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Abstract

Two-component signal transduction systems (TCSs) play fundamental roles in bacterial survival and pathogenesis and have been proposed as targets for the development of novel classes of antibiotics. A new coupled assay was developed and applied to analyse the kinetic mechanisms of three new kinds of inhibitors of TCS function. The assay exploits the biochemical properties of the cognate HpkA–DrrA histidine kinase–response regulator pair from and allows multiple turnovers of HpkA, linear formation of phosphorylated DrrA, and Michaelis–Menten analysis of inhibitors. The assay was validated in several ways, including confirmation of competitive inhibition by adenosine 5′-,-imidotriphosphate (AMP-PNP). The coupled assay, autophosphorylation and chemical cross-linking were used to determine the mechanisms by which several compounds inhibit TCS function. A cyanoacetoacetamide showed non-competitive inhibition with respect to ATP concentration in the coupled assay. The cyanoacetoacetamide also inhibited autophosphorylation of histidine kinases from other bacteria, indicating that the coupled assay could detect general inhibitors of histidine kinase function. Inhibition of HpkA autophosphorylation by this compound was probably caused by aggregation of HpkA, consistent with a previous model for other hydrophobic compounds. In contrast, ethodin was a potent inhibitor of the combined assay, did not inhibit HpkA autophosphorylation, but still led to aggregation of HpkA. These data suggest that ethodin bound to the HpkA kinase and inhibited transfer of the phosphoryl group to DrrA. A peptide corresponding to the phosphorylation site of DrrA appeared to inhibit TCS function by a mechanism similar to that of ethodin, except that autophosphorylation was inhibited at high peptide concentrations. The latter mechanism of inhibition of TCS function is unusual and its analysis demonstrates the utility of these approaches to the kinetic analyses of additional new classes of inhibitors of TCS function.

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2004-04-01
2019-10-15
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References

  1. Alloing, G., Granadel, C., Morrison, D. A. & Claverys, J. P. ( 1996; ). Competence pheromone, oligopeptide permease, and induction of competence in Streptococcus pneumoniae. Mol Microbiol 21, 471–478.[CrossRef]
    [Google Scholar]
  2. Barrett, J. F., Goldschmidt, R. M., Lawrence, L. E. & 19 other authors ( 1998; ). Antibacterial agents that inhibit two-component signal transduction systems. Proc Natl Acad Sci U S A 95, 5317–5322.[CrossRef]
    [Google Scholar]
  3. Beier, D. & Frank, R. ( 2000; ). Molecular characterization of two-component systems of Helicobacter pylori. J Bacteriol 182, 2068–2076.[CrossRef]
    [Google Scholar]
  4. Borkovich, K. A., Kaplan, N., Hess, J. F. & Simon, M. I. ( 1989; ). Transmembrane signal transduction in bacterial chemotaxis involves ligand-dependent activation of phosphate group transfer. Proc Natl Acad Sci U S A 86, 1208–1212.[CrossRef]
    [Google Scholar]
  5. Bornhorst, J. A. & Falke, J. J. ( 2000; ). Attractant regulation of the aspartate receptor-kinase complex: limited cooperative interations between receptors and effects of the receptor modification state. Biochemistry 39, 9486–9493.[CrossRef]
    [Google Scholar]
  6. Copeland, R. A. ( 2000; ). Enzymesa Practical Introduction to Structure, Mechanism, and Data Analysis. New York: Wiley-VCH.
  7. Fabret, C. & Hoch, J. A. ( 1998; ). A two-component signal transduction system essential for growth of Bacillus subtilis: implications for anti-infective therapy. J Bacteriol 180, 6375–6383.
    [Google Scholar]
  8. Goudreau, P. N., Lee, P. J. & Stock, A. M. ( 1998; ). Stabilization of the phospho-aspartyl residue in a two-component signal transduction system in Thermotoga maritima. Biochemistry 37, 14575–14584.[CrossRef]
    [Google Scholar]
  9. Groisman, E. A. ( 2001; ). The pleiotropic two-component regulatory system PhoP-PhoQ. J Bacteriol 183, 1835–1842.[CrossRef]
    [Google Scholar]
  10. Hilliard, J. J., Goldschmidt, R. M., Licata, L., Baum, E. Z. & Bush, K. ( 1999; ). Multiple mechanisms of action for inhibitors of histidine protein kinases from bacterial two-component systems. Antimicrob Agents Chemother 43, 1693–1699.
    [Google Scholar]
  11. Hoch, J. A. & Varughese, K. I. ( 2001; ). Keeping signals straight in phosphorelay signal transduction. J Bacteriol 183, 4941–4949.[CrossRef]
    [Google Scholar]
  12. Hubbard, J., Burnham, M. K. & Throup, J. P. ( 2003; ). Pathogenicity and histidine kinases: approaches toward the development of a new generation of antibiotics. In Histidine Kinases in Signal Transduction. Edited by M. Inouye & R. Dutta. San Diego, CA: Academic Press.
  13. Inouye, M. & Dutta, R. (editors) ( 2003; ). Histidine Kinases in Signal Transduction. San Diego, CA: Academic Press.
  14. Kallipolitis, B. H. & Ingmer, H. ( 2001; ). Listeria monocytogenes response regulators important for stress tolerance and pathogenesis. FEMS Microbiol Lett 204, 111–115.[CrossRef]
    [Google Scholar]
  15. Lange, R., Wagner, C., de Saizieu, A. & 7 other authors ( 1999; ). Domain organization and molecular characterization of 13 two-component systems identified by genome sequencing of Streptococcus pneumoniae. Gene 237, 223–234.[CrossRef]
    [Google Scholar]
  16. Lee, P. J. & Stock, A. M. ( 1996; ). Characterization of the genes and proteins of a two-component system from the hyperthermophilic bacterium Thermotoga maritima. J Bacteriol 178, 5579–5585.
    [Google Scholar]
  17. Lyon, G. J., Mayville, P., Muir, T. W. & Novick, R. P. ( 2000; ). Rational design of a global inhibitor of the virulence response in Staphylococcus aureus, based in part on localization of the site of inhibition to the receptor-histidine kinase, AgrC. Proc Natl Acad Sci U S A 97, 13330–13335.[CrossRef]
    [Google Scholar]
  18. Lyon, G. J., Wright, J. S., Muir, T. W. & Novick, R. P. ( 2002; ). Key determinants of receptor activation in the agr autoinducing peptides of Staphylococcus aureus. Biochemistry 41, 10095–10104.[CrossRef]
    [Google Scholar]
  19. Martin, P. K., Li, T., Sun, D., Biek, D. P. & Schmid, M. B. ( 1999; ). Role in cell permeability of an essential two-component system in Staphylococcus aureus. J Bacteriol 181, 3666–3673.
    [Google Scholar]
  20. Matsushita, M. & Janda, K. D. ( 2002; ). Histidine kinases as targets for new antimicrobial agents. Bioorg Med Chem 10, 855–867.[CrossRef]
    [Google Scholar]
  21. Ninfa, E. G., Stock, A., Mowbray, S. & Stock, J. ( 1991; ). Reconstitution of the bacterial chemotaxis signal transduction system from purified components. J Biol Chem 266, 9764–9770.
    [Google Scholar]
  22. Novak, R. & Tuomanen, E. ( 2003; ). Initiation of bacterial killing by two-component sensing of a “Death Peptide”: development of antiobiotic tolerance in Streptococcus pneumoniae. In Histidine Kinases in Signal Transduction, pp. 365–375. Edited by M. Inouye & R. Dutta. San Diego, CA: Academic Press.
  23. Novak, R., Henriques, B., Charpentier, E., Normark, S. & Tuomanen, E. ( 1999; ). Emergence of vancomycin tolerance in Streptococcus pneumoniae. Nature 399, 590–593.[CrossRef]
    [Google Scholar]
  24. Robertson, G. T., Zhao, J., Desai, B. V., Coleman, W. H., Nicas, T. I., Gilmour, R., Grinius, L., Morrison, D. A. & Winkler, M. E. ( 2002; ). Vancomycin tolerance induced by erythromycin but not by loss of vncRS, vex3, or pep27 function in Streptococcus pneumoniae. J Bacteriol 184, 6987–7000.[CrossRef]
    [Google Scholar]
  25. Rudolph, M. I., Cabanillas, A., Gomez, P., Garcia, M. A. & Villan, L. ( 1997; ). On the mechanism of action of ethodin in inducing myometrium contractions. Gen Pharmacol 28, 381–385.[CrossRef]
    [Google Scholar]
  26. Sachsenmaier, C. & Schachtele, C. ( 2002; ). Integrated technology platform protein kinases for drug development in oncology. Biotechniques Oct, Suppl., 101–106.
    [Google Scholar]
  27. Stephenson, K. & Hoch, J. A. ( 2002; ). Virulence- and antibiotic resistance-associated two-component signal transduction systems of Gram-positive pathogenic bacteria as targets for antimicrobial therapy. Pharmacol Ther 93, 293–305.[CrossRef]
    [Google Scholar]
  28. Stephenson, K., Yamaguchi, Y. & Hoch, J. A. ( 2000; ). The mechanism of action of inhibitors of bacterial two-component signal transduction systems. J Biol Chem 275, 38900–38904.[CrossRef]
    [Google Scholar]
  29. Stock, A. M., Robinson, V. L. & Goudreau, P. N. ( 2000; ). Two-component signal transduction. Annu Rev Biochem 69, 183–215.[CrossRef]
    [Google Scholar]
  30. Throup, J. P., Koretke, K. K., Bryant, A. P. & 9 other authors ( 2000; ). A genomic analysis of two-component signal transduction in Streptococcus pneumoniae. Mol Microbiol 35, 566–576.
    [Google Scholar]
  31. Walsh, C. ( 2003; ). AntibioticsActions, Origins, Resistance. Washington, DC: American Society for Microbiology.
  32. West, A. H. & Stock, A. M. ( 2001; ). Histidine kinases and response regulator proteins in two-component signaling systems. Trends Biochem Sci 26, 369–376.[CrossRef]
    [Google Scholar]
  33. Wright, G. D., Holman, T. R. & Walsh, C. T. ( 1993; ). Purification and characterization of VanR and the cytosolic domain of VanS: a two-component regulatory system required for vancomycin resistance in Enterococcus faecium BM4147. Biochemistry 32, 5057–5063.[CrossRef]
    [Google Scholar]
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vol. , part 4, pp. 885 - 896

