Melbourne Dental School - Theses

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Subject: Porphyromonas gingivalis × Author: HEATH, JACQUELINE ×

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    Characterisation of the function of PG1058 in Porphyromonas gingivalis
    HEATH, JACQUELINE ( 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.