Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1997 Feb;65(2):412–421. doi: 10.1128/iai.65.2.412-421.1997

Molecular characterization of a 6.6-kilodalton Borrelia burgdorferi outer membrane-associated lipoprotein (lp6.6) which appears to be downregulated during mammalian infection.

P Lahdenne 1, S F Porcella 1, K E Hagman 1, D R Akins 1, T G Popova 1, D L Cox 1, L I Katona 1, J D Radolf 1, M V Norgard 1
PMCID: PMC174610  PMID: 9009290

Abstract

Isolated outer membranes of Borrelia burgdorferi 297 were utilized to obtain partial amino acid sequence information for a low-molecular-weight, outer membrane-associated polypeptide. Degenerate oligonucleotide primers based upon this information were used to amplify a 100-bp probe for detection of the corresponding full-length gene within a B. burgdorferi total genomic library. The relevant open reading frame (ORF) encoded a polypeptide comprised of a 17-amino-acid putative signal peptide terminated by LFVAC, a probable consensus sequence for lipoprotein modification, and a mature protein of 51 amino acids (predicted molecular mass of 5.8 kDa). The DNA sequences of the corresponding ORFs in B. burgdorferi 297 and B31 were identical; the corresponding ORF in strain N40 differed by only one nucleotide. Assuming conventional processing and acylation, the molecular weight of the lipoprotein, designated lp6.6, is about 6,600. The lp6.6 gene, which was localized to the 49-kb linear plasmid of B. burgdorferi, subsequently was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase. Immunoblot analysis with monoclonal antibody 240.7 revealed that lp6.6 was identical to a low-molecular-weight, highly conserved B. burgdorferi lipoprotein reported previously (L. I. Katona, G. Beck, and G. S. Habicht, Infect. Immun. 60:4995-5003, 1992). Results of indirect immunofluorescence assays, growth inhibition assays, passive immunizations, and active immunizations indicated that this outer membrane-associated antigen is not surface exposed in B. burgdorferi. Particularly interesting was the finding that mice and rhesus monkeys chronically infected with B. burgdorferi failed to develop antibodies against this antigen. We propose that high-level expression of lp6.6 is associated with the arthropod phase of the spirochetal life cycle and that expression of the gene is downregulated during mammalian infection.

Full Text

The Full Text of this article is available as a PDF (610.1 KB).