Supplementary figuresS1–S4. See below for legends.

Purification of His-HpkA77 and His-DrrA expressed in . Proteins were expressed, purified by nickel-Sepharose chromatography, and analysed by 4–20 % SDS-PAGE. Gels were stained with Coomassie blue. Lane 1, purified His-HpkA77; lane 2, wide-range protein molecular mass standards (kDa); lane 3, purified His-DrrA; lane 4, multicolour protein molecular mass standards (kDa).

Autophosphorylation of His-HpkA77. Initial velocities of autophosphorylation were determined as described in Methods. The data are means from three independent experiments. The fit was obtained using a of 20 µM and a of 0.044 nM min (see Table 1 in main paper).

Effect of increasing His-HpkA77 concentrations on DrrA-P product formation in the HpkA–DrrA combined assay. The combined assay was performed as described in Methods. Reactions contained His-HpkA77 concentrations varying from 20 to 80 nM in the presence of 4 µM DrrA and 200 µM ATP. Data were analysed by linear regression.

[ P]DrrA formation as a function of ATP or DrrA concentration in the HpkA–DrrA combined assay. (a) Initial velocity versus ATP concentration. Rates were determined in reactions containing 20 nM His-HpkA77 and 4 µM His-DrrA and analysed by SDS-PAGE (see Methods). (b) Initial velocity versus His-DrrA concentration. Rates were determined in reactions containing 20 nM His-HpkA77 and 80 µM ATP and analysed by the filter format (see Methods). The data are means of at least three independent experiments. Curves were fitted to the Michaelis–Menten equation; apparent and values are listed in Table 1 in the main paper.



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