Disruption of the serine/threonine protein kinase H affects phthiocerol dimycocerosates synthesis in Free

Abstract

possesses a complex cell wall that is unique and essential for interaction of the pathogen with its human host. Emerging evidence suggests that the biosynthesis of complex cell-wall lipids is mediated by serine/threonine protein kinases (STPKs). Herein, we show, using radiolabelling, MS and immunostaining analyses, that targeted deletion of one of the STPKs, attenuates the production of phthiocerol dimycocerosates (PDIMs), a major virulence lipid. Comparative protein expression analysis revealed that proteins in the PDIM biosynthetic pathway are differentially expressed in a deleted strain. Furthermore, we analysed the composition of the major lipoglycans, lipoarabinomannan (LAM) and lipomannan (LM), and found a twofold higher LAM/LM ratio in the mutant strain. Thus, we provide experimental evidence that PknH contributes to the production and synthesis of cell-wall components.

Funding
This study was supported by the:
  • Canadian Institute of Health Research (CIHR) (Award MOP-106622 )
  • Medical Research Council
  • Wellcome Trust
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.062067-0
2013-04-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/159/4/726.html?itemId=/content/journal/micro/10.1099/mic.0.062067-0&mimeType=html&fmt=ahah

References

  1. Alibaud L., Rombouts Y., Trivelli X., Burguière A., Cirillo S. L. G., Cirillo J. D., Dubremetz J.-F., Guérardel Y., Lutfalla G., Kremer L. ( 2011). A Mycobacterium marinum TesA mutant defective for major cell wall-associated lipids is highly attenuated in Dictyostelium discoideum and zebrafish embryos. Mol Microbiol 80:919–934 [View Article] [PubMed]
    [Google Scholar]
  2. Astarie-Dequeker C., Le Guyader L., Malaga W., Seaphanh F.-K., Chalut C., Lopez A., Guilhot C. ( 2009). Phthiocerol dimycocerosates of M. tuberculosis participate in macrophage invasion by inducing changes in the organization of plasma membrane lipids. PLoS Pathog 5:e1000289 [View Article] [PubMed]
    [Google Scholar]
  3. Av-Gay Y., Everett M. ( 2000). The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis. . Trends Microbiol 8:238–244 [View Article] [PubMed]
    [Google Scholar]
  4. Azad A. K., Sirakova T. D., Rogers L. M., Kolattukudy P. E. ( 1996). Targeted replacement of the mycocerosic acid synthase gene in Mycobacterium bovis BCG produces a mutant that lacks mycosides. Proc Natl Acad Sci U S A 93:4787–4792 [View Article] [PubMed]
    [Google Scholar]
  5. Azad A. K., Sirakova T. D., Fernandes N. D., Kolattukudy P. E. ( 1997). Gene knockout reveals a novel gene cluster for the synthesis of a class of cell wall lipids unique to pathogenic mycobacteria. J Biol Chem 272:16741–16745 [View Article] [PubMed]
    [Google Scholar]
  6. Bach H., Mazor Y., Shaky S., Shoham-Lev A., Berdichevsky Y., Gutnick D. L., Benhar I. ( 2001). Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies. J Mol Biol 312:79–93 [View Article] [PubMed]
    [Google Scholar]
  7. Besra G. S. ( 1998). Preparation of cell-wall fractions from mycobacteria. Mycobacteria Protocols, Methods in Molecular Biology91–108 Parish T., Stoker N. G. Totowa, NJ, USA: Humana Press Inc; [View Article]
    [Google Scholar]
  8. Bligh E. G., Dyer W. J. ( 1959). A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917 [View Article] [PubMed]
    [Google Scholar]
  9. Camacho L. R., Ensergueix D., Perez E., Gicquel B., Guilhot C. ( 1999). Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis. Mol Microbiol 34:257–267 [View Article] [PubMed]
    [Google Scholar]
  10. Camacho L. R., Constant P., Raynaud C., Laneelle M. A., Triccas J. A., Gicquel B., Daffe M., Guilhot C. ( 2001). Analysis of the phthiocerol dimycocerosate locus of Mycobacterium tuberculosis. Evidence that this lipid is involved in the cell wall permeability barrier. J Biol Chem 276:19845–19854 [View Article] [PubMed]
    [Google Scholar]
  11. Chao J., Wong D., Zheng X., Poirier V., Bach H., Hmama Z., Av-Gay Y. ( 2010a). Protein kinase and phosphatase signaling in Mycobacterium tuberculosis physiology and pathogenesis. Biochim Biophys Acta 1804:620–627 [View Article] [PubMed]
    [Google Scholar]
  12. Chao J. D., Papavinasasundaram K. G., Zheng X., Chávez-Steenbock A., Wang X., Lee G. Q., Av-Gay Y. ( 2010b). Convergence of Ser/Thr and two-component signaling to coordinate expression of the dormancy regulon in Mycobacterium tuberculosis. . J Biol Chem 285:29239–29246 [View Article] [PubMed]
    [Google Scholar]
  13. Cole S. T., Brosch R., Parkhill J., Garnier T., Churcher C., Harris D., Gordon S. V., Eiglmeier K., Gas S. & other authors ( 1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544 [View Article] [PubMed]
    [Google Scholar]
  14. Constant P., Perez E., Malaga W., Lanéelle M.-A., Saurel O., Daffé M., Guilhot C. ( 2002). Role of the pks15/1 gene in the biosynthesis of phenolglycolipids in the Mycobacterium tuberculosis complex. Evidence that all strains synthesize glycosylated p-hydroxybenzoic methyl esters and that strains devoid of phenolglycolipids harbor a frameshift mutation in the pks15/1 gene. J Biol Chem 277:38148–38158 [View Article] [PubMed]
    [Google Scholar]
  15. Cox J. S., Chen B., McNeil M., Jacobs W. R. Jr ( 1999). Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice. Nature 402:79–83 [PubMed] [CrossRef]
    [Google Scholar]
  16. Daffé M., Laneelle M. A. ( 1988). Distribution of phthiocerol diester, phenolic mycosides and related compounds in mycobacteria. J Gen Microbiol 134:2049–2055 [PubMed]
    [Google Scholar]
  17. Dao D. N., Sweeney K., Hsu T., Gurcha S. S., Nascimento I. P., Roshevsky D., Besra G. S., Chan J., Porcelli S. A., Jacobs W. R. ( 2008). Mycolic acid modification by the mmaA4 gene of M. tuberculosis modulates IL-12 production. PLoS Pathog 4:e1000081 [View Article] [PubMed]
    [Google Scholar]
  18. Dobson G., Minnikin D. E., Minnikin S. E., Parlett M., Goodfellow M., Ridell M., Magnusson M. ( 1985). Systematics analysis of complex mycobacterial lipids. Chemical Methods in Bacterial systematics237–265 Goodfellow M., Minnikin D. E. London: Academic Press;
    [Google Scholar]
  19. Domenech P., Reed M. B. ( 2009). Rapid and spontaneous loss of phthiocerol dimycocerosate (PDIM) from Mycobacterium tuberculosis grown in vitro: implications for virulence studies. Microbiology 155:3532–3543 [View Article] [PubMed]
    [Google Scholar]
  20. Gupta M., Sajid A., Arora G., Tandon V., Singh Y. ( 2009). Forkhead-associated domain-containing protein Rv0019c and polyketide-associated protein PapA5, from substrates of serine/threonine protein kinase PknB to interacting proteins of Mycobacterium tuberculosis. . J Biol Chem 284:34723–34734 [View Article] [PubMed]
    [Google Scholar]
  21. Ioerger T. R., Feng Y., Ganesula K., Chen X., Dobos K. M., Fortune S., Jacobs W. R. Jr, Mizrahi V., Parish T. & other authors ( 2010). Variation among genome sequences of H37Rv strains of Mycobacterium tuberculosis from multiple laboratories. J Bacteriol 192:3645–3653 [View Article] [PubMed]
    [Google Scholar]
  22. Jain M., Cox J. S. ( 2005). Interaction between polyketide synthase and transporter suggests coupled synthesis and export of virulence lipid in M. tuberculosis. . PLoS Pathog 1:e2 [View Article] [PubMed]
    [Google Scholar]
  23. Jain M., Petzold C. J., Schelle M. W., Leavell M. D., Mougous J. D., Bertozzi C. R., Leary J. A., Cox J. S. ( 2007). Lipidomics reveals control of Mycobacterium tuberculosis virulence lipids via metabolic coupling. Proc Natl Acad Sci U S A 104:5133–5138 [View Article] [PubMed]
    [Google Scholar]
  24. Kirksey M. A., Tischler A. D., Siméone R., Hisert K. B., Uplekar S., Guilhot C., McKinney J. D. ( 2011). Spontaneous phthiocerol dimycocerosate-deficient variants of Mycobacterium tuberculosis are susceptible to gamma interferon-mediated immunity. Infect Immun 79:2829–2838 [View Article] [PubMed]
    [Google Scholar]
  25. Kremer L., Besra G. S. ( 2005). A waxy tale by Mycobacterium tuberculosis . Tuberculosis and the tubercle bacillus287–305 Cole S. T., Davies Eisenach K., McMurray D. N., Jacobs Jr W. R. Washington: American Society for Microbiology;
    [Google Scholar]
  26. Kremer L., Dover L. G., Carrère S., Nampoothiri K. M., Lesjean S., Brown A. K., Brennan P. J., Minnikin D. E., Locht C., Besra G. S. ( 2002). Mycolic acid biosynthesis and enzymic characterization of the β-ketoacyl-ACP synthase A-condensing enzyme from Mycobacterium tuberculosis. . Biochem J 364:423–430 [View Article] [PubMed]
    [Google Scholar]
  27. Minnikin D. E. ( 1982). Lipids: complex lipids their chemistry, biosynthesis and roles. The Biology of Mycobacteria95–184 Ratledge C., Stanford J. London, UK: Academic Press Ltd;
    [Google Scholar]
  28. Minnikin D. E., Kremer L., Dover L. G., Besra G. S. ( 2002). The methyl-branched fortifications of Mycobacterium tuberculosis. . Chem Biol 9:545–553 [View Article] [PubMed]
    [Google Scholar]
  29. Molle V., Kremer L., Girard-Blanc C., Besra G. S., Cozzone A. J., Prost J.-F. ( 2003). An FHA phosphoprotein recognition domain mediates protein EmbR phosphorylation by PknH, a Ser/Thr protein kinase from Mycobacterium tuberculosis. . Biochemistry 42:15300–15309 [View Article] [PubMed]
    [Google Scholar]
  30. Molle V., Brown A. K., Besra G. S., Cozzone A. J., Kremer L. ( 2006). The condensing activities of the Mycobacterium tuberculosis type II fatty acid synthase are differentially regulated by phosphorylation. J Biol Chem 281:30094–30103 [View Article] [PubMed]
    [Google Scholar]
  31. Onwueme K. C., Ferreras J. A., Buglino J., Lima C. D., Quadri L. E. N. ( 2004). Mycobacterial polyketide-associated proteins are acyltransferases: proof of principle with Mycobacterium tuberculosis PapA5. Proc Natl Acad Sci U S A 101:4608–4613 [View Article] [PubMed]
    [Google Scholar]
  32. Onwueme K. C., Vos C. J., Zurita J., Ferreras J. A., Quadri L. E. N. ( 2005). The dimycocerosate ester polyketide virulence factors of mycobacteria. Prog Lipid Res 44:259–302 [View Article] [PubMed]
    [Google Scholar]
  33. Papavinasasundaram K. G., Chan B., Chung J.-H., Colston M. J., Davis E. O., Av-Gay Y. ( 2005). Deletion of the Mycobacterium tuberculosis pknH gene confers a higher bacillary load during the chronic phase of infection in BALB/c mice. J Bacteriol 187:5751–5760 [View Article] [PubMed]
    [Google Scholar]
  34. Pérez J., Garcia R., Bach H., de Waard J. H., Jacobs W. R. Jr, Av-Gay Y., Bubis J., Takiff H. E. ( 2006). Mycobacterium tuberculosis transporter MmpL7 is a potential substrate for kinase PknD. Biochem Biophys Res Commun 348:6–12 [View Article] [PubMed]
    [Google Scholar]
  35. Rao A., Ranganathan A. ( 2004). Interaction studies on proteins encoded by the phthiocerol dimycocerosate locus of Mycobacterium tuberculosis. . Mol Genet Genomics 272:571–579 [View Article] [PubMed]
    [Google Scholar]
  36. Reed M. B., Domenech P., Manca C., Su H., Barczak A. K., Kreiswirth B. N., Kaplan G., Barry C. E. III ( 2004). A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response. Nature 431:84–87 [View Article] [PubMed]
    [Google Scholar]
  37. Rousseau C., Winter N., Pivert E., Bordat Y., Neyrolles O., Avé P., Huerre M., Gicquel B., Jackson M. ( 2004). Production of phthiocerol dimycocerosates protects Mycobacterium tuberculosis from the cidal activity of reactive nitrogen intermediates produced by macrophages and modulates the early immune response to infection. Cell Microbiol 6:277–287 [View Article] [PubMed]
    [Google Scholar]
  38. Sartain M. J., Dick D. L., Rithner C. D., Crick D. C., Belisle J. T. ( 2011). Lipidomic analyses of Mycobacterium tuberculosis based on accurate mass measurements and the novel “Mtb LipidDB”. J Lipid Res 52:861–872 [View Article] [PubMed]
    [Google Scholar]
  39. Schaeffer M. L., Agnihotri G., Volker C., Kallender H., Brennan P. J., Lonsdale J. T. ( 2001). Purification and biochemical characterization of the Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein synthases KasA and KasB. J Biol Chem 276:47029–47037 [View Article] [PubMed]
    [Google Scholar]
  40. Sendide K., Deghmane A.-E., Reyrat J.-M., Talal A., Hmama Z. ( 2004). Mycobacterium bovis BCG urease attenuates major histocompatibility complex class II trafficking to the macrophage cell surface. Infect Immun 72:4200–4209 [View Article] [PubMed]
    [Google Scholar]
  41. Sharma K., Gupta M., Pathak M., Gupta N., Koul A., Sarangi S., Baweja R., Singh Y. ( 2006). Transcriptional control of the mycobacterial embCAB operon by PknH through a regulatory protein, EmbR, in vivo. J Bacteriol 188:2936–2944 [View Article] [PubMed]
    [Google Scholar]
  42. Torrelles J. B., Schlesinger L. S. ( 2010). Diversity in Mycobacterium tuberculosis mannosylated cell wall determinants impacts adaptation to the host. Tuberculosis (Edinb) 90:84–93 [View Article] [PubMed]
    [Google Scholar]
  43. Trivedi O. A., Arora P., Sridharan V., Tickoo R., Mohanty D., Gokhale R. S. ( 2004). Enzymic activation and transfer of fatty acids as acyl-adenylates in mycobacteria. Nature 428:441–445 [View Article] [PubMed]
    [Google Scholar]
  44. Trivedi O. A., Arora P., Vats A., Ansari M. Z., Tickoo R., Sridharan V., Mohanty D., Gokhale R. S. ( 2005). Dissecting the mechanism and assembly of a complex virulence mycobacterial lipid. Mol Cell 17:631–643 [View Article] [PubMed]
    [Google Scholar]
  45. Veyron-Churlet R., Molle V., Taylor R. C., Brown A. K., Besra G. S., Zanella-Cléon I., Fütterer K., Kremer L. ( 2009). The Mycobacterium tuberculosis β-ketoacyl-acyl carrier protein synthase III activity is inhibited by phosphorylation on a single threonine residue. J Biol Chem 284:6414–6424 [View Article] [PubMed]
    [Google Scholar]
  46. Yu J., Tran V., Li M., Huang X., Niu C., Wang D., Zhu J., Wang J., Gao Q., Liu J. ( 2012). Both phthiocerol dimycocerosates and phenolic glycolipids are required for virulence of Mycobacterium marinum. . Infect Immun 80:1381–1389 [View Article] [PubMed]
    [Google Scholar]
  47. Zheng X., Papavinasasundaram K. G., Av-Gay Y. ( 2007). Novel substrates of Mycobacterium tuberculosis PknH Ser/Thr kinase. Biochem Biophys Res Commun 355:162–168 [View Article] [PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.062067-0
Loading
/content/journal/micro/10.1099/mic.0.062067-0
Loading

Data & Media loading...

Most cited Most Cited RSS feed