Selected References

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

  1. Aebersold R. H., Leavitt J., Saavedra R. A., Hood L. E., Kent S. B. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc Natl Acad Sci U S A. 1987 Oct;84(20):6970–6974. doi: 10.1073/pnas.84.20.6970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aebersold R. H., Teplow D. B., Hood L. E., Kent S. B. Electroblotting onto activated glass. High efficiency preparation of proteins from analytical sodium dodecyl sulfate-polyacrylamide gels for direct sequence analysis. J Biol Chem. 1986 Mar 25;261(9):4229–4238. [PubMed] [Google Scholar]
  3. Akins D. R., Porcella S. F., Popova T. G., Shevchenko D., Baker S. I., Li M., Norgard M. V., Radolf J. D. Evidence for in vivo but not in vitro expression of a Borrelia burgdorferi outer surface protein F (OspF) homologue. Mol Microbiol. 1995 Nov;18(3):507–520. doi: 10.1111/j.1365-2958.1995.mmi_18030507.x. [DOI] [PubMed] [Google Scholar]
  4. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  5. Andrews P. C., Dixon J. E. A procedure for in situ alkylation of cystine residues on glass fiber prior to protein microsequence analysis. Anal Biochem. 1987 Mar;161(2):524–528. doi: 10.1016/0003-2697(87)90484-2. [DOI] [PubMed] [Google Scholar]
  6. Aydintug M. K., Gu Y., Philipp M. T. Borrelia burgdorferi antigens that are targeted by antibody-dependent, complement-mediated killing in the rhesus monkey. Infect Immun. 1994 Nov;62(11):4929–4937. doi: 10.1128/iai.62.11.4929-4937.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Barbour A. G., Burgdorfer W., Grunwaldt E., Steere A. C. Antibodies of patients with Lyme disease to components of the Ixodes dammini spirochete. J Clin Invest. 1983 Aug;72(2):504–515. doi: 10.1172/JCI110998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Barbour A. G., Garon C. F. Linear plasmids of the bacterium Borrelia burgdorferi have covalently closed ends. Science. 1987 Jul 24;237(4813):409–411. doi: 10.1126/science.3603026. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Barbour A. G., Tessier S. L., Todd W. J. Lyme disease spirochetes and ixodid tick spirochetes share a common surface antigenic determinant defined by a monoclonal antibody. Infect Immun. 1983 Aug;41(2):795–804. doi: 10.1128/iai.41.2.795-804.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Barthold S. W., Bockenstedt L. K. Passive immunizing activity of sera from mice infected with Borrelia burgdorferi. Infect Immun. 1993 Nov;61(11):4696–4702. doi: 10.1128/iai.61.11.4696-4702.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Barthold S. W., Fikrig E., Bockenstedt L. K., Persing D. H. Circumvention of outer surface protein A immunity by host-adapted Borrelia burgdorferi. Infect Immun. 1995 Jun;63(6):2255–2261. doi: 10.1128/iai.63.6.2255-2261.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Barthold S. W., Persing D. H., Armstrong A. L., Peeples R. A. Kinetics of Borrelia burgdorferi dissemination and evolution of disease after intradermal inoculation of mice. Am J Pathol. 1991 Aug;139(2):263–273. [PMC free article] [PubMed] [Google Scholar]
  14. Barthold S. W., de Souza M. S., Janotka J. L., Smith A. L., Persing D. H. Chronic Lyme borreliosis in the laboratory mouse. Am J Pathol. 1993 Sep;143(3):959–971. [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Bledsoe H. A., Carroll J. A., Whelchel T. R., Farmer M. A., Dorward D. W., Gherardini F. C. Isolation and partial characterization of Borrelia burgdorferi inner and outer membranes by using isopycnic centrifugation. J Bacteriol. 1994 Dec;176(24):7447–7455. doi: 10.1128/jb.176.24.7447-7455.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Brusca J. S., McDowall A. W., Norgard M. V., Radolf J. D. Localization of outer surface proteins A and B in both the outer membrane and intracellular compartments of Borrelia burgdorferi. J Bacteriol. 1991 Dec;173(24):8004–8008. doi: 10.1128/jb.173.24.8004-8008.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Brusca J. S., Radolf J. D. Isolation of integral membrane proteins by phase partitioning with Triton X-114. Methods Enzymol. 1994;228:182–193. doi: 10.1016/0076-6879(94)28019-3. [DOI] [PubMed] [Google Scholar]
  19. Burgdorfer W. Vector/host relationships of the Lyme disease spirochete, Borrelia burgdorferi. Rheum Dis Clin North Am. 1989 Nov;15(4):775–787. [PubMed] [Google Scholar]
