1887

Abstract

Pathogenesis of is closely connected to its survival and replication within the host. Some pathogenic bacteria employ protein kinases that interfere with the cellular signalling network of host cells and promote bacterial survival. In this study, the and genes, which encode two putative protein kinases of HRv, protein kinase F (PknF) and protein kinase G (PknG), respectively, were cloned and expressed in . Purified PknF phosphorylated the peptide substrate myelin basic protein (MBP) at serine and threonine residues, while purified PknG phosphorylated only at serine residues. The activity of the two kinases was abrogated by mutation of the codon for the predicted ATP-binding-site lysine residue. Southern blot analysis revealed that homologues of the genes encoding the two kinases are present in HRa and BCG, but not in . Immunoblot analysis of various cellular fractions of HRv revealed that PknF is a transmembrane protein and that PknG is predominantly a cytosolic enzyme. The present study should aid in elucidating the role of these protein kinases in the pathogenesis of mycobacteria.

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2001-08-01
2019-12-10
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References

  1. Armstrong, J. A. & Hart, P. D. ( 1975; ). Phagosome-lysosome interactions in cultured macrophages infected with virulent tubercle bacilli. Reversal of the usual non-fusion pattern and observation on bactericidal survival. J Exp Med 142, 1-16.[CrossRef]
    [Google Scholar]
  2. Av-Gay, Y. & Davies, J. ( 1997; ). Components of eukaryotic like signaling pathways in Mycobacterium tuberculosis. Microb Comp Genomics 2, 63-73.[CrossRef]
    [Google Scholar]
  3. Av-Gay, Y. & Everett, M. ( 2000; ). The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis. Trends Microbiol 8, 238-244.[CrossRef]
    [Google Scholar]
  4. Av-Gay, Y., Jamil, S. & Drews, S. J. ( 1999; ). Expression and characterization of the Mycobacterium tuberculosis serine/threonine protein kinase PknB. Infect Immun 67, 5676-5682.
    [Google Scholar]
  5. Chen, C. & Okayama, H. ( 1987; ). High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7, 2745-2752.
    [Google Scholar]
  6. Chow, K., Ng, D., Stokes, R. & Johnson, P. ( 1994; ). Protein tyrosine phosphorylation in Mycobacterium tuberculosis. FEMS Microbiol Lett 124, 203-208.[CrossRef]
    [Google Scholar]
  7. Cole, S. T., Brosch, R., Parkhill, J. & 39 other authors ( 1998; ). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.[CrossRef]
    [Google Scholar]
  8. Ferrari, G., Langen, H., Naito, M. & Pieters, J. ( 1999; ). A coat protein on phagosomes involved in the intracellular survival of mycobacteria. Cell 97, 435-447.[CrossRef]
    [Google Scholar]
  9. Galyov, E. E., Hakansson, S., Forsberg, A. & Wolf-Watz, H. ( 1993; ). A secreted protein kinase of Yersinia pseudotuberculosis is an indispensable virulence determinant. Nature 361, 730-732.[CrossRef]
    [Google Scholar]
  10. Hakansson, S., Gaylov, E. E., Rosqvist, R. & Wolf-Watz, H. ( 1996; ). The Yersinia YpkA Ser/Thr kinase is translocated and subsequently targeted to the inner surface of the HeLa cell plasma membrane. Mol Microbiol 20, 593-603.[CrossRef]
    [Google Scholar]
  11. Hanks, S. & Quinn, A. M. ( 1991; ). Use of degenerate oligonucleotide probes to identify clones that encode protein kinases. Methods Enzymol 200, 38-62.
    [Google Scholar]
  12. Hanlon, W. A., Inouye, M. & Inouye, S. ( 1997; ). Pkn9, a Ser/Thr protein kinase involved in development of Myxococcus xanthus. Mol Microbiol 23, 459-471.[CrossRef]
    [Google Scholar]
  13. Harlow, E. & Lane, E. (1988). Antibodies: a Laboratory Manual, 1st edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  14. Hubler, L., Leventhal, P. S. & Bertics, P. J. ( 1992; ). Alteration of the kinetic properties of the epidermal growth factor receptor tyrosine kinase by basic proteins. Biochem J 281, 107-114.
    [Google Scholar]
  15. Koul, A., Choidas, A., Treder, M., Tyagi, A. K., Drlica, K., Singh, Y. & Ullrich, A. ( 2000; ). Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis. J Bacteriol 182, 5425-5432.[CrossRef]
    [Google Scholar]
  16. Kunkel, T. A., Bebenek, K. & McClary, J. ( 1991; ). Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol 204, 125-139.
    [Google Scholar]
  17. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[CrossRef]
    [Google Scholar]
  18. Lowrie, D. B. ( 1983; ). How macrophages kill tubercle bacilli. J Med Microbiol 16, 1-12.[CrossRef]
    [Google Scholar]
  19. Matsumoto, A. S., Hong, S. K., Ishizuka, H., Horinouchi, S. & Beppu, T. ( 1994; ). Phosphorylation of the AfsR protein involved in secondary metabolism in Streptomyces species by a eukaryotic-type protein kinase. Gene 146, 47-56.[CrossRef]
    [Google Scholar]
  20. Motley, S. T. & Lory, S. ( 1999; ). Functional characterization of a serine/threonine protein kinase of Pseudomonas aeruginosa. Infect Immun 67, 5386-5394.
    [Google Scholar]
  21. Munoz-Dorado, J., Inouye, S. & Inouye, M. ( 1991; ). A gene encoding a protein serine/threonine kinase is required for the normal development of M. xanthus, a gram-negative bacterium. Cell 67, 995-1006.[CrossRef]
    [Google Scholar]
  22. Nadvornik, R., Vomastek, T., Janecek, J., Technikova, Z. & Branny, P. ( 1999; ). pKg2, a novel transmembrane protein Ser/Thr kinase of Streptomyces granaticolor. J Bacteriol 181, 15-23.
    [Google Scholar]
  23. Parrish, N. M., Dick, J. D. & Bishai, W. R. ( 1998; ). Mechanism of latency in Mycobacterium tuberculosis. Trends Microbiol 6, 107-112.[CrossRef]
    [Google Scholar]
  24. Peirs, P., Witt, L. D., Braibant, M., Huygen, K. & Content, J. ( 1997; ). A serine/threonine protein kinase from Mycobacterium tuberculosis. Eur J Biochem 244, 604-612.[CrossRef]
    [Google Scholar]
  25. Prabhakaran, K., Harris, E. B. & Randhawa, B. ( 2000; ). Regulation by protein kinase of phagocytosis of Mycobacterium leprae by macrophages. J Med Microbiol 49, 339-342.
    [Google Scholar]
  26. Reyrat, J. M., Berthet, F. X. & Gicouel, B. ( 1995; ). The urease locus of Mycobacterium tuberculosis and its utilization for the demonstration of allelic exchange in Mycobacterium bovis bacillus Calmette-Guerin. Proc Natl Acad Sci USA 92, 8768-8772.[CrossRef]
    [Google Scholar]
  27. Sanger, F., Nicklen, S. & Coulson, A. R. ( 1977; ). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467.[CrossRef]
    [Google Scholar]
  28. Stock, J. B., Ninfa, A. J. & Stock, A. M. ( 1989; ). Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev 53, 450-490.
    [Google Scholar]
  29. Sturgill-Koszycki, S., Schlesinger, P. H., Chakraborty, P. & 7 other authors ( 1994; ). Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 263, 678–681.[CrossRef]
    [Google Scholar]
  30. Vincent, C., Doublet, P., Grangeasse, C., Vaganay, E., Cozzone, A. J. & Duclos, B. ( 1999; ). Cells of Escherichia coli contain a protein-tyrosine kinase, Wzc, and a phosphotyrosine-protein phosphatase, Wzb. J Bacteriol 181, 3472-3477.
    [Google Scholar]
  31. Yang, X., Kang, C. M., Brody, M. S. & Price, C. W. ( 1996; ). Opposing pairs of serine protein kinases and phosphatases transmit signals of environmental stress to activate a bacterial transcription factor. Genes Dev 10, 2265-2275.[CrossRef]
    [Google Scholar]
  32. Yarden, Y. & Ullrich, A. ( 1988; ). Growth factor receptor tyrosine kinases. Annu Rev Biochem 57, 443-478.[CrossRef]
    [Google Scholar]
  33. Zhang, C. ( 1996; ). Bacterial signalling involving eukaryotic-type protein kinases. Mol Microbiol 20, 9-15.[CrossRef]
    [Google Scholar]
  34. Zwick, E., Wallasch, C., Daub, H. & Ullrich, A. ( 1999; ). Distinct calcium-dependent pathways of epidermal growth factor receptor transactivation and PYK2 tyrosine phosphorylation in PC12 cells. J Biol Chem 274, 20989-20996.[CrossRef]
    [Google Scholar]
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