Moraxella (Branhamella) catarrhalis, has over the last decade, emerged as an important pathogen in otitis media and sinusitis in children and lower respiratory tract infections in adults, particularly those with COPD. The outer membrane proteins (OMPs) of gram-negative bacteria are important virulence factors and targets for the human immune response. Our understanding of the pathogenesis of infection by M. catarrhalis is limited because information on structure and function of its OMPs is lacking. The outer membrane of M. catarrhalis contains 10-20 OMPs with molecular masses (Mr) ranging from 20 to 100 kilodaltons (kD). Immunoblot assays using rabbit polyclonal serum raised against whole organisms and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of outer membrane preparations have shown the presence of a high molecular weight band (HMW-OMP) on many strains (Mr of 350-720 kD). A HMW-OMP band was detected in purified outer membrane preparations of 10 of 11 strains using SDS-PAGE and immunoblot assays. Proteinase K digestion of whole organism lysates subjected to SDS-PAGE revealed that the HMW-OMP was no longer detectable on Coomassie and silver-stained gels. These data indicate that the HMW-OMP is a protein. Many patients with infections due to M. catarrhalis have antibody to this protein when their serum is tested in immunoblot assay. This observation indicates that the HMW-OMP is expressed during human infection and serves as a potentially important target of the human immune response to infection. The HMW-OMP of 2 strains have been purified, and polyclonal antisera and monoclonal antibodies (mAb) developed. The polyclonal sera and the mAbs tested in immunoblot assay are able to detect the HMW-OMP of their respective homologous and most heterologous strains tested. Immunofluorescence studies with both mAbs show that their epitopes are surface exposed. These data provide the rationale for further study of the antigenic and molecular characteristics of the HMW-OMP of M. catarrhalis which is the focus of this proposal. The specific goals of this proposal are: i) The subunit(s) of the HMW-OMP will be determined by subjecting the protein to agents that disrupt quaternary structure fully. The N- terminal amino acid sequence(s) of the protein subunit(s) will be determined. ii) The genes encoding for the protein subunit(s) will be cloned and sequenced. Restriction fragment length polymorphism analysis will be performed to determine the degree of conservation f the gene among strains. iii) Using monoclonal antibodies and antibodies immunopurified from normal human serum to the HMW-OMP, the protein will be studied as a potential target of functional antibody in bactericidal and opsonophagocytosis assays. iv) Antigenic domains on the HMW-OMP will be mapped and the region of the molecule exposed on the surface of the intact bacterium will be identified.
| Heater, S J; Carrano, M W; Rains, D et al. (2000) Interaction of oxo-bridged vanadium(III) phenanthroline and bipyridine dimers with DNA. Inorg Chem 39:3881-9 |
