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Title: The role of outer membrane proteins of porphyromonas gingivalis in host-pathogen interactions
Author: Naylor, Kathryn Lucy
ISNI:       0000 0004 5991 6662
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2016
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The keystone periodontal pathogen, P. gingivalis, is a Gram-negative oral anaerobe that is strongly implicated as the prime etiological agent in the initiation and progression of periodontal disease. P. gingivalis contains a plethora of virulence factors, including fimbriae, proteases, lipopolysaccharides and outer membrane proteins that contribute to the pathogenesis of the disease. The bacterium also displays the ability to interact with host cells through the adherence and invasion both in vitro and in vivo. In this thesis new light has been shed on the role of the heterotrimeric outer membrane protein A (OmpA). Through the creation of ompA mutants and recombinant complementation plasmids, alongside the use of standard antibiotic protection assays in an oral epithelial cell line, this research has demonstrated the importance of OmpA, specifically the OmpA2 subunit, in the invasion of the host and in the ability of the bacteria to form biofilms. Structural analysis of the protein identified extracellular loops, which when synthetic versions were applied to host cells, demonstrated successful interruption of wild-type P. gingivalis adherence and invasion of the host, indicating a direct interaction of OmpA2 with oral epithelial cells. In particular, this research demonstrates that OmpA2-loop 4 plays an important role in the interaction with the host through significantly increased binding to host cells when applied to fluorescent latex beads. This work also characterised the putative periplasmic chaperone protein OmpH, identifying potential proteins that act as clients to this chaperone, including OmpA. Through the creation of an ompH1H2 knock out mutant, enzymatic analysis of various virulence factors was assessed, demonstrating a loss of gingipain activity, whilst also identifying a loss of membrane stability through the observation of an increase in outer membrane vesicle formation. Evidence is also presented for the role of OmpH protein in chaperoning OmpA across the periplasm to the outer membrane and this work has opened up the potential for novel work identifying other clients of this chaperone system. Overall, the work in this thesis has demonstrated that the OmpA protein, specifically OmpA2, is directly involved in the interaction with host oral epithelial cells and in biofilm formation, specifically demonstrating the direct role of the extracellular surface loops in this interaction. This work also hypothesises a role for the OmpH protein in chaperoning outer membrane proteins, with the identification of potential clients, such as OmpA and the gingipains. Finally, this work has developed an improved mutagenesis technique for the creation of P. gingivalis mutants based on a modified natural competence method.
Supervisor: Stafford, Graham ; Murdoch, Craig ; Douglas, Ian Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available