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. 1995 Dec 19;92(26):11998–12002. doi: 10.1073/pnas.92.26.11998

Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach.

M P Sory 1, A Boland 1, I Lambermont 1, G R Cornelis 1
PMCID: PMC40283  PMID: 8618831

Abstract

Pathogenic yersiniae secrete a set of antihost proteins, called Yops, by a type III secretion mechanism. Upon infection of cultured epithelial cells, extracellular Yersinia pseudotuberculosis and Yersinia enterocolitica translocate cytotoxin YopE across the host cell plasma membrane. Several lines of evidence suggest that tyrosine phosphatase YopH follows the same pathway. We analyzed internalization of YopE and YopH into murine PU5-1.8 macrophages by using recombinant Y. enterocolitica producing truncated YopE and YopH proteins fused to a calmodulin-dependent adenylate cyclase. The YopE-cyclase and YopH-cyclase hybrids were readily secreted by Y. enterocolitica. The N-terminal domain required for secretion was not longer than 15 residues of YopE and 17 residues of YopH. Internalization into eukaryotic cells, revealed by cAMP production, only required the N-terminal 50 amino acid residues of YopE and the N-terminal 71 amino acid residues of YopH. YopE and YopH are thus modular proteins composed of a secretion domain, a translocation domain, and an effector domain. Translocation of YopE and YopH across host cell's membranes was also dependent on the secretion of YopB and YopD by the same bacterium. The cyclase fusion approach could be readily extended to study the fate of other proteins secreted by invasive bacterial pathogens.

