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. 1991 Oct;59(10):3451–3462. doi: 10.1128/iai.59.10.3451-3462.1991

The immunodominant outer membrane antigen of Actinobacillus actinomycetemcomitans is located in the serotype-specific high-molecular-mass carbohydrate moiety of lipopolysaccharide.

R C Page 1, T J Sims 1, L D Engel 1, B J Moncla 1, B Bainbridge 1, J Stray 1, R P Darveau 1
PMCID: PMC258906  PMID: 1716610

Abstract

Most patients with juvenile periodontitis manifest serum antibodies, sometimes at very high titers, to antigens of Actinobacillus actinomycetemcomitans, but the antigens inducing the immune response have been only partly characterized. We separated A. actinomycetemcomitans serotype b cells into protein, lipopolysaccharide (LPS), and soluble polysaccharide fractions and characterized them. Coomassie blue- and silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels were used to detect protein and LPS components, and gas-liquid chromatography was used to determine their carbohydrate and fatty acid composition. Western blots, dot blots, and enzyme-linked immunosorbent assay inhibition with high-titer sera from juvenile periodontitis patients revealed which components were highest in antibody binding activity. These results showed that the major portion of the immunoglobulin G binding activity resides in the purified mannan-free LPS, with lesser amounts in the total protein fraction. Using Sephacryl S-300 chromatography, we separated LPS into high-molecular-mass components with high carbohydrate contents by gas-liquid chromatography and a low-molecular-mass component consisting mainly of lipid A and the inner core sugar heptulose. The results of quantitative dot blot assays and enzyme-linked immunosorbent assay inhibition show that the serotype-specific antibody binding activity is highly concentrated in the high-molecular-mass carbohydrate-rich LPS fraction and is almost completely absent in the low-molecular-weight lipid-rich fraction. Our observations contrast with previous reports that the predominant serotype antigen of A. actinomycetemcomitans resides in a mannan-rich polysaccharide isolated from spent culture medium. These observations support the conclusion that the immunodominant antigen of the outer membrane is the O antigen of the LPS.

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Selected References

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  1. Amano K., Nishihara T., Shibuya N., Noguchi T., Koga T. Immunochemical and structural characterization of a serotype-specific polysaccharide antigen from Actinobacillus actinomycetemcomitans Y4 (serotype b). Infect Immun. 1989 Oct;57(10):2942–2946. doi: 10.1128/iai.57.10.2942-2946.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Califano J. V., Schenkein H. A., Tew J. G. Immunodominant antigen of Actinobacillus actinomycetemcomitans Y4 in high-responder patients. Infect Immun. 1989 May;57(5):1582–1589. doi: 10.1128/iai.57.5.1582-1589.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Darveau R. P., Hancock R. E. Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J Bacteriol. 1983 Aug;155(2):831–838. doi: 10.1128/jb.155.2.831-838.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Doty S. L., Lopatin D. E., Syed S. A., Smith F. N. Humoral immune response to oral microorganisms in periodontitis. Infect Immun. 1982 Aug;37(2):499–505. doi: 10.1128/iai.37.2.499-505.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ebersole J. L., Sandoval M. N., Steffen M. J., Cappelli D. Serum antibody in Actinobacillus actinomycetemcomitans-infected patients with periodontal disease. Infect Immun. 1991 May;59(5):1795–1802. doi: 10.1128/iai.59.5.1795-1802.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ebersole J. L., Taubman M. A., Smith D. J., Genco R. J., Frey D. E. Human immune responses to oral micro-organisms. I. Association of localized juvenile periodontitis (LJP) with serum antibody responses to Actinobacillus actinomycetemcomitans. Clin Exp Immunol. 1982 Jan;47(1):43–52. [PMC free article] [PubMed] [Google Scholar]
  7. Ebersole J. L., Taubman M. A., Smith D. J., Hammond B. F., Frey D. E. Human immune responses to oral microorganisms. II. Serum antibody responses to antigens from Actinobacillus actinomycetemcomitans and the correlation with localized juvenile periodontitis. J Clin Immunol. 1983 Oct;3(4):321–331. doi: 10.1007/BF00915793. [DOI] [PubMed] [Google Scholar]
  8. Gunsolley J. C., Burmeister J. A., Tew J. G., Best A. M., Ranney R. R. Relationship of serum antibody to attachment level patterns in young adults with juvenile periodontitis or generalized severe periodontitis. J Periodontol. 1987 May;58(5):314–320. doi: 10.1902/jop.1987.58.5.314. [DOI] [PubMed] [Google Scholar]
  9. Hitchcock P. J., Brown T. M. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol. 1983 Apr;154(1):269–277. doi: 10.1128/jb.154.1.269-277.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Issekutz A. C. Removal of gram-negative endotoxin from solutions by affinity chromatography. J Immunol Methods. 1983 Jul 29;61(3):275–281. doi: 10.1016/0022-1759(83)90221-1. [DOI] [PubMed] [Google Scholar]
  11. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  12. 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]
  13. Listgarten M. A., Lai C. H., Evian C. I. Comparative antibody titers to Actinobacillus actinomycetemcomitans in juvenile periodontitis, chronic periodontitis and periodontally healthy subjects. J Clin Periodontol. 1981 Jun;8(3):155–164. doi: 10.1111/j.1600-051x.1981.tb02027.x. [DOI] [PubMed] [Google Scholar]
  14. McArthur W. P., Tsai C. C., Baehni P. C., Genco R. J., Taichman N. S. Leukotoxic effects of Actinobacillus actinomycetemcomitans. Modulation by serum components. J Periodontal Res. 1981 Mar;16(2):159–170. doi: 10.1111/j.1600-0765.1981.tb00962.x. [DOI] [PubMed] [Google Scholar]
  15. Moore W. E. Microbiology of periodontal disease. J Periodontal Res. 1987 Sep;22(5):335–341. doi: 10.1111/j.1600-0765.1987.tb01595.x. [DOI] [PubMed] [Google Scholar]
  16. Peterman J. H., Butler J. E. Application of theoretical considerations to the analysis of ELISA data. Biotechniques. 1989 Jun;7(6):608–615. [PubMed] [Google Scholar]
  17. Peterson A. A., McGroarty E. J. High-molecular-weight components in lipopolysaccharides of Salmonella typhimurium, Salmonella minnesota, and Escherichia coli. J Bacteriol. 1985 May;162(2):738–745. doi: 10.1128/jb.162.2.738-745.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ranney R. R., Yanni N. R., Burmeister J. A., Tew J. G. Relationship between attachment loss and precipitating serum antibody to Actinobacillus actinomycetemcomitans in adolescents and young adults having severe periodontal destruction. J Periodontol. 1982 Jan;53(1):1–7. doi: 10.1902/jop.1982.53.1.1. [DOI] [PubMed] [Google Scholar]
  19. Sims T. J., Moncla B. J., Darveau R. P., Page R. C. Antigens of Actinobacillus actinomycetemcomitans recognized by patients with juvenile periodontitis and periodontally normal subjects. Infect Immun. 1991 Mar;59(3):913–924. doi: 10.1128/iai.59.3.913-924.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Slots J., Reynolds H. S., Genco R. J. Actinobacillus actinomycetemcomitans in human periodontal disease: a cross-sectional microbiological investigation. Infect Immun. 1980 Sep;29(3):1013–1020. doi: 10.1128/iai.29.3.1013-1020.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Slots J., Zambon J. J., Rosling B. G., Reynolds H. S., Christersson L. A., Genco R. J. Actinobacillus actinomycetemcomitans in human periodontal disease. Association, serology, leukotoxicity, and treatment. J Periodontal Res. 1982 Sep;17(5):447–448. doi: 10.1111/j.1600-0765.1982.tb02022.x. [DOI] [PubMed] [Google Scholar]
  22. Syed S. A. Characteristics of Bacteroides asaccharolyticus from dental plaques of beagle dogs. J Clin Microbiol. 1980 May;11(5):522–526. doi: 10.1128/jcm.11.5.522-526.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tew J. G., Marshall D. R., Burmeister J. A., Ranney R. R. Relationship between gingival crevicular fluid and serum antibody titers in young adults with generalized and localized periodontitis. Infect Immun. 1985 Sep;49(3):487–493. doi: 10.1128/iai.49.3.487-493.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Tew J. G., Marshall D. R., Moore W. E., Best A. M., Palcanis K. G., Ranney R. R. Serum antibody reactive with predominant organisms in the subgingival flora of young adults with generalized severe periodontitis. Infect Immun. 1985 May;48(2):303–311. doi: 10.1128/iai.48.2.303-311.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Vincent J. W., Suzuki J. B., Falkler W. A., Jr, Cornett W. C. Reaction of human sera from juvenile periodontitis, rapidly progressive periodontitis, and adult periodontitis patients with selected periodontopathogens. J Periodontol. 1985 Aug;56(8):464–469. doi: 10.1902/jop.1985.56.8.464. [DOI] [PubMed] [Google Scholar]
  27. Westphal O., Jann K., Himmelspach K. Chemistry and immunochemistry of bacterial lipopolysaccharides as cell wall antigens and endotoxins. Prog Allergy. 1983;33:9–39. [PubMed] [Google Scholar]
  28. Wilson M. E., Schifferle R. E. Evidence that the serotype b antigenic determinant of Actinobacillus actinomycetemcomitans Y4 resides in the polysaccharide moiety of lipopolysaccharide. Infect Immun. 1991 Apr;59(4):1544–1551. doi: 10.1128/iai.59.4.1544-1551.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wright B. G., Rebers P. A. Procedure for determining heptose and hexose in lipopolysaccharides. Modification of the cysteine-sulfuric acid method. Anal Biochem. 1972 Oct;49(2):307–319. doi: 10.1016/0003-2697(72)90433-2. [DOI] [PubMed] [Google Scholar]
  30. Zambon J. J. Actinobacillus actinomycetemcomitans in human periodontal disease. J Clin Periodontol. 1985 Jan;12(1):1–20. doi: 10.1111/j.1600-051x.1985.tb01348.x. [DOI] [PubMed] [Google Scholar]
  31. Zambon J. J., Slots J., Genco R. J. Serology of oral Actinobacillus actinomycetemcomitans and serotype distribution in human periodontal disease. Infect Immun. 1983 Jul;41(1):19–27. doi: 10.1128/iai.41.1.19-27.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zambon J. J., Slots J., Miyasaki K., Linzer R., Cohen R., Levine M., Genco R. J. Purification and characterization of the serotype c antigen from Actinobacillus actinomycetemcomitans. Infect Immun. 1984 Apr;44(1):22–27. doi: 10.1128/iai.44.1.22-27.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zambon J. J., Umemoto T., De Nardin E., Nakazawa F., Christersson L. A., Genco R. J. Actinobacillus actinomycetemcomitans in the pathogenesis of human periodontal disease. Adv Dent Res. 1988 Nov;2(2):269–274. doi: 10.1177/08959374880020021101. [DOI] [PubMed] [Google Scholar]

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