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. 1988 Sep;170(9):4072–4082. doi: 10.1128/jb.170.9.4072-4082.1988

Antigenic relatedness and N-terminal sequence homology define two classes of periplasmic flagellar proteins of Treponema pallidum subsp. pallidum and Treponema phagedenis.

S J Norris 1, N W Charon 1, R G Cook 1, M D Fuentes 1, R J Limberger 1
PMCID: PMC211411  PMID: 3045083

Abstract

The periplasmic flagella of many spirochetes contain multiple proteins. In this study, two-dimensional electrophoresis, Western blotting (immunoblotting), immunoperoxidase staining, and N-terminal amino acid sequence analysis were used to characterize the individual periplasmic flagellar proteins of Treponema pallidum subsp. pallidum (Nichols strain) and T. phagedenis Kazan 5. Purified T. pallidum periplasmic flagella contained six proteins (Mrs = 37,000, 34,500, 33,000, 30,000, 29,000, and 27,000), whereas T. phagedenis periplasmic flagella contained a major 39,000-Mr protein and a group of two major and two minor 33,000- to 34,000-Mr polypeptide species; 37,000- and 30,000-Mr proteins were also present in some T. phagedenis preparations. Immunoblotting with monospecific antisera and monoclonal antibodies and N-terminal sequence analysis indicated that the major periplasmic flagellar proteins were divided into two distinct classes, designated class A and class B. Class A proteins consisted of the 37-kilodalton (kDa) protein of T. pallidum and the 39-kDa polypeptide of T. phagedenis; class B included the T. pallidum 34.5-, 33-, and 30-kDa proteins and the four 33- and 34-kDa polypeptide species of T. phagedenis. The proteins within each class were immunologically cross-reactive and possessed similar N-terminal sequences (67 to 95% homology); no cross-reactivity or sequence homology was evident between the two classes. Anti-class A or anti-class B antibodies did not react with the 29- or 27-kDa polypeptides of T. pallidum or the 37- and 30-kDa T. phagedenis proteins, indicating that these proteins are antigenically unrelated to the class A and class B proteins. The lack of complete N-terminal sequence homology among the major periplasmic flagellar proteins of each organism indicates that they are most likely encoded by separate structural genes. Furthermore, the N-terminal sequences of T. phagedenis and T. pallidum periplasmic flagellar proteins are highly conserved, despite the genetic dissimilarity of these two species.

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Selected References

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

  1. Bailey M. J., Cockayne A., Penn C. W. Production of murine monoclonal antibodies to the major axial filament polypeptide of Treponema pallidum. J Gen Microbiol. 1987 Jul;133(7):1805–1813. doi: 10.1099/00221287-133-7-1805. [DOI] [PubMed] [Google Scholar]
  2. Bharier M., Allis D. Purification and characterization of axial filaments from Treponema phagedenis biotype reiterii (the Reiter treponeme). J Bacteriol. 1974 Dec;120(3):1434–1442. doi: 10.1128/jb.120.3.1434-1442.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blanco D. R., Champion C. I., Miller J. N., Lovett M. A. Antigenic and structural characterization of Treponema pallidum (Nichols strain) endoflagella. Infect Immun. 1988 Jan;56(1):168–175. doi: 10.1128/iai.56.1.168-175.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blanco D. R., Radolf J. D., Lovett M. A., Miller J. N. The antigenic interrelationship between the endoflagella of Treponema phagedenis biotype Reiter and Treponema pallidum Nichols strain. I. Treponemicidal activity of cross-reactive endoflagellar antibodies against T. pallidum. J Immunol. 1986 Nov 1;137(9):2973–2979. [PubMed] [Google Scholar]
  5. 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]
  6. Brahamsha B., Greenberg E. P. Biochemical and cytological analysis of the complex periplasmic flagella from Spirochaeta aurantia. J Bacteriol. 1988 Sep;170(9):4023–4032. doi: 10.1128/jb.170.9.4023-4032.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bromley D. B., Charon N. W. Axial filament involvement in the motility of Leptospira interrogans. J Bacteriol. 1979 Mar;137(3):1406–1412. doi: 10.1128/jb.137.3.1406-1412.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Canale-Parola E. Motility and chemotaxis of spirochetes. Annu Rev Microbiol. 1978;32:69–99. doi: 10.1146/annurev.mi.32.100178.000441. [DOI] [PubMed] [Google Scholar]
  9. Cockayne A., Bailey M. J., Penn C. W. Analysis of sheath and core structures of the axial filament of Treponema pallidum. J Gen Microbiol. 1987 Jun;133(6):1397–1407. doi: 10.1099/00221287-133-6-1397. [DOI] [PubMed] [Google Scholar]
  10. DeLange R. J., Chang J. Y., Shaper J. H., Glazer A. N. Amino acid sequence of flagellin of Bacillus subtilis 168. III. Tryptic peptides, N-bromosuccinimide peptides, and the complete amino acid sequence. J Biol Chem. 1976 Feb 10;251(3):705–711. [PubMed] [Google Scholar]
  11. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  12. Gill P. R., Agabian N. The nucleotide sequence of the Mr = 28,500 flagellin gene of Caulobacter crescentus. J Biol Chem. 1983 Jun 25;258(12):7395–7401. [PubMed] [Google Scholar]
  13. Goldstein S. F., Charon N. W. Motility of the spirochete Leptospira. Cell Motil Cytoskeleton. 1988;9(2):101–110. doi: 10.1002/cm.970090202. [DOI] [PubMed] [Google Scholar]
  14. Hanff P. A., Norris S. J., Lovett M. A., Miller J. N. Purification of Treponema pallidum, Nichols strain, by Percoll density gradient centrifugation. Sex Transm Dis. 1984 Oct-Dec;11(4):275–286. doi: 10.1097/00007435-198410000-00003. [DOI] [PubMed] [Google Scholar]
  15. Hardy P. H., Jr, Fredericks W. R., Nell E. E. Isolation and antigenic characteristics of axial filaments from the Reiter Treponeme. Infect Immun. 1975 Feb;11(2):380–386. doi: 10.1128/iai.11.2.380-386.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hemmingsen S. M., Woolford C., van der Vies S. M., Tilly K., Dennis D. T., Georgopoulos C. P., Hendrix R. W., Ellis R. J. Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature. 1988 May 26;333(6171):330–334. doi: 10.1038/333330a0. [DOI] [PubMed] [Google Scholar]
  17. Hindersson P., Knudsen J. D., Axelsen N. H. Cloning and expression of treponema pallidum common antigen (Tp-4) in Escherichia coli K12. J Gen Microbiol. 1987 Mar;133(3):587–596. doi: 10.1099/00221287-133-3-587. [DOI] [PubMed] [Google Scholar]
  18. Hindersson P., Petersen C. S., Pedersen N. S., Høiby N., Axelsen N. H. Immunological cross-reaction between antigen Tp-4 of Treponema pallidum and an antigen common to a wide range of bacteria. Acta Pathol Microbiol Immunol Scand B. 1984 Aug;92(4):183–188. doi: 10.1111/j.1699-0463.1984.tb02818.x. [DOI] [PubMed] [Google Scholar]
  19. Holt S. C. Anatomy and chemistry of spirochetes. Microbiol Rev. 1978 Mar;42(1):114–160. doi: 10.1128/mr.42.1.114-160.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hopp T. P. Protein surface analysis. Methods for identifying antigenic determinants and other interaction sites. J Immunol Methods. 1986 Apr 3;88(1):1–18. doi: 10.1016/0022-1759(86)90045-1. [DOI] [PubMed] [Google Scholar]
  21. Hunkapiller M. W., Lujan E., Ostrander F., Hood L. E. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983;91:227–236. doi: 10.1016/s0076-6879(83)91019-4. [DOI] [PubMed] [Google Scholar]
  22. Joseph R., Canale-Parola E. Axial fibrils of anaerobic spirochetes: ultrastructure and chemical characteristics. Arch Mikrobiol. 1972;81(2):146–168. doi: 10.1007/BF00412325. [DOI] [PubMed] [Google Scholar]
  23. Jäckle H. Visualization of proteins after isoelectric focusing during two-dimensional gel electrophoresis. Anal Biochem. 1979 Sep 15;98(1):81–84. doi: 10.1016/0003-2697(79)90708-5. [DOI] [PubMed] [Google Scholar]
  24. Kuwajima G., Asaka J., Fujiwara T., Fujiwara T., Node K., Kondo E. Nucleotide sequence of the hag gene encoding flagellin of Escherichia coli. J Bacteriol. 1986 Dec;168(3):1479–1483. doi: 10.1128/jb.168.3.1479-1483.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lagenaur C., Agabian N. Physical characterization of Caulobacter crescentus flagella. J Bacteriol. 1976 Oct;128(1):435–444. doi: 10.1128/jb.128.1.435-444.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Limberger R. J., Charon N. W. Antiserum to the 33,000-dalton periplasmic-flagellum protein of "Treponema phagedenis" reacts with other treponemes and Spirochaeta aurantia. J Bacteriol. 1986 Nov;168(2):1030–1032. doi: 10.1128/jb.168.2.1030-1032.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Limberger R. J., Charon N. W. Treponema phagedenis has at least two proteins residing together on its periplasmic flagella. J Bacteriol. 1986 Apr;166(1):105–112. doi: 10.1128/jb.166.1.105-112.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Martin J. H., Savage D. C. Cloning, nucleotide sequence, and taxonomic implications of the flagellin gene of Roseburia cecicola. J Bacteriol. 1988 Jun;170(6):2612–2617. doi: 10.1128/jb.170.6.2612-2617.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Miao R., Fieldsteel A. H. Genetics of Treponema: relationship between Treponema pallidum and five cultivable treponemes. J Bacteriol. 1978 Jan;133(1):101–107. doi: 10.1128/jb.133.1.101-107.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nauman R. K., Holt S. C., Cox C. D. Purification, ultrastructure, and composition of axial filaments from Leptospira. J Bacteriol. 1969 Apr;98(1):264–280. doi: 10.1128/jb.98.1.264-280.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  32. Osborn M. J., Gander J. E., Parisi E., Carson J. Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J Biol Chem. 1972 Jun 25;247(12):3962–3972. [PubMed] [Google Scholar]
  33. Paster B. J., Canale-Parola E. Involvement of periplasmic fibrils in motility of spirochetes. J Bacteriol. 1980 Jan;141(1):359–364. doi: 10.1128/jb.141.1.359-364.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Penn C. W., Bailey M. J., Cockayne A. The axial filament antigen of Treponema pallidum. Immunology. 1985 Apr;54(4):635–641. [PMC free article] [PubMed] [Google Scholar]
  35. Petersen C. S., Pedersen N. S., Axelsen N. H. A simple method for the isolation of flagella from Treponema Reiter. Acta Pathol Microbiol Scand C. 1981 Dec;89(6):379–385. doi: 10.1111/j.1699-0463.1981.tb02716.x. [DOI] [PubMed] [Google Scholar]
  36. Radolf J. D., Blanco D. R., Miller J. N., Lovett M. A. Antigenic interrelationship between endoflagella of Treponema phagedenis biotype Reiter and Treponema pallidum (Nichols): molecular characterization of endoflagellar proteins. Infect Immun. 1986 Dec;54(3):626–634. doi: 10.1128/iai.54.3.626-634.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Trachtenberg S., DeRosier D. J., Aizawa S., Macnab R. M. Pairwise perturbation of flagellin subunits. The structural basis for the differences between plain and complex bacterial flagellar filaments. J Mol Biol. 1986 Aug 20;190(4):569–576. doi: 10.1016/0022-2836(86)90242-1. [DOI] [PubMed] [Google Scholar]
  39. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  40. Wei L. N., Joys T. M. Covalent structure of three phase-1 flagellar filament proteins of Salmonella. J Mol Biol. 1985 Dec 20;186(4):791–803. doi: 10.1016/0022-2836(85)90397-3. [DOI] [PubMed] [Google Scholar]
  41. Weissborn A., Steinmann H. M., Shapiro L. Characterization of the proteins of the Caulobacter crescentus flagellar filament. Peptide analysis and filament organization. J Biol Chem. 1982 Feb 25;257(4):2066–2074. [PubMed] [Google Scholar]

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