Functional analysis of the gingipains from Porphyromonas gingivalis and their role in periodontal disease

Periodontal disease is a major health burden with a huge economic cost. Every culture in the world exhibits signs of some form of the disease and periodontal diseases are a primary cause of tooth loss in man. Epidemiological studies have revealed that periodontal disease is a risk factor for other chronic diseases, including cardiovascular diseases, diabetes, autoimmune diseases, kidney disease, Alzheimer's disease and certain cancers, which suggests that periodontal disease can have a widespread impact on health in general. Periodontal disease is a complex, episodic disease characterized by inflammation of the gingiva and the supporting structures of the periodontium caused by the interaction of periodontopathogenic bacteria and host cells, which ultimately leads to the destruction of host tissues and the supporting structures of teeth. Among the various microbial species associated with the development of periodontal disease, Porphyromonas gingiva lis has been reported to be one of the most important causative agents. P. gingiva lis expresses a number of virulence factors, including cysteine proteases called the gingipains. The bacterium produces two arginine-specific cysteine proteases, HRgpA and RgpB, and one lysinespecific cysteine protease, Kgp. HRgpA and Kgp, respectively, are expressed as high molecular weight forms that contain both catalytic and adhesin subunits. The gingipains are key virulence factors of P. gingiva lis and contribute to the pathogenicity of P. gingiva/is through several mechanisms. The broad aim of this study was to examine these pathological mechanisms in greater molecular detail. The first aim was to investigate the ability of the gingipains to elicit pro-inflammatory cytokine production in activated macrophages, a pivotal immune cell that shapes local and systemic immune responses. The responses of differentiated macrophage-like cells to the gingipains of P. gingiva lis were examined and it was found that there was a marked upregulation of expression ofIL-l~ and GM-CSF after 24 hrs and a sharp increase in IL-l~ and GM-CSF production after 36 hrs, but that this expression was induced independently of the proteolytic activity of the gingipains. While, both the molecular target of the HA domains of the gingipains on the surface of macrophages and the signal transduction pathways within these cells remain to be identified, it seems clear that antagonizing these interactions may provide two potential avenues for future research that could prevent pro-inflammatory responses in tissue macrophages, which might mediate much of the unwanted chronic pro-inflammatory responses found in oral tissues following infection with P. gingiva lis. The second aim of this study was to elucidate the protease specificity of the gingipains using substrate phage display and kinetic analyses, to provide a more detailed understanding of the mechanisms through which the gingipains interact with substrates to degrade them, information which is important for the development of effective gingipain inhibitors. Overall, the substrate phage display results revealed that aside from the crucial S, position, for HRgpA the S2' and S3' subsites played the greatest role in determining HRgpA substrate specificity. In contrast, for RgpB and Kgp the S/ subsite, and the Sl' subsite, respectively, played the greatest role in determining RgpB and Kgp substrate specificity, respectively. Kinetic analysis, utilized to verify the relative importance of several amino acids at specific positions in the peptide sequence based on the substrate specificity profiles, confirmed that the S l' and S3 HRgpA subsites had a strong preferences for the predicted amino acids (glycine and valine, respectively). Furthermore, kinetic analysis verified that the Sl' and Ss RgpB subsites had a clear preference for the preferred amino acids, lysine and serine, respectively, and confirmed that the SI' and S2 subsites ofKgp appeared to have a strong preference for the predicted amino acids, glycine and isoleucine, respectively. Ultimately, excellent substrate sequences were identified for each of the gingipains. These sequences will provide an exceptional basis for the development of selective protein inhibitors of the gingipains. The final aim was to identify peptides that bind to the HA domains which could aid the development of antagonists that could be used for therapeutic intervention in chronic periodontal disease. Probing three random phage display libraries with HRgpA or Kgp resulted in the identification of 161 unique phage-displayed peptides that most likely bound to the C-terminal region of the high molecular weight gingipains. Overall, the only distinct consensus sequence (HxxPRGw) that was evident was for the HRgpA-affinity selected phage clones displaying disulphide-constrained heptapeptides. However, after six rounds of panning, seven of the dodecapeptide sequences panned on HRgpA and three dodecapeptides panned on Kgp appeared to contain an emerging, conserved HxRGH motif Interestingly, both the HxxPRGw and the HxRGH motifs shared two residues (RG) in common with the RGD motif common to integrin binding sites. Subsequently, it was determined that a number of the phage-displayed peptides retained their binding affinities for their target antigen after transformation into soluble, synthetic peptides. The results also suggested that structure within the synthetic, soluble peptides was also vitally important for these soluble peptides to bind to the HA domains. While future research needs to be carried out on the phage-displayed peptide sequences and their cognate synthetic peptides to develop these peptides for clinical applications, the peptide sequences identified in this study have the potential to be useful biochemical reagents, diagnostic tools or used in the development of therapeutics to aid in the treatment of periodontal disease. Collectively, work presented in this thesis has provided important insights into major virulence factors of P. gingiva lis, the gingipains, and has paved the way for the development of gingipain-directed therapeutics and inhibitors which may help to alleviate periodontal disease in the future.