Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1990 Feb;58(2):384–392. doi: 10.1128/iai.58.2.384-392.1990

The 34-kilodalton membrane immunogen of Treponema pallidum is a lipoprotein.

M A Swancutt 1, J D Radolf 1, M V Norgard 1
PMCID: PMC258467  PMID: 2404869

Abstract

Treponema pallidum subsp. pallidum and Escherichia coli incorporated exogenous [3H]palmitate into the 34-kilodalton (kDa) pathogen-specific antigen of T. pallidum. Radiolabeled fatty acid remained associated with the protein upon immunoprecipitation and after boiling in sodium dodecyl sulfate, acetone precipitation, and extensive extractions in organic solvents, suggesting that the fatty acid was covalently bound to the protein. Detection of [3H]palmitate after alkaline and acid hydrolyses confirmed the identity of the incorporated label. Globomycin inhibited maturation of the recombinant 34-kDa antigen, suggesting that E. coli uses the lipoprotein-specific signal peptidase II to process the treponemal antigen. Globomycin also inhibited processing of the 34-kDa antigen, as well as the 44.5- and 15-kDa antigens, in T. pallidum, implying that T. pallidum also possesses the lipoprotein export pathway common to both gram-negative and gram-positive bacteria. Ethanol inhibited processing of the 34-kDa antigen in minicells, suggesting that the 34-kDa antigen normally is translocated through the cytoplasmic membrane. Comparison of the Triton X-114 phase partitioning behavior of the 34-kDa antigen produced either by minicells or by a cell-free translation system indicated that the covalent attachment of fatty acid conferred hydrophobic biochemical properties to the 34-kDa antigen, consistent with the hypothesis that the attached lipid anchors the 34-kDa antigen into the membrane.

Full text

PDF
388

Images in this article

386Image
on p.386
387Image
on p.387
387Image
on p.387
388Image
on p.388
388Image
on p.388
389Image
on p.389
390Image
on p.390

