Date on Master's Thesis/Doctoral Dissertation

8-2017

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Interdisciplinary and Graduate Studies

Degree Program

Interdisciplinary Studies (Individualized Degree), PhD

Committee Chair

Lamont, Richard

Committee Member

Demuth, Donald

Committee Member

Lawrenz, Matthew

Committee Member

Potempa, Jan

Committee Member

Scott, David

Author's Keywords

bacterial pili; fimbriae; fimbrial assembly; porphyromonas gingivalis; periodontitis

Abstract

Porphyromonas gingivalis, an obligate anaerobic bacterium associated with chronic periodontitis, utilizes various virulence factors to achieve pathogenicity, one of which is the Mfa1 fimbriae. As a surface structure comprising Mfa1 major subunit along with accessory fimbrial proteins Mfa2-5, the Mfa1 fimbriae has been shown to mediate the adherence of P. gingivalis to antecedent bacterial colonizers of the oral cavity to cause increased virulence. However, the spatial relationships amongst the individual subunits and their assembly mechanism have remained unclear. Through immuno-electron microscopy, Mfa1-4 were localized on the surface of P. gingivalis with Mfa1 localizing throughout the fimbriae and Mfa2 in the base. Mfa3 and Mfa4 were both localized in the distal portion of the Mfa1 fimbriae. ELISA-based binding experiments with recombinant Mfa proteins and whole-cell ELISA experiments with wild-type and isogenic individual mfa mutants showed intricate interactions amongst Mfa proteins that implicated Mfa3 as the adaptor protein interlinking other fimbrial subunits with the fimbrial assembly initiating within the periplasm. Binding assays also demonstrated that the inter-subunit interactions occur independently of the proteolytic processing known to take place on the surface for Mfa1, Mfa3, and Mfa4 by an arginine specific protease Rgp. However, immunoblotting of Mfa1 proteins corresponding to pre- or post-Rgp processed forms indicated that the polymerization is initiated upon the proteolytic processing on the surface and that both N- and C-terminal regions of post-Rgp processed Mfa1 protein are crucial for its polymerization. Mfa1 polymerization assay with rgpA/B mutant also confirmed that the pre-Rgp processed form is unable to polymerize. Furthermore, substitutions of alternating hydrophobic amino acid residues in the terminal regions of recombinant Mfa1 with charged residues yielded Mfa1 proteins that failed to polymerize, reminiscent of a distinct polymerization present in type I and P pili systems of E. coli termed donor-strand exchange (DSE). Collectively, the fimbrial subunits may initiate the assembly within the periplasm prior to the proteolytic processing on the surface with Mfa3 serving as an adaptor protein between Mfa1 and other accessory fimbrial proteins and that the polymerization of Mfa1 necessitates a DSE-like process with the terminal regions forming the binding interface between Mfa1 subunits.

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