Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1983 Aug;72(2):677–684. doi: 10.1172/JCI111017

Purification and comparison of outer membrane protein P2 from Haemophilus influenzae type b isolates.

R S Munson Jr, J L Shenep, S J Barenkamp, D M Granoff
PMCID: PMC1129227  PMID: 6603479

Abstract

Haemophilus influenzae type b isolates have been subdivided based on differences in major outer membrane protein (OMP) profiles resolved on gradient and modified Laemmli sodium dodecyl sulfate-polyacrylamide gel electrophoresis systems. Although 21 subtypes have been observed, 86% of invasive isolates have one of five common subtypes and 71% of isolates have one of three subtypes. In isolates with two of the most common outer membrane subtypes, one major OMP has an apparent molecular weight of 37,000. In isolates with another common OMP subtype, a cross-reactive protein with an apparent molecular weight of 36,500 is observed. All three proteins have been designated P2. Protein P2 from these prototype isolates was solubilized with Zwittergent 3-14 and purified to homogeneity. Based on amino acid compositions, cyanogen bromide cleavage products, staphylococcal V8 protease, and chymotryptic peptide maps, the P2 protein from the three isolates has been highly conserved. Rabbit antibody prepared against P2 from one strain was cross-reactive with P2 isolated from the other two heterologous strains by Western blot. This antibody passively protected infant rats against type b Haemophilus infection caused by the homologous organism, but not against challenge by a strain with the heterologous 36,500 mol wt P2 protein. Thus, although the P2 protein from isolates with different OMP subtypes are closely related, the protection experiments suggest that determinants on the cell surface interacting with protective antibody may be strain or subtype specific.

Full text

PDF
678

Images in this article

680FIGURE 2
on p.680
680FIGURE 3
on p.680
681FIGURE 4
on p.681
681FIGURE 5
on p.681
682FIGURE 6
on p.682
682FIGURE 7
on p.682

Selected References

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

  1. 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]
  2. Barenkamp S. J., Munson R. S., Jr, Granoff D. M. Subtyping isolates of Haemophilus influenzae type b by outer-membrane protein profiles. J Infect Dis. 1981 May;143(5):668–676. doi: 10.1093/infdis/143.5.668. [DOI] [PubMed] [Google Scholar]
  3. Blake M. S., Gotschlich E. C. Purification and partial characterization of the major outer membrane protein of Neisseria gonorrhoeae. Infect Immun. 1982 Apr;36(1):277–283. doi: 10.1128/iai.36.1.277-283.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  5. Elder J. H., Pickett R. A., 2nd, Hampton J., Lerner R. A. Radioiodination of proteins in single polyacrylamide gel slices. Tryptic peptide analysis of all the major members of complex multicomponent systems using microgram quantities of total protein. J Biol Chem. 1977 Sep 25;252(18):6510–6515. [PubMed] [Google Scholar]
  6. Filip C., Fletcher G., Wulff J. L., Earhart C. F. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J Bacteriol. 1973 Sep;115(3):717–722. doi: 10.1128/jb.115.3.717-722.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Frasch C. E., Peppler M. S. Protection against group B Neisseria meningitidis disease: preparation of soluble protein and protein-polysaccharide immunogens. Infect Immun. 1982 Jul;37(1):271–280. doi: 10.1128/iai.37.1.271-280.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gonenne A., Ernst R. Solubilization of membrane proteins by sulfobetaines, novel zwitterionic surfactants. Anal Biochem. 1978 Jun 15;87(1):28–38. doi: 10.1016/0003-2697(78)90565-1. [DOI] [PubMed] [Google Scholar]
  9. Gulig P. A., McCracken G. H., Jr, Frisch C. F., Johnston K. H., Hansen E. J. Antibody response of infants to cell surface-exposed outer membrane proteins of Haemophilus influenzae type b after systemic Haemophilus disease. Infect Immun. 1982 Jul;37(1):82–88. doi: 10.1128/iai.37.1.82-88.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hancock R. E., Wieczorek A. A., Mutharia L. M., Poole K. Monoclonal antibodies against Pseudomonas aeruginosa outer membrane antigens: isolation and characterization. Infect Immun. 1982 Jul;37(1):166–171. doi: 10.1128/iai.37.1.166-171.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hansen E. J., Robertson S. M., Gulig P. A., Frisch C. F., Haanes E. J. Immunoprotection of rats against Haemophilus influenzae type B disease mediated by monoclonal antibody against a haemophilus outer-membrane protein. Lancet. 1982 Feb 13;1(8268):366–368. doi: 10.1016/s0140-6736(82)91394-0. [DOI] [PubMed] [Google Scholar]
  12. Kalb V. F., Jr, Bernlohr R. W. A new spectrophotometric assay for protein in cell extracts. Anal Biochem. 1977 Oct;82(2):362–371. doi: 10.1016/0003-2697(77)90173-7. [DOI] [PubMed] [Google Scholar]
  13. Kuo J. S., Monji N., Schwalbe R. S., McCoy D. W. A radioactive antigen-binding assay for the measurement of antibody to Haemophilus influenzae type b capsular polysaccharide. J Immunol Methods. 1981;43(1):35–47. doi: 10.1016/0022-1759(81)90034-x. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Osborn M. J., Wu H. C. Proteins of the outer membrane of gram-negative bacteria. Annu Rev Microbiol. 1980;34:369–422. doi: 10.1146/annurev.mi.34.100180.002101. [DOI] [PubMed] [Google Scholar]
  16. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  17. Robbins J. B., Schneerson R. Immunoadsorbents. Methods Enzymol. 1974;34:703–722. doi: 10.1016/s0076-6879(74)34093-1. [DOI] [PubMed] [Google Scholar]
  18. Schnaitman C. A. Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J Bacteriol. 1971 Oct;108(1):545–552. doi: 10.1128/jb.108.1.545-552.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Shenep J. L., Munson R. S., Jr, Granoff D. M. Human antibody responses to lipopolysaccharide after meningitis due to Haemophilus influenzae type b. J Infect Dis. 1982 Feb;145(2):181–190. doi: 10.1093/infdis/145.2.181. [DOI] [PubMed] [Google Scholar]
  20. South M. A. Lack of immune response to Hemophilus influenzae: immune paralysis or immaturity? J Pediatr. 1972 Feb;80(2):348–350. doi: 10.1016/s0022-3476(72)80622-x. [DOI] [PubMed] [Google Scholar]
  21. Swanson J. Studies on gonococcus infection. XVIII. 125I-labeled peptide mapping of the major protein of the gonococcal cell wall outer membrane. Infect Immun. 1979 Mar;23(3):799–810. doi: 10.1128/iai.23.3.799-810.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES