Abstract
In previous studies, Yersinia pestis YopM has been shown through mutational analysis to be necessary for virulence in mice and found to have homology with the thrombin-binding domain of the platelet receptor GPIb alpha. In this study, YopM was purified and shown by dot blot and chemical cross-linking tests to bind to human alpha-thrombin. No cross-linked product could be detected when human prothrombin was incubated with YopM. As a functional test of thrombin binding, it was shown that native but not boiled YopM inhibits thrombin-induced aggregation of human platelets. Control tests showed that YopM did not inactivate the platelets themselves, nor was its effect a nonspecific consequence of its very acidic isoelectric point. Microsequencing of YopM revealed an intact N terminus, indicating that functional YopM is not processed at the N terminus or secreted by a mechanism involving a cleavable signal sequence. Further characterization was made of an interesting effect on yopM expression that had been noticed in a previous study. A 1.5-kb HaeIII subclone overexpressed YopM in both Y. pestis and Escherichia coli compared with a larger clone containing the 5.3-kb HindIII-F fragment. To search for a possible regulator of YopM expression, the HindIII-F fragment was sequenced, revealing several open reading frames and three large repeated sequences. Deletional analysis showed that these were not involved in regulation of yopM. The data implicated a DNA structure 5' to yopM in moderating yopM expression.
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- Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bliska J. B., Guan K. L., Dixon J. E., Falkow S. Tyrosine phosphate hydrolysis of host proteins by an essential Yersinia virulence determinant. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1187–1191. doi: 10.1073/pnas.88.4.1187. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bolivar F., Backman K. Plasmids of Escherichia coli as cloning vectors. Methods Enzymol. 1979;68:245–267. doi: 10.1016/0076-6879(79)68018-7. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Bölin I., Wolf-Watz H. The plasmid-encoded Yop2b protein of Yersinia pseudotuberculosis is a virulence determinant regulated by calcium and temperature at the level of transcription. Mol Microbiol. 1988 Mar;2(2):237–245. doi: 10.1111/j.1365-2958.1988.tb00025.x. [DOI] [PubMed] [Google Scholar]
- Carlson S. L., Brooks W. H., Roszman T. L. Neurotransmitter-lymphocyte interactions: dual receptor modulation of lymphocyte proliferation and cAMP production. J Neuroimmunol. 1989 Sep;24(1-2):155–162. doi: 10.1016/0165-5728(89)90109-4. [DOI] [PubMed] [Google Scholar]
- Cornelis G. R., Sluiters C., Delor I., Geib D., Kaniga K., Lambert de Rouvroit C., Sory M. P., Vanooteghem J. C., Michiels T. ymoA, a Yersinia enterocolitica chromosomal gene modulating the expression of virulence functions. Mol Microbiol. 1991 May;5(5):1023–1034. doi: 10.1111/j.1365-2958.1991.tb01875.x. [DOI] [PubMed] [Google Scholar]
- Cornelis G., Vanootegem J. C., Sluiters C. Transcription of the yop regulon from Y. enterocolitica requires trans acting pYV and chromosomal genes. Microb Pathog. 1987 May;2(5):367–379. doi: 10.1016/0882-4010(87)90078-7. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Forsberg A., Wolf-Watz H. The virulence protein Yop5 of Yersinia pseudotuberculosis is regulated at transcriptional level by plasmid-plB1-encoded trans-acting elements controlled by temperature and calcium. Mol Microbiol. 1988 Jan;2(1):121–133. [PubMed] [Google Scholar]
- Gentile F., Raptis A., Knipling L. G., Wolff J. Extracellular cAMP formation from host cell ATP by Bordetella pertussis adenylate cyclase. Biochim Biophys Acta. 1988 Aug 19;971(1):63–71. doi: 10.1016/0167-4889(88)90162-0. [DOI] [PubMed] [Google Scholar]
- Goguen J. D., Yother J., Straley S. C. Genetic analysis of the low calcium response in Yersinia pestis mu d1(Ap lac) insertion mutants. J Bacteriol. 1984 Dec;160(3):842–848. doi: 10.1128/jb.160.3.842-848.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goldhammer A. R., Wolff J., Hope Cook G., Berkowitz S. A., Klee C. B., Manclark C. R., Hewlett E. L. Spurious protein activators of Bordetella pertussis adenylate cyclase. Eur J Biochem. 1981 Apr;115(3):605–609. doi: 10.1111/j.1432-1033.1981.tb06245.x. [DOI] [PubMed] [Google Scholar]
- 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]
- Hartman A. B., Venkatesan M., Oaks E. V., Buysse J. M. Sequence and molecular characterization of a multicopy invasion plasmid antigen gene, ipaH, of Shigella flexneri. J Bacteriol. 1990 Apr;172(4):1905–1915. doi: 10.1128/jb.172.4.1905-1915.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Holmsen H. Physiological functions of platelets. Ann Med. 1989 Feb;21(1):23–30. doi: 10.3109/07853898909149178. [DOI] [PubMed] [Google Scholar]
- Jakubowski J. A., Maraganore J. M. Inhibition of coagulation and thrombin-induced platelet activities by a synthetic dodecapeptide modeled on the carboxy-terminus of hirudin. Blood. 1990 Jan 15;75(2):399–406. [PubMed] [Google Scholar]
- Kisiel W., Hanahan D. J. Proteolysis of human factor II by factor Xa in the presence of hirudin. Biochem Biophys Res Commun. 1974 Jul 24;59(2):570–577. doi: 10.1016/s0006-291x(74)80018-5. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- Liu L. W., Ye J., Johnson A. E., Esmon C. T. Proteolytic formation of either of the two prothrombin activation intermediates results in formation of a hirugen-binding site. J Biol Chem. 1991 Dec 15;266(35):23633–23636. [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- 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]
- 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]
- Plano G. V., Barve S. S., Straley S. C. LcrD, a membrane-bound regulator of the Yersinia pestis low-calcium response. J Bacteriol. 1991 Nov;173(22):7293–7303. doi: 10.1128/jb.173.22.7293-7303.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Portnoy D. A., Moseley S. L., Falkow S. Characterization of plasmids and plasmid-associated determinants of Yersinia enterocolitica pathogenesis. Infect Immun. 1981 Feb;31(2):775–782. doi: 10.1128/iai.31.2.775-782.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Portnoy D. A., Wolf-Watz H., Bolin I., Beeder A. B., Falkow S. Characterization of common virulence plasmids in Yersinia species and their role in the expression of outer membrane proteins. Infect Immun. 1984 Jan;43(1):108–114. doi: 10.1128/iai.43.1.108-114.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Protsenko O. A., Anisimov P. I., Mozharov O. T., Konnov N. P., Popov Iu A. Vyiavlenie i kharakteristika plazmid chumnogo mikroba, determiniruiushchikh sintez pestitsina I, antigena fraktsiia I i ékzotoksina "myshinogo" toksina. Genetika. 1983 Jul;19(7):1081–1090. [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Rosqvist R., Skurnik M., Wolf-Watz H. Increased virulence of Yersinia pseudotuberculosis by two independent mutations. Nature. 1988 Aug 11;334(6182):522–524. doi: 10.1038/334522a0. [DOI] [PubMed] [Google Scholar]
- Rydel T. J., Ravichandran K. G., Tulinsky A., Bode W., Huber R., Roitsch C., Fenton J. W., 2nd The structure of a complex of recombinant hirudin and human alpha-thrombin. Science. 1990 Jul 20;249(4966):277–280. doi: 10.1126/science.2374926. [DOI] [PubMed] [Google Scholar]
- 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]
- Simonet M., Triadou P., Frehel C., Morel-Kopp M. C., Kaplan C., Berche P. Human platelet aggregation by Yersinia pseudotuberculosis is mediated by invasin. Infect Immun. 1992 Feb;60(2):366–373. doi: 10.1128/iai.60.2.366-373.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sodeinde O. A., Goguen J. D. Genetic analysis of the 9.5-kilobase virulence plasmid of Yersinia pestis. Infect Immun. 1988 Oct;56(10):2743–2748. doi: 10.1128/iai.56.10.2743-2748.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- 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]
- Straley S. C., Brubaker R. R. Cytoplasmic and membrane proteins of yersiniae cultivated under conditions simulating mammalian intracellular environment. Proc Natl Acad Sci U S A. 1981 Feb;78(2):1224–1228. doi: 10.1073/pnas.78.2.1224. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Straley S. C. The plasmid-encoded outer-membrane proteins of Yersinia pestis. Rev Infect Dis. 1988 Jul-Aug;10 (Suppl 2):S323–S326. doi: 10.1093/cid/10.supplement_2.s323. [DOI] [PubMed] [Google Scholar]
- Une T., Brubaker R. R. In vivo comparison of avirulent Vwa- and Pgm- or Pstr phenotypes of yersiniae. Infect Immun. 1984 Mar;43(3):895–900. doi: 10.1128/iai.43.3.895-900.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Venkatesan M. M., Buysse J. M., Hartman A. B. Sequence variation in two ipaH genes of Shigella flexneri 5 and homology to the LRG-like family of proteins. Mol Microbiol. 1991 Oct;5(10):2435–2445. doi: 10.1111/j.1365-2958.1991.tb02089.x. [DOI] [PubMed] [Google Scholar]
- Wasiewski W., Fasco M. J., Martin B. M., Detwiler T. C., Fenton J. W. Thrombin adsorption to surfaces and prevention with polyethylene glycol 6,000. Thromb Res. 1976 Jun;8(6):881–886. doi: 10.1016/0049-3848(76)90017-7. [DOI] [PubMed] [Google Scholar]
- Wolf-Watz H., Portnoy D. A., Bölin I., Falkow S. Transfer of the virulence plasmid of Yersinia pestis to Yersinia pseudotuberculosis. Infect Immun. 1985 Apr;48(1):241–243. doi: 10.1128/iai.48.1.241-243.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]