Selected References

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

  1. Bessler W., Resch K., Hancock E., Hantke K. Induction of lymphocyte proliferation and membrane changes by lipopeptide derivatives of the lipoprotein from the outer membrane of Escherichia coli. Z Immunitatsforsch Immunobiol. 1977 Apr;153(1):11–22. [PubMed] [Google Scholar]
  2. Biesert L., Scheuer W., Bessler W. G. Interaction of mitogenic bacterial lipoprotein and a synthetic analogue with mouse lymphocytes. Isolation and characterization of binding proteins. Eur J Biochem. 1987 Feb 2;162(3):651–657. doi: 10.1111/j.1432-1033.1987.tb10687.x. [DOI] [PubMed] [Google Scholar]
  3. Chamberlain N. R., Brandt M. E., Erwin A. L., Radolf J. D., Norgard M. V. Major integral membrane protein immunogens of Treponema pallidum are proteolipids. Infect Immun. 1989 Sep;57(9):2872–2877. doi: 10.1128/iai.57.9.2872-2877.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chamberlain N. R., DeOgny L., Slaughter C., Radolf J. D., Norgard M. V. Acylation of the 47-kilodalton major membrane immunogen of Treponema pallidum determines its hydrophobicity. Infect Immun. 1989 Sep;57(9):2878–2885. doi: 10.1128/iai.57.9.2878-2885.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chamberlain N. R., Radolf J. D., Hsu P. L., Sell S., Norgard M. V. Genetic and physicochemical characterization of the recombinant DNA-derived 47-kilodalton surface immunogen of Treponema pallidum subsp. pallidum. Infect Immun. 1988 Jan;56(1):71–78. doi: 10.1128/iai.56.1.71-78.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen R., Henning U. Nucleotide sequence of the gene for the peptidoglycan-associated lipoprotein of Escherichia coli K12. Eur J Biochem. 1987 Feb 16;163(1):73–77. doi: 10.1111/j.1432-1033.1987.tb10738.x. [DOI] [PubMed] [Google Scholar]
  7. Covarrubias L., Cervantes L., Covarrubias A., Soberón X., Vichido I., Blanco A., Kupersztoch-Portnoy Y. M., Bolivar F. Construction and characterization of new cloning vehicles. V. Mobilization and coding properties of pBR322 and several deletion derivatives including pBR327 and pBR328. Gene. 1981 Jan-Feb;13(1):25–35. doi: 10.1016/0378-1119(81)90040-8. [DOI] [PubMed] [Google Scholar]
  8. Cunningham T. M., Walker E. M., Miller J. N., Lovett M. A. Selective release of the Treponema pallidum outer membrane and associated polypeptides with Triton X-114. J Bacteriol. 1988 Dec;170(12):5789–5796. doi: 10.1128/jb.170.12.5789-5796.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Deich R. A., Metcalf B. J., Finn C. W., Farley J. E., Green B. A. Cloning of genes encoding a 15,000-dalton peptidoglycan-associated outer membrane lipoprotein and an antigenically related 15,000-dalton protein from Haemophilus influenzae. J Bacteriol. 1988 Feb;170(2):489–498. doi: 10.1128/jb.170.2.489-498.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dev I. K., Harvey R. J., Ray P. H. Inhibition of prolipoprotein signal peptidase by globomycin. J Biol Chem. 1985 May 25;260(10):5891–5894. [PubMed] [Google Scholar]
  11. Gonzales F. R., Leachman S., Norgard M. V., Radolf J. D., McCracken G. H., Jr, Evans C., Hansen E. J. Cloning and expression in Escherichia coli of the gene encoding the heat-modifiable major outer membrane protein of Haemophilus influenzae type b. Infect Immun. 1987 Dec;55(12):2993–3000. doi: 10.1128/iai.55.12.2993-3000.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hansen E. B., Pedersen P. E., Schouls L. M., Severin E., van Embden J. D. Genetic characterization and partial sequence determination of a Treponema pallidum operon expressing two immunogenic membrane proteins in Escherichia coli. J Bacteriol. 1985 Jun;162(3):1227–1237. doi: 10.1128/jb.162.3.1227-1237.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hantke K., Braun V. Covalent binding of lipid to protein. Diglyceride and amide-linked fatty acid at the N-terminal end of the murein-lipoprotein of the Escherichia coli outer membrane. Eur J Biochem. 1973 Apr;34(2):284–296. doi: 10.1111/j.1432-1033.1973.tb02757.x. [DOI] [PubMed] [Google Scholar]
  14. Hindersson P., Cockayne A., Schouls L. M., van Emden J. D. Immunochemical characterization and purification of Treponema pallidum antigen TpD expressed by Escherichia coli K12. Sex Transm Dis. 1986 Oct-Dec;13(4):237–244. doi: 10.1097/00007435-198610000-00006. [DOI] [PubMed] [Google Scholar]
  15. Hsu P. L., Chamberlain N. R., Orth K., Moomaw C. R., Zhang L. Q., Slaughter C. A., Radolf J. D., Sell S., Norgard M. V. Sequence analysis of the 47-kilodalton major integral membrane immunogen of Treponema pallidum. Infect Immun. 1989 Jan;57(1):196–203. doi: 10.1128/iai.57.1.196-203.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hussain M., Ichihara S., Mizushima S. Accumulation of glyceride-containing precursor of the outer membrane lipoprotein in the cytoplasmic membrane of Escherichia coli treated with globomycin. J Biol Chem. 1980 Apr 25;255(8):3707–3712. [PubMed] [Google Scholar]
  17. Ichihara S., Hussain M., Mizushima S. Characterization of new membrane lipoproteins and their precursors of Escherichia coli. J Biol Chem. 1981 Mar 25;256(6):3125–3129. [PubMed] [Google Scholar]
  18. Inukai M., Nakajima M., Osawa M., Haneishi T., Arai M. Globomycin, a new peptide antibiotic with spheroplast-forming activity. II. Isolation and physico-chemical and biological characterization. J Antibiot (Tokyo) 1978 May;31(5):421–425. doi: 10.7164/antibiotics.31.421. [DOI] [PubMed] [Google Scholar]
  19. Izui K., Nielsen J. B., Caulfield M. P., Lampen J. O. Large exopenicillinase, initial extracellular form detected in cultures of Bacillus licheniformis. Biochemistry. 1980 Apr 29;19(9):1882–1886. doi: 10.1021/bi00550a023. [DOI] [PubMed] [Google Scholar]
  20. Jones S. A., Marchitto K. S., Miller J. N., Norgard M. V. Monoclonal antibody with hemagglutination, immobilization, and neutralization activities defines an immunodominant, 47,000 mol wt, surface-exposed immunogen of Treponema pallidum (Nichols). J Exp Med. 1984 Nov 1;160(5):1404–1420. doi: 10.1084/jem.160.5.1404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lex A., Wiesmüller K. H., Jung G., Bessler W. G. A synthetic analogue of Escherichia coli lipoprotein, tripalmitoyl pentapeptide, constitutes a potent immune adjuvant. J Immunol. 1986 Oct 15;137(8):2676–2681. [PubMed] [Google Scholar]
  22. Lin J. J., Wu H. C. Biosynthesis and assembly of envelope lipoprotein in a glycerol-requiring mutant of Salmonella typhimurium. J Bacteriol. 1976 Mar;125(3):892–904. doi: 10.1128/jb.125.3.892-904.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Melchers F., Braun V., Galanos C. The lipoprotein of the outer membrane of Escherichia coli: a B-lymphocyte mitogen. J Exp Med. 1975 Aug 1;142(2):473–482. doi: 10.1084/jem.142.2.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nakajima M., Inukai M., Haneishi T., Terahara A., Arai M., Kinoshita T., Tamura C. Globomycin, a new peptide antibiotic with spheroplast-forming activity. III. Structural determination of globomycin. J Antibiot (Tokyo) 1978 May;31(5):426–432. doi: 10.7164/antibiotics.31.426. [DOI] [PubMed] [Google Scholar]
  25. Nakamura K., Inouye M. DNA sequence of the Serratia marcescens lipoprotein gene. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1369–1373. doi: 10.1073/pnas.77.3.1369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nielsen J. B., Caulfield M. P., Lampen J. O. Lipoprotein nature of Bacillus licheniformis membrane penicillinase. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3511–3515. doi: 10.1073/pnas.78.6.3511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Norgard M. V., Chamberlain N. R., Swancutt M. A., Goldberg M. S. Cloning and expression of the major 47-kilodalton surface immunogen of Treponema pallidum in Escherichia coli. Infect Immun. 1986 Nov;54(2):500–506. doi: 10.1128/iai.54.2.500-506.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Norgard M. V., Miller J. N. Cloning and expression of Treponema pallidum (Nichols) antigen genes in Escherichia coli. Infect Immun. 1983 Nov;42(2):435–445. doi: 10.1128/iai.42.2.435-445.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Orndorff P. E., Falkow S. Nucleotide sequence of pilA, the gene encoding the structural component of type 1 pili in Escherichia coli. J Bacteriol. 1985 Apr;162(1):454–457. doi: 10.1128/jb.162.1.454-457.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Palva E. T., Hirst T. R., Hardy S. J., Holmgren J., Randall L. Synthesis of a precursor to the B subunit of heat-labile enterotoxin in Escherichia coli. J Bacteriol. 1981 Apr;146(1):325–330. doi: 10.1128/jb.146.1.325-330.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pillai S., Baltimore D. Myristoylation and the post-translational acquisition of hydrophobicity by the membrane immunoglobulin heavy-chain polypeptide in B lymphocytes. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7654–7658. doi: 10.1073/pnas.84.21.7654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Radolf J. D., Chamberlain N. R., Clausell A., Norgard M. V. Identification and localization of integral membrane proteins of virulent Treponema pallidum subsp. pallidum by phase partitioning with the nonionic detergent triton X-114. Infect Immun. 1988 Feb;56(2):490–498. doi: 10.1128/iai.56.2.490-498.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Radolf J. D., Norgard M. V., Schulz W. W. Outer membrane ultrastructure explains the limited antigenicity of virulent Treponema pallidum. Proc Natl Acad Sci U S A. 1989 Mar;86(6):2051–2055. doi: 10.1073/pnas.86.6.2051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Robertson S. M., Kettman J. R., Miller J. N., Norgard M. V. Murine monoclonal antibodies specific for virulent Treponema pallidum (Nichols). Infect Immun. 1982 Jun;36(3):1076–1085. doi: 10.1128/iai.36.3.1076-1085.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stamm L. V., Hodinka R. L., Wyrick P. B., Bassford P. J., Jr Changes in the cell surface properties of Treponema pallidum that occur during in vitro incubation of freshly extracted organisms. Infect Immun. 1987 Sep;55(9):2255–2261. doi: 10.1128/iai.55.9.2255-2261.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Swancutt M. A., Riley B. S., Radolf J. D., Norgard M. V. Molecular characterization of the pathogen-specific, 34-kilodalton membrane immunogen of Treponema pallidum. Infect Immun. 1989 Nov;57(11):3314–3323. doi: 10.1128/iai.57.11.3314-3323.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Swancutt M. A., Twehous D. A., Norgard M. V. Monoclonal antibody selection and analysis of a recombinant DNA-derived surface immunogen of Treponema pallidum expressed in Escherichia coli. Infect Immun. 1986 Apr;52(1):110–119. doi: 10.1128/iai.52.1.110-119.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Takase I., Ishino F., Wachi M., Kamata H., Doi M., Asoh S., Matsuzawa H., Ohta T., Matsuhashi M. Genes encoding two lipoproteins in the leuS-dacA region of the Escherichia coli chromosome. J Bacteriol. 1987 Dec;169(12):5692–5699. doi: 10.1128/jb.169.12.5692-5699.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tokunaga M., Loranger J. M., Wu H. C. Prolipoprotein modification and processing enzymes in Escherichia coli. J Biol Chem. 1984 Mar 25;259(6):3825–3830. [PubMed] [Google Scholar]
  40. Tokunaga M., Tokunaga H., Wu H. C. Post-translational modification and processing of Escherichia coli prolipoprotein in vitro. Proc Natl Acad Sci U S A. 1982 Apr;79(7):2255–2259. doi: 10.1073/pnas.79.7.2255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Weinberg G. A., Towler D. A., Munson R. S., Jr Lipoproteins of Haemophilus influenzae type b. J Bacteriol. 1988 Sep;170(9):4161–4164. doi: 10.1128/jb.170.9.4161-4164.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yamaguchi K., Yu F., Inouye M. A single amino acid determinant of the membrane localization of lipoproteins in E. coli. Cell. 1988 May 6;53(3):423–432. doi: 10.1016/0092-8674(88)90162-6. [DOI] [PubMed] [Google Scholar]
  43. Zwizinski C., Wickner W. Purification and characterization of leader (signal) peptidase from Escherichia coli. J Biol Chem. 1980 Aug 25;255(16):7973–7977. [PubMed] [Google Scholar]
  44. van Embden J. D., van der Donk H. J., van Eijk R. V., van der Heide H. G., de Jong J. A., van Olderen M. F., Osterhaus A. B., Schouls L. M. Molecular cloning and expression of Treponema pallidum DNA in Escherichia coli K-12. Infect Immun. 1983 Oct;42(1):187–196. doi: 10.1128/iai.42.1.187-196.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES