1887

Abstract

The intestinal spirochaete causes colitis in a wide variety of host species. Little is known about the structure or protein constituents of the outer membrane (OM). To identify surface-exposed proteins in this species, membrane vesicles were isolated from strain 95-1000 cells by osmotic lysis in dHO followed by isopycnic centrifugation in sucrose density gradients. The membrane vesicles were separated into a high-density fraction (HDMV; =1·18 g cm) and a low-density fraction (LDMV; =1·12 g cm). Both fractions were free of flagella and soluble protein contamination. LDMV contained predominantly OM markers (lipo-oligosaccharide and a 29 kDa OM protein) and was used as a source of antigens to produce mAbs. Five -specific mAbs reacting with proteins with molecular masses of 23, 24, 35, 61 and 79 kDa were characterized. The 23 kDa protein was only partially soluble in Triton X-114, whereas the 24 and 35 kDa proteins were enriched in the detergent phase, implying that they were integral membrane proteins or lipoproteins. All three proteins were localized to the OM by immunogold labelling using specific mAbs. The gene encoding the abundant, surface-exposed 23 kDa protein was identified by screening a 95-1000 genome library with the mAb and was expressed in . Sequence analysis showed that it encoded a unique lipoprotein, designated BmpC. Recombinant BmpC partitioned predominantly in the OM fraction of strain SOLR. The mAb to BmpC was used to screen a collection of 13 genetically heterogeneous strains of isolated from five different host species. Interestingly, only strain 95-1000 was reactive with the mAb, indicating that either the surface-exposed epitope on BmpC is variable between strains or that the protein is restricted in its distribution within .

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26755-0
2004-04-01
2024-12-06
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/4/mic1501041.html?itemId=/content/journal/micro/10.1099/mic.0.26755-0&mimeType=html&fmt=ahah

References

  1. Blanco D. R., Reimann K., Skare J., Champion C. I., Foley D., Exner M. M., Hancock R. E. W., Miller J. N., Lovett M. A. 1994; Isolation of the outer membranes from Treponema pallidum and Treponema vincentii. J Bacteriol 176:6088–6099
    [Google Scholar]
  2. Bledsoe H. A., Carroll J. A., Whelchel T. R., Farmer M. A., Dorward D. W., Gherardini F. C. 1994; Isolation and partial characterization of Borrelia burgdorferi inner and outer membranes by using isopycnic centrifugation. J Bacteriol 176:7447–7455
    [Google Scholar]
  3. Caturegli P., Asanovich K. M., Walls J. J., Bakken J. S., Madigan J. E., Popov V. L., Dumler J. S. 2000; ankA: an Ehrlichia phagocytophila group gene encoding a cytoplasmic protein antigen with ankyrin repeats. Infect Immun 68:5277–5283 [CrossRef]
    [Google Scholar]
  4. Chatfield S. N., Fernie D. S., Beesley J., Penn C., Dougan G. 1988; Characterization of the cell envelope of Treponema hyodysenteriae. FEMS Microbiol Lett 55:303–308 [CrossRef]
    [Google Scholar]
  5. Cullen P. A., Lo M., Bulach D. M., Cordwell S. J., Adler B. 2002; Construction and evaluation of a plasmid vector for the expression of recombinant lipoproteins in Escherichia coli. Plasmid 49:18–29
    [Google Scholar]
  6. Cullen P. A., Coutts S. A. J., Cordwell S. J., Bulach D. M., Adler B. 2003; Characterization of a locus encoding four paralogous outer membrane lipoproteins of Brachyspira hyodysenteriae. Microbes Infect 5:275–283 [CrossRef]
    [Google Scholar]
  7. Cunningham T. M., Walker E. M., Miller J. N., Lovett M. A. 1988a; Selective release of the Treponema pallidum outer membrane and associated polypeptides with Triton X-114. J Bacteriol 170:5789–5796
    [Google Scholar]
  8. Cunningham T. M., Thomas D. D., Thompson S. D., Miller J. N., Lovett M. A. 1988b; Identification of Borrelia burgdorferi surface components by Triton X-114 phase partitioning. Ann N Y Acad Sci 539:376–378 [CrossRef]
    [Google Scholar]
  9. Duhamel G. E. 1996; Porcine colonic spirochaetosis caused by Serpulina pilosicoli. In Enteric Diseases, Misset Pigs pp. 10–13Edited by Evans A. Chaiwan, Hong Kong: Shiny International;
    [Google Scholar]
  10. Duhamel G. 2001; Comparative pathology and pathogenesis of naturally acquired and experimentally induced colonic spirochaetosis. Anim Health Res Rev 2:3–17
    [Google Scholar]
  11. Duhamel G., Trott D., Muniappa N., Mathiesen M., Tarasiuk K., Lee J., Hampson D. 1998; Canine intestinal spirochaetes consist of Serpulina pilosicoli and a newly identified group provisionally designated Serpulina canis sp. nov. J Clin Microbiol 36:2264–2270
    [Google Scholar]
  12. Gabe J. D., Chang R. J., Slomiany R., Andrews W. H., McCaman M. T. 1995; Isolation of extracytoplasmic proteins from Serpulina hyodysenteriae B204 and molecular cloning of the flaB1 gene encoding a 38-kilodalton flagella protein. Infect Immun 63:142–148
    [Google Scholar]
  13. Gabe J. D., Dragon E., Chang R. J., McCaman M. T. 1998; Identification of a linked set of genes in Serpulina hyodysenteriae (B204) predicted to encode closely related 39-kilodalton extracytoplasmic proteins. J Bacteriol 180:444–448
    [Google Scholar]
  14. Haake D. A. 2000; Spirochaetal lipoproteins and pathogenesis. Microbiology 146:1491–1504
    [Google Scholar]
  15. Haake D. A., Matsunaga J. 2002; Characterization of the Leptospiral outer membrane and description of three novel Leptospiral membrane proteins. Infect Immun 70:4936–4945 [CrossRef]
    [Google Scholar]
  16. Hampson D., Trott D. J. 1999; Spirochetal diarrhoea/porcine intestinal spirochetosis. In Diseases of Swine pp. 553–562Edited by Straw B., D'Allaire S., Mengling W., Taylor D. J. Ames: Iowa State University Press;
    [Google Scholar]
  17. Howell M. L., Alsabbagh E., Ma J. F. & 10 other authors; 2000; AnkB, a periplasmic ankyrin-like protein in Pseudomonas aeruginosa, is required for optimal catalase B (KatB) activity and resistance to hydrogen peroxide. J Bacteriol 182:4545–4556 [CrossRef]
    [Google Scholar]
  18. Inzana T., Apicella M. 1999; Use of a bilayer stacking gel to improve resolution of lipopolysaccharides and lipooligosaccharides in polyacrylamide gels. Electrophoresis 20:462–465 [CrossRef]
    [Google Scholar]
  19. Joens L. A., Marquez R. M., Halter M. 1993; Comparison of outer membrane fractions of Serpulina (Treponema) hyodysenteriae. Vet Microbiol 35:119–132 [CrossRef]
    [Google Scholar]
  20. Laemmli U. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
    [Google Scholar]
  21. Lee J. I., Hampson D. J. 1994; Genetic characterisation of intestinal spirochaetes and their association with disease. J Med Microbiol 40:365–371 [CrossRef]
    [Google Scholar]
  22. Lee B. J., Hampson D. J. 1995; A monoclonal antibody reacting with the cell envelope of spirochaetes isolated from cases of intestinal spirochaetosis in pigs and humans. FEMS Microbiol Lett 131:179–184 [CrossRef]
    [Google Scholar]
  23. Lee B. J., Hampson D. J. 1996; Production and characterisation of a monoclonal antibody to Serpulina hyodysenteriae. FEMS Microbiol Lett 136:193–197 [CrossRef]
    [Google Scholar]
  24. Lee B. J., La T., Mikosza A. S. J., Hampson D. J. 2000; Identification of the gene encoding BmpB, a 30 kDa outer envelope lipoprotein of Brachyspira (Serpulina) hyodysenteriae, and immunogenicity of recombinant BmpB in mice and pigs. Vet Microbiol 76:245–257 [CrossRef]
    [Google Scholar]
  25. Li Z., Dumas F., Dubreuil D., Jacques M. 1993; A species-specific periplasmic flagellae protein of Serpulina (Treponema) hyodysenteriae. Infect Immun 175:8000–8007
    [Google Scholar]
  26. Markwell M., Haas S., Bieber L., Tolbert N. 1978; A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87:206–210 [CrossRef]
    [Google Scholar]
  27. Matsudaira P. 1987; Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262:10035–10038
    [Google Scholar]
  28. McCaman M., Auer K., Foley W., Gabe J. 1999; Sequence characterization of two new members of a multi-gene family in Serpulina hyodysenteriae (B204) with homology to a 39 kDa surface-exposed protein: vspC and D. Vet Microbiol 68:273–283 [CrossRef]
    [Google Scholar]
  29. McCaman M. T., Auer K., Foley W., Gabe J. D. 2003; Brachyspira hyodysenteriae contains eight linked gene copies related to an expressed 39 kDa surface protein. Microbes Infect 5:1–6 [CrossRef]
    [Google Scholar]
  30. Muniappa N., Duhamel G. E., Mathiesen M. R., Bargar T. W. 1996; Light microscopic and ultrastructural changes in the ceca of chicks inoculated with human and canine Serpulina pilosicoli. Vet Pathol 33:542–550 [CrossRef]
    [Google Scholar]
  31. Ochiai S., Adachi Y., Mori K. 1997; Unification of the genera Serpulina and Brachyspira and proposals of Brachyspira hyodysenteriae comb. nov., Brachyspira innocens comb. nov. and Brachyspira pilosicoli comb. nov. Microbiol Immunobiol 41:445–452 [CrossRef]
    [Google Scholar]
  32. Oliver D. B., Beckwith J. 1982; Regulation of a membrane component required for protein secretion in Escherichia coli. Cell 30:311–319 [CrossRef]
    [Google Scholar]
  33. Oxberry S. L., Trott D. J., Hampson D. J. 1997; Serpulina pilosicoli water and water birds: potential sources of infection for humans and other animals. Epidemiol Infect 121:219–225
    [Google Scholar]
  34. Plaza H., Whelchel T. R., Garcynski S. F., Howerth E. W., Gherardini F. C. 1997; Purified outer membranes of Serpulina hyodysenteriae contain cholesterol. J Bacteriol 179:5414–5421
    [Google Scholar]
  35. Radolf J., Moomaw C., Slaughter C., Norgard M. 1989; Penicillin binding proteins and peptidoglycan of Treponema pallidum subsp. pallidum. Infect Immun 57:1248–1254
    [Google Scholar]
  36. Radolf J. D., Goldberg M. S., Bourell K., Baker S. I., Jones J. D., Norgard M. V. 1995a; Characterization of outer membranes isolated from Borrelia burgdorferi, the Lyme disease spirochete. Infect Immun 63:2154–2163
    [Google Scholar]
  37. Radolf J. D., Robinson E. J., Bourell K. W., Akins D. R., Porcella S. F., Weigel L. M., Jones J. D., Norgard M. V. 1995b; Characterization of outer membranes isolated from Treponema pallidum, the syphilis spirochete. Infect Immun 63:4244–4252
    [Google Scholar]
  38. Rayment S., Lee B., Hampson D., Livesley M. 1998; Identification of a gene encoding a putative pyruvate oxidoreductase in Serpulina pilosicoli. FEMS Microbiol Lett 166:121–126 [CrossRef]
    [Google Scholar]
  39. Sacco R. E., Trampel D. W., Wannemueler M. J. 1997; Experimental infection of mice with avian, porcine or human isolates of Serpulina pilosicoli. Infect Immun 65:5349–5353
    [Google Scholar]
  40. Seshadri R., Paulsen I. T., Eisen J. A.21 other authors 2003; Complete genome sequence of the Q-fever pathogen Coxiella burnetii. Proc Natl Acad Sci U S A 100:5455–5460 [CrossRef]
    [Google Scholar]
  41. Skare J. T., Shang E. S., Foley D. M.7 other authors 1995; Virulent strain associated outer membrane proteins of Borrelia burgdorferi. J Clin Invest 96:2380–2392 [CrossRef]
    [Google Scholar]
  42. Stanton T. B. 1987; Cholesterol metabolism by Treponema hyodysenteriae. Infect Immun 55:309–313
    [Google Scholar]
  43. Stanton T. B. 2002; The genus Brachyspira. In The ProkaryotesEdited by Dworkin M. New York: Springer;
    [Google Scholar]
  44. Stanton T. B., Jensen N. S. 1993; Purification and characterization of NADH oxidase from Serpulina (Treponema) hyodysenteriae. J Bacteriol 175:2980–2987
    [Google Scholar]
  45. Stanton T. B., Lebo D. F. 1988; Treponema hyodysenteriae growth under various culture conditions. Vet Microbiol 18:177–190 [CrossRef]
    [Google Scholar]
  46. Stephens C., Hampson D. J. 2002; Experimental infection of broiler breeder hens with the intestinal spirochaete Brachyspira (Serpulina) pilosicoli causes reduced egg production. Avian Pathol 31:169–175 [CrossRef]
    [Google Scholar]
  47. Tenaya I. W. M., Penhale W. J., Hampson D. J. 1998; Preparation of diagnostic polyclonal and monoclonal antibodies against outer envelope proteins of Serpulina pilosicoli. J Med Microbiol 47:317–324 [CrossRef]
    [Google Scholar]
  48. Thomas W., Sellwood R. 1993; Molecular cloning, expression, and DNA sequence analysis of the gene that encodes the 16-kilodalton outer membrane lipoprotein of Serpulina hyodysenteriae. Infect Immun 61:1136–1140
    [Google Scholar]
  49. Thomas W., Sellwood R., Lysons R. J. 1992; A 16-kilodalton lipoprotein of the outer membrane of Serpulina (Treponema) hyodysenteriae. Infect Immun 60:3111–3116
    [Google Scholar]
  50. Thomson J. R., Smith W. J., Murray B. P., McOrist S. 1997; Pathogenicity of three strains of Serpulina pilosicoli in pigs with a naturally acquired intestinal flora. Infect Immun 65:3693–3700
    [Google Scholar]
  51. Thomson J. R., Smith W. J., Murray B. P. 1998; Investigations into field cases of porcine colitis with particular reference to infection with Serpulina pilosicoli. Vet Rec 142:235–239 [CrossRef]
    [Google Scholar]
  52. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354 [CrossRef]
    [Google Scholar]
  53. Trampel D. W., Jensen N. S., Hoffman L. J. 1994; Cecal spirochetosis in commercial laying hens. Avian Dis 38:895–898 [CrossRef]
    [Google Scholar]
  54. Trivett-Moore N. L., Gilbert G. L., Law C., Trott D. J., Hampson D. J. 1998; Isolation of Serpulina pilosicoli from rectal biopsies showing evidence of intestinal spirochetosis. J Clin Microbiol 36:261–265
    [Google Scholar]
  55. Trott D. J., McLaren A. J., Hampson D. J. 1995; Pathogenicity of human and porcine intestinal spirochaetes in day-old specific pathogen free chicks: an animal model of intestinal spirochetosis. Infect Immun 63:3705–3710
    [Google Scholar]
  56. Trott D. J., Huxtable C. R., Hampson D. J. 1996a; Infection of newly weaned pigs with human and porcine strains of Serpulina pilosicoli. Infect Immun 64:4648–4654
    [Google Scholar]
  57. Trott D. J., Stanton T. A., Jensen N. S., Hampson D. J. 1996b; Phenotypic characteristics of Serpulina pilosicoli the agent of intestinal spirochaetosis. FEMS Microbiol Lett 142:209–214 [CrossRef]
    [Google Scholar]
  58. Trott D. J., Atyeo R. F., Lee J. I., Swayne D. A., Stoutenburg J. W., Hampson D. J. 1996c; Genetic relatedness amongst intestinal spirochaetes isolated from rats and birds. Lett Appl Microbiol 23:431–436 [CrossRef]
    [Google Scholar]
  59. Trott D. J., Stanton T. B., Jensen N. S., Duhamel G. E., Johnson J. L., Hampson D. J. 1996d; Serpulina pilosicoli sp. nov. the agent of porcine intestinal spirochetosis. Int J Syst Bacteriol 46:206–215 [CrossRef]
    [Google Scholar]
  60. Trott D. J., Combs B. G., Mikosza A. S. J., Oxberry S. L., Passey M., Taime J., Sehuko R., Alpers M. P., Hampson D. J. 1997; The prevalence of Serpulina pilosicoli in humans and domestic animals in the Eastern Highlands of Papua New Guinea. Epidemiol Infect 119:369–379 [CrossRef]
    [Google Scholar]
  61. Trott D. J., Alt D. P., Zuerner R. L., Wannemuehler M. J., Stanton T. B. 2001; The search for Brachyspira proteins that interact with the host. Anim Health Res Rev 2:19–30
    [Google Scholar]
  62. Tsai C., Frasch C. 1982; A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem 119:115–119 [CrossRef]
    [Google Scholar]
  63. van Belkum A., Scherer S., van Alphen L., Verbrugh H. 1998; Short sequence DNA repeats in prokaryotic genomes. Microbiol Mol Biol Rev 62:275–293
    [Google Scholar]
  64. Weigel L., Belisle J., Radolf J., Norgard M. 1994; Digoxigenin-ampicillin conjugate for detection of penicillin-binding proteins by chemiluminescence. Antimicrob Agents Chemother 38:330–336 [CrossRef]
    [Google Scholar]
  65. Weinstock G. M., Hardham J. M., McLeod M. P., Sodergren E. J., Norris S. J. 1998; The genome of Treponema pallidum: new light on the agent of syphilis. FEMS Microbiol Rev 22:323–332 [CrossRef]
    [Google Scholar]
  66. Zhang P., Cheng X., Duhamel G. E. 2000; Cloning and DNA sequence analysis of an immunogenic glucose/galactose MglB lipoprotein homologue from Brachyspira pilosicoli the agent of colonic spirochetosis. Infect Immun 68:4559–4565 [CrossRef]
    [Google Scholar]
  67. Zuerner R. L., Knudtson W., Bolin C. A., Trueba G. 1991; Characterization of outer membrane and secreted proteins of Leptospira interrogans serovar pomona. Microb Pathog 10:311–322 [CrossRef]
    [Google Scholar]
/content/journal/micro/10.1099/mic.0.26755-0
Loading
/content/journal/micro/10.1099/mic.0.26755-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error