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Selected References

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  1. Allaoui A., Woestyn S., Sluiters C., Cornelis G. R. YscU, a Yersinia enterocolitica inner membrane protein involved in Yop secretion. J Bacteriol. 1994 Aug;176(15):4534–4542. doi: 10.1128/jb.176.15.4534-4542.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bellalou J., Sakamoto H., Ladant D., Geoffroy C., Ullmann A. Deletions affecting hemolytic and toxin activities of Bordetella pertussis adenylate cyclase. Infect Immun. 1990 Oct;58(10):3242–3247. doi: 10.1128/iai.58.10.3242-3247.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bergman T., Erickson K., Galyov E., Persson C., Wolf-Watz H. The lcrB (yscN/U) gene cluster of Yersinia pseudotuberculosis is involved in Yop secretion and shows high homology to the spa gene clusters of Shigella flexneri and Salmonella typhimurium. J Bacteriol. 1994 May;176(9):2619–2626. doi: 10.1128/jb.176.9.2619-2626.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bliska J. B., Black D. S. Inhibition of the Fc receptor-mediated oxidative burst in macrophages by the Yersinia pseudotuberculosis tyrosine phosphatase. Infect Immun. 1995 Feb;63(2):681–685. doi: 10.1128/iai.63.2.681-685.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bliska J. B., Clemens J. C., Dixon J. E., Falkow S. The Yersinia tyrosine phosphatase: specificity of a bacterial virulence determinant for phosphoproteins in the J774A.1 macrophage. J Exp Med. 1992 Dec 1;176(6):1625–1630. doi: 10.1084/jem.176.6.1625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fields K. A., Plano G. V., Straley S. C. A low-Ca2+ response (LCR) secretion (ysc) locus lies within the lcrB region of the LCR plasmid in Yersinia pestis. J Bacteriol. 1994 Feb;176(3):569–579. doi: 10.1128/jb.176.3.569-579.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Glaser P., Ladant D., Sezer O., Pichot F., Ullmann A., Danchin A. The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia coli. Mol Microbiol. 1988 Jan;2(1):19–30. [PubMed] [Google Scholar]
  8. Hartland E. L., Green S. P., Phillips W. A., Robins-Browne R. M. Essential role of YopD in inhibition of the respiratory burst of macrophages by Yersinia enterocolitica. Infect Immun. 1994 Oct;62(10):4445–4453. doi: 10.1128/iai.62.10.4445-4453.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hermant D., Ménard R., Arricau N., Parsot C., Popoff M. Y. Functional conservation of the Salmonella and Shigella effectors of entry into epithelial cells. Mol Microbiol. 1995 Aug;17(4):781–789. doi: 10.1111/j.1365-2958.1995.mmi_17040781.x. [DOI] [PubMed] [Google Scholar]
  10. Jarvis K. G., Girón J. A., Jerse A. E., McDaniel T. K., Donnenberg M. S., Kaper J. B. Enteropathogenic Escherichia coli contains a putative type III secretion system necessary for the export of proteins involved in attaching and effacing lesion formation. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7996–8000. doi: 10.1073/pnas.92.17.7996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kaniga K., Tucker S., Trollinger D., Galán J. E. Homologs of the Shigella IpaB and IpaC invasins are required for Salmonella typhimurium entry into cultured epithelial cells. J Bacteriol. 1995 Jul;177(14):3965–3971. doi: 10.1128/jb.177.14.3965-3971.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Michiels T., Cornelis G. R. Secretion of hybrid proteins by the Yersinia Yop export system. J Bacteriol. 1991 Mar;173(5):1677–1685. doi: 10.1128/jb.173.5.1677-1685.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Michiels T., Cornelis G. Nucleotide sequence and transcription analysis of yop51 from Yersinia enterocolitica W22703. Microb Pathog. 1988 Dec;5(6):449–459. doi: 10.1016/0882-4010(88)90006-x. [DOI] [PubMed] [Google Scholar]
  14. Michiels T., Vanooteghem J. C., Lambert de Rouvroit C., China B., Gustin A., Boudry P., Cornelis G. R. Analysis of virC, an operon involved in the secretion of Yop proteins by Yersinia enterocolitica. J Bacteriol. 1991 Aug;173(16):4994–5009. doi: 10.1128/jb.173.16.4994-5009.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Michiels T., Wattiau P., Brasseur R., Ruysschaert J. M., Cornelis G. Secretion of Yop proteins by Yersiniae. Infect Immun. 1990 Sep;58(9):2840–2849. doi: 10.1128/iai.58.9.2840-2849.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Miller V. L., Falkow S. Evidence for two genetic loci in Yersinia enterocolitica that can promote invasion of epithelial cells. Infect Immun. 1988 May;56(5):1242–1248. doi: 10.1128/iai.56.5.1242-1248.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ménard R., Sansonetti P., Parsot C., Vasselon T. Extracellular association and cytoplasmic partitioning of the IpaB and IpaC invasins of S. flexneri. Cell. 1994 Nov 4;79(3):515–525. doi: 10.1016/0092-8674(94)90260-7. [DOI] [PubMed] [Google Scholar]
