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. 1995 Dec;63(12):4600–4605. doi: 10.1128/iai.63.12.4600-4605.1995

Antigenic determinants of the OmpC porin from Salmonella typhimurium.

S P Singh 1, S R Singh 1, Y U Williams 1, L Jones 1, T Abdullah 1
PMCID: PMC173661  PMID: 7591112

Abstract

The antigenic determinants of Salmonella typhimurium OmpC were investigated by the analysis of cyanogen bromide (CNBr)-generated porin peptides with antiporin monoclonal antibodies (MAbs). We identified six bands (f1 to f6) with estimated molecular masses of 35.5, 31.0, 25.0, 22.5, 13.8, and 10.0 kDa, respectively. In addition, two small fragments (f7 and f8; 3.0 to 6.0 kDa) were detected only infrequently. The OmpC monomer or its CNBr-generated peptides were electrophoretically transferred to a polyvinylidene difluoride membrane and then subjected to amino acid composition analysis and N-terminal sequencing. A comparison of the amino acid composition data with known compositions of Escherichia coli and Salmonella typhi OmpC showed some differences; however, the amino acid sequences of 71 residues identified in S. typhimurium showed 88 and 98% identity with OmpC from E. coli and S. typhi, respectively. The screening of CNBr peptides with the 12 anti-(S. typhimurium) OmpC MAbs by Western blot (immunoblot), in conjunction with the prediction of the OmpC folding pattern based on the known three-dimensional structure of E. coli OmpF, showed that four MAbs reacted with surface-exposed epitopes on loops L2, L8, and L4 to L7, four MAbs reacted with a region in the eyelet structure on loop L3, and four MAbs reacted with the buried epitopes on transmembrane beta strands. The MAbs reacting with surface-exposed loops showed no cross-reaction with E. coli OmpC, whose sequence has diverged extensively from that of S. typhi and (probably) S. typhimurium OmpC only in regions of the externally exposed loops. In contrast, MAbs reacting with transmembrane beta strands, whose sequence is strongly conserved, showed strong cross-reaction with E. coli OmpC. These results show that comparison with the E. coli OmpF structure predicts the folding pattern of S. typhimurium OmpC rather accurately and that evolutionary divergence in sequences is confined to the external loops. The possible roles of these surface-exposed and buried epitopes as potentially useful antigenic regions for diagnostic assays and vaccine development are discussed.

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Selected References

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  1. Bentley A. T., Klebba P. E. Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface. J Bacteriol. 1988 Mar;170(3):1063–1068. doi: 10.1128/jb.170.3.1063-1068.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Calderón I., Lobos S. R., Rojas H. A., Palomino C., Rodríguez L. H., Mora G. C. Antibodies to porin antigens of Salmonella typhi induced during typhoid infection in humans. Infect Immun. 1986 Apr;52(1):209–212. doi: 10.1128/iai.52.1.209-212.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Charbit A., Ronco J., Michel V., Werts C., Hofnung M. Permissive sites and topology of an outer membrane protein with a reporter epitope. J Bacteriol. 1991 Jan;173(1):262–275. doi: 10.1128/jb.173.1.262-275.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chatfield S. N., Strugnell R. A., Dougan G. Live Salmonella as vaccines and carriers of foreign antigenic determinants. Vaccine. 1989 Dec;7(6):495–498. doi: 10.1016/0264-410x(89)90271-5. [DOI] [PubMed] [Google Scholar]
  5. Cowan S. W., Rosenbusch J. P. Folding pattern diversity of integral membrane proteins. Science. 1994 May 13;264(5161):914–916. doi: 10.1126/science.8178151. [DOI] [PubMed] [Google Scholar]
  6. Cowan S. W., Schirmer T., Rummel G., Steiert M., Ghosh R., Pauptit R. A., Jansonius J. N., Rosenbusch J. P. Crystal structures explain functional properties of two E. coli porins. Nature. 1992 Aug 27;358(6389):727–733. doi: 10.1038/358727a0. [DOI] [PubMed] [Google Scholar]
  7. Di Padova F. E., Brade H., Barclay G. R., Poxton I. R., Liehl E., Schuetze E., Kocher H. P., Ramsay G., Schreier M. H., McClelland D. B. A broadly cross-protective monoclonal antibody binding to Escherichia coli and Salmonella lipopolysaccharides. Infect Immun. 1993 Sep;61(9):3863–3872. doi: 10.1128/iai.61.9.3863-3872.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Galanos C., Lüderitz O., Westphal O. A new method for the extraction of R lipopolysaccharides. Eur J Biochem. 1969 Jun;9(2):245–249. doi: 10.1111/j.1432-1033.1969.tb00601.x. [DOI] [PubMed] [Google Scholar]
  9. Garten W., Hindennach I., Henning U. The major proteins of the Escherichia coli outer cell-envelope membrane. Cyanogen bromide fragments of protein I, composition and order. Eur J Biochem. 1975 Dec 1;60(1):303–307. doi: 10.1111/j.1432-1033.1975.tb21004.x. [DOI] [PubMed] [Google Scholar]
  10. Gehring K. B., Nikaido H. Existence and purification of porin heterotrimers of Escherichia coli K12 OmpC, OmpF, and PhoE proteins. J Biol Chem. 1989 Feb 15;264(5):2810–2815. [PubMed] [Google Scholar]
  11. Gerbl-Rieger S., Peters J., Kellermann J., Lottspeich F., Baumeister W. Nucleotide and derived amino acid sequences of the major porin of Comamonas acidovorans and comparison of porin primary structures. J Bacteriol. 1991 Apr;173(7):2196–2205. doi: 10.1128/jb.173.7.2196-2205.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haase E. M., Campagnari A. A., Sarwar J., Shero M., Wirth M., Cumming C. U., Murphy T. F. Strain-specific and immunodominant surface epitopes of the P2 porin protein of nontypeable Haemophilus influenzae. Infect Immun. 1991 Apr;59(4):1278–1284. doi: 10.1128/iai.59.4.1278-1284.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Haase E. M., Yi K., Morse G. D., Murphy T. F. Mapping of bactericidal epitopes on the P2 porin protein of nontypeable Haemophilus influenzae. Infect Immun. 1994 Sep;62(9):3712–3722. doi: 10.1128/iai.62.9.3712-3722.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hackett J. Use of Salmonella for heterologous gene expression and vaccine delivery systems. Curr Opin Biotechnol. 1993 Oct;4(5):611–615. doi: 10.1016/0958-1669(93)90085-b. [DOI] [PubMed] [Google Scholar]
  15. Isibasi A., Ortiz-Navarrete V., Paniagua J., Pelayo R., González C. R., García J. A., Kumate J. Active protection of mice against Salmonella typhi by immunization with strain-specific porins. Vaccine. 1992;10(12):811–813. doi: 10.1016/0264-410x(92)90041-h. [DOI] [PubMed] [Google Scholar]
  16. Jap B. K., Walian P. J., Gehring K. Structural architecture of an outer membrane channel as determined by electron crystallography. Nature. 1991 Mar 14;350(6314):167–170. doi: 10.1038/350167a0. [DOI] [PubMed] [Google Scholar]
  17. Klebba P. E., Benson S. A., Bala S., Abdullah T., Reid J., Singh S. P., Nikaido H. Determinants of OmpF porin antigenicity and structure. J Biol Chem. 1990 Apr 25;265(12):6800–6810. [PubMed] [Google Scholar]
  18. Kuusi N., Nurminen M., Saxén H., Mäkelä P. H. Immunization with major outer membrane protein (porin) preparations in experimental murine salmonellosis: effect of lipopolysaccharide. Infect Immun. 1981 Nov;34(2):328–332. doi: 10.1128/iai.34.2.328-332.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lugtenberg B., Van Alphen L. Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram-negative bacteria. Biochim Biophys Acta. 1983 Mar 21;737(1):51–115. doi: 10.1016/0304-4157(83)90014-x. [DOI] [PubMed] [Google Scholar]
  20. Lupi N., Bourgois A., Bernadac A., Laboucarié S., Pagès J. M. Immunological analysis of porin polymorphism in Escherichia coli B and K-12. Mol Immunol. 1989 Nov;26(11):1027–1036. doi: 10.1016/0161-5890(89)90067-9. [DOI] [PubMed] [Google Scholar]
  21. Matsudaira P. Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem. 1987 Jul 25;262(21):10035–10038. [PubMed] [Google Scholar]
  22. Matsui K., Arai T. Specificity of protective immunity induced by porin from Salmonella typhimurium. Microbiologica. 1991 Apr;14(2):103–112. [PubMed] [Google Scholar]
  23. Mizuno T., Chou M. Y., Inouye M. A comparative study on the genes for three porins of the Escherichia coli outer membrane. DNA sequence of the osmoregulated ompC gene. J Biol Chem. 1983 Jun 10;258(11):6932–6940. [PubMed] [Google Scholar]
  24. Mutharia L. M., Hancock R. E. Characterization of two surface-localized antigenic sites on porin protein F of Pseudomonas aeruginosa. Can J Microbiol. 1985 Apr;31(4):381–386. doi: 10.1139/m85-073. [DOI] [PubMed] [Google Scholar]
  25. Muthukkumar S., Muthukkaruppan V. R. Mechanism of protective immunity induced by porin-lipopolysaccharide against murine salmonellosis. Infect Immun. 1993 Jul;61(7):3017–3025. doi: 10.1128/iai.61.7.3017-3025.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nandakumar K. S., Palanivel V., Muthukkaruppan V. Diagnosis of typhoid fever: detection of Salmonella typhi porins-specific antibodies by inhibition ELISA. Clin Exp Immunol. 1993 Nov;94(2):317–321. doi: 10.1111/j.1365-2249.1993.tb03450.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nikaido H. Porins and specific channels of bacterial outer membranes. Mol Microbiol. 1992 Feb;6(4):435–442. doi: 10.1111/j.1365-2958.1992.tb01487.x. [DOI] [PubMed] [Google Scholar]
  28. Nikaido H., Vaara M. Molecular basis of bacterial outer membrane permeability. Microbiol Rev. 1985 Mar;49(1):1–32. doi: 10.1128/mr.49.1.1-32.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pai S. R., Upshaw Y., Singh S. P. Characterization of monoclonal antibodies to the outer membrane protein (OmpD) of Salmonella typhimurium. Can J Microbiol. 1992 Nov;38(11):1102–1107. doi: 10.1139/m92-181. [DOI] [PubMed] [Google Scholar]
  30. Puente J. L., Alvarez-Scherer V., Gosset G., Calva E. Comparative analysis of the Salmonella typhi and Escherichia coli ompC genes. Gene. 1989 Nov 30;83(2):197–206. doi: 10.1016/0378-1119(89)90105-4. [DOI] [PubMed] [Google Scholar]
  31. Puente J. L., Juárez D., Bobadilla M., Arias C. F., Calva E. The Salmonella ompC gene: structure and use as a carrier for heterologous sequences. Gene. 1995 Apr 14;156(1):1–9. doi: 10.1016/0378-1119(94)00883-t. [DOI] [PubMed] [Google Scholar]
  32. Rawling E. G., Martin N. L., Hancock R. E. Epitope mapping of the Pseudomonas aeruginosa major outer membrane porin protein OprF. Infect Immun. 1995 Jan;63(1):38–42. doi: 10.1128/iai.63.1.38-42.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rutz J. M., Abdullah T., Singh S. P., Kalve V. I., Klebba P. E. Evolution of the ferric enterobactin receptor in gram-negative bacteria. J Bacteriol. 1991 Oct;173(19):5964–5974. doi: 10.1128/jb.173.19.5964-5974.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Singh S. P., Upshaw Y., Abdullah T., Singh S. R., Klebba P. E. Structural relatedness of enteric bacterial porins assessed with monoclonal antibodies to Salmonella typhimurium OmpD and OmpC. J Bacteriol. 1992 Mar;174(6):1965–1973. doi: 10.1128/jb.174.6.1965-1973.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Smit J., Kamio Y., Nikaido H. Outer membrane of Salmonella typhimurium: chemical analysis and freeze-fracture studies with lipopolysaccharide mutants. J Bacteriol. 1975 Nov;124(2):942–958. doi: 10.1128/jb.124.2.942-958.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Struyvé M., Visser J., Adriaanse H., Benz R., Tommassen J. Topology of PhoE porin: the 'eyelet' region. Mol Microbiol. 1993 Jan;7(1):131–140. doi: 10.1111/j.1365-2958.1993.tb01104.x. [DOI] [PubMed] [Google Scholar]
  37. Tabaraie B., Sharma B. K., Sharma P. R., Sehgal R., Ganguly N. K. Evaluation of Salmonella porins as a broad spectrum vaccine candidate. Microbiol Immunol. 1994;38(7):553–559. doi: 10.1111/j.1348-0421.1994.tb01822.x. [DOI] [PubMed] [Google Scholar]
  38. Tommassen J., Agterberg M., Janssen R., Spierings G. Use of the enterobacterial outer membrane protein PhoE in the development of new vaccines and DNA probes. Zentralbl Bakteriol. 1993 Apr;278(2-3):396–406. doi: 10.1016/s0934-8840(11)80856-x. [DOI] [PubMed] [Google Scholar]
  39. Weiss M. S., Kreusch A., Schiltz E., Nestel U., Welte W., Weckesser J., Schulz G. E. The structure of porin from Rhodobacter capsulatus at 1.8 A resolution. FEBS Lett. 1991 Mar 25;280(2):379–382. doi: 10.1016/0014-5793(91)80336-2. [DOI] [PubMed] [Google Scholar]
  40. van der Ley P., Struyvé M., Tommassen J. Topology of outer membrane pore protein PhoE of Escherichia coli. Identification of cell surface-exposed amino acids with the aid of monoclonal antibodies. J Biol Chem. 1986 Sep 15;261(26):12222–12225. [PubMed] [Google Scholar]

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