1887

Abstract

Proteinase–adhesin complexes of wild-type and RgpA and Kgp mutants were extracted using a Triton X-114 procedure and purified using arginine-affinity chromatography. The complexes were then characterized by peptide mass fingerprinting (PMF) and their equilibrium binding constants, immunogenicity and ability to induce protection as vaccines in the murine lesion model determined. The Triton X-114 procedure resulted in consistently higher yield and specific activity of the wild-type (wt) complex compared with that produced by the previously published sonication method. PMF and N-terminal sequencing of the purified wt complex showed that it consisted of the previously identified Arg-specific proteinase RgpA, the Lys-specific proteinase Kgp and adhesin domains RgpA, RgpA, RgpA, Kgp and Kgp. However, analysis of the 30 kDa band in the wt complex, previously suggested to be RgpA, indicated that this band contained C-terminally truncated Kgp (which has an identical N-terminus to RgpA) as well as the HagA* adhesin. Analysis of the Triton X-114 extracted complexes from the isogenic mutants (RgpA complex) and (Kgp complex) suggested that the Kgp complex consisted of Kgp, Kgp and Kgp/HagA and that the RgpA complex consisted of RgpA, RgpA, HagA*, RgpA and RgpA. Each of the complexes was found to have equilibrium binding constants ( ) in the nanomolar range for fibrinogen, fibronectin, haemoglobin, collagen type V and laminin. However, the Triton-wt complex exhibited significantly lower values for binding to each host protein compared with the sonication-wt complex, or the Triton-RgpA complex and Triton-Kgp complex. Furthermore, the Triton-wt complex induced a stronger antibody response to the A1 adhesins and tended to be more effective in providing protection in the mouse lesion model compared with the sonication-wt complex.

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2006-08-01
2024-12-07
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References

  1. Baker P. J, Evans R. T, Roopenian D. C. 1994; Oral infection with Porphyromonas gingivalis and induced alveolar bone loss in immunocompetent and severe combined immunodeficient mice. Arch Oral Biol 39:1035–1040 [CrossRef]
    [Google Scholar]
  2. Beck J. D, Offenbacher S, Williams R, Gibbs P, Garcia R. 1998; Periodontitis: a risk factor for coronary heart disease?. Ann Periodontol 3:127–141 [CrossRef]
    [Google Scholar]
  3. Bedi G. S, Williams T. 1994; Purification and characterization of a collagen-degrading protease from Porphyromonas gingivalis . J Biol Chem 269:599–606
    [Google Scholar]
  4. Bhogal P. S, Slakeski N, Reynolds E. C. 1997; A cell-associated protein complex of Porphyromonas gingivalis W50 composed of Arg- and Lys-specific cysteine proteinases and adhesins. Microbiology 143:2485–2495 [CrossRef]
    [Google Scholar]
  5. Bordier C. 1981; Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem 256:1604–1607
    [Google Scholar]
  6. Brooks C. S, Vuppala S. R, Jett A. M, Akins D. R. 2006; Identification of Borrelia burgdorferi outer surface proteins. Infect Immun 74:296–304 [CrossRef]
    [Google Scholar]
  7. Brown L. J, Johns B. A, Wall T. P. 2000; The economics of periodontal diseases. Periodontology 29:223–234
    [Google Scholar]
  8. Chen Z, Potempa J, Polanowski A, Wikstrom M, Travis J. 1992; Purification and characterization of a 50-kDa cysteine proteinase (gingipain) from Porphyromonas gingivalis . J Biol Chem 267:18896–18901
    [Google Scholar]
  9. Cohen J. 1969 Statistical Power Analysis for the Behavioral Sciences New York: Academic Press;
    [Google Scholar]
  10. Cullen P. A, Cordwell S. J, Bulach D. M, Haake D. A, Adler B. 2002; Global analysis of outer membrane proteins from Leptospira interrogans serovar Lai. Infect Immun 70:2311–2318 [CrossRef]
    [Google Scholar]
  11. Curtis M. A, Kuramitsu H. K, Lantz M, Macrina F. L, Nakayama K, Potempa J, Reynolds E. C, Aduse-Opoku J. 1999; Molecular genetics and nomenclature of proteases of Porphyromonas gingivalis . J Periodontal Res 34:464–472 [CrossRef]
    [Google Scholar]
  12. Evans R. T, Klausen B, Ramamurthy N. S, Golub L. M, Sfintescu C, Genco R. J. 1992; Periodontopathic potential of two strains of Porphyromonas gingivalis in gnotobiotic rats. Arch Oral Biol 37:813–819 [CrossRef]
    [Google Scholar]
  13. Fujimura S, Hirai K, Shibata Y, Nakayama K, Nakamura T. 1998; Comparative properties of envelope-associated arginine-gingipains and lysine-gingipain of Porphyromonas gingivalis . FEMS Microbiol Lett 163:173–179 [CrossRef]
    [Google Scholar]
  14. Iwahashi M, Ooshima A, Nakano R. 1996; Increase in the relative level of type V collagen during development and ageing of the placenta. J Clin Pathol 49:916–919 [CrossRef]
    [Google Scholar]
  15. Jain A, Serhan C, Stahl G. L, Van Dyke T. E, Batista E. L., Jr. 2003; Role for periodontitis in the progression of lipid deposition in an animal model. Infect Immun 71:6012–6018 [CrossRef]
    [Google Scholar]
  16. Knibbs R. N, Takagaki M, Blake D. A, Goldstein I. J. 1998; The role of valence on the high-affinity binding of Griffonia simplicifolia isolectins to type A human erythrocytes. Biochemistry 37:16952–16957 [CrossRef]
    [Google Scholar]
  17. Kontani M, Ono H, Shibata H, Okamura Y, Tanaka T, Fujiwara T, Kimura S, Hamada S. 1996; Cysteine protease of Porphyromonas gingivalis 381 enhances binding of fimbriae to cultured human fibroblasts and matrix proteins. Infect Immun 64:756–762
    [Google Scholar]
  18. Lamont R. J, Jenkinson H. F. 2000; Subgingival colonization by Porphyromonas gingivalis . Oral Microbiol Immunol 15:341–349 [CrossRef]
    [Google Scholar]
  19. Lin D, Smith M. A, Champagne C, Elter J, Beck J, Offenbacher S. 2003; Porphyromonas gingivalis infection during pregnancy increases maternal tumor necrosis factor alpha, suppresses maternal interleukin-10, and enhances fetal growth restriction and resorption in mice. Infect Immun 71:5156–5162 [CrossRef]
    [Google Scholar]
  20. Mercado F. B, Marshall R. I, Klestov A. C, Bartold P. M. 2001; Relationship between rheumatoid arthritis and periodontitis. J Periodontol 72:779–787 [CrossRef]
    [Google Scholar]
  21. Merker H. J, Bremer D, Barrach H. J, Gossrau R. 1987; The basement membrane of the persisting maternal blood vessels in the placenta of Callithrix jacchus . Anat Embryol 176:87–97 [CrossRef]
    [Google Scholar]
  22. Mikolajczyk J, Boatright K. M, Stennicke H. R, Nazif T, Potempa J, Bogyo M, Salvesen G. S. 2003; Sequential autolytic processing activates the zymogen of Arg-gingipain. J Biol Chem 278:10458–10464 [CrossRef]
    [Google Scholar]
  23. Miyakawa H, Honma K, Qi M, Kuramitsu H. K. 2004; Interaction of Porphyromonas gingivalis with low-density lipoproteins: implications for a role for periodontitis in atherosclerosis. J Periodontal Res 39:1–9 [CrossRef]
    [Google Scholar]
  24. Mortz E, Vorm O, Mann M, Roepstorff P. 1994; Identification of proteins in polyacrylamide gels by mass spectrometric peptide mapping combined with database search. Biol Mass Spectrom 23:249–261 [CrossRef]
    [Google Scholar]
  25. O'Brien-Simpson N. M, Paolini R. A, Reynolds E. C. 2000; RgpA-Kgp peptide-based immunogens provide protection against Porphyromonas gingivalis challenge in a murine lesion model. Infect Immun 68:4055–4063 [CrossRef]
    [Google Scholar]
  26. O'Brien-Simpson N, Veith P. D, Dashper S. G, Reynolds E. C. 2003; Porphyromonas gingivalis gingipains: the molecular teeth of a microbial vampire. Curr Protein Pept Sci 4:409–426 [CrossRef]
    [Google Scholar]
  27. O'Brien-Simpson N. M, Veith P. D, Dashper S. G, Reynolds E. C. 2004; Antigens of bacteria associated with periodontitis. Periodontology 35:101–134 [CrossRef]
    [Google Scholar]
  28. O'Brien-Simpson N, Pathirana R, Paolini R. A, Chen Y.-Y, Veith P. D, Tam V, Ally N, Pike R, Reynolds E. C. 2005; An immune response directed to proteinase and adhesin functional epitopes protects against Porphyromonas gingivalis -induced periodontal bone loss. J Immunol 175:3900–3989 [CrossRef]
    [Google Scholar]
  29. Offenbacher S, Jared H. L, O'Reilly P. G, Wells S. R, Salvi G. E, Lawrence H. P, Socransky S. S, Beck J. D. 1998; Potential pathogenic mechanisms of periodontitis associated pregnancy complications. Ann Periodontol 3:233–250 [CrossRef]
    [Google Scholar]
  30. Paramonov N, Rangarajan M, Hashim A, Gallagher A, Aduse-Opoku J, Slaney J. M, Hounsell E, Curtis M. A. 2005; Structural analysis of a novel anionic polysaccharide from Porphyromonas gingivalis strain W50 related to Arg-gingipain glycans. Mol Microbiol 58:847–863 [CrossRef]
    [Google Scholar]
  31. Patti J. M, Allen B. L, McGavin M. J, Hook M. 1994; MSCRAMM-mediated adherence of microorganisms to host tissues. Annu Rev Microbiol 48:585–617 [CrossRef]
    [Google Scholar]
  32. Persson G. R, Engel D, Whitney C, Darveau R, Weinberg A, Brunsvold M, Page R. C. 1994; Immunization against Porphyromonas gingivalis inhibits progression of experimental periodontitis in nonhuman primates. Infect Immun 62:1026–1031
    [Google Scholar]
  33. Pike R, McGraw W, Potempa J, Travis J. 1994; Lysine- and arginine-specific proteinases from Porphyromonas gingivalis . Isolation, characterization, and evidence for the existence of complexes with hemagglutinins. J Biol Chem 269:406–411
    [Google Scholar]
  34. Pike R. N, Potempa J, McGraw W, Coetzer T. H, Travis J. 1996; Characterization of the binding activities of proteinase-adhesin complexes from Porphyromonas gingivalis . J Bacteriol 178:2876–2882
    [Google Scholar]
  35. Polzin W. J, Lockrow E. G, Morishige W. K. 1997; A pilot study identifying type V collagenolytic activity in human amniotic fluid. Am J Perinatol 14:103–106 [CrossRef]
    [Google Scholar]
  36. Potempa J, Sroka A, Imamura T, Travis J. 2003; Gingipains, the major cysteine proteinases and virulence factors of Porphyromonas gingivalis : structure, function and assembly of multidomain protein complexes. Curr Protein Pept Sci 4:397–407 [CrossRef]
    [Google Scholar]
  37. Pucher J, Stewart J. 2004; Periodontal disease and diabetes mellitus. Curr Diab Rep 4:46–50 [CrossRef]
    [Google Scholar]
  38. Qiu X, Schroeder P, Bridon D. 1996; Identification and characterization of a C(K/R)TC motif as a common epitope present in all subtypes of hepatitis B surface antigen. J Immunol 156:3350–3356
    [Google Scholar]
  39. Radolf J. D, Chamberlain N. R, Clausell A, Norgard M. V. 1988; 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 56:490–498
    [Google Scholar]
  40. Rajapakse P. S, O'Brien-Simpson N. M, Slakeski N, Hoffmann B, Reynolds E. C. 2002; Immunization with the RgpA-Kgp proteinase-adhesin complexes of Porphyromonas gingivalis protects against periodontal bone loss in the rat periodontitis model. Infect Immun 70:2480–2486 [CrossRef]
    [Google Scholar]
  41. Schwarz-Linek U, Hook M, Potts J. R. 2004; The molecular basis of fibronectin-mediated bacterial adherence to host cells. Mol Microbiol 52:631–641 [CrossRef]
    [Google Scholar]
  42. Slakeski N, Bhogal P. S, O'Brien-Simpson N. M, Reynolds E. C. 1998; Characterization of a second cell-associated Arg-specific cysteine proteinase of Porphyromonas gingivalis and identification of an adhesin-binding motif involved in association of the prtR and prtK proteinases and adhesins into large complexes. Microbiology 144:1583–1592 [CrossRef]
    [Google Scholar]
  43. Slakeski N, Cleal S. M, Bhogal P. S, Reynolds E. C. 1999; Characterization of a Porphyromonas gingivalis gene prtK that encodes a lysine-specific cysteine proteinase and three sequence-related adhesins. Oral Microbiol Immunol 14:92–97 [CrossRef]
    [Google Scholar]
  44. Slots J. 1982; Importance of black-pigmented Bacteroides in human periodontal disease. Host parasite interactions in periodontal diseases. In Host-parasite Interactions in Periodontal Diseases pp  27–45 Edited by Genco R., Merganhagan S. E. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  45. Socransky S. S, Haffajee A. D. 2000; Dental biofilms: difficult therapeutic targets. Periodontology 28:12–55
    [Google Scholar]
  46. Socransky S. S, Haffajee A. D, Cugini M. A, Smith C, Kent R. L. Jr 1998; Microbial complexes in subgingival plaque. J Clin Periodontol 25:134–144 [CrossRef]
    [Google Scholar]
  47. Spahr A, Klein E, Khuseyinova N. 7 other authors 2006; Periodontal infections and coronary heart disease: role of periodontal bacteria and importance of total pathogen burden in the Coronary Event and Periodontal Disease (CORODONT) study. Arch Intern Med 166:554–559 [CrossRef]
    [Google Scholar]
  48. Suckau D, Resemann A, Schuerenberg M, Hufnagel P, Franzen J, Holle A. 2003; A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics. Anal Bioanal Chem 376:952–965 [CrossRef]
    [Google Scholar]
  49. Takii R, Kadowaki T, Baba A, Tsukuba T, Yamamoto K. 2005; A functional virulence complex composed of gingipains, adhesins, and lipopolysaccharide shows high affinity to host cells and matrix proteins and escapes recognition by host immune systems. Infect Immun 73:883–893 [CrossRef]
    [Google Scholar]
  50. van Winkelhoff A. J, Loos B. G, van der Reijden W. A, van der Velden U. 2002; Porphyromonas gingivalis , Bacteroides forsythus and other putative periodontal pathogens in subjects with and without periodontal destruction. J Clin Periodontol 29:1023–1028 [CrossRef]
    [Google Scholar]
  51. Veith P. D, Talbo G. H, Slakeski N, Dashper S. G, Moore C, Paolini R. A, Reynolds E. C. 2002; Major outer membrane proteins and proteolytic processing of RgpA and Kgp of Porphyromonas gingivalis W50. Biochem J 363:105–115 [CrossRef]
    [Google Scholar]
  52. Wu T, Trevisan M, Genco R. J, Falkner K. L, Dorn J. P, Sempos C. T. 2000; Examination of the relation between periodontal health status and cardiovascular risk factors: serum total and high density lipoprotein cholesterol, C-reactive protein, and plasma fibrinogen. Am J Epidemiol 151:273–282 [CrossRef]
    [Google Scholar]
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