromonas gingivalis, Treponema denticola and Tannerella forsythia. An OMV purification and enumeration protocol was optimised and used to determine the composition, general properties and major immunological stimulants associated with periodontal OMVs. This study includes an exploration into the immunological differences between multidrug-resistant and antibiotic-susceptible ESKAPE pathogens to demonstrate the advantages of the purification protocol above. Finally, the immunological effects of OMVs on innate immunity were explored by challenging oral host cells (epithelial/fibroblast/endothelial) and early innate immune responders (monocytes/macrophages) and evaluating their responses.
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ItemCharacterisation of the function of PG1058 in Porphyromonas gingivalis( 2016)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 post-translational modification with anionic lipopolysaccharide (A-LPS) and are attached to the OM. Comparative analyses of several Bacteroidetes genomes identified PG1058 as a candidate for a novel component of the T9SS. Inactivation of pg1058 in P. gingivalis resulted in slowed growth, loss of colonial pigmentation and surface-associated gingipain activity, a phenotype common to T9SS mutants. This study revealed normal cell architecture and no difference in OM solute permeability between the wild-type and pg1058- mutant. Thus the mutant phenotype was unlikely due to structural effects on cell division or perturbation of OM integrity causing aberrant assembly and function of the T9SS. T9SS substrates accumulated within the pg1058- mutant periplasm indicated perturbed T9SS function and the Kgp gingipain was shown to be absent from the cell surface, whilst A-LPS was present. This indicated that PG1058 is crucial for export of T9SS substrates but not for the export and surface-association of A-LPS. PG1058 was localised as an OM-associated periplasmic protein. Several proteins crucial to the T9SS and modification processes have been identified in the OM including PorT, PorV and PorU the CTD peptidase, that is itself a substrate of the T9SS. Increased abundance of PorT and PorV was seen in the pg1058- mutant, along with several other T9SS components. PorU accumulated in the periplasm with other T9SS substrates. The localisation and abundance of PG1058 in porV-, porT- and porU- mutants was not grossly altered. Bioinformatic analyses revealed that numerous Bacteroidetes possess multiple PG1058 homologues which correlated with multiple homologues of PorP, a known OM-associated T9SS component. In F. johnsoniae the PorP and PG1058 homologues were found adjacent in the genome suggesting a potential functional relationship between these proteins, with a role in substrate recognition and sorting being proposed. Several T9SS substrates are involved in host cell interactions and dysregulation of the host immune response. Susceptibility to macrophage phagocytosis, ability to bind to oral epithelial cells, ability to haemagglutinate red blood cells and ability to co-aggregate with the oral pathogen Treponema denticola were reduced but not abolished in the pg1058- and porV- T9SS mutants. Combined with a largely equivalent pro-inflammatory cytokine response, this suggests that P. gingivalis possesses surface-associated molecules in addition to the T9SS substrates which can influence human host interactions and thereby the virulence of P. gingivalis. Subtle differences in the response to the pg1058- and porV- mutants suggest that there are differences between these strains which warrant further investigation. This study confirms a role for PG1058 in the T9SS. Further investigation may indicate therapeutic targets within the T9SS to abate P. gingivalis virulence and periodontitis disease progression.
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ItemCommunity analyses of periodontal pathogens grown as a polymicrobial biofilm( 2012)Porphyromonas gingivalis, Treponema denticola and Tannerella have been implicated as the major aetiological agents in the clinical presentation of chronic periodontitis. These species have been proposed to be closely associated in the subgingival plaque polymicrobial biofilm (PBF). An investigation on how they interact when grown together as a PBF as compared to growth as monospecies planktonic culture was carried out. A PBF of these three species suitable for quantitative proteomic analysis was generated in a model biofilm fermentor whose design mimicked the conditions of in vivo subgingival plaque development in deep periodontal pockets. For comparison planktonic cultures of P. gingivalis and T. denticola were grown separately in continuous culture. For quantitative proteomic comparison between the two growth states, whole cell lysates were subjected to SDS-PAGE, followed by proteolytic H216O/ H218O labelling. The regulations of selected quantitated proteins were then verified by Western blotting. Two hundred and eighty-two P. gingivalis proteins and 313 T. denticola proteins were quantitated by liquid chromatography matrix-assisted laser desorption/ionisation time-of-flight tandem mass spectrometry (LC-MALDI TOF/TOF MS). The results suggest a change of strategy in iron acquisition by P. gingivalis due to the large increases in the abundance of HusA and HusB in the PBF while HmuY and other iron/haem transport systems decreased. Alteration of molecular stoichiometry of flagella subunits indicates T. denticola undergoing morphological and functional adaptations when grown in the PBF state. Significant changes in the abundance of peptidases and enzymes involved in glutamate and glycine catabolism suggest syntrophy. These findings indicate a close association between P. gingivalis and T. denticola that may play a role in disease pathogenesis when they are grown together as a PBF. The development and architecture of the PBF of the three bacterial species grown in flow cells was studied by conventional and low-vacuum scanning electron microscopy (SEM, LVSEM), as well as fluorescent in-situ hybridisation-confocal laser scanning microscopy (FISH-CLSM). The microscopy analyses revealed heterogeneous PBF architecture with spatially organised microcolonies and water channels. The temporospatial organisation of the three species during the PBF formation and development led to the proposal of a PBF development model where T. denticola is the primary colonizer, on which P. gingivalis attached and grew as large microcolonies intercalated with T. denticola. T. forsythia was largely excluded from the PBF, indicating antagonist interactions. Finally, MALDI-TOF MS reference spectra of the pure cultures of P. gingivalis, T. denticola and T. forsythia reference strains were established. An optimised protocol to yield mass spectra of acceptable quality involved using washed whole bacterial cells mixed with -cyano-4-hydroxycinnamic acid (CHCA) matrix in 2.5% trifluoroacetic acid (TFA)/50% acetonitrile (ACN). The acquired reference spectra showed a unique pattern for each reference strain with peaks within a mass range of m/z 2000 -13, 000. ClinProTools identified peaks that may be considered as potential biomarkers for each reference strain. The established protocol and MALDI-TOF reference mass spectra may serve as an application for the rapid identification and classification of oral microorganisms.