Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1995 Nov;63(11):4244–4252. doi: 10.1128/iai.63.11.4244-4252.1995

Characterization of outer membranes isolated from Treponema pallidum, the syphilis spirochete.

J D Radolf 1, E J Robinson 1, K W Bourell 1, D R Akins 1, S F Porcella 1, L M Weigel 1, J D Jones 1, M V Norgard 1
PMCID: PMC173603  PMID: 7591054

Abstract

Previous freeze-fracture electron microscopy (EM) studies have shown that the outer membrane (OM) of Treponema pallidum contains sparse transmembrane proteins. One strategy for molecular characterization of these rare OM proteins involves isolation of T. pallidum OMs. Here we describe a simple and extremely gentle method for OM isolation based upon isopycnic sucrose density gradient ultracentrifugation of treponemes following plasmolysis in 20% sucrose. Evidence that T. pallidum OMs were isolated included (i) the extremely low protein/lipid ratio of the putative OM fraction, (ii) a paucity of antigenic and/or biochemical markers for periplasmic, cytoplasmic membrane, and cytosolic compartments, and (iii) freeze-fracture EM demonstrating that the putative OMs contained intramembranous particles highly similar in size and density to those in native T. pallidum OMs. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the OMs contained a relatively small number of treponemal proteins, including several which did not appear to correspond to previously characterized T. pallidum antigens. Interestingly, these candidate rare OM proteins reacted poorly with syphilitic sera as determined by both conventional immunoblotting and enhanced chemiluminescence. Compared with whole cells, T. pallidum OMs were deficient in cardiolipin, the major lipoidal antigen reactive with antibodies in syphilitic sera. Also noteworthy was that other lipoidal constituents of OMs, including the recently discovered glycolipids, did not react with human syphilitic sera. These latter observations suggest that the poor antigenicity of virulent T. pallidum is a function of both the lipid composition and the low protein content of its OM.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Akins D. R., Purcell B. K., Mitra M. M., Norgard M. V., Radolf J. D. Lipid modification of the 17-kilodalton membrane immunogen of Treponema pallidum determines macrophage activation as well as amphiphilicity. Infect Immun. 1993 Apr;61(4):1202–1210. doi: 10.1128/iai.61.4.1202-1210.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  3. BLIGH E. G., DYER W. J. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. doi: 10.1139/o59-099. [DOI] [PubMed] [Google Scholar]
  4. Baker-Zander S. A., Shaffer J. M., Lukehart S. A. VDRL antibodies enhance phagocytosis of Treponema pallidum by macrophages. J Infect Dis. 1993 May;167(5):1100–1105. doi: 10.1093/infdis/167.5.1100. [DOI] [PubMed] [Google Scholar]
  5. Barbour A. G. Isolation and cultivation of Lyme disease spirochetes. Yale J Biol Med. 1984 Jul-Aug;57(4):521–525. [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Belisle J. T., Brandt M. E., Radolf J. D., Norgard M. V. Fatty acids of Treponema pallidum and Borrelia burgdorferi lipoproteins. J Bacteriol. 1994 Apr;176(8):2151–2157. doi: 10.1128/jb.176.8.2151-2157.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Birdsell D. C., Cota-Robles E. H. Production and ultrastructure of lysozyme and ethylenediaminetetraacetate-lysozyme spheroplasts of Escherichia coli. J Bacteriol. 1967 Jan;93(1):427–437. doi: 10.1128/jb.93.1.427-437.1967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Blanco D. R., Champion C. I., Exner M. M., Erdjument-Bromage H., Hancock R. E., Tempst P., Miller J. N., Lovett M. A. Porin activity and sequence analysis of a 31-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp1). J Bacteriol. 1995 Jun;177(12):3556–3562. doi: 10.1128/jb.177.12.3556-3562.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Blanco D. R., Giladi M., Champion C. I., Haake D. A., Chikami G. K., Miller J. N., Lovett M. A. Identification of Treponema pallidum subspecies pallidum genes encoding signal peptides and membrane-spanning sequences using a novel alkaline phosphatase expression vector. Mol Microbiol. 1991 Oct;5(10):2405–2415. doi: 10.1111/j.1365-2958.1991.tb02086.x. [DOI] [PubMed] [Google Scholar]
  11. Blanco D. R., Reimann K., Skare J., Champion C. I., Foley D., Exner M. M., Hancock R. E., Miller J. N., Lovett M. A. Isolation of the outer membranes from Treponema pallidum and Treponema vincentii. J Bacteriol. 1994 Oct;176(19):6088–6099. doi: 10.1128/jb.176.19.6088-6099.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bourell K. W., Schulz W., Norgard M. V., Radolf J. D. Treponema pallidum rare outer membrane proteins: analysis of mobility by freeze-fracture electron microscopy. J Bacteriol. 1994 Mar;176(6):1598–1608. doi: 10.1128/jb.176.6.1598-1608.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Cox D. L., Akins D. R., Porcella S. F., Norgard M. V., Radolf J. D. Treponema pallidum in gel microdroplets: a novel strategy for investigation of treponemal molecular architecture. Mol Microbiol. 1995 Mar;15(6):1151–1164. doi: 10.1111/j.1365-2958.1995.tb02288.x. [DOI] [PubMed] [Google Scholar]
  15. Cox D. L., Chang P., McDowall A. W., Radolf J. D. The outer membrane, not a coat of host proteins, limits antigenicity of virulent Treponema pallidum. Infect Immun. 1992 Mar;60(3):1076–1083. doi: 10.1128/iai.60.3.1076-1083.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Hardham J. M., Stamm L. V. Identification and characterization of the Treponema pallidum tpn50 gene, an ompA homolog. Infect Immun. 1994 Mar;62(3):1015–1025. doi: 10.1128/iai.62.3.1015-1025.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hardy P. H., Jr, Levin J. Lack of endotoxin in Borrelia hispanica and Treponema pallidum. Proc Soc Exp Biol Med. 1983 Oct;174(1):47–52. doi: 10.3181/00379727-174-41702. [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. Houston L. S., Cook R. G., Norris S. J. Isolation and characterization of a Treponema pallidum major 60-kilodalton protein resembling the groEL protein of Escherichia coli. J Bacteriol. 1990 Jun;172(6):2862–2870. doi: 10.1128/jb.172.6.2862-2870.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Isaacs R. D., Hanke J. H., Guzman-Verduzco L. M., Newport G., Agabian N., Norgard M. V., Lukehart S. A., Radolf J. D. Molecular cloning and DNA sequence analysis of the 37-kilodalton endoflagellar sheath protein gene of Treponema pallidum. Infect Immun. 1989 Nov;57(11):3403–3411. doi: 10.1128/iai.57.11.3403-3411.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jones J. D., Bourell K. W., Norgard M. V., Radolf J. D. Membrane topology of Borrelia burgdorferi and Treponema pallidum lipoproteins. Infect Immun. 1995 Jul;63(7):2424–2434. doi: 10.1128/iai.63.7.2424-2434.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Lafaye P., Lapresle C. Fixation of penicilloyl groups to albumin and appearance of anti-penicilloyl antibodies in penicillin-treated patients. J Clin Invest. 1988 Jul;82(1):7–12. doi: 10.1172/JCI113603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lugtenberg B., Van Alphen L. Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram-negative bacteria. Biochim Biophys Acta. 1983 Mar 21;737(1):51–115. doi: 10.1016/0304-4157(83)90014-x. [DOI] [PubMed] [Google Scholar]
  26. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Radolf J. D., Bourell K. W., Akins D. R., Brusca J. S., Norgard M. V. Analysis of Borrelia burgdorferi membrane architecture by freeze-fracture electron microscopy. J Bacteriol. 1994 Jan;176(1):21–31. doi: 10.1128/jb.176.1.21-31.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Radolf J. D., Goldberg M. S., Bourell K., Baker S. I., Jones J. D., Norgard M. V. Characterization of outer membranes isolated from Borrelia burgdorferi, the Lyme disease spirochete. Infect Immun. 1995 Jun;63(6):2154–2163. doi: 10.1128/iai.63.6.2154-2163.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Radolf J. D., Moomaw C., Slaughter C. A., Norgard M. V. Penicillin-binding proteins and peptidoglycan of Treponema pallidum subsp. pallidum. Infect Immun. 1989 Apr;57(4):1248–1254. doi: 10.1128/iai.57.4.1248-1254.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Radolf J. D. Role of outer membrane architecture in immune evasion by Treponema pallidum and Borrelia burgdorferi. Trends Microbiol. 1994 Sep;2(9):307–311. doi: 10.1016/0966-842x(94)90446-4. [DOI] [PubMed] [Google Scholar]
  36. Radolf J. D. Treponema pallidum and the quest for outer membrane proteins. Mol Microbiol. 1995 Jun;16(6):1067–1073. doi: 10.1111/j.1365-2958.1995.tb02332.x. [DOI] [PubMed] [Google Scholar]
  37. Schouls L. M., Ijsselmuiden O. E., Weel J., van Embden J. D. Overproduction and purification of Treponema pallidum recombinant-DNA-derived proteins TmpA and TmpB and their potential use in serodiagnosis of syphilis. Infect Immun. 1989 Sep;57(9):2612–2623. doi: 10.1128/iai.57.9.2612-2623.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schouls L. M., Mout R., Dekker J., van Embden J. D. Characterization of lipid-modified immunogenic proteins of Treponema pallidum expressed in Escherichia coli. Microb Pathog. 1989 Sep;7(3):175–188. doi: 10.1016/0882-4010(89)90053-3. [DOI] [PubMed] [Google Scholar]
  39. Scopio A., Johnson P., Laquerre A., Nelson D. R. Subcellular localization and chaperone activities of Borrelia burgdorferi Hsp60 and Hsp70. J Bacteriol. 1994 Nov;176(21):6449–6456. doi: 10.1128/jb.176.21.6449-6456.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Siakotos A. N. Analytical separation of nonlipid water soluble substances and gangliosides from other lipids by dextran gel column chromatography. J Am Oil Chem Soc. 1965 Nov;42(11):913–919. doi: 10.1007/BF02632444. [DOI] [PubMed] [Google Scholar]
  41. Spratt B. G., Cromie K. D. Penicillin-binding proteins of gram-negative bacteria. Rev Infect Dis. 1988 Jul-Aug;10(4):699–711. doi: 10.1093/clinids/10.4.699. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Walker E. M., Borenstein L. A., Blanco D. R., Miller J. N., Lovett M. A. Analysis of outer membrane ultrastructure of pathogenic Treponema and Borrelia species by freeze-fracture electron microscopy. J Bacteriol. 1991 Sep;173(17):5585–5588. doi: 10.1128/jb.173.17.5585-5588.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Walker E. M., Zampighi G. A., Blanco D. R., Miller J. N., Lovett M. A. Demonstration of rare protein in the outer membrane of Treponema pallidum subsp. pallidum by freeze-fracture analysis. J Bacteriol. 1989 Sep;171(9):5005–5011. doi: 10.1128/jb.171.9.5005-5011.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Weigel L. M., Belisle J. T., Radolf J. D., Norgard M. V. Digoxigenin-ampicillin conjugate for detection of penicillin-binding proteins by chemiluminescence. Antimicrob Agents Chemother. 1994 Feb;38(2):330–336. doi: 10.1128/aac.38.2.330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Weigel L. M., Radolf J. D., Norgard M. V. The 47-kDa major lipoprotein immunogen of Treponema pallidum is a penicillin-binding protein with carboxypeptidase activity. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11611–11615. doi: 10.1073/pnas.91.24.11611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wheeler C. M., Garcia Monco J. C., Benach J. L., Golightly M. G., Habicht G. S., Steere A. C. Nonprotein antigens of Borrelia burgdorferi. J Infect Dis. 1993 Mar;167(3):665–674. doi: 10.1093/infdis/167.3.665. [DOI] [PubMed] [Google Scholar]

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

RESOURCES