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Biomedical subjects

M C Maiden

Publications and source records attributed to M C Maiden.

46 records · Page 3Linked to original sources
PubMed1993

Expression of meningococcal epitopes in LamB of Escherichia coli and the stimulation of serosubtype-specific antibody responses.

The class 1 outer membrane protein (OMP), a major variable surface antigen of Neisseria meningitidis, is a component of novel meningococcal vaccines currently in field trials. Serological variants of the protein are also used to serosubtype meningococci. Most of the amino acid changes that give rise to antigenic variants of the protein occur in two variable regions (VR1 and VR2) that are thought to form loops on the cell surface. The polymerase chain reaction (PCR) was used to amplify the nucleotide sequences encoding VR1 and VR2 from the chromosomal DNA of N. meningitidis strain M1080. These were cloned in frame into the lamB gene of the Escherichia coli expression vector pAJC264. Whole-cell enzyme-linked immunosorbent assays (ELISAs), using monoclonal antibodies, and SDS-PAGE confirmed that, upon induction, strains of E. coli carrying these constructs expressed hybrid LamB proteins containing the N. meningitidis surface loops. These strains were used to immunize rabbits and the resultant polyclonal antisera reacted specifically with the class 1 OMP of reference strain M1080 (P1.7). Immunogold labelling of meningococcal cells and whole-cell dot-blot analyses with these antisera showed that the variable epitopes were exposed on the cell surface and confirmed that this approach could be used to obtain serosubtype-specific antisera. The binding profiles of the antisera were determined from their reactions with overlapping synthetic peptides and their reactivity compared with that of relevant serosubtype-specific monoclonal antibodies. This approach was used successfully to raise antisera against two other class 1 OMP VR2s. A fourth antiserum raised against a VR2, including the P1.1 epitope, was not subtype specific.

Amino Acid Sequence↗
PubMed1992

Analysis of the clonal relationships between strains of Neisseria meningitidis by pulsed field gel electrophoresis.

Fingerprint patterns were generated from strains of Neisseria meningitidis by digestion of chromosomal DNA samples with 'rare-site' restriction endonucleases and resolution of the resultant fragments by pulsed field gel electrophoresis (PFGE). The potential of this technique for the rapid establishment of the clonal relationships between different isolates of the meningococcus was investigated. The fingerprint patterns from various serogroup A strains, previously assigned to clonal subgroups on the basis of their electrophoretic types (ETs), were compared. Fingerprints generated with the endonucleases SfiI, SpeI and NheI each gave distinctive patterns for the clonal subgroups I-IV of serogroup A. Further, the endonucleases SpeI and, particularly, NheI were capable of resolving differences between various subgroup III strains isolated at different times and geographical locations. Strains isolated during the 'new wave' pandemic, which was associated with the Haj, from Europe, America, and Africa, had a characteristic fingerprint pattern and appeared to be distinct from 'old wave' pandemic strains. The PFGE technique is a relatively rapid and sensitive method for establishing clonal relationships among epidemic strains of N. meningitidis.

DNA, Bacterial↗
PubMed1992

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.

Antigenic Variation↗
PubMed1992

Molecular analysis of the serotyping antigens of Neisseria meningitidis.

Molecular approaches to the rapid analysis of the serotyping antigens of Neisseria meningitidis, the class 2 and 3 outer membrane proteins (OMPs), were developed, evaluated, and used to study 12 antigenic variants of these proteins. A primer set for the polymerase chain reaction (PCR) amplification of the genes encoding these antigens was devised. Low-stringency amplification of meningococcal chromosomal DNA with this primer set resulted in the amplification of two products from each strain, whereas at higher stringencies only one product was amplified in most strains. Southern hybridization techniques and restriction analyses were used to differentiate the PCR products amplified at high stringencies from strains expressing class 2 or class 3 OMPs; these PCR products were further characterized by the determination of their nucleotide sequences, confirming that they represented the amplified class 2 and class 3 OMP genes. Analyses of these and other nucleotide sequences enabled the construction of a phenogram illustrating the interrelationships between Neisseria OMP genes. The comparative analysis of deduced amino acid sequences revealed conserved and variable regions of the proteins; the latter probably correspond to surface loops on the protein and hence are potentially exposed to the immune system. Further analyses of the primary structures of these related porins from Neisseria species enabled construction of models of the secondary structure of these antigens and comparison of these models with those previously published. The methods reported in the present work are rapid reproducible procedures for the analysis of antigenic variants of these proteins.

Amino Acid Sequence↗
PubMed1992

Identification of meningococcal serosubtypes by polymerase chain reaction.

