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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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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ItemCharacterisation of innate T cells in response to oral bacterial infection( 2015)Chronic periodontitis is an inflammatory disease of the supporting tissues of teeth that is characterised by bone resorption and if left untreated can result in eventual tooth loss. The subgingival plaque bacteria Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia have been closely associated with chronic periodontitis. T cell immunity during periodontal infections has been well-documented, mainly involving the adaptive immunity which includes Th1 and Th2 responses. A small number of clinical studies have reported the presence of IL-17 and Th17-related cytokines in the gingival crevicular fluid and diseased tissues of periodontitis patients. However, the roles of innate T cells and Th17-related responses in disease progression is unclear. This study shows that NKT cells play a role in pro-inflammatory cytokine production, contributing to P. gingivalis-induced bone loss in the mouse periodontitis model. Inflammation and bone resorption was reduced in the absence of NKT cells or CD1d, the corresponding activating receptor for the TCR of NKT cells. As NKT cell responses were likely to be associated with glycolipid antigens, a major glycolipid of P. gingivalis was isolated, Pg-GL1, which was found to induce NKT cell and B cell activation. Results from knock-out mice studies suggested that NKT activation by Pg-GL1 occurs through a mixed requirement for TCR/CD1d-engagement and additional cytokines. Furthermore, Pg-GL1 was shown to induce the secretion of IL-1β, IL-6, IL-12(p40), IL-17, and G-CSF, cytokines that have been associated with inducing Th17-responses and, as contributing factors during periodontal inflammation and bone resorption. Similar to P. gingivalis, three major glycolipids (termed Tf-GL1, Tf-GL2, Tf-GL3) of T. forsythia were found to highly activate NKT cells and B cells. Alkaline treatment of T. forsythia glycolipids revealed that Tf-GL2 was alkali-resistant, while Tf-GL1 and Tf-GL3 were susceptible and their deacylated forms showed the presence of complex carbohydrates. Mass spectrometry analysis revealed a wide structural variation in the carbohydrate head-group and acyl chain lengths. Base-labile Tf-GL1 and Tf-GL3 were identified to be diacylgylcerolipids while base-resistant Tf-GL2 was proposed to be a glycosphingolipid. The structure of the third glycolipid, Tf-GL3, was predicted to be highly complex and thus may have contributed to its lower antigenicity compared with Tf-GL1 and Tf-GL2. When the stimulatory ability of T. forsythia glycolipids was further investigated, only Tf-GL2 was able to induce IL-17 production when cultured with purified NKT cells and BMDCs. As only Tf-GL2 was able to induce cytokine production, it was concluded that structural characteristics play an important role in antigen potency. When mice were orally infected with P. gingivalis, the CD27- γδ T cell sub-population in the maxillary epithelium was found to be activated after 14 days post-infection. Using pHrodo™ technology, it was shown that γδ T cells were able to phagocytose P. gingivalis, and this was confirmed using super resolution imaging. γδ T cells were then classified based on their ability to phagocytose bacteria and their expression of CD27, namely, “pHrodohi CD27-”, “pHrodomed/hi CD27+” and “pHrodolo CD27+” γδ T cell sub-populations. Naïve γδ T cells from thymus and spleen expressed a number of migratory markers and antigen presentation molecules, and the expression levels of these markers between CD27- and CD27+ γδ T cells were not significantly different. However, when γδ T cells were exposed to bacteria either through priming or in culture, the sub-populations of γδ T cells displayed differences in surface marker expression and cytokine secretion. Amongst the sub-populations, it was found that splenic γδ T cells that present a strong ability to phagocytose bacteria (pHrodohi or pHrodomed/hi) expressed high concentrations of cytokines and chemokines, which was most evidently observed when primed with heat-killed P. gingivalis. As the pHrodolo CD27+ γδ T cell sub-population expressed a high level of MHC molecules and were slower in phagocytosis, it was hypothesised that this sub-population is involved in antigen presentation, similar to dendritic cells. The results from this study provide a preliminary understanding of the functional roles of NKT and γδ T cells in response to oral bacteria and their bacterial products. As these innate T cells contribute to the induction of an inflammatory or non-inflammatory response, they are crucial in the determination of early responses during infection. As such, the expansion of this investigation may aid in the development of therapeutics to control inflammation and prevent the progression of chronic periodontitis.
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ItemCharacterisation of a novel regulatory protein, PgMntR from porphyromonas gingivalis( 2015)Porphyromonas gingivalis is a strictly anaerobic Gram-negative bacterium associated with the progression of chronic periodontitis. In the oral cavity, P. gingivalis is exposed to various oxidative stress conditions such as reactive oxygen species and unfavorable redox potentials before it can find a suitable environment to establish and proliferate. It has previously been shown that manganese is required for protection of P. gingivalis from oxidative stress generated by atmospheric oxygen and H2O2, and for intracellular survival of P. gingivalis in host cells. Thus manganese transport and its regulation are imperative to the survival of P. gingivalis. This laboratory has previously characterized a novel manganese transporter (FeoB2) found in P. gingivalis that utilizes GTP for energy. Encoded upstream of this transporter and within the same operon is the pgmntR gene. PgMntR is predicted to be a homologue of the manganese transport regulatory protein MntR, from the iron-dependent transcriptional regulator DtxR family. These proteins have domains involved in DNA binding, metal binding and dimerisation and some have an additional domain with structural homology to the E. coli FeoA, a small protein of unknown function. Interestingly, PgMntR has two FeoA domains indicating that PgMntR may have novel properties. To understand the role of PgMntR in P. gingivalis, recombinant PgMntR was expressed as a glutathione S-transferase or a 6xHis-tag fusion protein in E. coli and purified using affinity chromatography followed by ion-exchange and size exclusion chromatography. PgMntR formed a dimer under native conditions, in the presence or absence of manganese and under non-reducing or reducing conditions. All cysteine residues were free of disulphide bond formation indicating that dimerisation was due to non-covalent interactions. Titration of PgMntR with Mn2+ resulted in a linear fluorescence quenching up until three metal binding sites being fully occupied. This linear relationship between the fluorescence change and the Mn(II) binding suggested that all three Mn(II) occupied the metal binding sites at the same time. A dissociation constant of Kd ≤ 0.05 µM for Mn2+ binding to all the three sites was estimated from the titration curve. Binding three metal ions per monomer reveals that PgMntR is unique amongst the DtxR family. PgMntR bound two Fe(II) per monomer with distinct affinities, the lower binding affinity Site L had a dissociation constant KdL ≤ 0.05 µM, while the higher binding affinity Site H had a dissociation constant KdH << 0.05 µM. Although PgMntR was able to bind Mn(II) and Fe(II) with high affinity, it is unclear whether one or both of these metals activate PgMntR function in vivo. PgMntR binding to the promoter region of the manganese transporter FeoB2 was enhanced by Mn2+. However, high concentrations of ferrous ions induce the breakdown of the protein-DNA complex enabling transcription of the manganese transporter FeoB2. This different mode of action has not been found in any other MntR orthologues and is further evidence of the interplay between iron and manganese in P. gingivalis.