Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1996 Jul;64(7):2457–2466. doi: 10.1128/iai.64.7.2457-2466.1996

Epitope mapping of B-cell determinants on the 15-kilodalton lipoprotein of Treponema pallidum (Tpp15) with synthetic peptides.

R E Baughn 1, M Demecs 1, L H Taber 1, D M Musher 1
PMCID: PMC174098  PMID: 8698467

Abstract

The antigenicity of the 15-kDa lipoprotein of Treponema pallidum (Tpp15 or TpN15) was comprehensively evaluated in epitope-scanning studies with overlapping deca- and octapeptides and polygonal rabbit and human infant immunoglobulins (Igs) and antisera. This approach enabled us to identify potentially important regions and to determine the optimal dilutions of Igs or antisera for use in further studies. IgM and IgG from both species were capable of recognizing multiple, continuous epitopes. A total of 13 peptides, principally clustered in the central regions of the protein, were recognized by all syphilitic sera and Ig fractions. On the basis of window analyses, frequency profiles, and alanine substitution studies, five heptapeptides were selected for mimetic studies. Two of these five immunodominant, continuous epitopes initially appeared to be species specific; however, antisera elicited against mimetics of all five epitopes were polyspecific, recognizing similar motifs on several other treponemal proteins, including those of avirulent organisms. The only mimetic which yielded positive reactions with infant IgM and syphilitic sera in the absence of cross-reactions with rabbit antisera to avirulent treponemes was the variant of the VMYASSG motif. These findings are relevant to the development of simple, inexpensive assays for the serodiagnosis of active syphilis.

Full Text

The Full Text of this article is available as a PDF (436.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Baker-Zander S. A., Lukehart S. A. Antigenic cross-reactivity between Treponema pallidum and other pathogenic members of the family Spirochaetaceae. Infect Immun. 1984 Oct;46(1):116–121. doi: 10.1128/iai.46.1.116-121.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Batteiger B. E. The major outer membrane protein of a single Chlamydia trachomatis serovar can possess more than one serovar-specific epitope. Infect Immun. 1996 Feb;64(2):542–547. doi: 10.1128/iai.64.2.542-547.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baughn R. E. Antibody-independent interactions of fibronectin, C1q, and human neutrophils with Treponema pallidum. Infect Immun. 1986 Nov;54(2):456–464. doi: 10.1128/iai.54.2.456-464.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baughn R. E. Demonstration and immunochemical characterization of natural, autologous anti-idiotypic antibodies throughout the course of experimental syphilis. Infect Immun. 1990 Mar;58(3):766–773. doi: 10.1128/iai.58.3.766-773.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Baughn R. E., McNeely M. C., Jorizzo J. L., Musher D. M. Characterization of the antigenic determinants and host components in immune complexes from patients with secondary syphilis. J Immunol. 1986 Feb 15;136(4):1406–1414. [PubMed] [Google Scholar]
  7. Baughn R. E., Musher D. M. Isolation and preliminary characterization of circulating immune complexes from rabbits with experimental syphilis. Infect Immun. 1983 Nov;42(2):579–584. doi: 10.1128/iai.42.2.579-584.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Becker P. S., Akins D. R., Radolf J. D., Norgard M. V. Similarity between the 38-kilodalton lipoprotein of Treponema pallidum and the glucose/galactose-binding (MglB) protein of Escherichia coli. Infect Immun. 1994 Apr;62(4):1381–1391. doi: 10.1128/iai.62.4.1381-1391.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bell A. W., Buckel S. D., Groarke J. M., Hope J. N., Kingsley D. H., Hermodson M. A. The nucleotide sequences of the rbsD, rbsA, and rbsC genes of Escherichia coli K12. J Biol Chem. 1986 Jun 15;261(17):7652–7658. [PubMed] [Google Scholar]
  10. Berzins K., Perlmann H., Wåhlin B., Carlsson J., Wahlgren M., Udomsangpetch R., Björkman A., Patarroyo M. E., Perlmann P. Rabbit and human antibodies to a repeated amino acid sequence of a Plasmodium falciparum antigen, Pf 155, react with the native protein and inhibit merozoite invasion. Proc Natl Acad Sci U S A. 1986 Feb;83(4):1065–1069. doi: 10.1073/pnas.83.4.1065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bockenstedt L. K., Gee R. J., Mamula M. J. Self-peptides in the initiation of lupus autoimmunity. J Immunol. 1995 Apr 1;154(7):3516–3524. [PubMed] [Google Scholar]
  12. Borenstein L. A., Radolf J. D., Fehniger T. E., Blanco D. R., Miller J. N., Lovett M. A. Immunization of rabbits with recombinant Treponema pallidum surface antigen 4D alters the course of experimental syphilis. J Immunol. 1988 Apr 1;140(7):2415–2421. [PubMed] [Google Scholar]
  13. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  14. Briand J. P., Muller S., Van Regenmortel M. H. Synthetic peptides as antigens: pitfalls of conjugation methods. J Immunol Methods. 1985 Apr 8;78(1):59–69. doi: 10.1016/0022-1759(85)90329-1. [DOI] [PubMed] [Google Scholar]
  15. Broliden P. A., Rudén U., Ouattara A. S., de Thé G., Sølver E., Trojnar J., Wahren B. Specific synthetic peptides for detection of and discrimination between HIV-1 and HIV-2 infection. J Acquir Immune Defic Syndr. 1991;4(10):952–958. [PubMed] [Google Scholar]
  16. Champion C. I., Miller J. N., Borenstein L. A., Lovett M. A., Blanco D. R. Immunization with Treponema pallidum endoflagella alters the course of experimental rabbit syphilis. Infect Immun. 1990 Sep;58(9):3158–3161. doi: 10.1128/iai.58.9.3158-3161.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  18. Dassa E., Hofnung M. Sequence of gene malG in E. coli K12: homologies between integral membrane components from binding protein-dependent transport systems. EMBO J. 1985 Sep;4(9):2287–2293. doi: 10.1002/j.1460-2075.1985.tb03928.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Dobson S. R., Taber L. H., Baughn R. E. Recognition of Treponema pallidum antigens by IgM and IgG antibodies in congenitally infected newborns and their mothers. J Infect Dis. 1988 May;157(5):903–910. doi: 10.1093/infdis/157.5.903. [DOI] [PubMed] [Google Scholar]
  20. Doi H., Kitajima M., Watanabe I., Kikuchi Y., Matsuzawa F., Aikawa S., Takiguchi K., Ohno S. Diverse incidences of individual oligopeptides (dipeptidic to hexapeptidic) in proteins of human, bakers' yeast, and Escherichia coli origin registered in the Swiss-Prot data base. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2879–2883. doi: 10.1073/pnas.92.7.2879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fridell E., Cohen B. J., Wahren B. Evaluation of a synthetic-peptide enzyme-linked immunosorbent assay for immunoglobulin M to human parvovirus B19. J Clin Microbiol. 1991 Jul;29(7):1376–1381. doi: 10.1128/jcm.29.7.1376-1381.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Geysen H. M., Meloen R. H., Barteling S. J. Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc Natl Acad Sci U S A. 1984 Jul;81(13):3998–4002. doi: 10.1073/pnas.81.13.3998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Geysen H. M. Molecular technology: peptide epitope mapping and the pin technology. Southeast Asian J Trop Med Public Health. 1990 Dec;21(4):523–533. [PubMed] [Google Scholar]
  24. Geysen H. M., Rodda S. J., Mason T. J., Tribbick G., Schoofs P. G. Strategies for epitope analysis using peptide synthesis. J Immunol Methods. 1987 Sep 24;102(2):259–274. doi: 10.1016/0022-1759(87)90085-8. [DOI] [PubMed] [Google Scholar]
  25. Hensel U., Wellensiek H. J., Bhakdi S. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting as a serological tool in the diagnosis of syphilitic infections. J Clin Microbiol. 1985 Jan;21(1):82–87. doi: 10.1128/jcm.21.1.82-87.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hogg R. W., Voelker C., Von Carlowitz I. Nucleotide sequence and analysis of the mgl operon of Escherichia coli K12. Mol Gen Genet. 1991 Oct;229(3):453–459. doi: 10.1007/BF00267469. [DOI] [PubMed] [Google Scholar]
  27. Innis B. L., Thirawuth V., Hemachudha C. Identification of continuous epitopes of the envelope glycoprotein of dengue type 2 virus. Am J Trop Med Hyg. 1989 Jun;40(6):676–687. doi: 10.4269/ajtmh.1989.40.676. [DOI] [PubMed] [Google Scholar]
  28. James J. A., Gross T., Scofield R. H., Harley J. B. Immunoglobulin epitope spreading and autoimmune disease after peptide immunization: Sm B/B'-derived PPPGMRPP and PPPGIRGP induce spliceosome autoimmunity. J Exp Med. 1995 Feb 1;181(2):453–461. doi: 10.1084/jem.181.2.453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kitagawa T., Aikawa T. Enzyme coupled immunoassay of insulin using a novel coupling reagent. J Biochem. 1976 Jan;79(1):233–236. doi: 10.1093/oxfordjournals.jbchem.a131053. [DOI] [PubMed] [Google Scholar]
  30. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  31. Lachumanan R., Devi S., Cheong Y. M., Rodda S. J., Pang T. Epitope mapping of the Sta58 major outer membrane protein of Rickettsia tsutsugamushi. Infect Immun. 1993 Oct;61(10):4527–4531. doi: 10.1128/iai.61.10.4527-4531.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lewis L. L., Taber L. H., Baughn R. E. Evaluation of immunoglobulin M western blot analysis in the diagnosis of congenital syphilis. J Clin Microbiol. 1990 Feb;28(2):296–302. doi: 10.1128/jcm.28.2.296-302.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Linde A., Kallin B., Dillner J., Andersson J., Jägdahl L., Lindvall A., Wahren B. Evaluation of enzyme-linked immunosorbent assays with two synthetic peptides of Epstein-Barr virus for diagnosis of infectious mononucleosis. J Infect Dis. 1990 May;161(5):903–909. doi: 10.1093/infdis/161.5.903. [DOI] [PubMed] [Google Scholar]
  34. Liu F. T., Zinnecker M., Hamaoka T., Katz D. H. New procedures for preparation and isolation of conjugates of proteins and a synthetic copolymer of D-amino acids and immunochemical characterization of such conjugates. Biochemistry. 1979 Feb 20;18(4):690–693. doi: 10.1021/bi00571a022. [DOI] [PubMed] [Google Scholar]
  35. Lukehart S. A., Baker-Zander S. A., Gubish E. R., Jr Identification of Treponema pallidum antigens: comparison with a nonpathogenic treponeme. J Immunol. 1982 Aug;129(2):833–838. [PubMed] [Google Scholar]
  36. Lundkvist A., Björsten S., Niklasson B., Ahlborg N. Mapping of B-cell determinants in the nucleocapsid protein of Puumala virus: definition of epitopes specific for acute immunoglobulin G recognition in humans. Clin Diagn Lab Immunol. 1995 Jan;2(1):82–86. doi: 10.1128/cdli.2.1.82-86.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mamula M. J., Janeway C. A., Jr Do B cells drive the diversification of immune responses? Immunol Today. 1993 Apr;14(4):151–154. doi: 10.1016/0167-5699(93)90274-O. [DOI] [PubMed] [Google Scholar]
  38. Mamula M. J., Lin R. H., Janeway C. A., Jr, Hardin J. A. Breaking T cell tolerance with foreign and self co-immunogens. A study of autoimmune B and T cell epitopes of cytochrome c. J Immunol. 1992 Aug 1;149(3):789–795. [PubMed] [Google Scholar]
  39. Müller F., Moskophidis M., Borkhardt H. L. Detection of immunoglobulin M antibodies to Treponema pallidum in a modified enzyme-linked immunosorbent assay. Eur J Clin Microbiol. 1987 Feb;6(1):35–39. doi: 10.1007/BF02097187. [DOI] [PubMed] [Google Scholar]
  40. Norris S. J. Polypeptides of Treponema pallidum: progress toward understanding their structural, functional, and immunologic roles. Treponema Pallidum Polypeptide Research Group. Microbiol Rev. 1993 Sep;57(3):750–779. doi: 10.1128/mr.57.3.750-779.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ohno S. Many peptide fragments of alien antigens are homologous with host proteins, thus canalizing T-cell responses. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3065–3068. doi: 10.1073/pnas.88.8.3065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Purcell B. K., Chamberlain N. R., Goldberg M. S., Andrews L. P., Robinson E. J., Norgard M. V., Radolf J. D. Molecular cloning and characterization of the 15-kilodalton major immunogen of Treponema pallidum. Infect Immun. 1989 Dec;57(12):3708–3714. doi: 10.1128/iai.57.12.3708-3714.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Purcell B. K., Swancutt M. A., Radolf J. D. Lipid modification of the 15 kiloDalton major membrane immunogen of Treponema pallidum. Mol Microbiol. 1990 Aug;4(8):1371–1379. doi: 10.1111/j.1365-2958.1990.tb00716.x. [DOI] [PubMed] [Google Scholar]
  44. Richards F. M., Knowles J. R. Glutaraldehyde as a protein cross-linkage reagent. J Mol Biol. 1968 Oct 14;37(1):231–233. doi: 10.1016/0022-2836(68)90086-7. [DOI] [PubMed] [Google Scholar]
  45. Saurin W., Köster W., Dassa E. Bacterial binding protein-dependent permeases: characterization of distinctive signatures for functionally related integral cytoplasmic membrane proteins. Mol Microbiol. 1994 Jun;12(6):993–1004. doi: 10.1111/j.1365-2958.1994.tb01087.x. [DOI] [PubMed] [Google Scholar]
  46. Schulz G. E. A critical evaluation of methods for prediction of protein secondary structures. Annu Rev Biophys Biophys Chem. 1988;17:1–21. doi: 10.1146/annurev.bb.17.060188.000245. [DOI] [PubMed] [Google Scholar]
  47. Sánchez P. J., McCracken G. H., Jr, Wendel G. D., Olsen K., Threlkeld N., Norgard M. V. Molecular analysis of the fetal IgM response to Treponema pallidum antigens: implications for improved serodiagnosis of congenital syphilis. J Infect Dis. 1989 Mar;159(3):508–517. doi: 10.1093/infdis/159.3.508. [DOI] [PubMed] [Google Scholar]
  48. Topfer F., Gordon T., McCluskey J. Intra- and intermolecular spreading of autoimmunity involving the nuclear self-antigens La (SS-B) and Ro (SS-A). Proc Natl Acad Sci U S A. 1995 Jan 31;92(3):875–879. doi: 10.1073/pnas.92.3.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Versalovic J., Nash Z. D., Carinhas R., Musher D. M., Baughn R. E. Immunoglobulin class and subclass restriction of autoimmune responses in secondary syphilis. Clin Exp Immunol. 1990 Jun;80(3):381–386. doi: 10.1111/j.1365-2249.1990.tb03297.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Westhof E., Altschuh D., Moras D., Bloomer A. C., Mondragon A., Klug A., Van Regenmortel M. H. Correlation between segmental mobility and the location of antigenic determinants in proteins. Nature. 1984 Sep 13;311(5982):123–126. doi: 10.1038/311123a0. [DOI] [PubMed] [Google Scholar]
  51. Wicher V., Zabek J., Wicher K. Kinetics of pathogen-specific humoral response in Treponema pallidum-infected young and old inbred strain 2 guinea pigs. Clin Exp Immunol. 1989 Jul;77(1):144–150. [PMC free article] [PubMed] [Google Scholar]
  52. Wicher V., Zabek J., Wicher K. Pathogen-specific humoral response in Treponema pallidum-infected humans, rabbits, and guinea pigs. J Infect Dis. 1991 Apr;163(4):830–836. [PubMed] [Google Scholar]
  53. Zhong G. M., Reid R. E., Brunham R. C. Mapping antigenic sites on the major outer membrane protein of Chlamydia trachomatis with synthetic peptides. Infect Immun. 1990 May;58(5):1450–1455. doi: 10.1128/iai.58.5.1450-1455.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES