Denaturing gradient gel electrophoresis analysis for the detection of point mutations in the major outer-membrane protein gene Free

Abstract

Summary

Fifty clinical strains of were studied by denaturing gradient gel electrophoresis (DGGE) of bacterial DNA amplified by the polymerase chain reaction (PCR). The strains belonged to the three most commonly encountered serovars in developed countries—D, E and F. Six reference strains, including the serovar Da strain, were also studied. The DNA sequences explored encompassed the four variable domains (VDs) of , the gene encoding the major outer-membrane protein (MOMP). The corresponding regions in the MOMP contain the species-, subspecies- and serovar-specific epitopes. The four distinct serovars were clearly differentiated by specific migration pattern. No sequence variations were observed among strains of serovar F. However, variant strains within serovars D and E were found, which exhibited migration patterns different from those of the reference strains and these were sequenced directly. According to the observed sequence variations, serovar D strains could be divided into three stable representative groups (D, D1 and D2). Two variants were identified among serovar E strains. No biological differences were observed for the variant strains in terms of growth properties, ecology or pathogenicity. All the nucleotide substitutions detected in the VDs were non-synonymous at the protein level and, for the serovar D strains, could account for differences identified by specific monoclonal antibodies. These substitutions could be involved in antigenic drift, driven by the immune pressure of the host, leading to the emergence of serovariants. The data may explain, in part, chlamydial infection recurrences and could have critical implications for developing rational vaccine strategies.

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1995-07-01
2024-03-29
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References

