A novel porA-based real-time PCR for detection of meningococcal carriage

Real-time PCR based on the capsule transfer gene (ctrA) is a significant aid in the diagnosis of meningococcal infection but fails to detect a high proportion (60 %) of non-groupable strains associated with nasopharyngeal carriage. This study aimed to design a novel real-time (TaqMan) PCR that would detect more strains of meningococci and be suitable for large-scale carriage studies. Primer and probe sequences were based on the meningococcal porA gene and designed specifically to exclude the highly related porA pseudogene in Neisseria gonorrhoeae. The specificity of the assay was confirmed by testing strains of N. gonorrhoeae known to contain the porA pseudogene together with commensal strains of Neisseria lactamica and Neisseria sicca. None of these was detected in the assay. Neisseria meningitidis strains representing a wide range of serogroups together with non-groupable strains isolated from the nasopharynx were tested by ctrA assay and the novel porA-based TaqMan PCR. All carriage strains were detected by the porA-based assay including four that gave weak or no reaction with the ctrA assay. Comparison of ctrA and porA assays on 71 throat swabs obtained from university students showed that the porA assay detected meningococcal DNA in all samples that were ctrA positive plus three that were ctrA negative but culture positive. This novel porA-based TaqMan assay provides a highly specific method for detecting meningococcal DNA that is more sensitive than the ctrA assay for detecting meningococcal carriage and is particularly suitable for carriage studies where non-groupable strains and other Neisseria are present

Molecular cloning and expression of Neisseria meningitidis class 1 outer-membrane protein in Escherichia coli K-12

A genomic library of meningococcal DNA from a clinical isolate of Neisseria meningitidis was constructed in the expression vector lambda gt11. Outer membrane complex was prepared from the same strain and used to immunize rabbits to raise polyclonal anti-outer membrane complex serum. The amplified library was probed with this polyclonal serum, and seven expressing recombinants were isolated; further investigations indicated these to be identical. The expressed meningococcal gene in these recombinants was fused to vector beta-galactosidase and shown to encode epitopes present on the 42-kilodalton class 1 outer membrane protein. Estimation of the size of the recombinant fusion protein suggests that up to 40 kilodaltons of protein-coding sequence is present. The lambda gt11 recombinant contains a 3.4-kilobase DNA insert, which has been recloned into a plasmid and characterized by restriction endonuclease analysis. A restriction fragment from the insert, representing the protein-coding region hybridizes to a single 2.2-kilobase XbaI fragment from the homologous strain and to similar-sized XbaI fragments in other strains of meningococci, expressing antigenically distinct class 1 protein

Stable expression of meningococcal class 1 protein in an antigenically reactive form in outer membranes of Escherichia coli

The entire gene encoding the class 1 outer membrane protein of Neisseria meningitidis is located on a 2.2 kb fragment, obtained on digestion of chromosomal DNA with Xbal. This Xbal fragment from strain MC50 (subtype P1-16), which had previously been cloned in bacteriophage M13, has been transferred to the plasmid vector pMTL20. The resulting plasmid (pPORA100) was propagated in Escherichia coli (JM109) and cell lysates were subjected to SDS-PAGE. Western blotting with anti-class 1 protein antibodies revealed constitutive expression of a protein of 41kD, corresponding to the class 1 protein of the parent meningococcal strain, which was absent in the E. coli control Fractionation of E. coli cells carrying the recombinant plasmid revealed that the protein was exclusively located in the outer membrane, and N-terminal amino acid analysis of the expressed protein revealed that normal processing of the signal peptide had occurred. Immuno-gold electron microscopy showed that the protective epitope recognized by a P1-16 subtype-specific monoclonal antibody was exposed in an antigenically reactive form on the surface of E. coli cells carrying plasmid pPORA100. In contrast, expression in E. coli of a second plasmid (pPORA104) lacking the coding sequence for the first 15 amino acids of the signal peptide resulted in accumulation of recombinant class 1 protein only in the cytoplasm of the cells.Thus the presence of the meningococcal signal sequence ensures expression of this meningococcal porin protein in an antigenically native conformation in outer membranes of E. coli, while its absence results in expression of a soluble protein. Such constructs illustrate the potential use of recombinant DNA technology for the development of effective human vaccines against meningococcal infection

Point mutation in meningococcal por A gene associated with increased endemic disease

The por A gene, which encodes expression of meningococcal class 1 outer membrane protein, responsible for antigenic subtype specificity, has been cloned and sequenced in an isolate of Neisseria meningitidis (B:15:P1.7,16) from a patient in the Gloucester area with meningococcal meningitis. Comparison of the sequence with that of the equivalent gene from the P1.7,16 reference strain reveals a point mutation which generates a single aminoacid change in the epitope responsible for P1.16 specificity. Monoclonal antibodies with P1.16 specificity do not react with synthetic peptides that correspond to the altered epitope, and do not promote complement-mediated bactericidal killing of the isolate. Analysis of other strains shows widespread distribution of infections due to B:15:P1.7,16 meningococci with the altered epitope (P1.16b) in England and Wales

Variation within serovars of Neisseria gonorrhoeae detected by structural analysis of outer-membrane protein PIB and by pulsed-field gel electrophoresis

Microbiology14341415-1422MROB

A putative amino acid ABC transporter substrate-binding protein, NMB1612, from Neisseria meningitidis, induces murine bactericidal antibodies against meningococci expressing heterologous NMB1612 proteins

Abstract not availableMiao-Chiu Hung, María Victoria Humbert, Jay R. Laver, Renee Phillips1, John E. Heckels, Myron Christodoulide