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. 1997 Sep;65(9):3654–3661. doi: 10.1128/iai.65.9.3654-3661.1997

The Oms66 (p66) protein is a Borrelia burgdorferi porin.

J T Skare 1, T A Mirzabekov 1, E S Shang 1, D R Blanco 1, H Erdjument-Bromage 1, J Bunikis 1, S Bergström 1, P Tempst 1, B L Kagan 1, J N Miller 1, M A Lovett 1
PMCID: PMC175520  PMID: 9284133

Abstract

In this study we report the purification and characterization of a 66-kDa protein, designated Oms66, for outer membrane-spanning 66-kDa protein, that functions as a porin in the outer membrane (OM) of Borrelia burgdorferi. Oms66 was purified by fast-performance liquid chromatography and exhibited an average single-channel conductance of 9.62 +/- 0.37 nS in 1 M KCl, as evidenced by 581 individual insertional events in planar lipid bilayers. Electrophysiological characterization indicated that Oms66 was virtually nonselective between cations and anions and exhibited voltage-dependent closure with multiple substates. The amino acid sequence of tryptic peptides derived from purified Oms66 was identical to the deduced amino acid sequence of p66, a previously described surface-exposed protein of B. burgdorferi. Purified Oms66 was recognized by antiserum specific for p66 and serum from rabbits immune to challenge with virulent B. burgdorferi, indicating that p66 and Oms66 were identical proteins and that Oms66/p66 is an immunogenic protein in infected rabbits. In a methodology that reduces liposomal trapping and nonspecific interactions, native Oms66 was incorporated into liposomes, confirming that Oms66 is an outer membrane-spanning protein. Proteoliposomes containing Oms66 exhibited porin activity nearly identical to that of native, purified Oms66, indicating that reconstituted Oms66 retained native conformation. The use of proteoliposomes reconstituted with Oms66 and other Oms proteins provides an experimental system for determinating the relationship between conformation, protection, and biological function of these molecules.

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Selected References

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  1. 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]
  2. Benz R. Permeation of hydrophilic solutes through mitochondrial outer membranes: review on mitochondrial porins. Biochim Biophys Acta. 1994 Jun 29;1197(2):167–196. doi: 10.1016/0304-4157(94)90004-3. [DOI] [PubMed] [Google Scholar]
  3. Bergström S., Bundoc V. G., Barbour A. G. Molecular analysis of linear plasmid-encoded major surface proteins, OspA and OspB, of the Lyme disease spirochaete Borrelia burgdorferi. Mol Microbiol. 1989 Apr;3(4):479–486. doi: 10.1111/j.1365-2958.1989.tb00194.x. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Blanco D. R., Miller J. N., Lovett M. A. Surface antigens of the syphilis spirochete and their potential as virulence determinants. Emerg Infect Dis. 1997 Jan-Mar;3(1):11–20. doi: 10.3201/eid0301.970102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Bunikis J., Noppa L., Bergström S. Molecular analysis of a 66-kDa protein associated with the outer membrane of Lyme disease Borrelia. FEMS Microbiol Lett. 1995 Sep 1;131(2):139–145. doi: 10.1111/j.1574-6968.1995.tb07768.x. [DOI] [PubMed] [Google Scholar]
  9. Bunikis J., Noppa L., Ostberg Y., Barbour A. G., Bergström S. Surface exposure and species specificity of an immunoreactive domain of a 66-kilodalton outer membrane protein (P66) of the Borrelia spp. that cause Lyme disease. Infect Immun. 1996 Dec;64(12):5111–5116. doi: 10.1128/iai.64.12.5111-5116.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Burgdorfer W., Barbour A. G., Hayes S. F., Benach J. L., Grunwaldt E., Davis J. P. Lyme disease-a tick-borne spirochetosis? Science. 1982 Jun 18;216(4552):1317–1319. doi: 10.1126/science.7043737. [DOI] [PubMed] [Google Scholar]
  11. Burgdorfer W. Discovery of the Lyme disease spirochete and its relation to tick vectors. Yale J Biol Med. 1984 Jul-Aug;57(4):515–520. [PMC free article] [PubMed] [Google Scholar]
  12. Cowan S. W., Schirmer T., Rummel G., Steiert M., Ghosh R., Pauptit R. A., Jansonius J. N., Rosenbusch J. P. Crystal structures explain functional properties of two E. coli porins. Nature. 1992 Aug 27;358(6389):727–733. doi: 10.1038/358727a0. [DOI] [PubMed] [Google Scholar]
  13. Egli C., Leung W. K., Müller K. H., Hancock R. E., McBride B. C. Pore-forming properties of the major 53-kilodalton surface antigen from the outer sheath of Treponema denticola. Infect Immun. 1993 May;61(5):1694–1699. doi: 10.1128/iai.61.5.1694-1699.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fenno J. C., Müller K. H., McBride B. C. Sequence analysis, expression, and binding activity of recombinant major outer sheath protein (Msp) of Treponema denticola. J Bacteriol. 1996 May;178(9):2489–2497. doi: 10.1128/jb.178.9.2489-2497.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Foley D. M., Gayek R. J., Skare J. T., Wagar E. A., Champion C. I., Blanco D. R., Lovett M. A., Miller J. N. Rabbit model of Lyme borreliosis: erythema migrans, infection-derived immunity, and identification of Borrelia burgdorferi proteins associated with virulence and protective immunity. J Clin Invest. 1995 Aug;96(2):965–975. doi: 10.1172/JCI118144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jap B. K., Walian P. J. Biophysics of the structure and function of porins. Q Rev Biophys. 1990 Nov;23(4):367–403. doi: 10.1017/s003358350000559x. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Kropinski A. M., Parr T. R., Jr, Angus B. L., Hancock R. E., Ghiorse W. C., Greenberg E. P. Isolation of the outer membrane and characterization of the major outer membrane protein from Spirochaeta aurantia. J Bacteriol. 1987 Jan;169(1):172–179. doi: 10.1128/jb.169.1.172-179.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Luft B. J., Jiang W., Munoz P., Dattwyler R. J., Gorevic P. D. Biochemical and immunological characterization of the surface proteins of Borrelia burgdorferi. Infect Immun. 1989 Nov;57(11):3637–3645. doi: 10.1128/iai.57.11.3637-3645.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nikaido H. Transport across the bacterial outer membrane. J Bioenerg Biomembr. 1993 Dec;25(6):581–589. doi: 10.1007/BF00770245. [DOI] [PubMed] [Google Scholar]
  22. Pachner A. R., Steere A. C. The triad of neurologic manifestations of Lyme disease: meningitis, cranial neuritis, and radiculoneuritis. Neurology. 1985 Jan;35(1):47–53. doi: 10.1212/wnl.35.1.47. [DOI] [PubMed] [Google Scholar]
  23. Probert W. S., Allsup K. M., LeFebvre R. B. Identification and characterization of a surface-exposed, 66-kilodalton protein from Borrelia burgdorferi. Infect Immun. 1995 May;63(5):1933–1939. doi: 10.1128/iai.63.5.1933-1939.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. 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]
  28. Rudel T., Schmid A., Benz R., Kolb H. A., Lang F., Meyer T. F. Modulation of Neisseria porin (PorB) by cytosolic ATP/GTP of target cells: parallels between pathogen accommodation and mitochondrial endosymbiosis. Cell. 1996 May 3;85(3):391–402. doi: 10.1016/s0092-8674(00)81117-4. [DOI] [PubMed] [Google Scholar]
  29. Shang E. S., Exner M. M., Summers T. A., Martinich C., Champion C. I., Hancock R. E., Haake D. A. The rare outer membrane protein, OmpL1, of pathogenic Leptospira species is a heat-modifiable porin. Infect Immun. 1995 Aug;63(8):3174–3181. doi: 10.1128/iai.63.8.3174-3181.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Skare J. T., Champion C. I., Mirzabekov T. A., Shang E. S., Blanco D. R., Erdjument-Bromage H., Tempst P., Kagan B. L., Miller J. N., Lovett M. A. Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi. J Bacteriol. 1996 Aug;178(16):4909–4918. doi: 10.1128/jb.178.16.4909-4918.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Steere A. C., Bartenhagen N. H., Craft J. E., Hutchinson G. J., Newman J. H., Rahn D. W., Sigal L. H., Spieler P. N., Stenn K. S., Malawista S. E. The early clinical manifestations of Lyme disease. Ann Intern Med. 1983 Jul;99(1):76–82. doi: 10.7326/0003-4819-99-1-76. [DOI] [PubMed] [Google Scholar]
  33. Steere A. C., Batsford W. P., Weinberg M., Alexander J., Berger H. J., Wolfson S., Malawista S. E. Lyme carditis: cardiac abnormalities of Lyme disease. Ann Intern Med. 1980 Jul;93(1):8–16. doi: 10.7326/0003-4819-93-1-8. [DOI] [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. Steere A. C., Schoen R. T., Taylor E. The clinical evolution of Lyme arthritis. Ann Intern Med. 1987 Nov;107(5):725–731. doi: 10.7326/0003-4819-107-5-725. [DOI] [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. 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]
  38. Weinberg A., Holt S. C. Chemical and biological activities of a 64-kilodalton outer sheath protein from Treponema denticola strains. J Bacteriol. 1991 Nov;173(21):6935–6947. doi: 10.1128/jb.173.21.6935-6947.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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