  1. Grayston J. T., Wang S. P. New knowledge of Chlamydiae and the diseases they cause. J Infect Dis 1975; 132:87–105
    [Google Scholar]
  2. Wang S. P., Kuo C. C., Barnes R. C., Stephens R. S., Grayston J. T. Immunotyping of Chlamydia trachomatis with monoclonal antibodies. J Infect Dis 1985; 152:791–800
    [Google Scholar]
  3. Wang S. P., Grayston J. T. Three new serovars of Chlamydia trachomatis: Da. la. and L2a. J Infect Dis 1991; 163:403–405
    [Google Scholar]
  4. Zhang Y. X., Stewart S., Joseph T., Taylor H. R., Caldwell H. D. Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis. J Immunol 1991; 138:575–581
    [Google Scholar]
  5. Baehr W., Zhang Y. X., Joseph T. Mapping antigenic domains expressed by Chlamydia trachomatis major outer membrane protein genes. Proc Natl Acad Sci USA 1988; 85:4000–4004
    [Google Scholar]
  6. Stephens R. S., Wagar E. A., Schoolnik G. K. High-resolution mapping of serovar-specific and common antigenic determinants of the major outer membrane protein of Chlamydia trachomatis. J Exp Med 1988; 167:817–831
    [Google Scholar]
  7. Su H., Morrison R. P., Watkins N. G., Caldwell H. D. Identification and characterization of T helper cell epitopes of the major outer membrane protein of Chlamydia trachomatis. J Exp Med 1990; 172:203–212
    [Google Scholar]
  8. Dean D., Patton M., Stephens R. S. Direct sequence evaluation of the major outer membrane protein gene variant regions of Chlamydia trachomatis subtypes D. T. and L2`. Infect Immun 1991; 59:1579–1582
    [Google Scholar]
  9. Hamilton P. T., Malinowski D. P. Nucleotide sequence of the major outer membrane protein gene from Chlamydia trachomatis serovar H. Nucleic Acids Res 1989; 17:8366
    [Google Scholar]
  10. Hayes L. J., Clarke I. N. Nucleotide sequence of the major outer membrane protein gene of Chlamydia trachomatis strain A/SA1/OT. Nucleic Acids Res 1990; 18:6136
    [Google Scholar]
  11. Hayes L. J., Pickett M. A., Conlan J. W. The major outer-membrane proteins of Chlamydia trachomatis serovars A and B: intra-serovar amino acid changes do not alter specificities of serovar- and C subspecies-reactive antibody-binding domains. J Gen Microbiol 1990; 136:1559–1566
    [Google Scholar]
  12. Peterson E. M., Markoff B. A., de la Maza L. M. The major outer membrane protein nucleotide sequence of Chlamydia trachomatis, serovar E. Nucleic Acids Res 1990; 18:3414
    [Google Scholar]
  13. Pickett M. A., Ward M. E., Clarke I. N. Complete nucleotide sequence of the major outer membrane protein gene from Chlamydia trachomatis serovar LI. FEMS Microbiol Lett 1987; 42:185–190
    [Google Scholar]
  14. Sayada C., Denamur E., Elion J. Complete sequence of the major outer membrane protein-encoding gene of Chlamydia trachomatis serovar Da. Gene 1992; 120:129–130
    [Google Scholar]
  15. Stephens R. S., Sanchez-Pescador R., Wagar E. A., Irtouye C., Urdea M. S. Diversity of Chlamydia trachomatis major outer membrane protein genes. J Bacteriol 1987; 169:3879–3885
    [Google Scholar]
  16. Yuan Y., Zhang Y. X., Watkins N. G., Caldwell H. D. Nucleotide and deduced amino acid sequences for the four variable domains of the major outer membrane proteins of the 15 Chlamydia trachomatis serovars. Infect Immun 1989; 57:1040–1049
    [Google Scholar]
  17. Zhang Y. X., Morrison S. G., Caldwell H. D. The nucleotide sequence of major outer membrane protein gene of Chlamydia trachomatis serovar F. Nucleic Acids Res 1990; 18:1061
    [Google Scholar]
  18. Lucero M. E., Kuo C. C. Neutralization of Chlamydia trachomatis cell culture infection by serovar-specific monoclonal antibodies. Infect Immun 1985; 50:595–597
    [Google Scholar]
  19. Frost E. H., Deslandes S., Veilleux S., Bourgaux-Ramoisy D. Typing Chlamydia trachomatis by detection of restriction fragment length polymorphism in the gene encoding the major outer membrane protein. J Infect Dis 1991; 163:1103–1107
    [Google Scholar]
  20. Rodriguez P., Vekris A., de Barbeyrac B., Dutihl B., Bonnet J., Bebear C. Typing of Chlamydia trachomatis by restriction endonuclease analysis of the amplified major outer membrane protein gene. J Clin Microbiol 1991; 29:1132–1136
    [Google Scholar]
  21. Sayada C., Denamur E., Orfila J., Catalan F., Elion J. Rapid genotyping of the Chlamydia trachomatis major outer membrane protein by the polymerase chain reaction. FEMS Microbiol 1991; 83:73–78
    [Google Scholar]
  22. Dean D., Schachter J., Dawson C. R., Stephens R. S. Comparison of the major outer membrane protein variant sequence regions of the B/Ba isolates: a molecular epidemiologic approach to Chlamydia trachomatis infections. J Infect Dis 1992; 166:383–392
    [Google Scholar]
  23. Poole E., Lammont I. Chlamydia trachomatis serovar differentiation by direct sequence analysis of the variable segment 4 region of the major outer membrane protein gene. Infect Immun 1992; 60:1089–1094
    [Google Scholar]
  24. Fischer S. G., Lerman L. S. Separation of random fragments of DNA according to properties of their sequences. Proc Natl AcadSci USA 1980; 77:4420–4424
    [Google Scholar]
  25. Fischer S. G., Lerman L. S. DNA fragments differing by single-base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Proc Natl AcadSci USA 1983; 80:1579–1583
    [Google Scholar]
  26. Myers R. M., Fischer S. G., Lerman L. S., Maniatis T. Nearly all single base substitutions in DNA fragments joined to GC-clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Res 1985; 13:3131–3145
    [Google Scholar]
  27. Myers R. M., Maniatis T., Lerman L. S. Detection and localization of single base changes by denaturing gradient gel electrophoresis. Methods Enzymol 1987; 155:501–527
    [Google Scholar]
  28. Lerman L. S., Silverstein K. Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis. Methods Enzymol 1987; 155:482–501
    [Google Scholar]
  29. Sheffield V. C., Cox D. R., Lerman L. S., Myers R. M. Attachment of a 40-base pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci USA 1989; 86:232–236
    [Google Scholar]
  30. Barnes R. C., Rompalo A. M., Stamm W. E. Comparison of Chlamydia trachomatis serovars causing rectal and cervical infections. J Infect Dis 1987; 156:953–958
    [Google Scholar]
  31. Moncan T., Eb F., Orfila J. Monoclonal antibodies in serovar determination of 53 Chlamydia trachomatis isolates from Amiens, France. Res Microbiol 1990; 141:695–701
    [Google Scholar]
  32. Vretou E., Mentis A., Psarrou E., Tsoumaris L., Conidou G., & Spiliopolou D. Unusual prevalence of the rare serovar Da of Chlamydia trachomatis in Greece detected by monoclonal antibodies. Sex Transm Dis 1992; 19:78–83
    [Google Scholar]
  33. Mullis K. B. The polymerase chain reaction in an anemic mode: how to avoid cold oligodeoxyribonuclear fusion. PCR Methods Applic 1991; 1:1–4
    [Google Scholar]
  34. Rychlik W., Rhoads R. E. A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Res 1989; 17:8543–8551
    [Google Scholar]
  35. Attree O., Vidaud D., Vidaud M., Amselem S., Lavergne J. M., Goossens M. Mutations in the catalytic domain of human coagulation factor IX: rapid characterization by direct genomic sequencing of DNA fragments displaying an altered melting behavior. Genomics 1989; 4:266–272
    [Google Scholar]
  36. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977; 74:5463–5467
    [Google Scholar]
  37. Noll W. W., Collins M. Detection of human DNA polymorphisms with a simplified denaturing gradient gel electrophoresis technique. Proc Natl Acad Sci USA 1987; 84:3339–3343
    [Google Scholar]
  38. Traystman M. D., Higuchi M., Kasper C. K., Antonarakis S. E., Kazazian H. H. Use of denaturing gradient gel electrophoresis to detect point mutations in the factor VIII gene. Genomics 1990; 6:293–301
    [Google Scholar]
  39. Kogan S., Gitschier J. Mutations and a polymorphism in the factor VIII gene discovered by denaturing gradient gel electrophoresis. Proc Natl Acad Sci USA 1990; 87:2092–2096
    [Google Scholar]
  40. Cai S. P., Kan Y. W. Identification of the multiple ß-thalassemia mutations by denaturing gradient gel electrophoresis. J Clin Invest 1990; 85:550–553
    [Google Scholar]
  41. Tachdjian G., Benabdennebi M., Guidal C., Sayada C., Lapoumeroulie C., Elion J. Analysis of the 5′ flanking sequence of the gamma globin gene by denaturing gradient gel electrophoresis confirms the heterogeneity of the Bantu ß haplotype. Human Genet 1992; 90:23–26
    [Google Scholar]
  42. Ferec C., Audrezet M. P., Mercier B. Detection of over 98% cystic fibrosis mutations in a Celtic population. Nature Genetics 1992; 1:188–191
    [Google Scholar]
  43. Lampe M. F., Suchland R. J., Stamm W. E. Nucleotide sequence of the major outer membrane protein of the Chlamydia trachomatis D serovar and two D variants. In Bowie W. R., Caldwell H. D., Jones R. P. (eds) Chlamydial infections. Proceedings of the Seventh International Symposium on Human Chlamydial Infections Cambridge: Cambridge University Press; 199097–100
    [Google Scholar]
  44. van der Ploeg L. H. T., Gottesdiener K., Lee M. G. S. Antigenic variation in African trypanosomes. Trends Genet 1992; 8:452–457
    [Google Scholar]
  45. Zhong G., Reid R. E., Brunham R. C. Mapping antigenic sites on the major outer membrane protein of Chlamydia trachomatis with synthetic peptides. Infect Immun 1990; 58:1450–1455
    [Google Scholar]
  46. Suchland R. J., Stamm W. E. Simplified microtiter cell culture method for rapid immunotyping of Chlamydia trachomatis. J Clin Microbiol 1991; 29:1333–1338
    [Google Scholar]
  47. Anderson B., Ying J. H., Lewis D. E., Gibbs R. A. Rapid characterization of HIV-1 sequence diversity using denaturing gradient gel electrophoresis and direct automated DNA sequencing of PCR products. PCR Methods Applic 1993; 2:293–300
    [Google Scholar]
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