Molecular characterization of outer membrane protein E of Moraxella (Branhamella) catarrhalis.

摘要

Outer membrane protein E is a 50 kDa major protein of Moraxella (Branhamella) catarrhalis. The gene encoding OMP E was cloned and the nucleotide sequence was determined. An open reading frame of 1,377 bp encoding a protein of 460 amino acids residues was identified The molecular mass of OMP E deduced from the amino acid sequence was 47.03 kDa which correlates well with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. OMP E is synthesized as a precursor possessing a signal peptide of 25 amino acids cleaved by signal peptidase 1 during export to the outer membrane. The transcription initiation site was identified to be 78 nucleotides upstream of the ATG start codon. The predicted amino acids sequence was subjected to hydrophobicity analysis. This analysis indicates that OMP E, like other known porins, does not have long stretches of hydrophobic amino acids The hydropathy index of OMP E was calculated to be {dollar}-{dollar}0.69 indicating an overall hydrophilic composition. A homology search revealed borderline homology to FadL of E. coli (25.5% identity) and some porin proteins (20-25% identity). Restriction fragment length polymorphism analysis of the OMP E gene of 19 different strains of M. catarrhalis from diverse geographical and clinical sources, showed that the OMP E gene was highly conserved. Polyclonal and four monoclonal antibodies (MAbs) were generated to OMP E to study its antigenic structure. These antibodies recognized epitopes present in all strains tested. All MAbs were specific for M. catarrhalis, in that they did not react with any of eight gram-negative bacteria tested. Two MAbs, 1B3 and 9G10, recognized epitopes that are present on the surface of the intact bacterium. MAbs 1C11 and 7C10 recognized epitopes that are buried within the membrane. Proteinase K digestion of the whole bacterial cells showed that MAbs 1B3 and 9G10 recognized surface exposed epitopes located in the 17-kDa region of the amino terminus of OMP E molecule. OMP E was purified to homogeneity. The native structural size of OMP E was determined to be a trimer. These studies have demonstrated that OMP E has surface-exposed epitopes which are highly conserved among M. catarrhalis strains. Therefore OMP E is an excellent candidate as a vaccine antigen.