  18. Plano G. V., Straley S. C. Multiple effects of lcrD mutations in Yersinia pestis. J Bacteriol. 1993 Jun;175(11):3536–3545. doi: 10.1128/jb.175.11.3536-3545.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Raptis A., Knipling L., Wolff J. Dissociation of catalytic and invasive activities of Bordetella pertussis adenylate cyclase. Infect Immun. 1989 Jun;57(6):1725–1730. doi: 10.1128/iai.57.6.1725-1730.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rogel A., Schultz J. E., Brownlie R. M., Coote J. G., Parton R., Hanski E. Bordetella pertussis adenylate cyclase: purification and characterization of the toxic form of the enzyme. EMBO J. 1989 Sep;8(9):2755–2760. doi: 10.1002/j.1460-2075.1989.tb08417.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rosqvist R., Bölin I., Wolf-Watz H. Inhibition of phagocytosis in Yersinia pseudotuberculosis: a virulence plasmid-encoded ability involving the Yop2b protein. Infect Immun. 1988 Aug;56(8):2139–2143. doi: 10.1128/iai.56.8.2139-2143.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rosqvist R., Forsberg A., Rimpiläinen M., Bergman T., Wolf-Watz H. The cytotoxic protein YopE of Yersinia obstructs the primary host defence. Mol Microbiol. 1990 Apr;4(4):657–667. doi: 10.1111/j.1365-2958.1990.tb00635.x. [DOI] [PubMed] [Google Scholar]
  23. Rosqvist R., Forsberg A., Wolf-Watz H. Intracellular targeting of the Yersinia YopE cytotoxin in mammalian cells induces actin microfilament disruption. Infect Immun. 1991 Dec;59(12):4562–4569. doi: 10.1128/iai.59.12.4562-4569.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rosqvist R., Magnusson K. E., Wolf-Watz H. Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells. EMBO J. 1994 Feb 15;13(4):964–972. doi: 10.1002/j.1460-2075.1994.tb06341.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Simonet M., Richard S., Berche P. Electron microscopic evidence for in vivo extracellular localization of Yersinia pseudotuberculosis harboring the pYV plasmid. Infect Immun. 1990 Mar;58(3):841–845. doi: 10.1128/iai.58.3.841-845.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sory M. P., Cornelis G. R. Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells. Mol Microbiol. 1994 Nov;14(3):583–594. doi: 10.1111/j.1365-2958.1994.tb02191.x. [DOI] [PubMed] [Google Scholar]
  27. Sory M. P., Kaniga K., Goldenberg S., Cornelis G. R. Expression of the eukaryotic Trypanosoma cruzi CRA gene in Yersinia enterocolitica and induction of an immune response against CRA in mice. Infect Immun. 1992 Sep;60(9):3830–3836. doi: 10.1128/iai.60.9.3830-3836.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Starnbach M. N., Bevan M. J. Cells infected with Yersinia present an epitope to class I MHC-restricted CTL. J Immunol. 1994 Aug 15;153(4):1603–1612. [PMC free article] [PubMed] [Google Scholar]
  29. Stuckey J. A., Schubert H. L., Fauman E. B., Zhang Z. Y., Dixon J. E., Saper M. A. Crystal structure of Yersinia protein tyrosine phosphatase at 2.5 A and the complex with tungstate. Nature. 1994 Aug 18;370(6490):571–575. doi: 10.1038/370571a0. [DOI] [PubMed] [Google Scholar]
  30. Wattiau P., Bernier B., Deslée P., Michiels T., Cornelis G. R. Individual chaperones required for Yop secretion by Yersinia. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10493–10497. doi: 10.1073/pnas.91.22.10493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wattiau P., Cornelis G. R. SycE, a chaperone-like protein of Yersinia enterocolitica involved in Ohe secretion of YopE. Mol Microbiol. 1993 Apr;8(1):123–131. doi: 10.1111/j.1365-2958.1993.tb01209.x. [DOI] [PubMed] [Google Scholar]
  32. Woestyn S., Allaoui A., Wattiau P., Cornelis G. R. YscN, the putative energizer of the Yersinia Yop secretion machinery. J Bacteriol. 1994 Mar;176(6):1561–1569. doi: 10.1128/jb.176.6.1561-1569.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wolff J., Cook G. H., Goldhammer A. R., Berkowitz S. A. Calmodulin activates prokaryotic adenylate cyclase. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3841–3844. doi: 10.1073/pnas.77.7.3841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yahr T. L., Hovey A. K., Kulich S. M., Frank D. W. Transcriptional analysis of the Pseudomonas aeruginosa exoenzyme S structural gene. J Bacteriol. 1995 Mar;177(5):1169–1178. doi: 10.1128/jb.177.5.1169-1178.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Young V. B., Falkow S., Schoolnik G. K. The invasin protein of Yersinia enterocolitica: internalization of invasin-bearing bacteria by eukaryotic cells is associated with reorganization of the cytoskeleton. J Cell Biol. 1992 Jan;116(1):197–207. doi: 10.1083/jcb.116.1.197. [DOI] [PMC free article] [PubMed] [Google Scholar]

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