The polymerase chain reaction was used as the basis of a novel typing method for Neisseria meningitidis. Southern hybridization experiments demonstrated that it was possible to identify genes encoding different serological variants of the meningococcal class 1 outer membrane protein by probing with polymerase chain reaction products corresponding to known epitopes. A set of 14 defined variable regions was prepared in bacteriophage M13mp19 by the cloning of polymerase chain reaction products. The phage were dot blotted onto membrane filters, which were used as targets for hybridization of radiolabeled amplified class 1 outer membrane protein genes. Thus, the presence of many different subtype-specific epitopes could be investigated in one experiment. This technique was evaluated with a set of serological reference strains, mainly of serogroup B organisms, and provided an alternative, rapid, and comprehensive typing system that was capable of distinguishing known serosubtypes and also of defining currently untypeable strains independently of sodium dodecyl sulfate-polyacrylamide gel electrophoresis or serological analysis. An additional advantage of this technique was that in the case of an unknown serosubtype (i.e., one that did not hybridize with any of the known samples), the DNA amplified from the original sample could be used to determine the nucleotide sequence of the novel serosubtype and to clone the corresponding variable region into bacteriophage M13. It may be possible to develop this procedure for the diagnostic detection and typing of meningococci directly from clinical samples even when culture is not possible because of antibiotic treatment of an acute case.

Bacterial Outer Membrane Proteins↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1990

Homologous sugar transport proteins in Escherichia coli and their relatives in both prokaryotes and eukaryotes.

Separate proteins for proton-linked transport of D-xylose, L-arabinose, D-galactose, L-rhamnose and L-fucose into Escherichia coli are being studied. By cloning and sequencing the appropriate genes, the amino acid sequences of proteins for D-xylose/H+ symport (XylE), L-arabinose/H+ symport (AraE), and part of the protein for D-galactose/H+ symport (GalP) have been determined. These are homologous, with at least 28% identical amino acid residues conserved in the aligned sequences, although their primary sequences are not similar to those of other E. coli transport proteins for lactose, melibiose, or D-glucose. However, they are equally homologous to the passive D-glucose transport proteins from yeast, rat brain, rat adipocytes, human erythrocytes, human liver, and a human hepatoma cell line. The substrate specificity of GalP from E. coli is similar to that of the mammalian glucose transporters. Furthermore, the activities of GalP, AraE and the mammalian glucose transporters are all inhibited by cytochalasin B and N-ethylmaleimide. Conserved residues in the aligned sequences of the bacterial and mammalian transporters are identified, and the possible roles of some in sugar binding, cation binding, cytochalasin binding, and reaction with N-ethylmaleimide are discussed. Each protein is independently predicted to form 12 hydrophobic, membrane-spanning alpha-helices with a central hydrophilic segment, also comprised of alpha-helix. This unifying structural model of the sugar transporters shares features with other ion-linked transport proteins for citrate or tetracycline.

Amino Acid Sequence↗
PubMed1988

The cloning, DNA sequence, and overexpression of the gene araE coding for arabinose-proton symport in Escherichia coli K12.

A lambda placMu1 insertion was made into araE, the gene for arabinose-proton symport in Escherichia coli. A phage containing an araE'-'lacZ fusion was recovered from the lysogen and its restriction map compared with that of the 61-min region of the E. coli genome to establish the gene order thyA araE orf lysR lysA galR; araE was transcribed toward orf. A 4.8-kilobase SalI-EcoRI DNA fragment containing araE was subcloned from the phage lambda d(lysA+ galR+ araE+) into the plasmid vector pBR322. From this plasmid a 2.8-kilobase HincII-PvuII DNA fragment including araE was sequenced and also subcloned into the expression vector pAD284. The araE gene was 1416-base pairs long, encoding a hydrophobic protein of 472 amino acids with a calculated Mr of 51,683. The amino acid sequence was homologous with the xylose-proton symporter of E. coli and the glucose transporters from a human hepatoma HepG2 cell line, human erythrocytes, and rat brain. The overexpressed araE gene product was identified in Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of cell membranes as a protein of apparent Mr 35,000 +/- 1,150. Arabinose protected this protein against reaction with N-ethylmaleimide.

Amino Acid Sequence↗
PubMedDate not supplied

Mammalian and bacterial sugar transport proteins are homologous.

The uptake of a sugar across the boundary membrane is a primary event in the nutrition of most cells, but the hydrophobic nature of the transport proteins involved makes them difficult to characterize. Their amino-acid sequences can, however, be determined by cloning and sequencing the corresponding gene (or complementary DNA). We have determined the sequences of the arabinose-H+ and xylose-H+ membrane transport proteins of Escherichia coli. They are homologous with each other and, unexpectedly, with the glucose transporters of human hepatoma and rat brain cells. All four proteins share similarities with the E. coli citrate transporter. Comparisons of their sequences and hydropathic profiles yield insights into their structure, functionally important residues and possible evolutionary relationships. There is little apparent homology with the lactose-H+ (LacY) or melibiose-Na+ (MelB) transport proteins of E. coli.

Amino Acid Sequence↗