  20. Champion C. I., Blanco D. R., Skare J. T., Haake D. A., Giladi M., Foley D., Miller J. N., Lovett M. A. A 9.0-kilobase-pair circular plasmid of Borrelia burgdorferi encodes an exported protein: evidence for expression only during infection. Infect Immun. 1994 Jul;62(7):2653–2661. doi: 10.1128/iai.62.7.2653-2661.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Cox D. L., Akins D. R., Bourell K. W., Lahdenne P., Norgard M. V., Radolf J. D. Limited surface exposure of Borrelia burgdorferi outer surface lipoproteins. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7973–7978. doi: 10.1073/pnas.93.15.7973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Craft J. E., Fischer D. K., Shimamoto G. T., Steere A. C. Antigens of Borrelia burgdorferi recognized during Lyme disease. Appearance of a new immunoglobulin M response and expansion of the immunoglobulin G response late in the illness. J Clin Invest. 1986 Oct;78(4):934–939. doi: 10.1172/JCI112683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Dressler F., Whalen J. A., Reinhardt B. N., Steere A. C. Western blotting in the serodiagnosis of Lyme disease. J Infect Dis. 1993 Feb;167(2):392–400. doi: 10.1093/infdis/167.2.392. [DOI] [PubMed] [Google Scholar]
  26. Engstrom S. M., Shoop E., Johnson R. C. Immunoblot interpretation criteria for serodiagnosis of early Lyme disease. J Clin Microbiol. 1995 Feb;33(2):419–427. doi: 10.1128/jcm.33.2.419-427.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ferdows M. S., Barbour A. G. Megabase-sized linear DNA in the bacterium Borrelia burgdorferi, the Lyme disease agent. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5969–5973. doi: 10.1073/pnas.86.15.5969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Fikrig E., Barthold S. W., Kantor F. S., Flavell R. A. Protection of mice against the Lyme disease agent by immunizing with recombinant OspA. Science. 1990 Oct 26;250(4980):553–556. doi: 10.1126/science.2237407. [DOI] [PubMed] [Google Scholar]
  29. Hayashi S., Wu H. C. Lipoproteins in bacteria. J Bioenerg Biomembr. 1990 Jun;22(3):451–471. doi: 10.1007/BF00763177. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Jonsson M., Bergström S. Transcriptional and translational regulation of the expression of the major outer surface proteins in Lyme disease Borrelia strains. Microbiology. 1995 Jun;141(Pt 6):1321–1329. doi: 10.1099/13500872-141-6-1321. [DOI] [PubMed] [Google Scholar]
  32. Katona L. I., Beck G., Habicht G. S. Purification and immunological characterization of a major low-molecular-weight lipoprotein from Borrelia burgdorferi. Infect Immun. 1992 Dec;60(12):4995–5003. doi: 10.1128/iai.60.12.4995-5003.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  34. Lam T. T., Nguyen T. P., Montgomery R. R., Kantor F. S., Fikrig E., Flavell R. A. Outer surface proteins E and F of Borrelia burgdorferi, the agent of Lyme disease. Infect Immun. 1994 Jan;62(1):290–298. doi: 10.1128/iai.62.1.290-298.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Levine J. F., Wilson M. L., Spielman A. Mice as reservoirs of the Lyme disease spirochete. Am J Trop Med Hyg. 1985 Mar;34(2):355–360. doi: 10.4269/ajtmh.1985.34.355. [DOI] [PubMed] [Google Scholar]
  36. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  37. Montgomery R. R., Malawista S. E., Feen K. J., Bockenstedt L. K. Direct demonstration of antigenic substitution of Borrelia burgdorferi ex vivo: exploration of the paradox of the early immune response to outer surface proteins A and C in Lyme disease. J Exp Med. 1996 Jan 1;183(1):261–269. doi: 10.1084/jem.183.1.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Philipp M. T., Aydintug M. K., Bohm R. P., Jr, Cogswell F. B., Dennis V. A., Lanners H. N., Lowrie R. C., Jr, Roberts E. D., Conway M. D., Karaçorlu M. Early and early disseminated phases of Lyme disease in the rhesus monkey: a model for infection in humans. Infect Immun. 1993 Jul;61(7):3047–3059. doi: 10.1128/iai.61.7.3047-3059.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Platt T. Transcription termination and the regulation of gene expression. Annu Rev Biochem. 1986;55:339–372. doi: 10.1146/annurev.bi.55.070186.002011. [DOI] [PubMed] [Google Scholar]
  40. Porcella S. F., Popova T. G., Akins D. R., Li M., Radolf J. D., Norgard M. V. Borrelia burgdorferi supercoiled plasmids encode multicopy tandem open reading frames and a lipoprotein gene family. J Bacteriol. 1996 Jun;178(11):3293–3307. doi: 10.1128/jb.178.11.3293-3307.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. 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]
  44. Roberts E. D., Bohm R. P., Jr, Cogswell F. B., Lanners H. N., Lowrie R. C., Jr, Povinelli L., Piesman J., Philipp M. T. Chronic lyme disease in the rhesus monkey. Lab Invest. 1995 Feb;72(2):146–160. [PubMed] [Google Scholar]
  45. Roehrig J. T., Piesman J., Hunt A. R., Keen M. G., Happ C. M., Johnson B. J. The hamster immune response to tick-transmitted Borrelia burgdorferi differs from the response to needle-inoculated, cultured organisms. J Immunol. 1992 Dec 1;149(11):3648–3653. [PubMed] [Google Scholar]
  46. Sadziene A., Thompson P. A., Barbour A. G. In vitro inhibition of Borrelia burgdorferi growth by antibodies. J Infect Dis. 1993 Jan;167(1):165–172. doi: 10.1093/infdis/167.1.165. [DOI] [PubMed] [Google Scholar]
  47. Sadziene A., Wilske B., Ferdows M. S., Barbour A. G. The cryptic ospC gene of Borrelia burgdorferi B31 is located on a circular plasmid. Infect Immun. 1993 May;61(5):2192–2195. doi: 10.1128/iai.61.5.2192-2195.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Sambri V., Moroni A., Massaria F., Brocchi E., De Simone F., Cevenini R. Immunological characterization of a low molecular mass polypeptidic antigen of Borrelia burgdorferi. FEMS Microbiol Immunol. 1991 Nov;3(6):345–349. doi: 10.1111/j.1574-6968.1991.tb04260.x. [DOI] [PubMed] [Google Scholar]
  49. Schaible U. E., Gern L., Wallich R., Kramer M. D., Prester M., Simon M. M. Distinct patterns of protective antibodies are generated against Borrelia burgdorferi in mice experimentally inoculated with high and low doses of antigen. Immunol Lett. 1993 May;36(2):219–226. doi: 10.1016/0165-2478(93)90056-8. [DOI] [PubMed] [Google Scholar]
  50. Schwan T. G., Piesman J., Golde W. T., Dolan M. C., Rosa P. A. Induction of an outer surface protein on Borrelia burgdorferi during tick feeding. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2909–2913. doi: 10.1073/pnas.92.7.2909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schwartz J. J., Gazumyan A., Schwartz I. rRNA gene organization in the Lyme disease spirochete, Borrelia burgdorferi. J Bacteriol. 1992 Jun;174(11):3757–3765. doi: 10.1128/jb.174.11.3757-3765.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Sinsky R. J., Piesman J. Ear punch biopsy method for detection and isolation of Borrelia burgdorferi from rodents. J Clin Microbiol. 1989 Aug;27(8):1723–1727. doi: 10.1128/jcm.27.8.1723-1727.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stevenson B., Schwan T. G., Rosa P. A. Temperature-related differential expression of antigens in the Lyme disease spirochete, Borrelia burgdorferi. Infect Immun. 1995 Nov;63(11):4535–4539. doi: 10.1128/iai.63.11.4535-4539.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Stevenson B., Tilly K., Rosa P. A. A family of genes located on four separate 32-kilobase circular plasmids in Borrelia burgdorferi B31. J Bacteriol. 1996 Jun;178(12):3508–3516. doi: 10.1128/jb.178.12.3508-3516.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Szczepanski A., Benach J. L. Lyme borreliosis: host responses to Borrelia burgdorferi. Microbiol Rev. 1991 Mar;55(1):21–34. doi: 10.1128/mr.55.1.21-34.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wallich R., Brenner C., Kramer M. D., Simon M. M. Molecular cloning and immunological characterization of a novel linear-plasmid-encoded gene, pG, of Borrelia burgdorferi expressed only in vivo. Infect Immun. 1995 Sep;63(9):3327–3335. doi: 10.1128/iai.63.9.3327-3335.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wallich R., Simon M. M., Hofmann H., Moter S. E., Schaible U. E., Kramer M. D. Molecular and immunological characterization of a novel polymorphic lipoprotein of Borrelia burgdorferi. Infect Immun. 1993 Oct;61(10):4158–4166. doi: 10.1128/iai.61.10.4158-4166.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. de Silva A. M., Telford S. R., 3rd, Brunet L. R., Barthold S. W., Fikrig E. Borrelia burgdorferi OspA is an arthropod-specific transmission-blocking Lyme disease vaccine. J Exp Med. 1996 Jan 1;183(1):271–275. doi: 10.1084/jem.183.1.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. von Heijne G. Analysis of the distribution of charged residues in the N-terminal region of signal sequences: implications for protein export in prokaryotic and eukaryotic cells. EMBO J. 1984 Oct;3(10):2315–2318. doi: 10.1002/j.1460-2075.1984.tb02132.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES