Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1996 Apr;64(4):1413–1419. doi: 10.1128/iai.64.4.1413-1419.1996

Identification and characterization of epitopes on the 120-kilodalton surface protein antigen of Rickettsia prowazekii with synthetic peptides.

W M Ching 1, H Wang 1, B Jan 1, G A Dasch 1
PMCID: PMC173934  PMID: 8606109

Abstract

The 120-kDa surface protein antigens (SPAs) of typhus rickettsiae are highly immunogenic and have been shown to be responsible for the species-specific serological reactions of the typhus group rickettsiae. To study the immunochemistry of these proteins, overlapping decapeptides encompassing the whole protein were synthesized on derivatized polyethylene pins. A modified enzyme-linked immunosorbent assay was used to identify epitopes recognized by rabbit hyperimmune antisera to Rickettsia prowazekii SPA. Eight distinct epitopes were mapped by this method in three regions. Four of the epitopes, which were located in the carboxyterminus of mature processed SPA, were strongly competitively inhibited by native folded SPA but not by intact rickettsiae, suggesting that they were on the SPA surface but not exposed on the rickettsial surface. Three of these epitopes were present on both R. prowazekii and Rickettsia typhi SPAs. The immunoreactivities of five epitopes were further characterized by synthesizing modified peptides. Glycine substitution experiments determined the critical residues in the epitopes. The dependence of binding of the peptide epitopes to the polyclonal antisera was mapped to single residues. The limited number and weak reactivity of linear peptide epitopes observed with human and rabbit sera, possibly due to a lack of the methylated amino acids which are present in rickettsia-derived SPA, suggest that the present approach will not provide useful synthetic antigens for diagnosis of typhus infections.

Full Text

The Full Text of this article is available as a PDF (248.0 KB).

Selected References

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

  1. Alexander H., Alexander S., Getzoff E. D., Tainer J. A., Geysen H. M., Lerner R. A. Altering the antigenicity of proteins. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3352–3356. doi: 10.1073/pnas.89.8.3352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blaser M. J., Pei Z. Pathogenesis of Campylobacter fetus infections: critical role of high-molecular-weight S-layer proteins in virulence. J Infect Dis. 1993 Feb;167(2):372–377. doi: 10.1093/infdis/167.2.372. [DOI] [PubMed] [Google Scholar]
  3. Carl M., Dasch G. A. The importance of the crystalline surface layer protein antigens of rickettsiae in T-cell immunity. J Autoimmun. 1989 Jun;2 (Suppl):81–91. doi: 10.1016/0896-8411(89)90119-4. [DOI] [PubMed] [Google Scholar]
  4. Carl M., Dobson M. E., Ching W. M., Dasch G. A. Characterization of the gene encoding the protective paracrystalline-surface-layer protein of Rickettsia prowazekii: presence of a truncated identical homolog in Rickettsia typhi. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8237–8241. doi: 10.1073/pnas.87.21.8237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carter J. M. Epitope mapping of a protein using the Geysen (PEPSCAN) procedure. Methods Mol Biol. 1994;36:207–223. doi: 10.1385/0-89603-274-4:207. [DOI] [PubMed] [Google Scholar]
  6. Ching W. M., Carl M., Dasch G. A. Mapping of monoclonal antibody binding sites on CNBr fragments of the S-layer protein antigens of Rickettsia typhi and Rickettsia prowazekii. Mol Immunol. 1992 Jan;29(1):95–105. doi: 10.1016/0161-5890(92)90161-p. [DOI] [PubMed] [Google Scholar]
  7. Ching W. M., Dasch G. A., Carl M., Dobson M. E. Structural analyses of the 120-kDa serotype protein antigens of typhus group rickettsiae. Comparison with other S-layer proteins. Ann N Y Acad Sci. 1990;590:334–351. doi: 10.1111/j.1749-6632.1990.tb42241.x. [DOI] [PubMed] [Google Scholar]
  8. Ching W. M., Wychowski C., Beach M. J., Wang H., Davies C. L., Carl M., Bradley D. W., Alter H. J., Feinstone S. M., Shih J. W. Interaction of immune sera with synthetic peptides corresponding to the structural protein region of hepatitis C virus. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3190–3194. doi: 10.1073/pnas.89.8.3190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  10. Churilla A., Ching W. M., Dasch G. A., Carl M. Human T lymphocyte recognition of cyanogen bromide fragments of the surface protein of Rickettsia typhi. Ann N Y Acad Sci. 1990;590:215–220. doi: 10.1111/j.1749-6632.1990.tb42222.x. [DOI] [PubMed] [Google Scholar]
  11. Dasch G. A. Isolation of species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii for immunodiagnosis and immunoprophylaxis. J Clin Microbiol. 1981 Sep;14(3):333–341. doi: 10.1128/jcm.14.3.333-341.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dasch G. A., Samms J. R., Williams J. C. Partial purification and characterization of the major species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii identified by rocket immunoelectrophoresis. Infect Immun. 1981 Jan;31(1):276–288. doi: 10.1128/iai.31.1.276-288.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Eremeeva M. E., Balayeva N. M., Raoult D. Serological response of patients suffering from primary and recrudescent typhus: comparison of complement fixation reaction, Weil-Felix test, microimmunofluorescence, and immunoblotting. Clin Diagn Lab Immunol. 1994 May;1(3):318–324. doi: 10.1128/cdli.1.3.318-324.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Gilmore R. D., Jr, Cieplak W., Jr, Policastro P. F., Hackstadt T. The 120 kilodalton outer membrane protein (rOmp B) of Rickettsia rickettsii is encoded by an unusually long open reading frame: evidence for protein processing from a large precursor. Mol Microbiol. 1991 Oct;5(10):2361–2370. doi: 10.1111/j.1365-2958.1991.tb02082.x. [DOI] [PubMed] [Google Scholar]
  16. Gilmore R. D., Jr, Joste N., McDonald G. A. Cloning, expression and sequence analysis of the gene encoding the 120 kD surface-exposed protein of Rickettsia rickettsii. Mol Microbiol. 1989 Nov;3(11):1579–1586. doi: 10.1111/j.1365-2958.1989.tb00143.x. [DOI] [PubMed] [Google Scholar]
  17. Hackstadt T., Messer R., Cieplak W., Peacock M. G. Evidence for proteolytic cleavage of the 120-kilodalton outer membrane protein of rickettsiae: identification of an avirulent mutant deficient in processing. Infect Immun. 1992 Jan;60(1):159–165. doi: 10.1128/iai.60.1.159-165.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hahn M. J., Kim K. K., Kim I., Chang W. H. Cloning and sequence analysis of the gene encoding the crystalline surface layer protein of Rickettsia typhi. Gene. 1993 Oct 29;133(1):129–133. doi: 10.1016/0378-1119(93)90237-w. [DOI] [PubMed] [Google Scholar]
  19. Kay W. W., Phipps B. M., Ishiguro E. E., Olafson R. W., Trust T. J. Surface layer virulence A-proteins from Aeromonas salmonicida strains. Can J Biochem Cell Biol. 1984 Nov;62(11):1064–1071. doi: 10.1139/o84-137. [DOI] [PubMed] [Google Scholar]
  20. Palmer E. L., Martin M. L., Mallavia L. Ultrastucture of the surface of Rickettsia prowazeki and Rickettsia akari. Appl Microbiol. 1974 Oct;28(4):713–716. doi: 10.1128/am.28.4.713-716.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Raoult D., Dasch G. A. The line blot: an immunoassay for monoclonal and other antibodies. Its application to the serotyping of gram-negative bacteria. J Immunol Methods. 1989 Dec 20;125(1-2):57–65. doi: 10.1016/0022-1759(89)90078-1. [DOI] [PubMed] [Google Scholar]
  22. Rodda S. J., Geysen H. M., Mason T. J., Schoofs P. G. The antibody response to myoglobin--I. Systematic synthesis of myoglobin peptides reveals location and substructure of species-dependent continuous antigenic determinants. Mol Immunol. 1986 Jun;23(6):603–610. doi: 10.1016/0161-5890(86)90096-9. [DOI] [PubMed] [Google Scholar]
  23. Shemyakin M. M., Ovchinnikov Y. A., Ivanov V. T. Topochemical investigations of peptide systems. Angew Chem Int Ed Engl. 1969 Jul;8(7):492–499. doi: 10.1002/anie.196904921. [DOI] [PubMed] [Google Scholar]
  24. Wade D., Boman A., Wåhlin B., Drain C. M., Andreu D., Boman H. G., Merrifield R. B. All-D amino acid-containing channel-forming antibiotic peptides. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4761–4765. doi: 10.1073/pnas.87.12.4761. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES