The Role of the Outer Membrane Proteins of P. gingivalis in Host-Pathogen Interactions

Objectives: Porphyromonas gingivalis is considered a keystone pathogen in periodontal disease. As part of its pathogenic features its ability to interact with host epithelial cells is central. During the invasion process, surface associated proteins, e.g. fimbriae, are crucial. Work in Sheffield identified a signature set of differentially regulated genes identified in an invasive subtype of the natural P. gingivalis population. These included the outer membrane protein encoding ompA and associated ompH genes. The aim of this study is to investigate the role of ompA and other signature set genes in interaction with oral epithelial cells and biofilm formation.
Methods: Using knockout mutagenesis, strains lacking the ompA1-2 operon and individual ompA genes alongside an ompH1-2 mutant were created. The effects on invasion and biofilm formation were investigated using antibiotic protection assays and biofilm staining respectively. For protein-protein interaction studies, binding of purified recombinant truncated OmpA protein and biotinylated OmpA peptides with oral cells is being investigated using fluorescence microscopy, cross-linking, and affinity chromatography to isolate interacting partners that will be identified by Mass Spectroscopy.
Results: ompA1-2 and ompA2 gene knockouts display reduced attachment and invasion of epithelial cells, while ompA1 does not. Biofilm formation by the mutants mirrors these data with a 20-fold reduction in the ompA1-2 mutant and two-fold reduction for ompA2. Meanwhile, a ∆ompH mutant displays altered cellular protein profile (SDS-PAGE) and is being investigated similarly. To investigate direct OmpA-human interactions we are using fluorescent antibodies with a recombinant soluble form of OmpA2 and biotinylated predicted OmpA2 surface loop peptides. In addition, these peptides reduce interaction of P. gingivalis with oral cells in vitro.
Conclusions: These results illustrate a role for OmpA2 and its predicted surface loops in cellular interactions and biofilm formation and highlight a role for ompH in assembly of OmpA and other surface proteins.