Characterization of the gene encoding Omp85, a novel outer membrane protein of Neisseria gonorrhoeae and introduction of genes into Neisseria gonorrhoeae.

摘要

A survey of gonococcal outer membrane proteins using an expression library screened with anti-outer membrane antibody identified sixteen proteins. Among them was an 85,000 Dalton (Omp85) protein that was homologous to Haemophilus influenzae protective surface protein D15 and the protective antigen Oma87 of Pasteurella multocida. Analogous proteins were present in Brucella abortus (Omp1), Helicobacter pylori, and some strains of Escherichia coli. These data suggested Omp85 was a member of a family of highly conserved proteins which may be important in establishing and/or maintaining mucosal infections. This protein was constitutively and universally expressed in all gonococcal strains tested. The genes encoding the Omp85 protein from N. gonorrhoeae and Neisseria meningitidis were cloned and sequenced. Both the gonococcal and meningococcal proteins were found to possess a typical leader sequence and terminal Phe residue, characteristic of outer membrane proteins. The gonococcal protein was 792 amino acids in length while the meningococcal protein was 797 amino acids in length. Insertional inactivation of the gene in N. gonorrhoeae resulted in a merodiploid state, suggesting the essential nature of the gene.; Since inactivation of omp85 appeared not to be possible, an alternative approach to study function would be to express Omp85 in a closely related species which naturally lacks Omp85, such as Branhamella (Moraxella) catarrhalis. Since very little is known about the transformation parameters or the genetics of this species, a pilot procedure using N. gonorrhoeae was developed. The 16S rDNA locus was used as the target sequence for recombination with a plasmid construct containing the gonococcal promoter aniA and the bacterial luciferase reporter gene luxAB, inserted within a cloned portion of gonococcal 16S sequence. This construct successfully recombined into the gonococcal 16S rDNA locus. The transformant, 1400.1, emitted light in the presence of the substrate n-decyl aldehyde. An integration vector for further manipulations of this kind in N. gonorrhoeae was constructed.