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. 1996 Jul 23;93(15):7973–7978. doi: 10.1073/pnas.93.15.7973

Limited surface exposure of Borrelia burgdorferi outer surface lipoproteins.

D L Cox 1, D R Akins 1, K W Bourell 1, P Lahdenne 1, M V Norgard 1, J D Radolf 1
PMCID: PMC38859  PMID: 8755587

Abstract

We used novel immunofluorescence strategies to demonstrate that outer surface proteins (Osps) A, B and C of Borrelia burgdorferi have limited surface exposure, finding that contradicts the prevailing viewpoint that these antigens are exclusively surface exposed. Light labeling was observed when antibodies to OspA or OspB were added to motile organisms, whereas intense fluorescence was observed when the same slides were methanol-fixed and reprobed. Modest labeling also was observed when spirochetes encapsulated in agarose beads (gel microdroplets) were incubated with antibodies to these same two antigens. This contrasted with the intense fluorescence observed when encapsulated spirochetes were probed in the presence of 0.06% Triton X-100, which selectively removed outer membranes. Proteinase K (PK) treatment of encapsulated spirochetes abrogated surface labeling. However, PK-treated spirochetes fluoresced intensely after incubation with antibodies to OspA or OspB in the presence of detergent, confirming the existence of large amounts of subsurface Osp antigens. Modest surface labeling once again was detected when PK-treated spirochetes were reprobed after overnight incubation, a result consistent with the existence of a postulated secretory apparatus that shuttles lipoproteins to the borrelial surface. Last, experiments with the OspC-expressing B. burgdorferi strain 297 revealed that this antigen was barely detectable on spirochetal surfaces even though it was a major constituent of isolated outer mem- branes. We propose a model of B. burgdorferi molecular architecture that helps to explain spirochetal persistence during chronic Lyme disease.

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Selected References

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

  1. 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]
  2. Barbour A. G., Hayes S. F. Biology of Borrelia species. Microbiol Rev. 1986 Dec;50(4):381–400. doi: 10.1128/mr.50.4.381-400.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barbour A. G., Tessier S. L., Hayes S. F. Variation in a major surface protein of Lyme disease spirochetes. Infect Immun. 1984 Jul;45(1):94–100. doi: 10.1128/iai.45.1.94-100.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. Brandt M. E., Riley B. S., Radolf J. D., Norgard M. V. Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins. Infect Immun. 1990 Apr;58(4):983–991. doi: 10.1128/iai.58.4.983-991.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Coleman J. L., Benach J. L. Isolation of antigenic components from the Lyme disease spirochete: their role in early diagnosis. J Infect Dis. 1987 Apr;155(4):756–765. doi: 10.1093/infdis/155.4.756. [DOI] [PubMed] [Google Scholar]
  11. Coleman J. L., Rogers R. C., Rosa P. A., Benach J. L. Variations in the ospB gene of Borrelia burgdorferi result in differences in monoclonal antibody reactivity and in production of escape variants. Infect Immun. 1994 Jan;62(1):303–307. doi: 10.1128/iai.62.1.303-307.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Dunn J. J., Lade B. N., Barbour A. G. Outer surface protein A (OspA) from the Lyme disease spirochete, Borrelia burgdorferi: high level expression and purification of a soluble recombinant form of OspA. Protein Expr Purif. 1990 Nov;1(2):159–168. doi: 10.1016/1046-5928(90)90011-m. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Fikrig E., Tao H., Kantor F. S., Barthold S. W., Flavell R. A. Evasion of protective immunity by Borrelia burgdorferi by truncation of outer surface protein B. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4092–4096. doi: 10.1073/pnas.90.9.4092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fung B. P., McHugh G. L., Leong J. M., Steere A. C. Humoral immune response to outer surface protein C of Borrelia burgdorferi in Lyme disease: role of the immunoglobulin M response in the serodiagnosis of early infection. Infect Immun. 1994 Aug;62(8):3213–3221. doi: 10.1128/iai.62.8.3213-3221.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gassmann G. S., Kramer M., Göbel U. B., Wallich R. Nucleotide sequence of a gene encoding the Borrelia burgdorferi flagellin. Nucleic Acids Res. 1989 May 11;17(9):3590–3590. doi: 10.1093/nar/17.9.3590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. Norris S. J., Carter C. J., Howell J. K., Barbour A. G. Low-passage-associated proteins of Borrelia burgdorferi B31: characterization and molecular cloning of OspD, a surface-exposed, plasmid-encoded lipoprotein. Infect Immun. 1992 Nov;60(11):4662–4672. doi: 10.1128/iai.60.11.4662-4672.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Preac-Mursic V., Wilske B., Patsouris E., Jauris S., Will G., Soutschek E., Rainhardt S., Lehnert G., Klockmann U., Mehraein P. Active immunization with pC protein of Borrelia burgdorferi protects gerbils against B. burgdorferi infection. Infection. 1992 Nov-Dec;20(6):342–349. doi: 10.1007/BF01710681. [DOI] [PubMed] [Google Scholar]
  24. Probert W. S., LeFebvre R. B. Protection of C3H/HeN mice from challenge with Borrelia burgdorferi through active immunization with OspA, OspB, or OspC, but not with OspD or the 83-kilodalton antigen. Infect Immun. 1994 May;62(5):1920–1926. doi: 10.1128/iai.62.5.1920-1926.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. 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]
  29. 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]
  30. 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]
  31. 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]
  32. 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]
  33. Skare J. T., Shang E. S., Foley D. M., Blanco D. R., Champion C. I., Mirzabekov T., Sokolov Y., Kagan B. L., Miller J. N., Lovett M. A. Virulent strain associated outer membrane proteins of Borrelia burgdorferi. J Clin Invest. 1995 Nov;96(5):2380–2392. doi: 10.1172/JCI118295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Steere A. C. Lyme disease. N Engl J Med. 1989 Aug 31;321(9):586–596. doi: 10.1056/NEJM198908313210906. [DOI] [PubMed] [Google Scholar]
  35. Suk K., Das S., Sun W., Jwang B., Barthold S. W., Flavell R. A., Fikrig E. Borrelia burgdorferi genes selectively expressed in the infected host. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4269–4273. doi: 10.1073/pnas.92.10.4269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Wilske B., Preac-Mursic V., Jauris S., Hofmann A., Pradel I., Soutschek E., Schwab E., Will G., Wanner G. Immunological and molecular polymorphisms of OspC, an immunodominant major outer surface protein of Borrelia burgdorferi. Infect Immun. 1993 May;61(5):2182–2191. doi: 10.1128/iai.61.5.2182-2191.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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