Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1995 Nov;33(11):2826–2832. doi: 10.1128/jcm.33.11.2826-2832.1995

Identification and characterization of an immunogenic outer membrane protein of Campylobacter jejuni.

A Burnens 1, U Stucki 1, J Nicolet 1, J Frey 1
PMCID: PMC228588  PMID: 8576327

Abstract

We cloned and expressed in Escherichia coli a gene encoding an 18-kDa outer membrane protein (Omp18) from Campylobacter jejuni ATCC 29428. The nucleotide sequence of the gene encoding Omp18 was determined, and an open reading frame of 165 amino acids was revealed. The amino acid sequence had the typical features of a leader sequence and a signal peptidase II cleavage site at the N-terminal part of Omp18. Moreover, the sequence had a high degree of similarity to the peptidoglycan-associated outer membrane lipoprotein P6 of Haemophilus influenzae and the peptidoglycan-associated lipoprotein PAL of E. coli. Southern blot analysis in which the cloned gene was used as a probe revealed genes similar to that encoding Omp18 in all species of the thermophilic group of campylobacters as well as Campylobacter sputorum. All campylobacters tested expressed a protein with a molecular mass identical to that of Omp18. The protein reacted immunologically with polyclonal antibodies directed against Omp18 from C. jejuni. PCR amplification of the gene encoding Omp18 with specific primers and subsequent restriction enzyme analysis of the amplified DNA fragments showed that the gene for Omp18 is highly conserved in C. jejuni strains isolated from humans, dogs, cats, calves, and chickens but is different in other Campylobacter species. In order to obtain pure recombinant Omp18 protein for serological assays, the cloned gene for Omp18 was genetically modified by replacing the signal sequence with a DNA segment encoding six adjacent histidine residues. Expression of this construct in E. coli allowed purification of the modified protein (Omp18-6xHis) by metal chelation chromatography. Sera from patients with past C. jejuni infection reacted positively with Omp18-6xHis, while sera from healthy blood donors showed no reaction with this antigen. Omp18, which is an outer membrane protein belonging to the family of PALs is well conserved in C. jejuni and is highly immunogenic. It is therefore a good candidate as an antigen for the serological diagnosis of past C. jejuni infections.

Full Text

The Full Text of this article is available as a PDF (581.4 KB).

Selected References

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

  1. Alm R. A., Guerry P., Power M. E., Trust T. J. Variation in antigenicity and molecular weight of Campylobacter coli VC167 flagellin in different genetic backgrounds. J Bacteriol. 1992 Jul;174(13):4230–4238. doi: 10.1128/jb.174.13.4230-4238.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alm R. A., Guerry P., Trust T. J. Distribution and polymorphism of the flagellin genes from isolates of Campylobacter coli and Campylobacter jejuni. J Bacteriol. 1993 May;175(10):3051–3057. doi: 10.1128/jb.175.10.3051-3057.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carlone G. M., Thomas M. L., Rumschlag H. S., Sottnek F. O. Rapid microprocedure for isolating detergent-insoluble outer membrane proteins from Haemophilus species. J Clin Microbiol. 1986 Sep;24(3):330–332. doi: 10.1128/jcm.24.3.330-332.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Engleberg N. C., Howe D. C., Rogers J. E., Arroyo J., Eisenstein B. I. Characterization of a Legionella pneumophila gene encoding a lipoprotein antigen. Mol Microbiol. 1991 Aug;5(8):2021–2029. doi: 10.1111/j.1365-2958.1991.tb00824.x. [DOI] [PubMed] [Google Scholar]
  5. Green B. A., Vazquez M. E., Zlotnick G. W., Quigley-Reape G., Swarts J. D., Green I., Cowell J. L., Bluestone C. D., Doyle W. J. Evaluation of mixtures of purified Haemophilus influenzae outer membrane proteins in protection against challenge with nontypeable H. influenzae in the chinchilla otitis media model. Infect Immun. 1993 May;61(5):1950–1957. doi: 10.1128/iai.61.5.1950-1957.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hemilä H. Sequence of a PAL-related lipoprotein from Bacillus subtilis. FEMS Microbiol Lett. 1991 Jul 15;66(1):37–41. doi: 10.1016/0378-1097(91)90417-9. [DOI] [PubMed] [Google Scholar]
  7. Jin T., Penner J. L. Role of the 92.5-kilodalton outer membrane protein of Campylobacter jejuni in serological reactions. J Clin Microbiol. 1988 Dec;26(12):2480–2483. doi: 10.1128/jcm.26.12.2480-2483.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kervella M., Pagès J. M., Pei Z., Grollier G., Blaser M. J., Fauchère J. L. Isolation and characterization of two Campylobacter glycine-extracted proteins that bind to HeLa cell membranes. Infect Immun. 1993 Aug;61(8):3440–3448. doi: 10.1128/iai.61.8.3440-3448.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kessel T., Omland T. Akutt epiglottitt hos barn forårsaket av Haemophilus influenzae type b. Nord Med. 1970 Jan 8;83(2):40–42. [PubMed] [Google Scholar]
  10. 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]
  11. Lazzaroni J. C., Portalier R. The excC gene of Escherichia coli K-12 required for cell envelope integrity encodes the peptidoglycan-associated lipoprotein (PAL). Mol Microbiol. 1992 Mar;6(6):735–742. doi: 10.1111/j.1365-2958.1992.tb01523.x. [DOI] [PubMed] [Google Scholar]
  12. Lior H., Woodward D. L., Edgar J. A., Laroche L. J., Gill P. Serotyping of Campylobacter jejuni by slide agglutination based on heat-labile antigenic factors. J Clin Microbiol. 1982 May;15(5):761–768. doi: 10.1128/jcm.15.5.761-768.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Logan S. M., Harris L. A., Trust T. J. Isolation and characterization of Campylobacter flagellins. J Bacteriol. 1987 Nov;169(11):5072–5077. doi: 10.1128/jb.169.11.5072-5077.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Mishu B., Ilyas A. A., Koski C. L., Vriesendorp F., Cook S. D., Mithen F. A., Blaser M. J. Serologic evidence of previous Campylobacter jejuni infection in patients with the Guillain-Barré syndrome. Ann Intern Med. 1993 Jun 15;118(12):947–953. doi: 10.7326/0003-4819-118-12-199306150-00006. [DOI] [PubMed] [Google Scholar]
  16. Murphy T. F., Nelson M. B., Apicella M. A. The P6 outer membrane protein of nontypeable Haemophilus influenzae as a vaccine antigen. J Infect Dis. 1992 Jun;165 (Suppl 1):S203–S205. doi: 10.1093/infdis/165-supplement_1-s203. [DOI] [PubMed] [Google Scholar]
  17. Müller M. Proteolysis in protein import and export: signal peptide processing in eu- and prokaryotes. Experientia. 1992 Feb 15;48(2):118–129. doi: 10.1007/BF01923506. [DOI] [PubMed] [Google Scholar]
  18. Nachamkin I., Bohachick K., Patton C. M. Flagellin gene typing of Campylobacter jejuni by restriction fragment length polymorphism analysis. J Clin Microbiol. 1993 Jun;31(6):1531–1536. doi: 10.1128/jcm.31.6.1531-1536.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nachamkin I., Hart A. M. Western blot analysis of the human antibody response to Campylobacter jejuni cellular antigens during gastrointestinal infection. J Clin Microbiol. 1985 Jan;21(1):33–38. doi: 10.1128/jcm.21.1.33-38.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nachamkin I., Yang X. H. Local immune responses to the Campylobacter flagellin in acute Campylobacter gastrointestinal infection. J Clin Microbiol. 1992 Feb;30(2):509–511. doi: 10.1128/jcm.30.2.509-511.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Pei Z. H., Ellison R. T., 3rd, Blaser M. J. Identification, purification, and characterization of major antigenic proteins of Campylobacter jejuni. J Biol Chem. 1991 Sep 5;266(25):16363–16369. [PubMed] [Google Scholar]
  23. Pei Z., Blaser M. J. PEB1, the major cell-binding factor of Campylobacter jejuni, is a homolog of the binding component in gram-negative nutrient transport systems. J Biol Chem. 1993 Sep 5;268(25):18717–18725. [PubMed] [Google Scholar]
  24. Pitkänen T., Pönkä A., Pettersson T., Kosunen T. U. Campylobacter enteritis in 188 hospitalized patients. Arch Intern Med. 1983 Feb;143(2):215–219. doi: 10.1001/archinte.1983.00350020033007. [DOI] [PubMed] [Google Scholar]
  25. Power M. E., Alm R. A., Trust T. J. Biochemical and antigenic properties of the Campylobacter flagellar hook protein. J Bacteriol. 1992 Jun;174(12):3874–3883. doi: 10.1128/jb.174.12.3874-3883.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Salvi R. J., Ahroon W., Saunders S. S., Arnold S. A. Evoked potentials: computer-automated threshold-tracking procedure using an objective detection criterion. Ear Hear. 1987 Jun;8(3):151–156. [PubMed] [Google Scholar]
  28. Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tibor A., Weynants V., Denoel P., Lichtfouse B., De Bolle X., Saman E., Limet J. N., Letesson J. J. Molecular cloning, nucleotide sequence, and occurrence of a 16.5-kilodalton outer membrane protein of Brucella abortus with similarity to pal lipoproteins. Infect Immun. 1994 Sep;62(9):3633–3639. doi: 10.1128/iai.62.9.3633-3639.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Vandamme P., Daneshvar M. I., Dewhirst F. E., Paster B. J., Kersters K., Goossens H., Moss C. W. Chemotaxonomic analyses of Bacteroides gracilis and Bacteroides ureolyticus and reclassification of B. gracilis as Campylobacter gracilis comb. nov. Int J Syst Bacteriol. 1995 Jan;45(1):145–152. doi: 10.1099/00207713-45-1-145. [DOI] [PubMed] [Google Scholar]
  33. Yamaguchi K., Yu F., Inouye M. A single amino acid determinant of the membrane localization of lipoproteins in E. coli. Cell. 1988 May 6;53(3):423–432. doi: 10.1016/0092-8674(88)90162-6. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES