Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1991 Sep;59(9):3191–3198. doi: 10.1128/iai.59.9.3191-3198.1991

The e (P4) outer membrane protein of Haemophilus influenzae: biologic activity of anti-e serum and cloning and sequencing of the structural gene.

B A Green 1, J E Farley 1, T Quinn-Dey 1, R A Deich 1, G W Zlotnick 1
PMCID: PMC258152  PMID: 1715322

Abstract

Outer membrane proteins of nontypeable (NT) Haemophilus influenzae are among the major candidates for inclusion in vaccines against these organisms. This article reports the purification of the e (P4) lipoprotein of H. influenzae and the subsequent production of antiserum directed against this protein. The anti-e polyclonal serum cross-reacted with e protein in multiple clinical NT H. influenzae isolates. Monoclonal antibody analysis of e protein showed at least one surface-exposed epitope to be conserved among NT H. influenzae strains. Anti-e serum also had bactericidal activity against multiple clinical isolates of NT H. influenzae. These results are in contrast to previous reports in the literature that purified P4 protein did not elicit biologically active antibodies. Anti-e antibodies exhibited synergistic bactericidal activity directed against NT H. influenzae when mixed with antibodies directed against another Haemophilus lipoprotein, PCP. This bactericidal synergy was observed against a variety of NT clinical isolates. We also report the cloning of the Haemophilus e lipoprotein, or hel, gene encoding the e protein and its expression and processing in Escherichia coli. The nucleotide sequence of the gene and deduced amino acid sequence of the protein are given. These results demonstrate that e protein is a viable candidate to be a component of a vaccine against NT H. influenzae.

Full text

PDF
3196

Images in this article

3193Image
on p.3193
3193Image
on p.3193
3193Image
on p.3193
3194Image
on p.3194
3195Image
on p.3195

Selected References

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

  1. Barenkamp S. J. Protection by serum antibodies in experimental nontypable Haemophilus influenzae otitis media. Infect Immun. 1986 May;52(2):572–578. doi: 10.1128/iai.52.2.572-578.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  3. Chanyangam M., Smith A. L., Moseley S. L., Kuehn M., Jenny P. Contribution of a 28-kilodalton membrane protein to the virulence of Haemophilus influenzae. Infect Immun. 1991 Feb;59(2):600–608. doi: 10.1128/iai.59.2.600-608.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Deich R. A., Anilionis A., Fulginiti J., Metcalf B. J., Quataert S., Quinn-Dey T., Zlotnick G. W., Green B. A. Antigenic conservation of the 15,000-dalton outer membrane lipoprotein PCP of Haemophilus influenzae and biologic activity of anti-PCP antisera. Infect Immun. 1990 Oct;58(10):3388–3393. doi: 10.1128/iai.58.10.3388-3393.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Deich R. A., Green B. A. Mobilization of Haemophilus influenzae chromosomal markers by an Escherichia coli F' factor. J Bacteriol. 1987 May;169(5):1905–1910. doi: 10.1128/jb.169.5.1905-1910.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Deich R. A., Metcalf B. J., Finn C. W., Farley J. E., Green B. A. Cloning of genes encoding a 15,000-dalton peptidoglycan-associated outer membrane lipoprotein and an antigenically related 15,000-dalton protein from Haemophilus influenzae. J Bacteriol. 1988 Feb;170(2):489–498. doi: 10.1128/jb.170.2.489-498.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gonzales F. R., Leachman S., Norgard M. V., Radolf J. D., McCracken G. H., Jr, Evans C., Hansen E. J. Cloning and expression in Escherichia coli of the gene encoding the heat-modifiable major outer membrane protein of Haemophilus influenzae type b. Infect Immun. 1987 Dec;55(12):2993–3000. doi: 10.1128/iai.55.12.2993-3000.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Granoff D. M., Munson R. S., Jr Prospects for prevention of Haemophilus influenzae type b disease by immunization. J Infect Dis. 1986 Mar;153(3):448–461. doi: 10.1093/infdis/153.3.448. [DOI] [PubMed] [Google Scholar]
  9. Green B. A., Metcalf B. J., Quinn-Dey T., Kirkley D. H., Quataert S. A., Deich R. A. A recombinant non-fatty acylated form of the Hi-PAL (P6) protein of Haemophilus influenzae elicits biologically active antibody against both nontypeable and type b H. influenzae. Infect Immun. 1990 Oct;58(10):3272–3278. doi: 10.1128/iai.58.10.3272-3278.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Green B. A., Quinn-Dey T., Zlotnick G. W. Biologic activities of antibody to a peptidoglycan-associated lipoprotein of Haemophilus influenzae against multiple clinical isolates of H. influenzae type b. Infect Immun. 1987 Dec;55(12):2878–2883. doi: 10.1128/iai.55.12.2878-2883.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Holmans P. L., Loftus T. A., Hansen E. J. Cloning and surface expression in Escherichia coli of a structural gene encoding a surface protein of Haemophilus influenzae type b. Infect Immun. 1985 Oct;50(1):236–242. doi: 10.1128/iai.50.1.236-242.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  15. Maniatis T., Hardison R. C., Lacy E., Lauer J., O'Connell C., Quon D., Sim G. K., Efstratiadis A. The isolation of structural genes from libraries of eucaryotic DNA. Cell. 1978 Oct;15(2):687–701. doi: 10.1016/0092-8674(78)90036-3. [DOI] [PubMed] [Google Scholar]
  16. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  17. Munson R. S., Jr, Granoff D. M. Purification and partial characterization of outer membrane proteins P5 and P6 from Haemophilus influenzae type b. Infect Immun. 1985 Sep;49(3):544–549. doi: 10.1128/iai.49.3.544-549.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Munson R., Jr, Grass S. Purification, cloning, and sequence of outer membrane protein P1 of Haemophilus influenzae type b. Infect Immun. 1988 Sep;56(9):2235–2242. doi: 10.1128/iai.56.9.2235-2242.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Munson R., Jr, Tolan R. W., Jr Molecular cloning, expression, and primary sequence of outer membrane protein P2 of Haemophilus influenzae type b. Infect Immun. 1989 Jan;57(1):88–94. doi: 10.1128/iai.57.1.88-94.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Murphy T. F., Bartos L. C., Campagnari A. A., Nelson M. B., Apicella M. A. Antigenic characterization of the P6 protein of nontypable Haemophilus influenzae. Infect Immun. 1986 Dec;54(3):774–779. doi: 10.1128/iai.54.3.774-779.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Murphy T. F., Bartos L. C., Rice P. A., Nelson M. B., Dudas K. C., Apicella M. A. Identification of a 16,600-dalton outer membrane protein on nontypeable Haemophilus influenzae as a target for human serum bactericidal antibody. J Clin Invest. 1986 Oct;78(4):1020–1027. doi: 10.1172/JCI112656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nelson M. B., Apicella M. A., Murphy T. F., Vankeulen H., Spotila L. D., Rekosh D. Cloning and sequencing of Haemophilus influenzae outer membrane protein P6. Infect Immun. 1988 Jan;56(1):128–134. doi: 10.1128/iai.56.1.128-134.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Thomas W. R., Callow M. G., Dilworth R. J., Audesho A. A. Expression in Escherichia coli of a high-molecular-weight protective surface antigen found in nontypeable and type b Haemophilus influenzae. Infect Immun. 1990 Jun;58(6):1909–1913. doi: 10.1128/iai.58.6.1909-1913.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Weinberg G. A., Towler D. A., Munson R. S., Jr Lipoproteins of Haemophilus influenzae type b. J Bacteriol. 1988 Sep;170(9):4161–4164. doi: 10.1128/jb.170.9.4161-4164.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wu H. C., Tokunaga M. Biogenesis of lipoproteins in bacteria. Curr Top Microbiol Immunol. 1986;125:127–157. doi: 10.1007/978-3-642-71251-7_9. [DOI] [PubMed] [Google Scholar]
  30. Zlotnick G. W., Sanfilippo V. T., Mattler J. A., Kirkley D. H., Boykins R. A., Seid R. C., Jr Purification and characterization of a peptidoglycan-associated lipoprotein from Haemophilus influenzae. J Biol Chem. 1988 Jul 15;263(20):9790–9794. [PubMed] [Google Scholar]
  31. van Alphen L., Eijk P., Geelen-van den Broek L., Dankert J. Immunochemical characterization of variable epitopes of outer membrane protein P2 of nontypeable Haemophilus influenzae. Infect Immun. 1991 Jan;59(1):247–252. doi: 10.1128/iai.59.1.247-252.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES