Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1996 May;64(5):1631–1637. doi: 10.1128/iai.64.5.1631-1637.1996

Structural domains of Porphyromonas gingivalis recombinant fimbrillin that mediate binding to salivary proline-rich protein and statherin.

A Amano 1, A Sharma 1, J Y Lee 1, H T Sojar 1, P A Raj 1, R J Genco 1
PMCID: PMC173972  PMID: 8613371

Abstract

Fimbriae (the oligomeric form of fimbrillin) are considered important in the adherence and colonization of Porphyromonas gingivalis in the oral cavity. In the present study, we have identified the structural domains of P. gingivalis fimbrillin that mediate the binding to salivary proline-rich protein 1 (PRP1) and statherin. A series of synthetic fimbrillin peptides were used to localize the active fimbrillin domains involved in the binding to PRP1 and statherin. The binding of 125I-labeled 41-r-Fim (whole-length recombinant fimbrillin, amino acid [aa] residues 1 to 337) to PRP1-coated hydroxyapatite beads (HAP) was strongly inhibited by the fimbrillin C-terminal peptides corresponding to aa residues 266 to 286 and 318 to 337 (peptides 266-286, and 318-337, respectively), while the binding to statherin was inhibited by C-terminal peptides 266-286, 293-306 and 307-326. Peptide 126-146 also showed a weak inhibitory effect, about half that of other active peptides, on the binding to both PRP1 and statherin. P. gingivalis whole-cell binding to PRP1- or statherin-coated HAP was inhibited by more than 80% by the same active peptides. To confirm that the C-terminal portion of fimbrillin includes domains responsible for the binding, two C-terminally truncated variants of recombinant fimbrillin were generated and purified. These were designated 34.5-r-Fim, corresponding to aa residues 1 to 286, and 32-r-Fim, corresponding to aa residues 1 to 265. 125I-34.5-r-Fim revealed 35 and 34% loss of binding ability to PRP1 and statherin, respectively. 125I-32-r-Fim had significantly less binding ability to PRP1 and statherin than 125I-34.5-r-Fim, which was reduced 78 and 73%, respectively. Whole-cell binding to PRP1-, statherin-, or whole saliva-coated HAP was inhibited up to 100% by 41-r-Fim, while 32-r-Fim also showed considerable inhibition, possibly due to the region of aa 126 to 146. Collectively, these results suggest that there are separate and multiple binding sites for PRP1 and statherin in the P. gingivalis fimbrillin, and the combination of all of these binding sites may be indispensable in establishing stable bacterial adherence to saliva-coated surfaces in the oral cavity.

Full Text

The Full Text of this article is available as a PDF (425.9 KB).

Selected References

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

  1. Amano A., Sojar H. T., Lee J. Y., Sharma A., Levine M. J., Genco R. J. Salivary receptors for recombinant fimbrillin of Porphyromonas gingivalis. Infect Immun. 1994 Aug;62(8):3372–3380. doi: 10.1128/iai.62.8.3372-3380.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Brant E. E., Sojar H. T., Sharma A., Bedi G. S., Genco R. J., De Nardin E. Identification of linear antigenic sites on the Porphyromonas gingivalis 43-kDa fimbrillin subunit. Oral Microbiol Immunol. 1995 Jun;10(3):146–150. doi: 10.1111/j.1399-302x.1995.tb00135.x. [DOI] [PubMed] [Google Scholar]
  4. Clark W. B., Bammann L. L., Gibbons R. J. Comparative estimates of bacterial affinities and adsorption sites on hydroxyapatite surfaces. Infect Immun. 1978 Mar;19(3):846–853. doi: 10.1128/iai.19.3.846-853.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dahlén G. G. Black-pigmented gram-negative anaerobes in periodontitis. FEMS Immunol Med Microbiol. 1993 Mar;6(2-3):181–192. doi: 10.1111/j.1574-695X.1993.tb00323.x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Fujiwara T., Morishima S., Takahashi I., Hamada S. Molecular cloning and sequencing of the fimbrilin gene of Porphyromonas gingivalis strains and characterization of recombinant proteins. Biochem Biophys Res Commun. 1993 Nov 30;197(1):241–247. doi: 10.1006/bbrc.1993.2467. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Hamada S., Fujiwara T., Morishima S., Takahashi I., Nakagawa I., Kimura S., Ogawa T. Molecular and immunological characterization of the fimbriae of Porphyromonas gingivalis. Microbiol Immunol. 1994;38(12):921–930. doi: 10.1111/j.1348-0421.1994.tb02148.x. [DOI] [PubMed] [Google Scholar]
  10. Kelly C. G., Todryk S., Kendal H. L., Munro G. H., Lehner T. T-cell, adhesion, and B-cell epitopes of the cell surface Streptococcus mutans protein antigen I/II. Infect Immun. 1995 Sep;63(9):3649–3658. doi: 10.1128/iai.63.9.3649-3658.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Lee J. Y., Sojar H. T., Bedi G. S., Genco R. J. Porphyromonas (Bacteroides) gingivalis fimbrillin: size, amino-terminal sequence, and antigenic heterogeneity. Infect Immun. 1991 Jan;59(1):383–389. doi: 10.1128/iai.59.1.383-389.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lee J. Y., Sojar H. T., Bedi G. S., Genco R. J. Synthetic peptides analogous to the fimbrillin sequence inhibit adherence of Porphyromonas gingivalis. Infect Immun. 1992 Apr;60(4):1662–1670. doi: 10.1128/iai.60.4.1662-1670.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Naito Y., Tohda H., Okuda K., Takazoe I. Adherence and hydrophobicity of invasive and noninvasive strains of Porphyromonas gingivalis. Oral Microbiol Immunol. 1993 Aug;8(4):195–202. doi: 10.1111/j.1399-302x.1993.tb00559.x. [DOI] [PubMed] [Google Scholar]
  17. Quehenberger O., Prossnitz E. R., Cavanagh S. L., Cochrane C. G., Ye R. D. Multiple domains of the N-formyl peptide receptor are required for high-affinity ligand binding. Construction and analysis of chimeric N-formyl peptide receptors. J Biol Chem. 1993 Aug 25;268(24):18167–18175. [PubMed] [Google Scholar]
  18. Ramasubbu N., Reddy M. S., Bergey E. J., Haraszthy G. G., Soni S. D., Levine M. J. Large-scale purification and characterization of the major phosphoproteins and mucins of human submandibular-sublingual saliva. Biochem J. 1991 Dec 1;280(Pt 2):341–352. doi: 10.1042/bj2800341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sharma A., Sojar H. T., Lee J. Y., Genco R. J. Expression of a functional Porphyromonas gingivalis fimbrillin polypeptide in Escherichia coli: purification, physicochemical and immunochemical characterization, and binding characteristics. Infect Immun. 1993 Aug;61(8):3570–3573. doi: 10.1128/iai.61.8.3570-3573.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sojar H. T., Lee J. Y., Bedi G. S., Cho M. I., Genco R. J. Purification, characterization and immunolocalization of fimbrial protein from Porphyromonas (bacteroides) gingivalis. Biochem Biophys Res Commun. 1991 Mar 15;175(2):713–719. doi: 10.1016/0006-291x(91)91624-l. [DOI] [PubMed] [Google Scholar]
  21. Washington O. R., Deslauriers M., Stevens D. P., Lyford L. K., Haque S., Yan Y., Flood P. M. Generation and purification of recombinant fimbrillin from Porphyromonas (Bacteroides) gingivalis 381. Infect Immun. 1993 Mar;61(3):1040–1047. doi: 10.1128/iai.61.3.1040-1047.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES