Method: Using knockout mutagenesis, individual ompA genes (we already have a double ompA1-2 deletion strain) and the signature set will be mutated and the effects on invasion and biofilm formation investigated using antibiotic protection assays and Crystal Violet staining respectively. For protein-protein interaction studies, binding of recombinant OmpA protein, purified using affinity chromatography, with human oral epithelial cells (OK-F6) is being investigated using fluorescence microscopy followed by cross-linking, affinity chromatography. Interacting partners will be identified by Mass Spectroscopy.
Results: A recombinant soluble form of OmpA has been successfully expressed and purified. Using fluorescent antibodies, we are examining interaction of OmpA with OK-F6 cells and developing methods to purify interacting partners using cross-linking. Knockout mutants for the individual ompA genes have been successfully created. The invasion and biofilm formation abilities of single mutants in comparison to the ompA1-2 deletion strain (5-fold reduction in invasion; 20-fold reduction in biofilm formation compared to wild-type) is being examined.
Conclusion: These results illustrate a role for OmpA in invasion and biofilm formation and highlight a role for individual genes in the ompA operon and others in the signature gene set in invasion.