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. 1995 Jun;63(6):2269–2276. doi: 10.1128/iai.63.6.2269-2276.1995

Virulence plasmid-encoded YopK is essential for Yersinia pseudotuberculosis to cause systemic infection in mice.

A Holmström 1, R Rosqvist 1, H Wolf-Watz 1, A Forsberg 1
PMCID: PMC173296  PMID: 7768608

Abstract

The virulence plasmid common to pathogenic Yersinia species encodes a number of secreted proteins denoted Yops (Yersinia outer proteins). Here, we identify and characterize a novel plasmid-encoded virulence determinant of Yersinia pseudotuberculosis, YopK. The yopK gene was found to be conserved among the three pathogenic Yersinia species and to be homologous to the previously described yopQ and yopK genes of Y. enterocolitica and Y. pestis, respectively. Similar to the other Yops, YopK expression and secretion were shown to be regulated by temperature and by the extracellular Ca2+ concentration; thus, yopK is part of the yop regulon. In addition, YopK secretion was mediated by the specific Yop secretion system. In Y. pseudotuberculosis, YopK was shown neither to have a role in this bacterium's ability to resist phagocytosis by macrophages nor to cause cytotoxicity in HeLa cells. YopK was, however, shown to be required for the bacterium to cause a systemic infection in both intraperitoneally and orally infected mice. Characterization of the infection kinetics showed that, similarly to the wild-type strain, the yopK mutant strain colonized and persisted in the Peyer's patches of orally infected mice. A yopE mutant which is impaired in cytotoxicity and in antiphagocytosis was, however, found to be rapidly cleared from these lymphoid organs. Neither the yopK nor the yopE mutant strain could overcome the primary host defense and reach the spleen. This finding implies that YopK acts at a different level during the infections process than the antiphagocytic YopE cytotoxin does.

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Selected References

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  1. BURROWS T. W., BACON G. A. The basis of virulence in Pasteurella pestis: the development of resistance to phagocytosis in vitro. Br J Exp Pathol. 1956 Jun;37(3):286–299. [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Bölin I., Norlander L., Wolf-Watz H. Temperature-inducible outer membrane protein of Yersinia pseudotuberculosis and Yersinia enterocolitica is associated with the virulence plasmid. Infect Immun. 1982 Aug;37(2):506–512. doi: 10.1128/iai.37.2.506-512.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bölin I., Portnoy D. A., Wolf-Watz H. Expression of the temperature-inducible outer membrane proteins of yersiniae. Infect Immun. 1985 Apr;48(1):234–240. doi: 10.1128/iai.48.1.234-240.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bölin I., Wolf-Watz H. Molecular cloning of the temperature-inducible outer membrane protein 1 of Yersinia pseudotuberculosis. Infect Immun. 1984 Jan;43(1):72–78. doi: 10.1128/iai.43.1.72-78.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. China B., Michiels T., Cornelis G. R. The pYV plasmid of Yersinia encodes a lipoprotein, YlpA, related to TraT. Mol Microbiol. 1990 Sep;4(9):1585–1593. doi: 10.1111/j.1365-2958.1990.tb02070.x. [DOI] [PubMed] [Google Scholar]
  9. Cohn D. H., Mileham A. J., Simon M. I., Nealson K. H., Rausch S. K., Bonam D., Baldwin T. O. Nucleotide sequence of the luxA gene of Vibrio harveyi and the complete amino acid sequence of the alpha subunit of bacterial luciferase. J Biol Chem. 1985 May 25;260(10):6139–6146. [PubMed] [Google Scholar]
  10. Conchas R. F., Carniel E. A highly efficient electroporation system for transformation of Yersinia. Gene. 1990 Mar 1;87(1):133–137. doi: 10.1016/0378-1119(90)90505-l. [DOI] [PubMed] [Google Scholar]
  11. Cornelis G., Sluiters C., de Rouvroit C. L., Michiels T. Homology between virF, the transcriptional activator of the Yersinia virulence regulon, and AraC, the Escherichia coli arabinose operon regulator. J Bacteriol. 1989 Jan;171(1):254–262. doi: 10.1128/jb.171.1.254-262.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Forsberg A. J., Pavitt G. D., Higgins C. F. Use of transcriptional fusions to monitor gene expression: a cautionary tale. J Bacteriol. 1994 Apr;176(7):2128–2132. doi: 10.1128/jb.176.7.2128-2132.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Forsberg A., Bölin I., Norlander L., Wolf-Watz H. Molecular cloning and expression of calcium-regulated, plasmid-coded proteins of Y. pseudotuberculosis. Microb Pathog. 1987 Feb;2(2):123–137. doi: 10.1016/0882-4010(87)90104-5. [DOI] [PubMed] [Google Scholar]
  14. Forsberg A., Rosqvist R. In vivo expression of virulence genes of Yersinia pseudotuberculosis. Infect Agents Dis. 1993 Aug;2(4):275–278. [PubMed] [Google Scholar]
  15. Forsberg A., Rosqvist R., Wolf-Watz H. Regulation and polarized transfer of the Yersinia outer proteins (Yops) involved in antiphagocytosis. Trends Microbiol. 1994 Jan;2(1):14–19. doi: 10.1016/0966-842x(94)90339-5. [DOI] [PubMed] [Google Scholar]
  16. Forsberg A., Viitanen A. M., Skurnik M., Wolf-Watz H. The surface-located YopN protein is involved in calcium signal transduction in Yersinia pseudotuberculosis. Mol Microbiol. 1991 Apr;5(4):977–986. doi: 10.1111/j.1365-2958.1991.tb00773.x. [DOI] [PubMed] [Google Scholar]
  17. Forsberg A., Wolf-Watz H. Genetic analysis of the yopE region of Yersinia spp.: identification of a novel conserved locus, yerA, regulating yopE expression. J Bacteriol. 1990 Mar;172(3):1547–1555. doi: 10.1128/jb.172.3.1547-1555.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Galyov E. E., Håkansson S., Forsberg A., Wolf-Watz H. A secreted protein kinase of Yersinia pseudotuberculosis is an indispensable virulence determinant. Nature. 1993 Feb 25;361(6414):730–732. doi: 10.1038/361730a0. [DOI] [PubMed] [Google Scholar]
  19. Galyov E. E., Håkansson S., Wolf-Watz H. Characterization of the operon encoding the YpkA Ser/Thr protein kinase and the YopJ protein of Yersinia pseudotuberculosis. J Bacteriol. 1994 Aug;176(15):4543–4548. doi: 10.1128/jb.176.15.4543-4548.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Grützkau A., Hanski C., Hahn H., Riecken E. O. Involvement of M cells in the bacterial invasion of Peyer's patches: a common mechanism shared by Yersinia enterocolitica and other enteroinvasive bacteria. Gut. 1990 Sep;31(9):1011–1015. doi: 10.1136/gut.31.9.1011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  22. Guan K. L., Dixon J. E. Protein tyrosine phosphatase activity of an essential virulence determinant in Yersinia. Science. 1990 Aug 3;249(4968):553–556. doi: 10.1126/science.2166336. [DOI] [PubMed] [Google Scholar]
  23. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  24. Hanski C., Kutschka U., Schmoranzer H. P., Naumann M., Stallmach A., Hahn H., Menge H., Riecken E. O. Immunohistochemical and electron microscopic study of interaction of Yersinia enterocolitica serotype O8 with intestinal mucosa during experimental enteritis. Infect Immun. 1989 Mar;57(3):673–678. doi: 10.1128/iai.57.3.673-678.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Heesemann J., Algermissen B., Laufs R. Genetically manipulated virulence of Yersinia enterocolitica. Infect Immun. 1984 Oct;46(1):105–110. doi: 10.1128/iai.46.1.105-110.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lambert de Rouvroit C., Sluiters C., Cornelis G. R. Role of the transcriptional activator, VirF, and temperature in the expression of the pYV plasmid genes of Yersinia enterocolitica. Mol Microbiol. 1992 Feb;6(3):395–409. [PubMed] [Google Scholar]
  27. Leung K. Y., Reisner B. S., Straley S. C. YopM inhibits platelet aggregation and is necessary for virulence of Yersinia pestis in mice. Infect Immun. 1990 Oct;58(10):3262–3271. doi: 10.1128/iai.58.10.3262-3271.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Leung K. Y., Straley S. C. The yopM gene of Yersinia pestis encodes a released protein having homology with the human platelet surface protein GPIb alpha. J Bacteriol. 1989 Sep;171(9):4623–4632. doi: 10.1128/jb.171.9.4623-4632.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. 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]
  32. Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mulder B., Michiels T., Simonet M., Sory M. P., Cornelis G. Identification of additional virulence determinants on the pYV plasmid of Yersinia enterocolitica W227. Infect Immun. 1989 Aug;57(8):2534–2541. doi: 10.1128/iai.57.8.2534-2541.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Perry R. D., Haddix P., Atkins E. B., Soughers T. K., Straley S. C. Regulation of expression of V antigen and outer membrane proteins in Yersinia pestis. Contrib Microbiol Immunol. 1987;9:173–178. [PubMed] [Google Scholar]
  35. 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]
  36. 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]
  37. 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]
  38. 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]
  39. Salmond G. P., Reeves P. J. Membrane traffic wardens and protein secretion in gram-negative bacteria. Trends Biochem Sci. 1993 Jan;18(1):7–12. doi: 10.1016/0968-0004(93)90080-7. [DOI] [PubMed] [Google Scholar]
  40. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Sodeinde O. A., Sample A. K., Brubaker R. R., Goguen J. D. Plasminogen activator/coagulase gene of Yersinia pestis is responsible for degradation of plasmid-encoded outer membrane proteins. Infect Immun. 1988 Oct;56(10):2749–2752. doi: 10.1128/iai.56.10.2749-2752.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Straley S. C., Bowmer W. S. Virulence genes regulated at the transcriptional level by Ca2+ in Yersinia pestis include structural genes for outer membrane proteins. Infect Immun. 1986 Feb;51(2):445–454. doi: 10.1128/iai.51.2.445-454.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Straley S. C., Cibull M. L. Differential clearance and host-pathogen interactions of YopE- and YopK- YopL- Yersinia pestis in BALB/c mice. Infect Immun. 1989 Apr;57(4):1200–1210. doi: 10.1128/iai.57.4.1200-1210.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Straley S. C., Plano G. V., Skrzypek E., Haddix P. L., Fields K. A. Regulation by Ca2+ in the Yersinia low-Ca2+ response. Mol Microbiol. 1993 Jun;8(6):1005–1010. doi: 10.1111/j.1365-2958.1993.tb01644.x. [DOI] [PubMed] [Google Scholar]
  46. Wattiau P., Cornelis G. R. Identification of DNA sequences recognized by VirF, the transcriptional activator of the Yersinia yop regulon. J Bacteriol. 1994 Jul;176(13):3878–3884. doi: 10.1128/jb.176.13.3878-3884.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  48. Yother J., Chamness T. W., Goguen J. D. Temperature-controlled plasmid regulon associated with low calcium response in Yersinia pestis. J Bacteriol. 1986 Feb;165(2):443–447. doi: 10.1128/jb.165.2.443-447.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]

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