Role of outer membrane proteins in serum resistance of A. actinomycetemcomitans and A. aphrophilus

The aim of this project was to study how outer membrane proteins contribute to serum resistance in A. actinomycetemcomitans and A. aphrophilus. Genes encoding Omp100 (A. actinomycetemcomitans) and OmpA (A. actinomycetemcomitans and A. aphrophilus) were knocked out through homologous recombination. Serum resistance was tested by incubating the bacterial strains in normal human serum (NHS; 50%) for 2h at 37°C. As control heat-inactivated (56°C) NHS was used. LC-MS/MS analysis was used to identify proteins in selected bands from SDS-PAGE. Whereas Omp100 appeared not to contribute significantly to serum resistance, we observed that the ompA mutants of both species had a markedly reduced capability to survive in 50% NHS. In presence of Mg-EGTA the ompA mutants survived clearly better in NHS suggesting that OmpA may act through the classical pathway of the complement system. Interestingly, when incubating A. actinomycetemcomitans ompA mutants in NHS, derivatives were obtained that had regained serum resistance. LC-MS/MS analysis revealed that these strains produced high levels of an OmpA-like protein (76% amino acid identity). Further experiments to investigate how OmpA and the OmpA-like protein may interact with complement system are being conducted.