Date on Master's Thesis/Doctoral Dissertation

12-2016

Document Type

Master's Thesis

Degree Name

M.S.

Department

Oral Biology

Degree Program

Oral Biology, MS

Committee Chair

Demuth, Donald R.

Committee Co-Chair (if applicable)

Steinbach-Rankins, Jill

Committee Member

Steinbach-Rankins, Jill

Committee Member

Palmer, Kenneth

Committee Member

Scott, David

Author's Keywords

BAR peptide; nanoparticles; multivalency; fusion proteins; porphyromonas gingivalis; biofilms

Abstract

Studies suggest that P. gingivalis functions as a keystone pathogen and interacts with primary colonizers in the supragingival biofilm such as S. gordonii. This interaction contributes to the initial colonization of the oral cavity by P. gingivalis and thus represents a potential target for therapeutic intervention. We have identified a peptide (BAR) derived from the streptococcal SspB protein that functions to inhibit P. gingivalis adherence to S. gordonii. In addition, we showed that nanoparticles (NPs) functionalized with BAR inhibit this interaction more potently than free soluble peptide, possibly by promoting interaction with P. gingivalis at higher valency than free peptide and increasing the avidity of the interaction. Two approaches were used to assess the valency of BAR- P. gingivalis interaction. First NPs were conjugated with various defined amounts of BAR. The resulting NPs were tested for inhibition of P. gingivalis adherence using a two-species biofilm model and the results were compared with inhibition by free peptide. Nanoparticle preparations were synthesized in the presence of increasing amounts of fluorescently labeled or unlabeled BAR. We found that peptide bound to nanoparticles increased in a dose dependent manner ranging from 1.20 µg BAR/mg of NPs to 5.87 µg BAR/mg of NPs. We tested them for inhibition in the biofilm assay. We observed dose-dependent efficacy based on the amount of BAR peptide on the nanoparticle surface. Valency of BAR peptide directly correlated to increased inhibition. Second, inhibition of adherence was also determined using BAR-antibody fusion proteins in dimer and tetramer form. Peptide-antibody fusions were produced using a plant based production platform and tested as above. We designed nucleic acid constructs that encoded BAR-antibody fusion proteins containing two or four molar equivalents of BAR. The fusion protein containing two equivalents of BAR was successfully expressed whereas the protein containing 4 BAR equivalents appeared to be toxic to cells expressing the protein. The fusion protein containing two BAR molecules showed a dose-dependent increase in the percent inhibition as the amount of the BAR peptide increased However, the Ab-BAR fusion peptide was not more potent than soluble BAR peptide. Increasing the valency of the BAR-P. gingivalis interaction may pave the way for development of more potent therapeutics that target the initial colonization of the oral cavity by P. gingivalis.