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. 1990 Jun;9(6):1991–1999. doi: 10.1002/j.1460-2075.1990.tb08327.x

Extracellular transport of cholera toxin B subunit using Neisseria IgA protease beta-domain: conformation-dependent outer membrane translocation.

T Klauser 1, J Pohlner 1, T F Meyer 1
PMCID: PMC551908  PMID: 2189728

Abstract

The beta-domain of the Neisseria IgA protease precursor (Iga) provides the essential transport function for the protease across the outer membrane. To investigate the secretion function of the beta-domain (Iga beta), we engineered hybrid proteins between Iga beta and the non-toxic 12 kd cholera toxin B subunit (CtxB) and examined their targeting behaviour in Salmonella typhimurium. We show that CtxB-Iga beta hybrid proteins integrate into the outer membrane, leading to the exposition of the CtxB moiety on the cell surface. Exposed CtxB can be degraded by externally added proteases like trypsin, but can also be specifically cleaved off from membrane-associated Iga beta by purified IgA protease. We further demonstrate that folding of the CtxB moiety at the periplasmic side of the outer membrane interferes with its translocation. Prevention of disulphide-induced folding in periplasmic CtxB renders the protein moiety competent for outer membrane transport. Iga beta may be of general interest as an export vehicle for even larger proteins from Gram-negative bacteria.

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Selected References

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

  1. Agterberg M., Adriaanse H., Tommassen J. Use of outer membrane protein PhoE as a carrier for the transport of a foreign antigenic determinant to the cell surface of Escherichia coli K-12. Gene. 1987;59(1):145–150. doi: 10.1016/0378-1119(87)90275-7. [DOI] [PubMed] [Google Scholar]
  2. Bochkareva E. S., Lissin N. M., Girshovich A. S. Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein. Nature. 1988 Nov 17;336(6196):254–257. doi: 10.1038/336254a0. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Charbit A., Boulain J. C., Ryter A., Hofnung M. Probing the topology of a bacterial membrane protein by genetic insertion of a foreign epitope; expression at the cell surface. EMBO J. 1986 Nov;5(11):3029–3037. doi: 10.1002/j.1460-2075.1986.tb04602.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen E. Y., Seeburg P. H. Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA. 1985 Apr;4(2):165–170. doi: 10.1089/dna.1985.4.165. [DOI] [PubMed] [Google Scholar]
  6. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  7. Chirico W. J., Waters M. G., Blobel G. 70K heat shock related proteins stimulate protein translocation into microsomes. Nature. 1988 Apr 28;332(6167):805–810. doi: 10.1038/332805a0. [DOI] [PubMed] [Google Scholar]
  8. Collier D. N., Bankaitis V. A., Weiss J. B., Bassford P. J., Jr The antifolding activity of SecB promotes the export of the E. coli maltose-binding protein. Cell. 1988 Apr 22;53(2):273–283. doi: 10.1016/0092-8674(88)90389-3. [DOI] [PubMed] [Google Scholar]
  9. Crooke E., Guthrie B., Lecker S., Lill R., Wickner W. ProOmpA is stabilized for membrane translocation by either purified E. coli trigger factor or canine signal recognition particle. Cell. 1988 Sep 23;54(7):1003–1011. doi: 10.1016/0092-8674(88)90115-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eilers M., Hwang S., Schatz G. Unfolding and refolding of a purified precursor protein during import into isolated mitochondria. EMBO J. 1988 Apr;7(4):1139–1145. doi: 10.1002/j.1460-2075.1988.tb02923.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eilers M., Schatz G. Binding of a specific ligand inhibits import of a purified precursor protein into mitochondria. Nature. 1986 Jul 17;322(6076):228–232. doi: 10.1038/322228a0. [DOI] [PubMed] [Google Scholar]
  12. Freudl R. Insertion of peptides into cell-surface-exposed areas of the Escherichia coli OmpA protein does not interfere with export and membrane assembly. Gene. 1989 Oct 30;82(2):229–236. doi: 10.1016/0378-1119(89)90048-6. [DOI] [PubMed] [Google Scholar]
  13. Freudl R., Schwarz H., Stierhof Y. D., Gamon K., Hindennach I., Henning U. An outer membrane protein (OmpA) of Escherichia coli K-12 undergoes a conformational change during export. J Biol Chem. 1986 Aug 25;261(24):11355–11361. [PubMed] [Google Scholar]
  14. Halter R., Pohlner J., Meyer T. F. IgA protease of Neisseria gonorrhoeae: isolation and characterization of the gene and its extracellular product. EMBO J. 1984 Jul;3(7):1595–1601. doi: 10.1002/j.1460-2075.1984.tb02016.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Halter R., Pohlner J., Meyer T. F. Mosaic-like organization of IgA protease genes in Neisseria gonorrhoeae generated by horizontal genetic exchange in vivo. EMBO J. 1989 Sep;8(9):2737–2744. doi: 10.1002/j.1460-2075.1989.tb08415.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hardy S. J., Holmgren J., Johansson S., Sanchez J., Hirst T. R. Coordinated assembly of multisubunit proteins: oligomerization of bacterial enterotoxins in vivo and in vitro. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7109–7113. doi: 10.1073/pnas.85.19.7109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hirst T. R., Holmgren J. Conformation of protein secreted across bacterial outer membranes: a study of enterotoxin translocation from Vibrio cholerae. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7418–7422. doi: 10.1073/pnas.84.21.7418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hirst T. R., Holmgren J. Transient entry of enterotoxin subunits into the periplasm occurs during their secretion from Vibrio cholerae. J Bacteriol. 1987 Mar;169(3):1037–1045. doi: 10.1128/jb.169.3.1037-1045.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Holmgren J. Actions of cholera toxin and the prevention and treatment of cholera. Nature. 1981 Jul 30;292(5822):413–417. doi: 10.1038/292413a0. [DOI] [PubMed] [Google Scholar]
  21. Howard S. P., Buckley J. T. Protein export by a gram-negative bacterium: production of aerolysin by Aeromonas hydrophila. J Bacteriol. 1985 Mar;161(3):1118–1124. doi: 10.1128/jb.161.3.1118-1124.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Klose M., MacIntyre S., Schwarz H., Henning U. The influence of amino substitutions within the mature part of an Escherichia coli outer membrane protein (OmpA) on assembly of the polypeptide into its membrane. J Biol Chem. 1988 Sep 15;263(26):13297–13302. [PubMed] [Google Scholar]
  23. 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]
  24. Lederberg E. M., Cohen S. N. Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J Bacteriol. 1974 Sep;119(3):1072–1074. doi: 10.1128/jb.119.3.1072-1074.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lockman H., Kaper J. B. Nucleotide sequence analysis of the A2 and B subunits of Vibrio cholerae enterotoxin. J Biol Chem. 1983 Nov 25;258(22):13722–13726. [PubMed] [Google Scholar]
  26. Ludwig D. S., Holmes R. K., Schoolnik G. K. Chemical and immunochemical studies on the receptor binding domain of cholera toxin B subunit. J Biol Chem. 1985 Oct 15;260(23):12528–12534. [PubMed] [Google Scholar]
  27. Mekalanos J. J., Swartz D. J., Pearson G. D., Harford N., Groyne F., de Wilde M. Cholera toxin genes: nucleotide sequence, deletion analysis and vaccine development. Nature. 1983 Dec 8;306(5943):551–557. doi: 10.1038/306551a0. [DOI] [PubMed] [Google Scholar]
  28. Meyer T. F., Mlawer N., So M. Pilus expression in Neisseria gonorrhoeae involves chromosomal rearrangement. Cell. 1982 Aug;30(1):45–52. doi: 10.1016/0092-8674(82)90010-1. [DOI] [PubMed] [Google Scholar]
  29. Misra R., Benson S. A. Genetic identification of the pore domain of the OmpC porin of Escherichia coli K-12. J Bacteriol. 1988 Aug;170(8):3611–3617. doi: 10.1128/jb.170.8.3611-3617.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. OSBORN M. J., ROSEN S. M., ROTHFIELD L., ZELEZNICK L. D., HORECKER B. L. LIPOPOLYSACCHARIDE OF THE GRAM-NEGATIVE CELL WALL. Science. 1964 Aug 21;145(3634):783–789. doi: 10.1126/science.145.3634.783. [DOI] [PubMed] [Google Scholar]
  31. Pearson G. D., Mekalanos J. J. Molecular cloning of Vibrio cholerae enterotoxin genes in Escherichia coli K-12. Proc Natl Acad Sci U S A. 1982 May;79(9):2976–2980. doi: 10.1073/pnas.79.9.2976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pohlner J., Halter R., Beyreuther K., Meyer T. F. Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease. 1987 Jan 29-Feb 4Nature. 325(6103):458–462. doi: 10.1038/325458a0. [DOI] [PubMed] [Google Scholar]
  33. Pohlner J., Halter R., Meyer T. F. Neisseria gonorrhoeae IgA protease. Secretion and implications for pathogenesis. Antonie Van Leeuwenhoek. 1987;53(6):479–484. doi: 10.1007/BF00415506. [DOI] [PubMed] [Google Scholar]
  34. Randall L. L., Hardy S. J. Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in E. coli. Cell. 1986 Sep 12;46(6):921–928. doi: 10.1016/0092-8674(86)90074-7. [DOI] [PubMed] [Google Scholar]
  35. Ribi H. O., Ludwig D. S., Mercer K. L., Schoolnik G. K., Kornberg R. D. Three-dimensional structure of cholera toxin penetrating a lipid membrane. Science. 1988 Mar 11;239(4845):1272–1276. doi: 10.1126/science.3344432. [DOI] [PubMed] [Google Scholar]
  36. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  37. Schenkman S., Tsugita A., Schwartz M., Rosenbusch J. P. Topology of phage lambda receptor protein. Mapping targets of proteolytic cleavage in relation to binding sites for phage or monoclonal antibodies. J Biol Chem. 1984 Jun 25;259(12):7570–7576. [PubMed] [Google Scholar]
  38. Stern A., Brown M., Nickel P., Meyer T. F. Opacity genes in Neisseria gonorrhoeae: control of phase and antigenic variation. Cell. 1986 Oct 10;47(1):61–71. doi: 10.1016/0092-8674(86)90366-1. [DOI] [PubMed] [Google Scholar]
  39. Tommassen J., Leunissen J., van Damme-Jongsten M., Overduin P. Failure of E. coli K-12 to transport PhoE-LacZ hybrid proteins out of the cytoplasm. EMBO J. 1985 Apr;4(4):1041–1047. doi: 10.1002/j.1460-2075.1985.tb03736.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vogel H., Jähnig F. Models for the structure of outer-membrane proteins of Escherichia coli derived from raman spectroscopy and prediction methods. J Mol Biol. 1986 Jul 20;190(2):191–199. doi: 10.1016/0022-2836(86)90292-5. [DOI] [PubMed] [Google Scholar]
  41. van der Ley P., Amesz H., Tommassen J., Lugtenberg B. Monoclonal antibodies directed against the cell-surface-exposed part of PhoE pore protein of the Escherichia coli K-12 outer membrane. Eur J Biochem. 1985 Mar 1;147(2):401–407. doi: 10.1111/j.1432-1033.1985.tb08764.x. [DOI] [PubMed] [Google Scholar]

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