Abstract
P6 is an outer membrane protein of Haemophilus influenzae that is antigenically conserved and considered a candidate component of future H. influenzae vaccines. P6 contains a surface-exposed epitope recognized by monoclonal antibody (MAb) 3B9. This epitope has been shown to be distinct from that recognized by the P6-specific MAbs 7F3 and 4G4 in a competitive inhibition enzyme-linked immunosorbent assay (ELISA). MAb 3B9 did not bind to synthetic P6-specific sequential and overlapping hexameric peptides. Five peptides made to correspond to P6 sequences with high probabilities of surface exposure did not inhibit binding of MAb 3B9 to P6. An antiserum to one of the peptides, designated SP66, inhibited binding of MAb 3B9 to P6. A rabbit antiserum to P6 bound to sequential hexameric peptides, Gly-87AsnThrAspGluArgGlyThr-94, which were in the SP66 region of P6. This antiserum inhibited the binding of P6 to MAb 3B9 in a competitive inhibition ELISA. P6 mutations with His and Ala substitutions at residues Thr-88 and Asn-89 still bound MAb 3B9. MAb 3B9 reacted with Escherichia coli OmpA and Salmonella typhimurium OmpA. Sequence comparisons of P6 with these proteins indicated that the residue in the SP66 region responsible for binding is either Gly-87, Asp-90, or Gly-93. Mercaptoethanol reduction abolished MAb 3B9 binding to E. coli OmpA and S. typhimurium OmpA. In these proteins, immediately downstream of the second cysteine, there is an ArgArg dipeptide which is identical to and aligns with Arg-147Arg-148 in P6. This dipeptide has a high probability of surface exposure in P6. Mutagenesis of the Arg-147Arg-148 to an AlaAla dipeptide in P6 abolished binding of MAb 3B9, demonstrating that it was either a portion of the epitope or important in the protein folding necessary for expression of this epitope. This study demonstrates that MAb 3B9 recognizes a conserved conformational determinant on the surface of H. influenzae that is composed of two discontinuous regions of P6.
Full Text
The Full Text of this article is available as a PDF (606.1 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Abdillahi H., Poolman J. T. Typing of group-B Neisseria meningitidis with monoclonal antibodies in the whole-cell ELISA. J Med Microbiol. 1988 Jul;26(3):177–180. [PubMed] [Google Scholar]
- 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]
- Apicella M. A., Shero M., Jarvis G. A., Griffiss J. M., Mandrell R. E., Schneider H. Phenotypic variation in epitope expression of the Neisseria gonorrhoeae lipooligosaccharide. Infect Immun. 1987 Aug;55(8):1755–1761. doi: 10.1128/iai.55.8.1755-1761.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barenkamp S. J., Bodor F. F. Development of serum bactericidal activity following nontypable Haemophilus influenzae acute otitis media. Pediatr Infect Dis J. 1990 May;9(5):333–339. doi: 10.1097/00006454-199005000-00006. [DOI] [PubMed] [Google Scholar]
- Beck-Sickinger A. G., Rotering H., Wiesmüller K. H., Dorner F., Jung G. Mapping of antigenic and immunogenic sites of Haemophilus influenzae outer membrane protein P6 using synthetic lipopeptides. Biol Chem Hoppe Seyler. 1994 Mar;375(3):173–182. doi: 10.1515/bchm3.1994.375.3.173. [DOI] [PubMed] [Google Scholar]
- Beher M. G., Schnaitman C. A., Pugsley A. P. Major heat-modifiable outer membrane protein in gram-negative bacteria: comparison with the ompA protein of Escherichia coli. J Bacteriol. 1980 Aug;143(2):906–913. doi: 10.1128/jb.143.2.906-913.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen R., Henning U. Nucleotide sequence of the gene for the peptidoglycan-associated lipoprotein of Escherichia coli K12. Eur J Biochem. 1987 Feb 16;163(1):73–77. doi: 10.1111/j.1432-1033.1987.tb10738.x. [DOI] [PubMed] [Google Scholar]
- 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]
- Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Duchêne M., Schweizer A., Lottspeich F., Krauss G., Marget M., Vogel K., von Specht B. U., Domdey H. Sequence and transcriptional start site of the Pseudomonas aeruginosa outer membrane porin protein F gene. J Bacteriol. 1988 Jan;170(1):155–162. doi: 10.1128/jb.170.1.155-162.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Faden H., Bernstein J., Brodsky L., Stanievich J., Krystofik D., Shuff C., Hong J. J., Ogra P. L. Otitis media in children. I. The systemic immune response to nontypable Hemophilus influenzae. J Infect Dis. 1989 Dec;160(6):999–1004. doi: 10.1093/infdis/160.6.999. [DOI] [PubMed] [Google Scholar]
- Faden H., Brodsky L., Bernstein J., Stanievich J., Krystofik D., Shuff C., Hong J. J., Ogra P. L. Otitis media in children: local immune response to nontypeable Haemophilus influenzae. Infect Immun. 1989 Nov;57(11):3555–3559. doi: 10.1128/iai.57.11.3555-3559.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Freudl R., Cole S. T. Cloning and molecular characterization of the ompA gene from Salmonella typhimurium. Eur J Biochem. 1983 Aug 15;134(3):497–502. doi: 10.1111/j.1432-1033.1983.tb07594.x. [DOI] [PubMed] [Google Scholar]
- 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]
- Gotschlich E. C., Seiff M., Blake M. S. The DNA sequence of the structural gene of gonococcal protein III and the flanking region containing a repetitive sequence. Homology of protein III with enterobacterial OmpA proteins. J Exp Med. 1987 Feb 1;165(2):471–482. doi: 10.1084/jem.165.2.471. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Hansen E. J., Frisch C. F., Johnston K. H. Detection of antibody-accessible proteins on the cell surface of Haemophilus influenzae type b. Infect Immun. 1981 Sep;33(3):950–953. doi: 10.1128/iai.33.3.950-953.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harabuchi Y., Faden H., Yamanaka N., Duffy L., Wolf J., Krystofik D. Human milk secretory IgA antibody to nontypeable Haemophilus influenzae: possible protective effects against nasopharyngeal colonization. J Pediatr. 1994 Feb;124(2):193–198. doi: 10.1016/s0022-3476(94)70302-7. [DOI] [PubMed] [Google Scholar]
- Harabuchi Y., Faden H., Yamanaka N., Duffy L., Wolf J., Krystofik D. Nasopharyngeal colonization with nontypeable Haemophilus influenzae and recurrent otitis media. Tonawanda/Williamsville Pediatrics. J Infect Dis. 1994 Oct;170(4):862–866. doi: 10.1093/infdis/170.4.862. [DOI] [PubMed] [Google Scholar]
- Henning U., Sonntag I., Hindennach I. Mutants (ompA) affecting a major outer membrane protein of Escherichia coli K12. Eur J Biochem. 1978 Dec;92(2):491–498. doi: 10.1111/j.1432-1033.1978.tb12771.x. [DOI] [PubMed] [Google Scholar]
- Joiner K. A., Scales R., Warren K. A., Frank M. M., Rice P. A. Mechanism of action of blocking immunoglobulin G for Neisseria gonorrhoeae. J Clin Invest. 1985 Nov;76(5):1765–1772. doi: 10.1172/JCI112167. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kennett R. H. Cell fusion. Methods Enzymol. 1979;58:345–359. doi: 10.1016/s0076-6879(79)58149-x. [DOI] [PubMed] [Google Scholar]
- Klose M., Schwarz H., MacIntyre S., Freudl R., Eschbach M. L., Henning U. Internal deletions in the gene for an Escherichia coli outer membrane protein define an area possibly important for recognition of the outer membrane by this polypeptide. J Biol Chem. 1988 Sep 15;263(26):13291–13296. [PubMed] [Google Scholar]
- Kyd J. M., Taylor D., Cripps A. W. Conservation of immune responses to proteins isolated by preparative polyacrylamide gel electrophoresis from the outer membrane of nontypeable Haemophilus influenzae. Infect Immun. 1994 Dec;62(12):5652–5658. doi: 10.1128/iai.62.12.5652-5658.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Ludwig B., Schmid A., Marre R., Hacker J. Cloning, genetic analysis, and nucleotide sequence of a determinant coding for a 19-kilodalton peptidoglycan-associated protein (Ppl) of Legionella pneumophila. Infect Immun. 1991 Aug;59(8):2515–2521. doi: 10.1128/iai.59.8.2515-2521.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- MacIntyre S., Freudl R., Degen M., Hindennach I., Henning U. The signal sequence of an Escherichia coli outer membrane protein can mediate translocation of a not normally secreted protein across the plasma membrane. J Biol Chem. 1987 Jun 15;262(17):8416–8422. [PubMed] [Google Scholar]
- 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]
- Murphy T. F., Apicella M. A. Nontypable Haemophilus influenzae: a review of clinical aspects, surface antigens, and the human immune response to infection. Rev Infect Dis. 1987 Jan-Feb;9(1):1–15. doi: 10.1093/clinids/9.1.1. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Murphy T. F., Kirkham C., Sikkema D. J. Neonatal, urogenital isolates of biotype 4 nontypeable Haemophilus influenzae express a variant P6 outer membrane protein molecule. Infect Immun. 1992 May;60(5):2016–2022. doi: 10.1128/iai.60.5.2016-2022.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Murphy T. F., Nelson M. B., Dudas K. C., Mylotte J. M., Apicella M. A. Identification of a specific epitope of Haemophilus influenzae on a 16,600-dalton outer membrane protein. J Infect Dis. 1985 Dec;152(6):1300–1307. doi: 10.1093/infdis/152.6.1300. [DOI] [PubMed] [Google Scholar]
- 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]
- Nelson M. B., Munson R. S., Jr, Apicella M. A., Sikkema D. J., Molleston J. P., Murphy T. F. Molecular conservation of the P6 outer membrane protein among strains of Haemophilus influenzae: analysis of antigenic determinants, gene sequences, and restriction fragment length polymorphisms. Infect Immun. 1991 Aug;59(8):2658–2663. doi: 10.1128/iai.59.8.2658-2663.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Robertson S. M., Frisch C. F., Gulig P. A., Kettman J. R., Johnston K. H., Hansen E. J. Monoclonal antibodies directed against a cell surface-exposed outer membrane protein of Haemophilus influenzae type b. Infect Immun. 1982 Apr;36(1):80–88. doi: 10.1128/iai.36.1.80-88.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sikkema D. J., Nelson M. B., Apicella M. A., Murphy T. F. Outer membrane protein P6 of Haemophilus influenzae binds to its own gene. Mol Microbiol. 1992 Feb;6(4):547–554. doi: 10.1111/j.1365-2958.1992.tb01499.x. [DOI] [PubMed] [Google Scholar]
- Spinola S. M., Griffiths G. E., Bogdan J., Menegus M. A. Characterization of an 18,000-molecular-weight outer membrane protein of Haemophilus ducreyi that contains a conserved surface-exposed epitope. Infect Immun. 1992 Feb;60(2):385–391. doi: 10.1128/iai.60.2.385-391.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]