Melbourne Dental School - Theses
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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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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.