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. 1996 May;178(10):2818–2824. doi: 10.1128/jb.178.10.2818-2824.1996

Involvement of arginine-specific cysteine proteinase (Arg-gingipain) in fimbriation of Porphyromonas gingivalis.

K Nakayama 1, F Yoshimura 1, T Kadowaki 1, K Yamamoto 1
PMCID: PMC178016  PMID: 8631669

Abstract

Arginine-specific cysteine proteinase (Arg-gingipain [RGP], a major proteinase secreted from the oral anaerobic bacterium Porphyromonas gingivalis, is encoded by two separate genes (rgpA and rgpB) on the P. gingivalis chromosome and widely implicated as an important virulence factor in the pathogenesis of periodontal disease (K. Nakayama, T. Kadowaki, K. Okamoto, and K. Yamamoto, J. Biol. Chem. 270:23619-23626, 1995). In this study, we investigated the role of RGP in the formation of P. gingivalis fimbriae which are thought to mediate adhesion of the organism to the oral surface by use of the rgp mutants. Electron microscopic observation revealed that the rgpA rgpB double (RGP-null) mutant possessed very few fimbriae on the cell surface, whereas the number of fimbriae of the rgpA or rgpB mutant was similar to that of the wild-type parent strain. The rgpB+ revertants that were isolated from the double mutant and recovered 20 to 40% of RGP activity of the wild-type parent possessed as many fimbriae as the wild-type parent, indicating that RGP significantly contributes to the fimbriation of P. gingivalis as well as to the degradation of various host proteins, disturbance of host defense mechanisms, and hemagglutination. Immunoblot analysis of cell extracts of these mutants with antifimbrilin antiserum revealed that the rgpA rgpB double mutant produced small amounts of two immunoreactive proteins with molecular masses of 45 and 43 kDa, corresponding to those of the precursor and mature forms of fimbrilin, respectively. The result suggests that RGP may function as a processing proteinase for fimbrilin maturation. In addition, a precursor form of the 75-kDa protein, one of the major outer membrane proteins of P. gingivalis, was accumulated in the rgpA rgpB double mutant but not in the single mutants and the revertants, suggesting an extensive role for RGP in the maturation of some of the cell surface proteins.

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Selected References

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  1. Chen Z., Potempa J., Polanowski A., Wikstrom M., Travis J. Purification and characterization of a 50-kDa cysteine proteinase (gingipain) from Porphyromonas gingivalis. J Biol Chem. 1992 Sep 15;267(26):18896–18901. [PubMed] [Google Scholar]
  2. Ciborowski P., Nishikata M., Allen R. D., Lantz M. S. Purification and characterization of two forms of a high-molecular-weight cysteine proteinase (porphypain) from Porphyromonas gingivalis. J Bacteriol. 1994 Aug;176(15):4549–4557. doi: 10.1128/jb.176.15.4549-4557.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dickinson D. P., Kubiniec M. A., Yoshimura F., Genco R. J. Molecular cloning and sequencing of the gene encoding the fimbrial subunit protein of Bacteroides gingivalis. J Bacteriol. 1988 Apr;170(4):1658–1665. doi: 10.1128/jb.170.4.1658-1665.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Fujimura S., Nakamura T. Purification and characterization of a 43-kDa protease of Bacteroides gingivalis. Oral Microbiol Immunol. 1990 Dec;5(6):360–362. doi: 10.1111/j.1399-302x.1990.tb00441.x. [DOI] [PubMed] [Google Scholar]
  5. Hamada N., Watanabe K., Sasakawa C., Yoshikawa M., Yoshimura F., Umemoto T. Construction and characterization of a fimA mutant of Porphyromonas gingivalis. Infect Immun. 1994 May;62(5):1696–1704. doi: 10.1128/iai.62.5.1696-1704.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kadowaki T., Yoneda M., Okamoto K., Maeda K., Yamamoto K. Purification and characterization of a novel arginine-specific cysteine proteinase (argingipain) involved in the pathogenesis of periodontal disease from the culture supernatant of Porphyromonas gingivalis. J Biol Chem. 1994 Aug 19;269(33):21371–21378. [PubMed] [Google Scholar]
  7. 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]
  8. Lawson D. A., Meyer T. F. Biochemical characterization of Porphyromonas (Bacteroides) gingivalis collagenase. Infect Immun. 1992 Apr;60(4):1524–1529. doi: 10.1128/iai.60.4.1524-1529.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lippke J. A., Strzempko M. N., Raia F. F., Simon S. L., French C. K. Isolation of intact high-molecular-weight DNA by using guanidine isothiocyanate. Appl Environ Microbiol. 1987 Oct;53(10):2588–2589. doi: 10.1128/aem.53.10.2588-2589.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lugtenberg B., Meijers J., Peters R., van der Hoek P., van Alphen L. Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Lett. 1975 Oct 15;58(1):254–258. doi: 10.1016/0014-5793(75)80272-9. [DOI] [PubMed] [Google Scholar]
  11. Malek R., Fisher J. G., Caleca A., Stinson M., van Oss C. J., Lee J. Y., Cho M. I., Genco R. J., Evans R. T., Dyer D. W. Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats. J Bacteriol. 1994 Feb;176(4):1052–1059. doi: 10.1128/jb.176.4.1052-1059.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mayrand D., Holt S. C. Biology of asaccharolytic black-pigmented Bacteroides species. Microbiol Rev. 1988 Mar;52(1):134–152. doi: 10.1128/mr.52.1.134-152.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nakayama K., Kadowaki T., Okamoto K., Yamamoto K. Construction and characterization of arginine-specific cysteine proteinase (Arg-gingipain)-deficient mutants of Porphyromonas gingivalis. Evidence for significant contribution of Arg-gingipain to virulence. J Biol Chem. 1995 Oct 6;270(40):23619–23626. doi: 10.1074/jbc.270.40.23619. [DOI] [PubMed] [Google Scholar]
  14. Nakayama K. Rapid viability loss on exposure to air in a superoxide dismutase-deficient mutant of Porphyromonas gingivalis. J Bacteriol. 1994 Apr;176(7):1939–1943. doi: 10.1128/jb.176.7.1939-1943.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ogawa T., Mori H., Yasuda K., Hasegawa M. Molecular cloning and characterization of the genes encoding the immunoreactive major cell-surface proteins of Porphyromonas gingivalis. FEMS Microbiol Lett. 1994 Jul 1;120(1-2):23–30. doi: 10.1111/j.1574-6968.1994.tb07002.x. [DOI] [PubMed] [Google Scholar]
  16. Okamoto K., Misumi Y., Kadowaki T., Yoneda M., Yamamoto K., Ikehara Y. Structural characterization of argingipain, a novel arginine-specific cysteine proteinase as a major periodontal pathogenic factor from Porphyromonas gingivalis. Arch Biochem Biophys. 1995 Feb 1;316(2):917–925. doi: 10.1006/abbi.1995.1123. [DOI] [PubMed] [Google Scholar]
  17. Ono M., Okuda K., Takazoe I. Purification and characterization of a thiol-protease from Bacteroides gingivalis strain 381. Oral Microbiol Immunol. 1987 Jun;2(2):77–81. doi: 10.1111/j.1399-302x.1987.tb00294.x. [DOI] [PubMed] [Google Scholar]
  18. Onoe T., Hoover C. I., Nakayama K., Ideka T., Nakamura H., Yoshimura F. Identification of Porphyromonas gingivalis prefimbrilin possessing a long leader peptide: possible involvement of trypsin-like protease in fimbrilin maturation. Microb Pathog. 1995 Nov;19(5):351–364. doi: 10.1016/s0882-4010(96)80006-4. [DOI] [PubMed] [Google Scholar]
  19. Otsuka M., Endo J., Hinode D., Nagata A., Maehara R., Sato M., Nakamura R. Isolation and characterization of protease from culture supernatant of Bacteroides gingivalis. J Periodontal Res. 1987 Nov;22(6):491–498. doi: 10.1111/j.1600-0765.1987.tb02060.x. [DOI] [PubMed] [Google Scholar]
  20. Pavloff N., Potempa J., Pike R. N., Prochazka V., Kiefer M. C., Travis J., Barr P. J. Molecular cloning and structural characterization of the Arg-gingipain proteinase of Porphyromonas gingivalis. Biosynthesis as a proteinase-adhesin polyprotein. J Biol Chem. 1995 Jan 20;270(3):1007–1010. doi: 10.1074/jbc.270.3.1007. [DOI] [PubMed] [Google Scholar]
  21. Pike R., McGraw W., Potempa J., Travis J. Lysine- and arginine-specific proteinases from Porphyromonas gingivalis. Isolation, characterization, and evidence for the existence of complexes with hemagglutinins. J Biol Chem. 1994 Jan 7;269(1):406–411. [PubMed] [Google Scholar]
  22. Potempa J., Pike R., Travis J. The multiple forms of trypsin-like activity present in various strains of Porphyromonas gingivalis are due to the presence of either Arg-gingipain or Lys-gingipain. Infect Immun. 1995 Apr;63(4):1176–1182. doi: 10.1128/iai.63.4.1176-1182.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Smalley J. W., Birss A. J., Shuttleworth C. A. The degradation of type I collagen and human plasma fibronectin by the trypsin-like enzyme and extracellular membrane vesicles of Bacteroides gingivalis W50. Arch Oral Biol. 1988;33(5):323–329. doi: 10.1016/0003-9969(88)90065-9. [DOI] [PubMed] [Google Scholar]
  24. Smalley J. W., Birss A. J. Trypsin-like enzyme activity of the extracellular membrane vesicles of Bacteroides gingivalis W50. J Gen Microbiol. 1987 Oct;133(10):2883–2894. doi: 10.1099/00221287-133-10-2883. [DOI] [PubMed] [Google Scholar]
  25. Sorsa T., Uitto V. J., Suomalainen K., Turto H., Lindy S. A trypsin-like protease from Bacteroides gingivalis: partial purification and characterization. J Periodontal Res. 1987 Sep;22(5):375–380. doi: 10.1111/j.1600-0765.1987.tb01602.x. [DOI] [PubMed] [Google Scholar]
  26. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  27. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Watanabe K., Takasawa T., Yoshimura F., Ozeki M., Kawanami M., Kato H. Molecular cloning and expression of a major surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis in Escherichia coli. FEMS Microbiol Lett. 1992 Apr 1;71(1):47–55. doi: 10.1016/0378-1097(92)90540-5. [DOI] [PubMed] [Google Scholar]
  29. Yoshimura F., Takahashi K., Nodasaka Y., Suzuki T. Purification and characterization of a novel type of fimbriae from the oral anaerobe Bacteroides gingivalis. J Bacteriol. 1984 Dec;160(3):949–957. doi: 10.1128/jb.160.3.949-957.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Yoshimura F., Takahashi Y., Hibi E., Takasawa T., Kato H., Dickinson D. P. Proteins with molecular masses of 50 and 80 kilodaltons encoded by genes downstream from the fimbrilin gene (fimA) are components associated with fimbriae in the oral anaerobe Porphyromonas gingivalis. Infect Immun. 1993 Dec;61(12):5181–5189. doi: 10.1128/iai.61.12.5181-5189.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Yoshimura F., Watanabe K., Takasawa T., Kawanami M., Kato H. Purification and properties of a 75-kilodalton major protein, an immunodominant surface antigen, from the oral anaerobe Bacteroides gingivalis. Infect Immun. 1989 Nov;57(11):3646–3652. doi: 10.1128/iai.57.11.3646-3652.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. van Winkelhoff A. J., van Steenbergen T. J., de Graaff J. The role of black-pigmented Bacteroides in human oral infections. J Clin Periodontol. 1988 Mar;15(3):145–155. doi: 10.1111/j.1600-051x.1988.tb01561.x. [DOI] [PubMed] [Google Scholar]

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