Cloning, sequencing, characterisation and implications for vaccine design of the novel dihydrolipoyl acetyltransferase of Neisseria meningitidis.
A lambdaZap-II expression library of Neisseria meningitidis was screened with a rabbit polyclonal antiserum (R-70) raised against c. 70-kDa proteins purified from outer membrane vesicles by elution from preparative 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 analysed. Four open reading frames (ORFs) were identified, three of which showed a high degree of homology with the pyruvate dehydrogenase (E1p), dihydrolipoyl acetyltransferase (E2p) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase complex (PDHC) of a number of prokaryotic and eukaryotic species. Sequence analysis indicated that the meningococcal E2p (Men-E2p) contains two N-terminal lipoyl domains, an E1/E3 binding domain and a catalytic domain. The domains are separated by hinge regions rich in alanine, proline and charged residues. Another lipoyl domain with high sequence similarity to the Men-E2p lipoyl domain was found at the N-terminal of the E3 component. A further ORF, coding for a 16.5-kDa protein, was found between the ORFs encoding the E2p and E3 components. The identity and functional characteristics of the expressed and purified heterologous Men-E2p were confirmed 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 antiserum raised against Men-E2p reacted with protein extracts of A. vinelandii, Escherichia coli and N. gonorrhoeae and with the lipoyl and catalytic domains of E2p obtained by limited proteolysis. In contrast, the original R-70 antiserum reacted almost exclusively with the lipoyl domain, indicating the strong immunogenicity of this domain. Antibodies to Men-E2p were detected in patients and animals (rabbits and mice) infected with homologous or heterologous meningococci or other neisserial species. These results have important implications for the understanding of PDHC and the design of future outer membrane vesicle-based vaccines.
Ala' Aldeen
D A
DA
Department of Microbiology, University Hospital, Queen's Medical Centre, Nottingham.
Westphal
A H
AH
De Kok
A
A
Weston
V
V
Atta
M S
MS
Baldwin
T J
TJ
Bartley
J
J
Borriello
S P
SP
D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001428:Bacterial Vaccines / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D008589:Meningococcal Infections / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D016366:Open Reading Frames / Q000235:genetics; D011768:Pyruvate Dehydrogenase Complex / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D011817:Rabbits; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D004269:DNA, Bacterial; D007106:Immune Sera; D011768:Pyruvate Dehydrogenase Complex; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase