Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1985 Oct;50(1):15–21. doi: 10.1128/iai.50.1.15-21.1985

Antigenic heterogeneity of outer membrane proteins of nontypable Haemophilus influenzae is a basis for a serotyping system.

T F Murphy, M A Apicella
PMCID: PMC262126  PMID: 3876283

Abstract

A serotyping system for nontypable Haemophilus influenzae (NTHI) was developed by using isolated outer membrane protein (OMP) preparations and rabbit antisera. OMPs of 23 strains were isolated by molecular sieve chromatography of outer membranes in 1.5% sodium deoxycholate buffer. These OMP preparations were relatively free of lipopolysaccharide as determined by silver staining of sodium dodecyl sulfate gels and by dot assay with a monoclonal antibody which is specific for the lipid A of H. influenzae. Three antisera raised to whole organisms were used to serotype 21 of 23 strains with a kinetic enzyme-linked immunosorbent assay. Digestion of OMP preparations with proteinase K removed greater than 90% of the antigenic reactivity, indicating that the system is based on OMP antigens. Marked antigenic heterogeneity of OMPs exists among strains of NTHI. By determining the pattern of serological reactivity of OMPs with the three antisera, isolates were divided into groups based on antigenic differences. Six serotypes were identified. This OMP serotyping system is based on multiple antigenic determinants. Future studies will focus on identifying serotype-specific epitopes to further refine this serological classification scheme for NTHI.

Full text

PDF
17

Images in this article

17Image
on p.17
17Image
on p.17

Selected References

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

  1. Anderson P., Flesher A., Shaw S., Harding A. L., Smith D. H. Phenotypic and genetic variation in the susceptibility of Haemophilus influenzae type b to antibodies to somatic antigens. J Clin Invest. 1980 Apr;65(4):885–891. doi: 10.1172/JCI109741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Apicella M. A., Dudas K. C., Campagnari A., Rice P., Mylotte J. M., Murphy T. F. Antigenic heterogeneity of lipid A of Haemophilus influenzae. Infect Immun. 1985 Oct;50(1):9–14. doi: 10.1128/iai.50.1.9-14.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barenkamp S. J., Munson R. S., Jr, Granoff D. M. Outer membrane protein and biotype analysis of pathogenic nontypable Haemophilus influenzae. Infect Immun. 1982 May;36(2):535–540. doi: 10.1128/iai.36.2.535-540.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berk S. L., Holtsclaw S. A., Wiener S. L., Smith J. K. Nontypeable Haemophilus influenzae in the elderly. Arch Intern Med. 1982 Mar;142(3):537–539. [PubMed] [Google Scholar]
  5. Burans J. P., Lynn M., Solotorovsky M. Induction of active immunity with membrane fractions from Haemophilus influenzae type b. Infect Immun. 1983 Jul;41(1):285–293. doi: 10.1128/iai.41.1.285-293.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Danielsson D., Sandström E. Serology of Neisseria gonorrhoeae. Demonstration by co-agglutination and immunoelectrophoresis of antigenic differences associated with colour/opacity colonial variants. Acta Pathol Microbiol Scand B. 1980 Feb;88(1):39–46. doi: 10.1111/j.1699-0463.1980.tb02601.x. [DOI] [PubMed] [Google Scholar]
  7. Ebeling W., Hennrich N., Klockow M., Metz H., Orth H. D., Lang H. Proteinase K from Tritirachium album Limber. Eur J Biochem. 1974 Aug 15;47(1):91–97. doi: 10.1111/j.1432-1033.1974.tb03671.x. [DOI] [PubMed] [Google Scholar]
  8. Ellner P. D., Shahidi A. Postpartum bacteremia due to Haemophilus influenzae. Report of two cases. Obstet Gynecol. 1969 Sep;34(3):403–405. [PubMed] [Google Scholar]
  9. Erwin A. L., Kenny G. E. Haemophilus influenzae type b isolates show antigenic variation in a major outer membrane protein. Infect Immun. 1984 Nov;46(2):570–577. doi: 10.1128/iai.46.2.570-577.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gnehm H. E., Pelton S. I., Gulati S., Rice P. A. Characterization of antigens from nontypable Haemophilus influenzae recognized by human bactericidal antibodies. Role of Haemophilus outer membrane proteins. J Clin Invest. 1985 May;75(5):1645–1658. doi: 10.1172/JCI111872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gulig P. A., Frisch C. F., Hansen E. J. A set of two monoclonal antibodies specific for the cell surface-exposed 39K major outer membrane protein of Haemophilus influenzae type b defines all strains of this pathogen. Infect Immun. 1983 Nov;42(2):516–524. doi: 10.1128/iai.42.2.516-524.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hansen M. V., Musher D. M., Baughn R. E. Outer membrane proteins of nontypable Haemophilus influenzae and reactivity of paired sera from infected patients with their homologous isolates. Infect Immun. 1985 Mar;47(3):843–846. doi: 10.1128/iai.47.3.843-846.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Johnston K. H., Gotschlich E. C. Isolation and characterization of the outer membrane of Neisseria gonorrhoeae. J Bacteriol. 1974 Jul;119(1):250–257. doi: 10.1128/jb.119.1.250-257.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Klein J. O. Microbiology and antimicrobial treatment of otitis media. Ann Otol Rhinol Laryngol Suppl. 1981 May-Jun;90(3 Pt 3):30–36. doi: 10.1177/00034894810903s209. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Leive L. Release of lipopolysaccharide by EDTA treatment of E. coli. Biochem Biophys Res Commun. 1965 Nov 22;21(4):290–296. doi: 10.1016/0006-291x(65)90191-9. [DOI] [PubMed] [Google Scholar]
  19. Loeb M. R., Smith D. H. Outer membrane protein composition in disease isolates of Haemophilus influenzae: pathogenic and epidemiological implications. Infect Immun. 1980 Dec;30(3):709–717. doi: 10.1128/iai.30.3.709-717.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Munson R. S., Jr, Shenep J. L., Barenkamp S. J., Granoff D. M. Purification and comparison of outer membrane protein P2 from Haemophilus influenzae type b isolates. J Clin Invest. 1983 Aug;72(2):677–684. doi: 10.1172/JCI111017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Murphy T. F., Dudas K. C., Mylotte J. M., Apicella M. A. A subtyping system for nontypable Haemophilus influenzae based on outer-membrane proteins. J Infect Dis. 1983 May;147(5):838–846. doi: 10.1093/infdis/147.5.838. [DOI] [PubMed] [Google Scholar]
  22. Musher D. M., Hague-Park M., Baughn R. E., Wallace R. J., Jr, Cowley B. Opsonizing and bactericidal effects of normal human serum on nontypable Haemophilus influenzae. Infect Immun. 1983 Jan;39(1):297–304. doi: 10.1128/iai.39.1.297-304.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Musher D. M., Kubitschek K. R., Crennan J., Baughn R. E. Pneumonia and acute febrile tracheobronchitis due to haemophilus influenzae. Ann Intern Med. 1983 Oct;99(4):444–450. doi: 10.7326/0003-4819-99-4-444. [DOI] [PubMed] [Google Scholar]
  24. Rice P. A., Kasper D. L. Characterization of gonococcal antigens responsible for induction of bactericidal antibody in disseminated infection. J Clin Invest. 1977 Nov;60(5):1149–1158. doi: 10.1172/JCI108867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rice P. A., Kasper D. L. Characterization of serum resistance of Neisseria gonorrhoeae that disseminate. Roles of blocking antibody and gonococcal outer membrane proteins. J Clin Invest. 1982 Jul;70(1):157–167. doi: 10.1172/JCI110589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sandström E., Danielsson D. Serology of Neisseria gonorrhoeae. Characterization of rabbit hyperimmune antisera by line-rocket immunoelectrophoresis for use in coagglutination. Acta Pathol Microbiol Scand B. 1980 Feb;88(1):17–26. doi: 10.1111/j.1699-0463.1980.tb02599.x. [DOI] [PubMed] [Google Scholar]
  27. Sandström E., Danielsson D. Serology of Neisseria gonorrhoeae. Classification by co-agglutination. Acta Pathol Microbiol Scand B. 1980 Feb;88(1):27–38. doi: 10.1111/j.1699-0463.1980.tb02600.x. [DOI] [PubMed] [Google Scholar]
  28. Simon H. B., Southwick F. S., Moellering R. C., Jr, Sherman E. Hemophilus influenzae in hospitalized adults: current perspectives. Am J Med. 1980 Aug;69(2):219–226. doi: 10.1016/0002-9343(80)90381-2. [DOI] [PubMed] [Google Scholar]
  29. Stull T. L., Jacobs R. F., Haas J. E., Roberts M. C., Wilson C. B., Smith A. L. Human serum bactericidal activity against Haemophilus influenzae type b. J Gen Microbiol. 1984 Mar;130(3):665–672. doi: 10.1099/00221287-130-3-665. [DOI] [PubMed] [Google Scholar]
  30. Tam M. R., Buchanan T. M., Sandström E. G., Holmes K. K., Knapp J. S., Siadak A. W., Nowinski R. C. Serological classification of Neisseria gonorrhoeae with monoclonal antibodies. Infect Immun. 1982 Jun;36(3):1042–1053. doi: 10.1128/iai.36.3.1042-1053.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tsai C. M., Frasch C. E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem. 1982 Jan 1;119(1):115–119. doi: 10.1016/0003-2697(82)90673-x. [DOI] [PubMed] [Google Scholar]
  32. Tsang V. C., Wilson B. C., Peralta J. M. Quantitative, single-tube, kinetic-dependent enzyme-linked immunosorbent assay (k-ELISA). Methods Enzymol. 1983;92:391–403. doi: 10.1016/0076-6879(83)92033-5. [DOI] [PubMed] [Google Scholar]
  33. Wallace R. J., Jr, Baker C. J., Quinones F. J., Hollis D. G., Weaver R. E., Wiss K. Nontypable Haemophilus influenzae (biotype 4) as a neonatal, maternal, and genital pathogen. Rev Infect Dis. 1983 Jan-Feb;5(1):123–136. doi: 10.1093/clinids/5.1.123. [DOI] [PubMed] [Google Scholar]
  34. Wallace R. J., Jr, Musher D. M., Septimus E. J., McGowan J. E., Jr, Quinones F. J., Wiss K., Vance P. H., Trier P. A. Haemophilus influenzae infections in adults: characterization of strains by serotypes, biotypes, and beta-lactamase production. J Infect Dis. 1981 Aug;144(2):101–106. doi: 10.1093/infdis/144.2.101. [DOI] [PubMed] [Google Scholar]

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

RESOURCES