Comparison of the class 1 outer membrane proteins of eight serological reference strains of Neisseria meningitidis

Primers suitable for the amplification of the gene encoding the class 1 outer membrane protein of Neisseria meningitidis by the polymerase chain reaction (PCR) were designed from published DNA sequences and used to study the gene in eight meningococcal strains of different serogroup, serotype and subtype. At high annealing stringency one product, shown to correspond to the class 1 protein gene, was amplified from each strain. For three strains an additional smaller product, provisionally identified as the gene encoding the class 3 outer membrane protein, was amplified at lower annealing stringencies. Nucleotide sequence analysis of the PCR products corresponding to the class 1 proteins established the differences in the primary structure of the proteins between each of the subtypes and other outer-membrane proteins from Neisseria spp. These differences impose constraints on possible structural models of these proteins. Most amino acid sequence variation occurred in two domains of between 8 and 17 amino acids; there was an additional region which varied mainly between classes of outer membrane protein and there were nine conserved regions. Using appropriate primers it was possible to distinguish between class 1 outer membrane protein genes from strains of different subtypes by the PCR.     

Heat-modifiable outer membrane proteins of Neisseria meningitidis and their organization within the membrane.

Neisseria meningitidis group B serotype 2 strain M986 contains two predominant outer membrane proteins, with apparent molecular weights of 41,000 (protein b) and 28,000 (protein e). Heating of outer membrane vesicles at 56 degrees C for 20 min caused much of b** to disaggregate and denature into b (41,000 daltons). In contrast, protein e could be rapidly solubilized by SDS at room temperature into its monomeric state (e*), but it was not converted to its final higher apparent molecular weight of 28,000 (e) unless heated at 100 degrees C for 2 min. We propose that protein b exists in the membrane as trimers or tetramers in a transmembrane configuration and that protein e exists as subunits on the exterior surface of the outer membrane and has a highly ordered tertiary structure.     

Antigenic cross-reactivity between outer membrane proteins of Neisseria meningitidis and commensal Neisseria species

Two mouse sera against outer membrane proteins from a pathogenic Neisseria meningitidis strain and a commensal N. lactamica strain and two human sera from patients recovering from meningococcal meningitis were used to identify antigens common to pathogenic and commensal Neisseria species. Two major antigens of 55 kDa and 32 kDa, present in all N. meningitidis and N. lactamica strains tested, were demonstrable with all the sera used; the 55-kDa protein was iron-regulated. Demonstration of other common antigens was dependent on the serum used: a 65-kDa antigen was visualised with the human and the mouse anti-N. lactamica sera; a 37-kDa antigen identified as the meningococcal ferric binding protein (FbpA) was only detected with the mouse sera, and two antigens of 83 kDa and 15 kDa were only shown with the mouse anti-N. meningitidis serum. The results demonstrate the existence of several outer membrane antigens common to N. lactamica and N. meningitidis strains, in agreement with the hypothesis that natural immunity against meningitis is partially acquired through colonisation by commensal species, and open new perspectives for the design of vaccine formulations and the development of strategies for vaccination against meningitis.     

Association of iron-regulated outer membrane proteins of Neisseria meningitidis with the RmpM (class 4) protein

The RmpM (class 4) protein of Neisseria meningitidis has previously been shown to be associated with the outer membrane porins. In the present study, we demonstrate that this protein forms complexes with the lactoferrin receptor LbpA, the transferrin receptor TbpA and the siderophore receptor FrpB as well. This complexation apparently resulted in a stabilization of oligomeric forms of these iron-regulated proteins. In vitro experiments further revealed a reduced ability to acquire iron from human lactoferrin in the rmpM mutant. Furthermore, all TonB-dependent receptors investigated here appeared to exist as oligomers (probably dimers), suggesting that this is a general feature of this class of proteins.     

Five structural classes of major outer membrane proteins in Neisseria meningitidis

Group B Neisseria meningitidis is thus far subdivided into 15 protein serotypes based on antigenically different major outer membrane proteins. Most serotypes have three or four major proteins in their outer membranes. Comparative structural analysis by chymotryptic 125I-peptide mapping was performed on these major proteins from the prototype strains as well as from six non-serotypable strains. The major outer membrane proteins from each of the serotypes were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the Laemmli system. Individual proteins within the gel slices were radioiodinated and digested with chymotrypsin, and then their 125I-peptides were separated by electrophoresis and chromatography on cellulose thin-layer plates. The peptide maps obtained by autoradiography were categorized into five different structural classes which correlated with the apparent molecular weights of proteins, i.e., 46 +/- 1K, 41 +/- 1K, 38 +/- 1K, 33 +/- 1K, and 28 +/- 1K. Each of the major outer membrane proteins within a strain had a distinctly different chymotryptic peptide map, indicating significant differences in the primary structure of these proteins. In contrast, outer membrane proteins of the same or very similar molecular weight from different serotype strains had similar, occasionally identical peptide maps, indicating a high degree of structural homology. The unique peptides from proteins of the same structural classes were often hydrophilic, whereas common peptides were often hydrophobic, suggesting that the serotype determinants reside within the variable hydrophilic regions of major outer membrane proteins.     

Human T cell recognition of the Mycobacterium leprae LSR antigen: epitopes and HLA restriction

We have in this work mapped epitopes and HLA molecules used in human T cell recognition of the Mycobacterium leprae LSR protein antigen. HLA typed healthy subjects immunized with heat killed M. leprae were used as donors to establish antigen reactive CD4+ T cell lines which were screened for proliferative responses against overlapping synthetic peptides covering the C-terminal part of the antigen sequence. By using this approach we were able to identify two epitope regions represented by peptide 2 (aa 29-40) and peptide 6 (aa 49-60), of which the former was mapped in detail by defining the N- and C-terminal amino acid positions necessary for T cell recognition of the core epitope. MHC restriction analysis showed that peptide 2 was presented to T cells by allogeneic cells coexpressing HLA-DR4 and DRw53 or DR7 and DRw53. In contrast, peptide 6 was presented to T cells only in the context of HLA-DR5 molecules. In conclusion, the M. leprae LSR protein antigen can be recognized by human T cells in the context of multiple HLA-DR molecules, of which none are reported to be associated with the susceptibility to develop leprosy. The results obtained are in support of using the LSR antigen in subunit vaccine design.     

Bioinformatic analysis of outer membrane proteome of Neisseria meningitidis and Neisseria lactamica.

Two-dimensional electrophoresis (isoelectric focusing/SDS-PAGE) and Western-blotting techniques were used to analyze and compare common and/or specific outer-membrane proteins and antigens from Neisseria meningitidis and Neisseria lactamica. Bioinformatic image analyses of proteome and immunoproteome maps indicated the presence of numerous proteins and several antigens shared by N. meningitidis and N. lactamica, although the inter-strain variation in the maps was of similar magnitude to the inter-species variation, and digital comparison of the maps did not reveal proteins found to be identical by MALDI-TOF fingerprinting analysis. PorA and RmpM, two relevant outer-membrane antigens, manifested as various spots at several different positions. While some of these were common to all the strains analyzed, others were exclusive to N. meningitidis and their electrophoretic mobilities were different than expected. One such spot, with a molecular mass of 19 kDa, may be the C-terminal fragment of RmpM (RmpM-Cter). The results demonstrate that computer-driven analysis based exclusively on spot positions in the proteome or immunoproteome maps is not a reliable approach to predict the identity of proteins or antigens; rather, other identification techniques are necessary to obtain accurate comparisons.     

Identification and characterisation of a novel conserved outer membrane protein from Neisseria meningitidis

We have identified a homologue of the adhesin AIDA-I of Escherichia coli in Neisseria meningitidis. This gene was designated nhhA (Neisseria hia homologue), as analysis of the complete coding sequence revealed that it is more closely related to the adhesins Hia and Hsf of Haemophilus influenzae. The sequence of nhhA was determined from 10 strains, and found to be highly conserved. Studies of the localisation by Western immunoblot analysis of total cell proteins and outer membrane complex preparations and by immunogold electron microscopy revealed that NhhA is located in the outer membrane. A strain survey showed that nhhA is present in 85/85 strains of N. meningitidis representative of all the major disease-associated serogroups, based on Southern blot analysis. It is expressed in the majority of strains tested by Western immunoblot.     

Iron and outer membrane proteins in the susceptibility of Neisseria meningitidis to human serum

The proportion of carrier-isolated Neisseria meningitidis strains sensitive to human serum (37.2%) was found to be significantly higher than that of case-isolated ones (4.1%), although the difference is too low to consider serum-resistance responsible for invasion in this microorganism. Serum-susceptibility was not related to the existence of specific outer membrane proteins, as is the case of N. gonorrhoeae. Iron restriction induced iron-regulated outer membrane proteins in each strain (but not the same proteins in all strains) but without any detectable effect on serum-susceptibility. Iron excess was also unable to induce changes in the susceptibility of N. meningitidis to human serum.     

Analysis of Moraxella catarrhalis outer membrane antigens cross-reactive with Neisseria meningitidis and Neisseria lactamica

Mouse sera against outer membrane proteins from Moraxella catarrhalis, Neisseria meningitidis and Neisseria lactamica, and human sera from both healthy individuals and patients convalescing from meningococcal meningitis were used to identify cross-reactive antigens. Mouse anti-N. meningitidis and anti-N. lactamica sera recognized 77, 62 and 32 kDa outer membrane antigens in M. catarrhalis strains; on the contrary, the meningococcal porin PorB (38-42 kDa) was recognized by one of the two anti-M. catarrhalis sera. Human sera from both healthy individuals and patients convalescing from meningococcal meningitis also showed cross-reactive antibodies against these proteins. The existence of cross-reactive antigens in M. catarrhalis and N. meningitidis (as well as in N. lactamica) could favor the development of natural immunization against both pathogens.     

Mapping of T cell epitopes using recombinant antigens and synthetic peptides.

Two complementary approaches were used to determine the epitope specificity of clonal and polyclonal human T lymphocytes reactive with the 65-kd antigen of Mycobacterium leprae. A recombinant DNA sublibrary constructed from portions of the 65-kd gene was used to map T cell determinants within amino acid sequences 101-146 and 409-526. Independently, potential T cell epitopes within the protein were predicted based on an empirical analysis of specific patterns in the amino acid sequence. Of six peptides that were predicted and subsequently synthesised, two (112-132 and 437-459) were shown to contain human T cell epitopes. This corroborated and refined the results obtained using the recombinant DNA sublibrary. Both of these regions are identical in M. leprae and M. tuberculosis and are distinct from the known B cell epitopes of the 65-kd protein. This combination of recombinant DNA technology and peptide chemistry may prove valuable in analysis of the cellular immune response to infectious agents.     

Structural comparison of Neisseria gonorrhoeae outer membrane proteins.

Outer membranes from opaque colonia variants of Neisseria gonorrhoeae P9 contain a major outer membrane protein (protein I) together with one or more of a series of heat-modifiable proteins (proteins II). Proteins I. II, and IIa have been isolated by detergent extraction of outer membranes. Amino acid analysis showed proteins II and IIa to have a very similar composition. Cyanogen bromide cleavage of proteins II and IIa produced a pair of fragments with identical molecular weight and a pari which differed by an amount (0.5K) equivalent to the difference between the intact proteins. Tryptic peptide maps of 125I-labeled proteins II, IIa, and IIb showed many similarities, with only a few peptides unique to any one protein. Peptide maps of protein IIa from cells which had been surface labeled showed that the unique peptides were exposed on the surface. The heat-modifiable proteins thus appear to form a family of proteins with closely related structure probably differing in that part which is exposed on the bacterial surface.     

Cross-linking analysis of Neisseria gonorrhoeae outer membrane proteins.

The arrangement of proteins in the outer membrane of Neisseria gonorrhoeae was investigated through the use of cleavable chemical cross-linking reagents and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cross-linking of isolated outer membranes yielded dimers and trimers of the major outer membrane protein. In addition, data were obtained suggesting that a stable interaction exists between the major protein I and protein II, the second most prevalent protein in the gonococcal outer membrane.     

Sequence variation in class 1 outer membrane protein in Neisseria meningitidis isolated from patients with meningococcal infection and close household contacts

Abstract Aspergillus oryzae , which is widely used for Japanese traditional fermentation, produced at least two lipolytic enzymes (L1 and L2). Southern hybridization analysis of restriction enzyme-digested genomic DNA fragments of Aspergillus oryzae with 23-mer oligonucleotides synthesized according to the amino acid sequence of the enzyme L1 as probes suggested that there is single copy of the L1 gene in the genome. DNA fragments containing the L1 gene were cloned in Escherichia coli . Nucleotide sequencing of the DNA fragments revealed an open reading frame consisting of 213 amino acid residues. It had three putative introns whose sizes were 52 bp, 48 bp and 53 bp, respectively. Putative CAAT and TATA boxes were found at positions −147 and −100 from A (+1) of the translational initiation codon, and a polyadenylation site at 158 bp downstream of the stop codon. The deduced amino acid sequence of the L1 gene was highly similar to those of cutinases from phytopathogenic fungi. Thus, it is interesting to note that the non-phytopathogenic fungus, A. oryzae , produces cutinase, which seems to play an important role in flavor formation.     

The class 1 outer membrane protein of Neisseria meningitidis: gene sequence and structural and immunological similarities to gonococcal porins

The class 1 protein is a major protein of the outer membrane of Neisseria meningitidis, and an important immunodeterminant in humans. The complete nucleotide sequence for the structural gene of a class 1 protein has been determined. The sequence predicts a protein of 374 amino acids, preceded by a typical signal peptide of 19 residues. The hydropathy profile of the predicted protein sequence resembles that of the Escherichia coli and gonococcal porins. The predicted protein sequence of the class 1 protein exhibits considerable structural similarity to the gonococcal porins PIA and PIB. Western blot studies also reveal immunologically conserved domains between the class 1 protein, PIA and PIB. A restriction fragment from the class 1 gene hybridizes to gonococcal genomic fragments in Southern blots. In addition to the class 1 gene coding region there is a large open reading frame on the opposite strand.     

Prediction and characterization of T-cell epitopes for epitope vaccine design from outer membrane protein of Neisseria meningitidis serogroup B

Neisseria meningitidis serogroup B (MC58) is a leading cause of meningitis and septicaemia, principally infects the infants and adolescents. No vaccine is available for the prevention of these infections because the serogroup B capsular polysaccharide is unable to stimulate an immune response, due to its similarity with polysialic acid. To overcome these obstacles, we proposed to develop a peptide based epitope vaccine from outer membrane protein contained in outer membrane vesicles (OMV) based on our computational analysis. In OMV a total of 236 proteins were identified, only 15 (6.4%) of which were predicted to be located in outer membrane. The major requirement is the identification and selection of T-cell epitopes that act as a vaccine target. We have selected 13 out of 15 outer membrane proteins from OMV proteins. Due to similarity of the fkpA and omp85 with the human FKBP2 and SAMM50 protein, we removed these two sequences from the analysis as their presence in the vaccine is likely to elicit an autoimmune response. ProPred and ProPred1 were used to predict promiscuous helper T Lymphocytes (HTL) and cytotoxic T Lymphocytes (CTL) epitopes and MHCPred for their binding affinity in N. meningitidis serogroup B (MC58), respectively. Binding peptides (epitopes) are distinguished from nonbinding peptides in properties such as amino acid preference on the basis of amino acid composition. By using this dataset, we compared physico-chemical and structural properties at amino acid level through amino acid composition, computed from ProtParam server. Results indicate that porA, porB, opc, rmpM, mtrE and nspA are more suitable vaccine candidates. The predicted peptides are expected to be useful in the design of multi-epitope vaccines without compromising the human population coverage.     

Role of horizontal genetic exchange in the antigenic variation of the class 1 outer membrane protein of Neisseria meningitidis

The nucleotide sequences of the genes encoding the class 1 outer membrane protein of Neisseria meningitidis (PorA) from 15 meningococcal isolates have been examined. These strains, isolated over a number of years, represented a variety of serological types, clonal groups, and geographical locations. Analysis of the aligned nucleotide sequences showed that the known serological relationships between these proteins were not necessarily reflected throughout the nucleotide sequences of their genes. The uneven distribution of base substitutions, revealed by a comparison of the informative bases, suggested that these genes possessed a mosaic structure. This structure probably resulted from the horizontal transfer of DNA between strains and would have contributed to both the generation and the spread of novel antigenic variants of the protein. In addition, the nucleotide differences between porA genes from different strains were not consistent with the nucleotide sequence divergence of the whole chromosome, as indicated by pulsed-field gel electrophoresis (PFGE) fingerprinting techniques: some strains with divergent PFGE fingerprints shared porA genes with extensive regions of nucleotide sequence identity and, conversely, some strains with similar chromosome structures possessed porA genes with different nucleotide sequences and serological properties. This suggested that entire genes had been exchanged between strains. Given that the meningococcal class 1 OMP is a major component in novel vaccines, some of which are currently undergoing field trials, the potential of horizontal genetic exchange to generate antigenic diversity has implications for the design of such vaccines.     

Detection of IgG and IgM to meningococcal outer membrane proteins in relation to carriage of Neisseria meningitidis or Neisseria lactamica

Carriage of non-serogroupable Neisseria meningitidis or Neisseria lactamica induces antibodies protective against meningococcal disease. Antibodies directed against outer membrane proteins are bactericidal and the serotype and subtype outer membrane protein antigens are being examined for their value as vaccine candidates for serogroup B disease. The aim of this study was to examine the effect of carriage of these two Neisseria species among children and young adults on induction of antibodies to outer membrane components from strains causing disease in Greece. Among 53 patients with meningococcal disease, IgG or IgM antibodies were detected by ELISA in 9 of 13 (69%) from whom the bacteria were isolated and 27 of 40 (67%) who were culture-negative. For military recruits (n = 604), the proportion of carriers of meningococci with IgM or IgG to outer membrane proteins was higher than non-carriers, P < 0.05 and P = 0.000000, respectively. Among school children (n = 319), the proportion with IgM or IgG to outer membrane proteins for carriers of meningococci was higher compared with non-carriers, P = 0.000000 and P = 0000043, respectively. Carriage of N. lactamica was not associated with the presence of either IgM or IgG to the outer membrane proteins in the children. The higher proportion of children (50%) with IgM to outer membrane proteins compared with recruits (10%) might reflect more recent exposure and primary immune responses to the bacteria. The lack of association between antibodies to outer membrane proteins and carriage of N. lactamica could reflect observations that the majority of N. lactamica isolates from Greece and other countries do not react with monoclonal typing reagents. Bactericidal antibodies to meningococci associated with high levels of IgG to N. lactamica were found in a previous study; these are thought to be directed to antigens other than outer membrane proteins or capsules and imply antigens such as lipo-oligosaccharide are involved in induction of antibodies cross-reactive with meningococci.     

The class 1 outer membrane protein of Neisseria meningitidis produced in Bacillus subtilis can give rise to protective immunity

The class 1 outer membrane protein of Neisseria meningitidis B:15:P1.7, 16 was expressed in Bacillus subtilis in high yield as intracellular aggregates. These were easy to isolate and the protein (called BacP1) could be solubilized under denaturing conditions. Sera of mice immunized with thus-solubilized BacP1 contained high titres of antibodies that reacted with the class 1 protein of the meningococcal envelope in immunoblots but did not react with native meningococcal envelope in enzyme immunoassays (EIA) or with intact meningococci in bactericidal assays. However, when the BacP1 protein was complexed with heterologous (Salmonella) lipopolysaccharide, the ensuing sera reacted with meningococcal envelope preparations in both EIA and immunoblots, showed subtype-specific bactericidal activity, and were protective in an infant rat meningitis model.     

Chemical analysis of major outer membrane proteins of Neisseria meningitidis: comparison of serotypes 2 and 11

Most of the 15 protein serotypes found in group B Neisseria meningitidis have distinct major outer membrane protein patterns when examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by the Weber-Osborn system. Both serotypes 2 and 11 contain major outer membrane proteins with apparent molecular weights of 41,000 and 28,000 (41K and 28K). The 41K and 28K proteins were purified from the prototype strains of these two serotypes (M986 type 2 and M136 type 11) by preparative slab SDS-PAGE and were chemically characterized. No hexosamine was found in the purified 41K and 28K proteins. Although the two 41K proteins had similar amino acid compositions, their mobilities in Laemmli SDS-PAGE and their fragmentation patterns on SDS-PAGE after cyanogen bromide cleavage were different. The two 28K proteins differed in their amino acid composition, mobilities in Laemmli SDS-PAGE, and cyanogen bromide cleavage products. Peptide maps following chymotrypsin digestion of radioiodinated 41K and 28K proteins revealed distinct peptide maps for all four proteins. Comparison of the peptide maps of two 41K or two 28K proteins indicated that most of the unique peptides were hydrophilic, whereas most of the common peptides were hydrophobic. These results indicated that both of the 41K proteins and the 28K proteins from serotypes 2 and 11 were chemically different, although the proteins having the same molecular weights appeared to share common peptides.