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. 1991 Apr;59(4):1521–1528. doi: 10.1128/iai.59.4.1521-1528.1991

Molecular cloning and characterization of a 35.5-kilodalton lipoprotein of Treponema pallidum.

C L Hubbard 1, F C Gherardini 1, P J Bassford Jr 1, L V Stamm 1
PMCID: PMC257871  PMID: 1848530

Abstract

A clone expressing a 35.5-kDa recombinant treponemal protein was isolated from a genomic DNA library constructed from Treponema pallidum street strain 14. Polyclonal antiserum raised against the recombinant protein reacted with a corresponding native protein of comparable size in T. pallidum that is specific to the pathogenic treponemes. Radiolabeling of the recombinant protein with [3H]palmitate demonstrated that it is lipid modified. Like other recently characterized T. pallidum lipoproteins, the 35.5-kDa lipoprotein partitioned into the detergent phase from T. pallidum cells fractionated with Triton X-114, suggesting that it is an integral membrane protein. Processing of the recombinant 35.5-kDa lipoprotein from a precursor form to a smaller mature form was not evident in pulse-chase experiments. However, pretreatment of Escherichia coli cells expressing the 35.5-kDa lipoprotein with inhibitors of protein processing or translocation revealed the existence of a higher-molecular-mass precursor. Gene fusion studies with the transposon TnphoA demonstrated the presence of an export signal in the 35.5-kDa lipoprotein that promotes the extracytoplasmic localization of a 35.5-kDa lipoprotein-PhoA hybrid.

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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., Thomas C. M., Cockayne A., Strugnell R. A., Penn C. W. Cloning and expression of Treponema pallidum antigens in Escherichia coli. J Gen Microbiol. 1989 Sep;135(9):2365–2378. doi: 10.1099/00221287-135-9-2365. [DOI] [PubMed] [Google Scholar]
  2. Baker-Zander S. A., Fohn M. J., Lukehart S. A. Development of cellular immunity to individual soluble antigens of Treponema pallidum during experimental syphilis. J Immunol. 1988 Dec 15;141(12):4363–4369. [PubMed] [Google Scholar]
  3. 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]
  4. Baseman J. B., Nichols J. C., Mogerley S. Capacity of virulent Treponema pallidum (Nichols) for deoxyribonucleic acid synthesis. Infect Immun. 1979 Feb;23(2):392–397. doi: 10.1128/iai.23.2.392-397.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blanco D. R., Walker E. M., Haake D. A., Champion C. I., Miller J. N., Lovett M. A. Complement activation limits the rate of in vitro treponemicidal activity and correlates with antibody-mediated aggregation of Treponema pallidum rare outer membrane protein. J Immunol. 1990 Mar 1;144(5):1914–1921. [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. Cockayne A., Strugnell R. A., Bailey M. J., Penn C. W. Comparative antigenic analysis of Treponema pallidum laboratory and street strains. J Gen Microbiol. 1989 Aug;135(8):2241–2247. doi: 10.1099/00221287-135-8-2241. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Dallas W. S., Ray P. H., Leong J., Benedict C. D., Stamm L. V., Bassford P. J., Jr Identification and purification of a recombinant Treponema pallidum basic membrane protein antigen expressed in Escherichia coli. Infect Immun. 1987 May;55(5):1106–1115. doi: 10.1128/iai.55.5.1106-1115.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Fieldsteel A. H., Cox D. L., Moeckli R. A. Cultivation of virulent Treponema pallidum in tissue culture. Infect Immun. 1981 May;32(2):908–915. doi: 10.1128/iai.32.2.908-915.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fitzgerald J. J., Johnson R. C., Smith M. Accidental laboratory infection with Treponema pallidum, Nichols strain. J Am Vener Dis Assoc. 1976 Dec;3(2 Pt 1):76–78. [PubMed] [Google Scholar]
  16. Gutierrez C., Barondess J., Manoil C., Beckwith J. The use of transposon TnphoA to detect genes for cell envelope proteins subject to a common regulatory stimulus. Analysis of osmotically regulated genes in Escherichia coli. J Mol Biol. 1987 May 20;195(2):289–297. doi: 10.1016/0022-2836(87)90650-4. [DOI] [PubMed] [Google Scholar]
  17. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  18. Hohn B., Collins J. A small cosmid for efficient cloning of large DNA fragments. Gene. 1980 Nov;11(3-4):291–298. doi: 10.1016/0378-1119(80)90069-4. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Inouye S., Soberon X., Franceschini T., Nakamura K., Itakura K., Inouye M. Role of positive charge on the amino-terminal region of the signal peptide in protein secretion across the membrane. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3438–3441. doi: 10.1073/pnas.79.11.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Miao R. M., Fieldsteel A. H. Genetic relationship between Treponema pallidum and Treponema pertenue, two noncultivable human pathogens. J Bacteriol. 1980 Jan;141(1):427–429. doi: 10.1128/jb.141.1.427-429.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Norris S. J., Sell S. Antigenic complexity of Treponema pallidum: antigenicity and surface localization of major polypeptides. J Immunol. 1984 Nov;133(5):2686–2692. [PubMed] [Google Scholar]
  24. Nunes-Edwards P. L., Thiermann A. B., Bassford P. J., Jr, Stamm L. V. Identification and characterization of the protein antigens of Leptospira interrogans serovar hardjo. Infect Immun. 1985 May;48(2):492–497. doi: 10.1128/iai.48.2.492-497.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Oliver D. B., Cabelli R. J., Dolan K. M., Jarosik G. P. Azide-resistant mutants of Escherichia coli alter the SecA protein, an azide-sensitive component of the protein export machinery. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8227–8231. doi: 10.1073/pnas.87.21.8227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Penn C. W., Cockayne A., Bailey M. J. The outer membrane of Treponema pallidum: biological significance and biochemical properties. J Gen Microbiol. 1985 Sep;131(9):2349–2357. doi: 10.1099/00221287-131-9-2349. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. 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]
  31. SAITO H., MIURA K. I. PREPARATION OF TRANSFORMING DEOXYRIBONUCLEIC ACID BY PHENOL TREATMENT. Biochim Biophys Acta. 1963 Aug 20;72:619–629. [PubMed] [Google Scholar]
  32. 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]
  33. Soberon X., Covarrubias L., Bolivar F. Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325. Gene. 1980 May;9(3-4):287–305. doi: 10.1016/0378-1119(90)90328-o. [DOI] [PubMed] [Google Scholar]
  34. Stamm L. V., Bassford P. J., Jr Cellular and extracellular protein antigens of Treponema pallidum synthesized during in vitro incubation of freshly extracted organisms. Infect Immun. 1985 Mar;47(3):799–807. doi: 10.1128/iai.47.3.799-807.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stamm L. V., Folds J. D., Bassford P. J., Jr Expression of Treponema pallidum antigens in Escherichia coli K-12. Infect Immun. 1982 Jun;36(3):1238–1241. doi: 10.1128/iai.36.3.1238-1241.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Stamm L. V., Kerner T. C., Jr, Bankaitis V. A., Bassford P. J., Jr Identification and preliminary characterization of Treponema pallidum protein antigens expressed in Escherichia coli. Infect Immun. 1983 Aug;41(2):709–721. doi: 10.1128/iai.41.2.709-721.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stamm L. V., Stapleton J. T., Bassford P. J., Jr In vitro assay to demonstrate high-level erythromycin resistance of a clinical isolate of Treponema pallidum. Antimicrob Agents Chemother. 1988 Feb;32(2):164–169. doi: 10.1128/aac.32.2.164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Swancutt M. A., Radolf J. D., Norgard M. V. The 34-kilodalton membrane immunogen of Treponema pallidum is a lipoprotein. Infect Immun. 1990 Feb;58(2):384–392. doi: 10.1128/iai.58.2.384-392.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. 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]
  43. Wicher K., van Embden J. D., Schouls L. M., Zabek J., Jakubowski A., Wicher V. Immunogenicity of three recombinant Treponema pallidum antigens examined in guinea pigs. Int Arch Allergy Appl Immunol. 1989;89(2-3):128–135. doi: 10.1159/000234935. [DOI] [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]

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