Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1988 Mar;170(3):1063–1068. doi: 10.1128/jb.170.3.1063-1068.1988

Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface.

A T Bentley 1, P E Klebba 1
PMCID: PMC210874  PMID: 2830227

Abstract

We studied the reactivity of 66 anti-Escherichia coli B/r porin monoclonal antibodies (MAbs) with several E. coli and Salmonella typhimurium strains. Western immunoblots showed complete immunological cross-reactivity between E. coli B/r and K-12; among 34 MAbs which recognized porin in immunoblots of denatured outer membranes of E. coli B/r, all reacted with OmpF in denatured outer membranes of E. coli K-12. Extensive reactivity, although less than that for strain B/r (31 of 34 MAbs), occurred for porin from a wild-type isolate, E. coli O8:K27. Only one of the MAbs reacted with porin in denatured outer membranes of S. typhimurium. Even with immunochemical amplification of the Western immunoblot technique, only six MAbs recognized S. typhimurium porin (OmpD), demonstrating that there is significant immunological divergence between the porins of these species. Antibody binding to the bacterial surface, which was analyzed by cytofluorimetry, was strongly influenced by lipopolysaccharide (LPS) structure. An intact O antigen, as in E. coli O8:K27, blocked adsorption of all 20 MAbs in the test panel. rfa+ E. coli K-12, without an O antigen but with an intact LPS core, bound seven MAbs. When assayed against a series of rfa E. coli K-12 mutants, the number of MAbs that recognized porin surface epitopes increased sequentially as the LPS core became shorter. A total of 17 MAbs bound porin in a deep rough rfaD strain. Similar results were obtained with S. typhimurium. None of the anti-E. coli B/r porin MAbs adsorbed to a smooth strain, but three antibodies recognized porin on deep rough (rfaF, rfaE) mutants. These data define six distinct porin surface epitopes that are shielded by LPS from reaction with antibodies.

Full text

PDF
1063

Images in this article

Selected References

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

  1. Alexander H., Johnson D. A., Rosen J., Jerabek L., Green N., Weissman I. L., Lerner R. A. Mimicking the alloantigenicity of proteins with chemically synthesized peptides differing in single amino acids. Nature. 1983 Dec 15;306(5944):697–699. doi: 10.1038/306697a0. [DOI] [PubMed] [Google Scholar]
  2. Bavoil P., Nikaido H., von Meyenburg K. Pleiotropic transport mutants of Escherichia coli lack porin, a major outer membrane protein. Mol Gen Genet. 1977 Dec 14;158(1):23–33. doi: 10.1007/BF00455116. [DOI] [PubMed] [Google Scholar]
  3. Beher M. G., Schnaitman C. A. Regulation of the OmpA outer membrane protein of Escherichia coli. J Bacteriol. 1981 Sep;147(3):972–985. doi: 10.1128/jb.147.3.972-985.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blake M. S., Johnston K. H., Russell-Jones G. J., Gotschlich E. C. A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem. 1984 Jan;136(1):175–179. doi: 10.1016/0003-2697(84)90320-8. [DOI] [PubMed] [Google Scholar]
  5. Coleman W. G., Jr, Leive L. Two mutations which affect the barrier function of the Escherichia coli K-12 outer membrane. J Bacteriol. 1979 Sep;139(3):899–910. doi: 10.1128/jb.139.3.899-910.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eriksson-Grennberg K. R., Nordström K., Englund P. Resistance of Escherichia coli to penicillins. IX. Genetics and physiology of class II ampicillin-resistant mutants that are galactose negative or sensitive to bacteriophage C21, or both. J Bacteriol. 1971 Dec;108(3):1210–1223. doi: 10.1128/jb.108.3.1210-1223.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Getzoff E. D., Geysen H. M., Rodda S. J., Alexander H., Tainer J. A., Lerner R. A. Mechanisms of antibody binding to a protein. Science. 1987 Mar 6;235(4793):1191–1196. doi: 10.1126/science.3823879. [DOI] [PubMed] [Google Scholar]
  8. Gmeiner J., Schlecht S. Molecular composition of the outer membrane of Escherichia coli and the importance of protein-lipopolysaccharide interactions. Arch Microbiol. 1980 Sep;127(2):81–86. doi: 10.1007/BF00428010. [DOI] [PubMed] [Google Scholar]
  9. Graham A. C., Stocker B. A. Genetics of sensitivity of Salmonella species to colicin M and bacteriophages T5, T1, and ES18. J Bacteriol. 1977 Jun;130(3):1214–1223. doi: 10.1128/jb.130.3.1214-1223.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hofstra H., Dankert J. Major outer membrane proteins: common antigens in enterobacteriaceae species. J Gen Microbiol. 1980 Jul;119(1):123–131. doi: 10.1099/00221287-119-1-123. [DOI] [PubMed] [Google Scholar]
  11. Hofstra H., Van Tol J. D., Dankert J. Cross-reactivity of major outer membrane proteins of Enterobacteriaceae, studied by crossed immunoelectrophoresis. J Bacteriol. 1980 Jul;143(1):328–337. doi: 10.1128/jb.143.1.328-337.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Inokuchi K., Mutoh N., Matsuyama S., Mizushima S. Primary structure of the ompF gene that codes for a major outer membrane protein of Escherichia coli K-12. Nucleic Acids Res. 1982 Nov 11;10(21):6957–6968. doi: 10.1093/nar/10.21.6957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kadam S. K., Rehemtulla A., Sanderson K. E. Cloning of rfaG, B, I, and J genes for glycosyltransferase enzymes for synthesis of the lipopolysaccharide core of Salmonella typhimurium. J Bacteriol. 1985 Jan;161(1):277–284. doi: 10.1128/jb.161.1.277-284.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kearney J. F., Radbruch A., Liesegang B., Rajewsky K. A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines. J Immunol. 1979 Oct;123(4):1548–1550. [PubMed] [Google Scholar]
  15. Klebba P. E., McIntosh M. A., Neilands J. B. Kinetics of biosynthesis of iron-regulated membrane proteins in Escherichia coli. J Bacteriol. 1982 Mar;149(3):880–888. doi: 10.1128/jb.149.3.880-888.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lindberg A. A. Bacteriophage receptors. Annu Rev Microbiol. 1973;27:205–241. doi: 10.1146/annurev.mi.27.100173.001225. [DOI] [PubMed] [Google Scholar]
  17. Lüderitz O., Galanos C., Risse H. J., Ruschmann E., Schlecht S., Schmidt G., Schulte-Holthausen H., Wheat R., Westphal O., Schlosshardt J. Structural relationship of Salmonella O and R antigens. Ann N Y Acad Sci. 1966 Jun 30;133(2):349–374. doi: 10.1111/j.1749-6632.1966.tb52376.x. [DOI] [PubMed] [Google Scholar]
  18. Mayer H., Rapin A. M., Schmidt G., Boman H. G. Immunochemical studies on lipopolysaccharides from wild-type and mutants of Escherichia coli K-12. Eur J Biochem. 1976 Jul 1;66(2):357–368. doi: 10.1111/j.1432-1033.1976.tb10525.x. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Mutharia L. M., Hancock R. E. Surface localization of Pseudomonas aeruginosa outer membrane porin protein F by using monoclonal antibodies. Infect Immun. 1983 Dec;42(3):1027–1033. doi: 10.1128/iai.42.3.1027-1033.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nikaido H. Outer membrane of Salmonella typhimurium. Transmembrane diffusion of some hydrophobic substances. Biochim Biophys Acta. 1976 Apr 16;433(1):118–132. doi: 10.1016/0005-2736(76)90182-6. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Nikaido H., Wu H. C. Amino acid sequence homology among the major outer membrane proteins of Escherichia coli. Proc Natl Acad Sci U S A. 1984 Feb;81(4):1048–1052. doi: 10.1073/pnas.81.4.1048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nurminen M., Lounatmaa K., Sarvas M., Mäkelä P. H., Nakae T. Bacteriophage-resistant mutants of Salmonella typhimurium deficient in two major outer membrane proteins. J Bacteriol. 1976 Aug;127(2):941–955. doi: 10.1128/jb.127.2.941-955.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Osborn M. J. Biosynthesis and structure of the core region of the lipopolysaccharide in Salmonella typhimurium. Ann N Y Acad Sci. 1966 Jun 30;133(2):375–383. doi: 10.1111/j.1749-6632.1966.tb52377.x. [DOI] [PubMed] [Google Scholar]
  26. Overbeeke N., Van Scharrenburg G., Lugtenberg B. Antigenic relationships between pore proteins of Escherichia coli K12. Eur J Biochem. 1980 Sep;110(1):247–254. doi: 10.1111/j.1432-1033.1980.tb04862.x. [DOI] [PubMed] [Google Scholar]
  27. Prehm P., Stirm S., Jann B., Jann K., Boman H. G. Cell-wall lipopolysaccharides of ampicillin-resistant mutants of Escherichia coli K-12. Eur J Biochem. 1976 Jul 1;66(2):369–377. doi: 10.1111/j.1432-1033.1976.tb10526.x. [DOI] [PubMed] [Google Scholar]
  28. Prehm P., Stirm S., Jann B., Jann K. Cell-wall lipopolysaccharide from Escherichia coli B. Eur J Biochem. 1975 Aug 1;56(1):41–55. doi: 10.1111/j.1432-1033.1975.tb02205.x. [DOI] [PubMed] [Google Scholar]
  29. Reske K., Jann K. The O8 antigen of Escherichia coli. Structure of the polysaccharide chain. Eur J Biochem. 1972 Dec 4;31(2):320–328. doi: 10.1111/j.1432-1033.1972.tb02536.x. [DOI] [PubMed] [Google Scholar]
  30. Roantree R. J., Kuo T. T., MacPhee D. G. The effect of defined lipopolysaccharide core defects upon antibiotic resistances of Salmonella typhimurium. J Gen Microbiol. 1977 Dec;103(2):223–234. doi: 10.1099/00221287-103-2-223. [DOI] [PubMed] [Google Scholar]
  31. Sanderson K. E., MacAlister T., Costerton J. W., Cheng K. J. Permeability of lipopolysaccharide-deficient (rough) mutants of Salmonella typhimurium to antibiotics, lysozyme, and other agents. Can J Microbiol. 1974 Aug;20(8):1135–1145. doi: 10.1139/m74-176. [DOI] [PubMed] [Google Scholar]
  32. Sato T., Yura T. Chromosomal location and expression of the structural gene for major outer membrane protein Ia of Escherichia coli K-12 and of the homologous gene of Salmonella typhimurium. J Bacteriol. 1979 Aug;139(2):468–477. doi: 10.1128/jb.139.2.468-477.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schmidt G., Jann B., Jann K. Immunochemistry of R lipopolysaccharides of Escherichia coli. Studies on R mutants with an incomplete core, derived from E. coli O8:K27. Eur J Biochem. 1970 Oct;16(2):382–392. doi: 10.1111/j.1432-1033.1970.tb01092.x. [DOI] [PubMed] [Google Scholar]
  34. Shinnick T. M., Sutcliffe J. G., Green N., Lerner R. A. Synthetic peptide immunogens as vaccines. Annu Rev Microbiol. 1983;37:425–446. doi: 10.1146/annurev.mi.37.100183.002233. [DOI] [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. Steele B., Boman H. G. Capsular material and morphology of some ampicillin sensitive and resistant strains of Escherichia coli K12. Acta Pathol Microbiol Scand B Microbiol Immunol. 1970;78(1):59–74. doi: 10.1111/j.1699-0463.1970.tb04270.x. [DOI] [PubMed] [Google Scholar]
  37. Stocker B. A., Nurminen M., Mäkelä P. H. Mutants defective in the 33K outer membrane protein of Salmonella typhimurium. J Bacteriol. 1979 Aug;139(2):376–383. doi: 10.1128/jb.139.2.376-383.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tokunaga H., Tokunaga M., Nakae T. Characterization of porins from the outer membrane of Salmonella typhimurium. 1. Chemical analysis. Eur J Biochem. 1979 Apr;95(3):433–439. doi: 10.1111/j.1432-1033.1979.tb12982.x. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Yu F., Ichihara S., Mizushima S. A major outer membrane protein (O-8) of Escherichia coli K-12 exists as a trimer in sodium dodecyl sulfate solution. FEBS Lett. 1979 Apr 1;100(1):71–74. doi: 10.1016/0014-5793(79)81133-3. [DOI] [PubMed] [Google Scholar]
  41. van der Ley P., Kuipers O., Tommassen J., Lugtenberg B. O-antigenic chains of lipopolysaccharide prevent binding of antibody molecules to an outer membrane pore protein in Enterobacteriaceae. Microb Pathog. 1986 Feb;1(1):43–49. doi: 10.1016/0882-4010(86)90030-6. [DOI] [PubMed] [Google Scholar]

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

RESOURCES