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M C Maiden

Publications and source records attributed to M C Maiden.

At least 37 records · Page 2Linked to original sources
PubMed1999

Comparative analysis of two meningococcal immunotyping monoclonal antibodies by resonant mirror biosensor and antibody gene sequencing.

Lipooligosaccharide (LOS) is a major surface component of the cell walls of Neisseria meningitidis, which is important for its roles in pathogenesis and antigenic variation, as a target for immunological typing, and as a possible vaccine component. Although the structures of many antigenic variants have been determined, routine immunological typing of these molecules remains problematic. Resonant mirror analysis was combined with gene sequencing to characterize two monoclonal antibodies (MAbs) used in typing panels that were raised against the same LOS immunotype, L3,7,9. The two MAbs (MAb 4A8-B2 and MAb 9-2-L379) were of the same immunoglobulin subtype, but while MAb 9-2-L379 was more than a 1,000-fold more sensitive in immunotyping assays of both whole meningococcal cells and purified LOS, MAb 4A8-B2 was more specific for immunotype L3,7,9. The differences in sensitivity were a consequence of MAb 9-2-L379 having a 44-fold-faster association constant than MAb 4A8-B2. Comparison of the amino acid sequences of the variable chains of the MAbs revealed that they had very similar heavy chains (81% amino acid sequence identity) but diverse light chains (54% sequence identity). The differential binding kinetics and specificities observed with these MAbs were probably due to differences in the epitopes recognized, and these were probably a consequence of the different immunization protocols used in their production.

Amino Acid Sequence↗
PubMed1999

Structural and evolutionary inference from molecular variation in Neisseria porins.

The porin proteins of the pathogenic Neisseria species, Neisseria gonorrhoeae and Neisseria meningitidis, are important as serotyping antigens, putative vaccine components, and for their proposed role in the intracellular colonization of humans. A three-dimensional structural homology model for Neisseria porins was generated from Escherichia coli porin structures and N. meningitidis PorA and PorB sequences. The Neisseria sequences were readily assembled into the 16-strand beta-barrel fold characteristic of porins, despite relatively low sequence identity with the Escherichia proteins. The model provided information on the spatial relationships of variable regions of peptide sequences in the PorA and PorB trimers and insights relevant to the use of these proteins in vaccines. The nucleotide sequences of the porin genes from a number of other Neisseria species were obtained by PCR direct sequencing and from GenBank. Alignment and analysis of all available Neisseria porin sequences by use of the structurally conserved regions derived from the PorA and PorB structural models resulted in the recovery of an improved phylogenetic signal. Phylogenetic analyses were consistent with an important role for horizontal genetic exchange in the emergence of different porin classes and confirmed the close evolutionary relationships of the porins from N. meningitidis, N. gonorrhoeae, Neisseria lactamica, and Neisseria polysaccharea. Only members of this group contained three conserved lysine residues which form a potential GTP binding site implicated in pathogenesis. The model placed these residues on the inside of the pore, in close proximity, consistent with their role in regulating pore function when inserted into host cells.

Amino Acid Sequence↗
PubMed1999

Population genetic and evolutionary approaches to analysis of Neisseria meningitidis isolates belonging to the ET-5 complex.

Periodically, new disease-associated variants of the human pathogen Neisseria meningitidis arise. These meningococci diversify during spread, and related isolates recovered from different parts of the world have different genetic and antigenic characteristics. An example is the ET-5 complex, members of which were isolated globally from the mid-1970s onwards. Isolates from a hyperendemic outbreak of meningococcal disease in Worcester, England, during the late 1980s were characterized by multilocus sequence typing and sequence determination of antigen genes. These data established that the Worcester outbreak was caused by ET-5 complex meningococci which were not closely related to the ET-5 complex bacteria responsible for a hyperendemic outbreak in the nearby town of Stroud during the years preceding the Worcester outbreak. A comparison with other ET-5 complex meningococci established that there were at least three distinct globally distributed subpopulations within the ET-5 complex, characterized by particular housekeeping and antigen gene alleles. The Worcester isolates belonged to one of these subpopulations, the Stroud isolates belonged to another, and at least one representative of the third subpopulation identified in this work was isolated elsewhere in the United Kingdom. The sequence data demonstrated that ET-5 variants have arisen by multiple complex pathways involving the recombination of antigen and housekeeping genes and de novo mutation of antigen genes. The data further suggest that either the ET-5 complex has been in existence for many years, evolving and spreading relatively slowly until its disease-causing potential was recognized, or it has evolved and spread rapidly since its first identification in the 1970s, with each of the subpopulations attaining a distribution spanning several continents.

Amino Acid Sequence↗
PubMed1999

Multilocus sequence typing and antigen gene sequencing in the investigation of a meningococcal disease outbreak.

Multilocus sequence typing and antigen gene sequencing were used to investigate an outbreak of meningococcal disease in a university in the United Kingdom. The data obtained showed that five distinct Neisseria meningitidis strains belonging to the ET-37 complex were present in the student population during the outbreak. Three of these strains were not associated with invasive disease, and two distinct strains caused invasive disease, including several fatalities. The initial case of the disease cluster was caused by a strain distinct from that responsible for at least two subsequent cases and two cases remote from the university, which were epidemiologically linked to the outbreak. These observations were consistent with pulsed-field gel electrophoresis data, but the sequence data alone were sufficient to resolve the strains involved in the disease cluster. Interpretation of the nucleotide sequence data was more straightforward than interpretation of the fingerprint patterns, and the sequence data provided information on the genetic differences among the isolates.

Bacterial Outer Membrane Proteins↗
PubMed1998

Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms.

Traditional and molecular typing schemes for the characterization of pathogenic microorganisms are poorly portable because they index variation that is difficult to compare among laboratories. To overcome these problems, we propose multilocus sequence typing (MLST), which exploits the unambiguous nature and electronic portability of nucleotide sequence data for the characterization of microorganisms. To evaluate MLST, we determined the sequences of approximately 470-bp fragments from 11 housekeeping genes in a reference set of 107 isolates of Neisseria meningitidis from invasive disease and healthy carriers. For each locus, alleles were assigned arbitrary numbers and dendrograms were constructed from the pairwise differences in multilocus allelic profiles by cluster analysis. The strain associations obtained were consistent with clonal groupings previously determined by multilocus enzyme electrophoresis. A subset of six gene fragments was chosen that retained the resolution and congruence achieved by using all 11 loci. Most isolates from hyper-virulent lineages of serogroups A, B, and C meningococci were identical for all loci or differed from the majority type at only a single locus. MLST using six loci therefore reliably identified the major meningococcal lineages associated with invasive disease. MLST can be applied to almost all bacterial species and other haploid organisms, including those that are difficult to cultivate. The overwhelming advantage of MLST over other molecular typing methods is that sequence data are truly portable between laboratories, permitting one expanding global database per species to be placed on a World-Wide Web site, thus enabling exchange of molecular typing data for global epidemiology via the Internet.

Bacterial Typing Techniques↗
PubMed1998

Amplification of the meningococcal porB gene for non-culture serotype characterization.

Since 1992, the proportion of culture-confirmed meningococcal infections compared with numbers of notified cases of meningococcal disease has decreased in England and Wales. As most meningococcal strain characterization methods depend on a clinical isolate, this has resulted in a loss of epidemiological information. To address this problem, and to aid nonculture diagnosis, a semi-nested PCR protocol for the amplification of the meningococcal porB gene from clinical specimens was developed. This gene encodes the meningococcal serotyping antigen; strain typing data was provided by hybridization of allele-specific oligonucleotide probes to the digoxigenin-labelled porB amplicon in a 'PCR ELISA'. This assay was specific for meningococcal DNA and sensitivities of 0.81 for cerebrospinal fluid (CSF), 0.57 for serum, and 0.62 for whole blood taken from patients with proven meningococcal infection were obtained.

Bacterial Outer Membrane Proteins↗
PubMed1998

Molecular variation of meningococcal serotype 4 antigen genes.

Changes in the frequency of serogroup B non serotypable (B:NT) meningococci isolated in England and Wales were investigated by T-track fingerprint analysis, DNA nucleotide sequence determination, and serotyping by whole cell ELISA and dot blot assay. Seventy-three per cent of the isolates designated as B:NT by the Meningococcal Reference Unit (MRU) dot blot assay during 1993-4, expressed variants of the serotyping antigen, PorB, that were serotype 4 by whole cell ELISA. T-track fingerprint patterns of these and other 'serotype 4' isolates revealed five distinct porB alleles which were shown by nucleotide sequence determination to encode different peptide sequences. Differential binding of the 'serotype 4' mAbs MN14G21 and 5DC4C8G8 in whole cell ELISA and dot blot assays was the result, (i) of differences in the peptide sequence of predicted surface loop I and (ii) an amino acid deletion in predicted loop VI of the PorB protein.

Amino Acid Sequence↗
PubMed1998

A gonococcal porA pseudogene: implications for understanding the evolution and pathogenicity of Neisseria gonorrhoeae.

Members of the genus Neisseria, including the human pathogens Neisseria meningitidis and Neisseria gonorrhoeae, express at least one member of a family of related porins. N. meningitidis is the only species known to express a second porin, the meningococcal serosubtyping antigen PorA, the most divergent member of this family. Unexpectedly, a porA gene was identified in the gonococcal genome. Both the gonococcal and meningococcal porA loci were adjacent to a homologue of the Escherichia coli greA gene, although the IS1106 element downstream of porA in some meningococci was absent in the gonococcus. Almost identical porA loci were present in four unrelated gonococcal isolates and clinical specimens from patients with gonorrhoea. Lack of PorA expression in the gonococcus resulted from mutations in the promoter region, which prevented transcription, and frameshift mutations in the coding region of the porA gene. Hybridization and amplification experiments, showing the absence of a porA gene in seven other Neisseria species, suggested that porA was acquired by a common ancestor of the gonococcus and meningococcus but inactivated in the gonococcus on speciation. This implies that, while advantageous during colonization of the upper respiratory tract, this protein has no function in, or hinders, colonization of the urogenital tract.

Amino Acid Sequence↗
PubMed1998

Horizontal genetic exchange, evolution, and spread of antibiotic resistance in bacteria.

Some transformable bacteria have acquired target-mediated antibiotic resistance by horizontal genetic exchange of fragments of chromosomal genes. The resistant strains express variants of the antibiotic target that are metabolically active but exhibit a lowered affinity for the antibiotic. The alleles encoding these resistant proteins are mosaics comprising DNA derived from the host and other bacteria, often members of a different species. Examples include penicillin-resistant penicillin-binding proteins (PBPs) in Streptococcus pneumoniae and the pathogenic Neisseria species and sulfonamide-resistant dihydropterate synthase in Neisseria meningitidis. Distinct mosaic alleles encoding antibiotic resistance have arisen on multiple occasions, indicating the mobility of chromosomal genes in these species. Mosaic genes can arise at any chromosomal locus, and S. pneumoniae organisms with high-level penicillin resistance have acquired mosaic PBP genes at three bacterial bpb loci. Furthermore, horizontal genetic exchange permits movement of alleles among bacterial lineages, increasing the opportunities for the spread of antibiotic resistance.

Animals↗
PubMed1998

Heterogeneity of the PorB protein in serotype 22 Neisseria meningitidis.

The genetic diversity of porB genes from meningococcal isolates characterized as serotype 22 was investigated by gene sequencing. This procedure identified seven distinct porB sequences, demonstrating variation in the PorB protein recognized by the serotype 22 monoclonal antibody. This is consistent with the genetic heterogeneity of serotype 22 meningococci reported previously.

Amino Acid Sequence↗
PubMed1996

The maintenance of strain structure in populations of recombining infectious agents.

Using mathematical models that combine population genetic and epidemiological processes, we resolve the paradox that many important pathogens appear to persist as discrete strains despite the constant exchange of genetic material. We show that dominant polymorphic determinants (that is, those that elicit the most effective immune responses) will be organized into nonoverlapping combinations as a result of selection by the host immune system, thereby defining a set of discrete independently transmitted strains. By analysing 222 isolates of Neisseria meningitidis, we show that two highly polymorphic epitopes of the outer membrane protein PorA exist in nonoverlapping combinations as predicted by this general framework. The model indicates that dominant polymorphic determinants will be in linkage disequilibrium, despite frequent genetic exchange, even though they may be encoded by several unlinked genes. This suggests that the detection of nonrandom associations between epitope regions can be employed as a novel strategem for identifying dominant polymorphic antigens.

Genes, Dominant↗
PubMed1996

Antigenic diversity of meningococcal outer membrane protein PorA has implications for epidemiological analysis and vaccine design.

The currently used serological subtyping scheme for the pathogen Neisseria meningitidis is not comprehensive, a proportion of isolates are reported as not subtypeable (NST), and few isolates are fully characterized with two subtypes for each strain. To establish the reasons for this and to assess the effectiveness of DNA-based subtyping schemes, dot blot hybridization and nucleotide sequence analyses were used to characterize the genes encoding antigenic variants of the meningococcal subtyping antigen, the PorA protein. A total of 233 strains, including 174 serologically NST and 59 partially or completely subtyped meningococcal strains, were surveyed. The NST isolates were chosen to be temporally and geographically representative of NST strains, isolated in England and Wales, and submitted to the Meningococcal Reference Unit in the period 1989 to 1991. The DNA-based analyses demonstrated that all of the strains examined possessed a porA gene. Some of these strains were serologically NST because of a lack of monoclonal antibodies against certain PorA epitopes; in other cases, strains expressed minor variants of known PorA epitopes that did not react with monoclonal antibodies in serological assays. Lack of expression remained a possible explanation for serological typing failure in some cases. These findings have important implications for epidemiological analysis and vaccine design and demonstrate the need for genetic characterization, rather than phenotypic characterization using monoclonal antibodies, for the identification of meningococcal strains.

Amino Acid Sequence↗
PubMed1996

The porA alleles are identical in subgroup III serogroup A Neisseria meningitidis strains isolated in China in the 1960s and 1980s.

The porA genes from serogroup A, subgroup III strains isolated in the People's Republic of China in 1966 and in 1984 and 1985 were amplified by PCR at an annealing temperature of 75 degrees C. The DNA sequences (5 strains) and the restriction patterns generated by MspI (14 strains) were identical, unlike the results reported by Peixuan et al. (Z. Peixuan, H. Xujing, and X. Li, J. Clin. Microbiol. 33:458-462, 1995). Furthermore, PCR products which were amplified at an annealing temperature of 60 degrees C, as described by Peixuan et al., were heterogeneous in our study.

Alleles↗
PubMed1994

The porA gene in serogroup A meningococci: evolutionary stability and mechanism of genetic variation.

Molecular analyses were applied to the genes encoding variants of the serosubtyping antigen, the class 1 outer membrane protein (PorA), from 55 serogroup A Neisseria meningitidis strains. These genes were evolutionarily stable and exhibited a limited range of genetic variation, primarily generated by recombination. Translation of the gene sequences revealed a total of 19 distinct amino acid sequences in the variable regions of the protein, 6 of which were not recognized by currently available serosubtyping monoclonal antibodies. Knowledge of these amino acid sequences permitted a rational re-assignment of serosubtype names. Comparison of the complete genes with porA gene sequences from serogroup B and C meningococci showed that serogroup A possessed a limited number of the possible porA genes from a globally distributed gene pool. Each serogroup A subgroup was characterized by one of four porA gene types, probably acquired upon subgroup divergence, which was stable over periods of decades and during epidemiological spread. Comparison with other variable genes (pil and iga) indicated that the three alleles were independently assorted within the subgroup, suggesting that their gene types were older than the subgroups in which they occurred.

Alleles↗
PubMed1993

Population genetics of a transformable bacterium: the influence of horizontal genetic exchange on the biology of Neisseria meningitidis.

Information of the biochemistry and genetics of bacterial species, usually obtained by the study of single isolates, is enhanced by studies of populations of bacteria. Recent advances in molecular technology, particularly polymerase chain reaction-based nucleotide sequence analysis, provide powerful tools for the study of population genetics. Data obtained by such techniques indicate that, while some bacterial species have a clonal population structure, others are non-clonal or panmictic. Clonal populations are a consequence of asexual reproduction by binary fission; panmictic population structures result from 'horizontal' exchange of genetic material between clones. A consequence of horizontal genetic exchange is mosaic gene structures, recognisable by comparisons of nucleotide sequences. In transformable bacteria, for example the human pathogen Neisseria meningitidis, several different genes, including the gene encoding the class 1 outer membrane protein, a major surface antigen, are mosaics. This genetic process has implications both for vaccine design and in the interpretation of epidemiological data.

Bacterial Outer Membrane Proteins↗