Construction of porA Mutants

  • Peter van der Ley
  • Loek van Alphen
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 66)


The PorA or class 1 protein is one of the major meningococcal outermembrane proteins (OMPs). It is one of the two porins found in this organism, the other one being the PorB or class 2/3 protein. It folds into a 16-stranded β-barrel structure, which is now well-established for bacterial porins, in which seven loops are exposed at the cell surface and the remaining one forms the constriction of the pore (1,2). There are approx 20 different serosubtypes of PorA (3), based on sequence variability in the longest surface-exposed loops 1 and 4 (see Fig. 1). In addition, minor sequence variations within individual subtypes have been observed. As a result, some subtypes such as P1.10 actually constitute a family of variants differing by single amino acid substitutions, which may affect antibody recognition; for other subtypes such as P1.4 the number of variants is more limited (4,5). Several studies with experimental outer membrane-derived vaccines have shown that PorA is a major inducer of bactericidal antibodies (6, 7, 8), making it a crucial component of any meningococcal vaccine. These antibodies are highly subtype-specific. Epidemic strains tend to be clonal and mainly express a single PorA subtype that changes only slowly over time (9). In hyperendemic situations, more variation is found but it is generally still possible to select a limited number of PorA subtypes that will cover most of the strains (10). However, PorA variation in both time and geography means that it is unlikely that a universal once-and-for-all meningococcal vaccine based on this protein alone can ever be made. This necessitates the use of vaccine strains with flexible PorA composition, in which new variants can be inserted into established production strains when required by new epidemiological circumstances. This chapter will describe methods to construct isogenic meningococcal strains with altered porA genes, which can be used both for vaccine production and as test strains to determine the precise epitope specificity of bactericidal antibodies directed against the various loops of PorA.
Fig. 1.

Topology model for PorA protein. Residues shown in boldface represent the surface-exposed P1.5 and P1.2 epitopes in loop 1 and 4. Residues marked with an asterisk represent the points of insertion into the KpnI site in loop 5 or 6.


  1. 1.
    van der Ley, P., Heckels, J. E., Virji, M., Hoogerhout, P., and Poolman, J. T. (1991) Topology of outer membrane porins in pathogenic Neisseria spp. Infect. Immun. 59, 2963–2971.PubMedGoogle Scholar
  2. 2.
    Derrick, J. P., Urwin, R., Suker, J., Feavers, I. M., and Maiden, M. C. J. (1999) Structural and evolutionary inference from molecular variation in Neisseria porins. Infect. Immun. 67, 2406–2413.PubMedGoogle Scholar
  3. 3.
    Sacchi, C. T., Lemos, A. P. S., Brandt, M. E., Whitney, A. M., Melles, C. E. A., Solari, C. A., et al. (1998) Proposed standardization of Neisseria meningitidis PorA variable-region typing nomenclature. Clin. Diagn. Lab. Immunol. 5, 845–855.PubMedGoogle Scholar
  4. 4.
    Suker, J., Feavers, I. M., and Maiden, M. C. J. (1996) Monoclonal antibody recognition of members of the meningococcal P1.10 variable region family: implications for serological typing and vaccine design. Microbiology 142, 63–69.CrossRefPubMedGoogle Scholar
  5. 5.
    Bart, A., Dankert, J., and van der Ende, A. (1999) Antigenic variation of the class 1 outer membrane protein in hyperendemic Neisseria meningitidis strains in The Netherlands. Infect. Immun. 67, 3842–3846.PubMedGoogle Scholar
  6. 6.
    Rosenqvist, E., Høiby, E. A., Wedege, E., Bryn, K., Kolberg, J., Klem, A., et al. (1995) Human antibody responses to meningococcal outer membrane antigens after three doses of the Norwegian group B meningococcal vaccine. Infect. Immun. 63, 4642–4652.PubMedGoogle Scholar
  7. 7.
    Tappero, J., Lagos, R., Ballesteros, A. M., Plikaytis, B., Williams, D., Dykes, J., et al. (1999) Immunogenicity of 2 serogroup B outer-membrane protein meningococcal vaccines. A randomized controlled trial in Chile. JAMA 281, 1520–1527.CrossRefPubMedGoogle Scholar
  8. 8.
    Cartwright, K., Morris, R., Rümke, H., Fox, A., Borrow, R., Begg, N., et al. (1999) Immunogenicity and reactogenicity in UK infants of a novel meningococcal vesicle vaccine containing multiple class 1 (PorA) outer membrane proteins. Vaccine 17, 2612–2619.CrossRefPubMedGoogle Scholar
  9. 9.
    Suker, J., Feavers, I. M., Achtman, M., Morelli, G., Wang, J.-F., and Maiden, M. C. J. (1994) The porA gene in serogroup A meningococci: evolutionary stability and mechanism of genetic variation. Mol. Microbiol. 12, 253–265.CrossRefPubMedGoogle Scholar
  10. 10.
    Scholten, R. J. P. M., Bijlmer, H. A., Poolman, J. T., Kuipers, B., Caugant, D. A., van Alphen, L., et al. (1993) Meningococcal disease in the Netherlands, 1958–1990: A steady increase in the incidence sinds 1982 partially caused by new serotypes and subtypes of Neisseria meningitidis. Clin. Infect. Dis. 16, 237–246.PubMedGoogle Scholar
  11. 11.
    van der Ley, P., van der Biezen, J., Hohenstein, P., Peeters, C., and Poolman, J. T. (1993) Use of transformation to construct antigenic hybrids of the class 1 outer membrane protein in Neisseria meningitidis. Infect. Immun. 61, 4217–4224.PubMedGoogle Scholar
  12. 12.
    Rouppe van der Voort, E., van der Ley, P., van der Biezen, J., George, S., Tunnela, O., van Dijken, H., et al. (1996) Specificity of human bactericidal antibodies against PorA P1.7,16 induced with a hexavalent meningococcal outer membrane vesicle vaccine. Infect. Immun. 64, 2745–2751.Google Scholar
  13. 13.
    van der Ley, P., van der Biezen, J., and Poolman, J. T. (1995) Construction of Neisseria meningitidis strains carrying multiple chromosomal copies of the porA gene for use in the production of a multivalent outer mebrane vesivle vaccine. Vaccine 13, 401–407.CrossRefPubMedGoogle Scholar
  14. 14.
    van der Ende, A., Hopman, C. T. P., Zaat, B., Oude Essink, B., Berkhout, B., and Dankert, J. (1995) Variable expression of class 1 outer membrane protein in Neisseria meningitidis strains is caused by variation in the spacing between −10 and −35 regions of the promoter. J. Bacteriol. 177, 2475–2480.PubMedGoogle Scholar
  15. 15.
    van der Ende, A., Hopman, C. T. P., and Dankert, J. (1999) Deletion of porA by recombination between clusters of repetitive extragenic palindromic sequences in Neisseria meningitidis. Infect. Immun. 67, 2928–2934.Google Scholar
  16. 16.
    Tommassen, J., Vermeij, P., Struyvé, M., Benz, R., and Poolman, J. T. (1990) Isolation of Neisseria meningitidis mutants deficient in class 1 (PorA) and class 3 (PorB) outer membrane proteins. Infect. Immun. 58, 1355–1359.PubMedGoogle Scholar
  17. 17.
    Goodman, S.D. and Scocca, J. J. (1988) Identification and arrangement of the DNA sequence recognized in specific transformation of Neisseria gonorrhoeae. Proc. Natl. Acad. Sci. USA 85, 6982–6986.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2001

Authors and Affiliations

  • Peter van der Ley
    • 1
  • Loek van Alphen
    • 1
  1. 1.Laboratory of Vaccine ResearchNational Institute of Public Health and the EnvironmentBilthovenThe Netherlands

Personalised recommendations