CLONING, SEQUENCING, CHARACTERIZATION AND IMPLICATIONS FOR VACCINE DESIGN OF THE NOVEL DIHYDROLIPOYL ACETYLTRANSFERASE OF NEISSERIA-MENINGITIDIS

A lambda Zap-II expression library of Neisseria meningitidis was scree ned with a rabbit polyclonal antiserum (R-70) raised against c. 70-kDa proteins purified from outer membrane vesicles by elution from prepar ative SDS-polyacrylamide gels, Selected clones were isolated, further purified, and their recombinant pBluescript SKII plasmids were excised , The cloned DNA insert was sequenced from positive clones and analyse d, Four open reading frames (ORFs) were identified, three of which sho wed a high degree of homology with the pyruvate dehydrogenase (E1p), d ihydrolipoyl acetyltransferase (E2p) and dihydrolipoyl dehydrogenase ( E3) components of the pyruvate dehydrogenase complex (PDHC) of a numbe r of prokaryotic and eukaryotic species, Sequence analysis indicated t hat the meningococcal E2p (Men-E2p) contains two N-terminal lipoyl dom ains, an E1/E3 binding domain and a catalytic domain, The domains are separated by hinge regions rich in alanine, proline and charged residu es, Another lipoyl domain with high sequence similarity to the Men-E2p lipoyl domain was found at the N-terminal of the E3 component. A furt her ORF, coding for a 16.5-kDa protein, was found between the ORFs enc oding the E2p and E3 components, The identity and functional character istics of the expressed and purified heterologous Men-E2p were confirm ed as dihydrolipoyl acetyltransferase by immunological and biochemical assays, N-terminal amino-acid analysis confirmed the sequence of the DNA-derived mature protein, Purified Men-E2p reacted with monospecific antisera raised against the whole E2p molecule and against the lipoyl domain of the Azotobacter vinelandii E2p, Conversely, rabbit antiseru m raised against Men-E2p reacted with protein extracts of A, vinelandi i, Escherichia coli and N. gonorrhoeae and with the lipoyl and catalyt ic domains of E2p obtained by limited proteolysis, In contrast, the or iginal R-70 antiserum reacted almost exclusively with the lipoyl domai n, indicating the strong immunogenicity of this domain, Antibodies to Men-E2p were detected in patients and animals (rabbits and mice) infec ted with homologous or heterologous meningococci or other neisserial s pecies, These results have important implications for the understandin g of PDHC and the design of future outer membrane vesicle-based vaccin es.