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Phosphorylation of KasB Regulates Virulence and Acid-Fastness in


Acid-fast staining has been used since 1882 as the hallmark diagnostic test for detecting Mycobacterium tuberculosis, the causative agent of tuberculosis. It has been attributed to the presence of a waxy cell envelope, and primarily to its key components, mycolic acids. Here, we report a new mechanism of regulation in which phosphorylation of KasB, involved in the completion of full-length mycolic acids, leads to shortened mycolic acids and loss of acid-fast staining. Moreover, a M. tuberculosis mutant strain mimicking constitutive phosphorylation of KasB is severely attenuated for growth in both immunocompetent and immunosuppressed mice and fails to cause mortality and pathophysiological symptoms. These results emphasize the critical role of kinase-dependent phosphorylation in the pathogenesis of M. tuberculosis by controlling the mycolic acid chain length. Our study demonstrates the importance of a regulatory mechanism governing acid-fastness and virulence of M. tuberculosis.


Vyšlo v časopise: Phosphorylation of KasB Regulates Virulence and Acid-Fastness in. PLoS Pathog 10(5): e32767. doi:10.1371/journal.ppat.1004115
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1004115

Souhrn

Acid-fast staining has been used since 1882 as the hallmark diagnostic test for detecting Mycobacterium tuberculosis, the causative agent of tuberculosis. It has been attributed to the presence of a waxy cell envelope, and primarily to its key components, mycolic acids. Here, we report a new mechanism of regulation in which phosphorylation of KasB, involved in the completion of full-length mycolic acids, leads to shortened mycolic acids and loss of acid-fast staining. Moreover, a M. tuberculosis mutant strain mimicking constitutive phosphorylation of KasB is severely attenuated for growth in both immunocompetent and immunosuppressed mice and fails to cause mortality and pathophysiological symptoms. These results emphasize the critical role of kinase-dependent phosphorylation in the pathogenesis of M. tuberculosis by controlling the mycolic acid chain length. Our study demonstrates the importance of a regulatory mechanism governing acid-fastness and virulence of M. tuberculosis.


Zdroje

1. ZhouP, LongQ, ZhouY, WangH, XieJ (2012) Mycobacterium tuberculosis two-component systems and implications in novel vaccines and drugs. Crit Rev Eukaryot Gene Expr 22: 37–52.

2. ColeST, BroschR, ParkhillJ, GarnierT, ChurcherC, et al. (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393: 537–544.

3. Av-GayY, EverettM (2000) The eukaryotic-like Ser/Thr protein kinases of Mycobacterium tuberculosis. Trends Microbiol 8: 238–244.

4. PrisicS, DankwaS, SchwartzD, ChouMF, LocasaleJW, et al. (2010) Extensive phosphorylation with overlapping specificity by Mycobacterium tuberculosis serine/threonine protein kinases. Proc Natl Acad Sci U S A 107: 7521–7526.

5. ChaoJ, WongD, ZhengX, PoirierV, BachH, et al. (2010) Protein kinase and phosphatase signaling in Mycobacterium tuberculosis physiology and pathogenesis. Biochim Biophys Acta 1804: 620–627.

6. MolleV, KremerL (2010) Division and cell envelope regulation by Ser/Thr phosphorylation: Mycobacterium shows the way. Mol Microbiol 75: 1064–1077.

7. WehenkelA, BellinzoniM, GranaM, DuranR, VillarinoA, et al. (2008) Mycobacterial Ser/Thr protein kinases and phosphatases: physiological roles and therapeutic potential. Biochim Biophys Acta 1784: 193–202.

8. MolleV, BrownAK, BesraGS, CozzoneAJ, KremerL (2006) The condensing activities of the Mycobacterium tuberculosis type II fatty acid synthase are differentially regulated by phosphorylation. J Biol Chem 281: 30094–30103.

9. Veyron-ChurletR, Zanella-CleonI, Cohen-GonsaudM, MolleV, KremerL (2010) Phosphorylation of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein reductase MabA regulates mycolic acid biosynthesis. J Biol Chem 285: 12714–12725.

10. MolleV, GultenG, VilchezeC, Veyron-ChurletR, Zanella-CleonI, et al. (2010) Phosphorylation of InhA inhibits mycolic acid biosynthesis and growth of Mycobacterium tuberculosis. Mol Microbiol 78: 1591–1605.

11. KhanS, NagarajanSN, ParikhA, SamantarayS, SinghA, et al. (2010) Phosphorylation of enoyl-acyl carrier protein reductase InhA impacts mycobacterial growth and survival. J Biol Chem 285: 37860–37871.

12. SlamaN, LeibaJ, EynardN, DaffeM, KremerL, et al. (2011) Negative regulation by Ser/Thr phosphorylation of HadAB and HadBC dehydratases from Mycobacterium tuberculosis type II fatty acid synthase system. Biochem Biophys Res Commun 412: 401–406.

13. LacaveC, LaneelleMA, DaffeM, MontrozierH, LaneelleG (1989) Mycolic acid metabolic filiation and location in Mycobacterium aurum and Mycobacterium phlei. Eur J Biochem 181: 459–466.

14. BhattA, FujiwaraN, BhattK, GurchaSS, KremerL, et al. (2007) Deletion of kasB in Mycobacterium tuberculosis causes loss of acid-fastness and subclinical latent tuberculosis in immunocompetent mice. Proc Natl Acad Sci U S A 104: 5157–5162.

15. Veyron-ChurletR, MolleV, TaylorRC, BrownAK, BesraGS, et al. (2009) The Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III activity is inhibited by phosphorylation on a single threonine residue. J Biol Chem 284: 6414–6424.

16. CanovaMJ, KremerL, MolleV (2009) The Mycobacterium tuberculosis GroEL1 chaperone is a substrate of Ser/Thr protein kinases. J Bacteriol 191: 2876–2883.

17. CorralesRM, MolleV, LeibaJ, MoureyL, de ChastellierC, et al. (2012) Phosphorylation of mycobacterial PcaA inhibits mycolic acid cyclopropanation: consequences for intracellular survival and for phagosome maturation block. J Biol Chem 287: 26187–26199.

18. MolleV, LeibaJ, Zanella-CleonI, BecchiM, KremerL (2010) An improved method to unravel phosphoacceptors in Ser/Thr protein kinase-phosphorylated substrates. Proteomics 10: 3910–3915.

19. SridharanS, WangL, BrownAK, DoverLG, KremerL, et al. (2007) X-Ray Crystal Structure of Mycobacterium tuberculosis beta-Ketoacyl Acyl Carrier Protein Synthase II (mtKasB). J Mol Biol 366: 469–480.

20. LucknerSR, MachuttaCA, TongePJ, KiskerC (2009) Crystal structures of Mycobacterium tuberculosis KasA show mode of action within cell wall biosynthesis and its inhibition by thiolactomycin. Structure 17: 1004–1013.

21. KangCM, NyayapathyS, LeeJY, SuhJW, HussonRN (2008) Wag31, a homologue of the cell division protein DivIVA, regulates growth, morphology and polar cell wall synthesis in mycobacteria. Microbiology 154: 725–735.

22. CottinV, Van LindenA, RichesDW (1999) Phosphorylation of tumor necrosis factor receptor CD120a (p55) by p42(mapk/erk2) induces changes in its subcellular localization. J Biol Chem 274: 32975–32987.

23. VilchezeC, WangF, AraiM, HazbonMH, ColangeliR, et al. (2006) Transfer of a point mutation in Mycobacterium tuberculosis inhA resolves the target of isoniazid. Nat Med 12: 1027–1029.

24. GaoLY, LavalF, LawsonEH, GrogerRK, WoodruffA, et al. (2003) Requirement for kasB in Mycobacterium mycolic acid biosynthesis, cell wall impermeability and intracellular survival: implications for therapy. Mol Microbiol 49: 1547–1563.

25. GlickmanMS, CahillSM, JacobsWRJr (2001) The Mycobacterium tuberculosis cmaA2 gene encodes a mycolic acid trans-cyclopropane synthetase. J Biol Chem 276: 2228–2233.

26. Veyron-ChurletR, BigotS, GuerriniO, VerdouxS, MalagaW, et al. (2005) The biosynthesis of mycolic acids in Mycobacterium tuberculosis relies on multiple specialized elongation complexes interconnected by specific protein-protein interactions. J Mol Biol 353: 847–858.

27. Veyron-ChurletR, GuerriniO, MoureyL, DaffeM, ZerbibD (2004) Protein-protein interactions within the Fatty Acid Synthase-II system of Mycobacterium tuberculosis are essential for mycobacterial viability. Mol Microbiol 54: 1161–1172.

28. NovoDJ, PerlmutterNG, HuntRH, ShapiroHM (2000) Multiparameter flow cytometric analysis of antibiotic effects on membrane potential, membrane permeability, and bacterial counts of Staphylococcus aureus and Micrococcus luteus. Antimicrob Agents Chemother 44: 827–834.

29. ShapiroHM (2008) Flow cytometry of bacterial membrane potential and permeability. Methods Mol Med 142: 175–186.

30. CamachoLR, EnsergueixD, PerezE, GicquelB, GuilhotC (1999) Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis. Mol Microbiol 34: 257–267.

31. DomenechP, ReedMB, BarryCE3rd (2005) Contribution of the Mycobacterium tuberculosis MmpL protein family to virulence and drug resistance. Infect Immun 73: 3492–3501.

32. DeshayesC, BachH, EuphrasieD, AttarianR, CoureuilM, et al. (2010) MmpS4 promotes glycopeptidolipids biosynthesis and export in Mycobacterium smegmatis. Mol Microbiol 78: 989–1003.

33. SinghA, JainS, GuptaS, DasT, TyagiAK (2003) mymA operon of Mycobacterium tuberculosis: its regulation and importance in the cell envelope. FEMS Microbiol Lett 227: 53–63.

34. HatziosSK, SchelleMW, HolsclawCM, BehrensCR, BotyanszkiZ, et al. (2009) PapA3 is an acyltransferase required for polyacyltrehalose biosynthesis in Mycobacterium tuberculosis. J Biol Chem 284: 12745–12751.

35. FernandezP, Saint-JoanisB, BariloneN, JacksonM, GicquelB, et al. (2006) The Ser/Thr protein kinase PknB is essential for sustaining mycobacterial growth. J Bacteriol 188: 7778–7784.

36. KangCM, AbbottDW, ParkST, DascherCC, CantleyLC, et al. (2005) The Mycobacterium tuberculosis serine/threonine kinases PknA and PknB: substrate identification and regulation of cell shape. Genes Dev 19: 1692–1704.

37. PapavinasasundaramKG, ChanB, ChungJH, ColstonMJ, DavisEO, et al. (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.

38. CowleyS, KoM, PickN, ChowR, DowningKJ, et al. (2004) The Mycobacterium tuberculosis protein serine/threonine kinase PknG is linked to cellular glutamate/glutamine levels and is important for growth in vivo. Mol Microbiol 52: 1691–1702.

39. SlaydenRA, BarryCE3rd (2002) The role of KasA and KasB in the biosynthesis of meromycolic acids and isoniazid resistance in Mycobacterium tuberculosis. Tuberculosis (Edinb) 82: 149–160.

40. CantaloubeS, Veyron-ChurletR, HaddacheN, DaffeM, ZerbibD (2011) The Mycobacterium tuberculosis FAS-II dehydratases and methyltransferases define the specificity of the mycolic acid elongation complexes. PLoS One 6: e29564.

41. YamadaH, BhattA, DanevR, FujiwaraN, MaedaS, et al. (2012) Non-acid-fastness in Mycobacterium tuberculosis DeltakasB mutant correlates with the cell envelope electron density. Tuberculosis (Edinb) 92: 351–357.

42. RousseauC, NeyrollesO, BordatY, GirouxS, SirakovaTD, et al. (2003) Deficiency in mycolipenate- and mycosanoate-derived acyltrehaloses enhances early interactions of Mycobacterium tuberculosis with host cells. Cell Microbiol 5: 405–415.

43. TrebucqA (2004) Revisiting sputum smear microscopy. Int J Tuberc Lung Dis 8: 805.

44. SeilerP, UlrichsT, BandermannS, PradlL, JorgS, et al. (2003) Cell-wall alterations as an attribute of Mycobacterium tuberculosis in latent infection. J Infect Dis 188: 1326–1331.

45. JacksonM, CrickDC, BrennanPJ (2000) Phosphatidylinositol is an essential phospholipid of mycobacteria. J Biol Chem 275: 30092–30099.

46. CanovaMJ, KremerL, MolleV (2008) pETPhos: a customized expression vector designed for further characterization of Ser/Thr/Tyr protein kinases and their substrates. Plasmid 60: 149–153.

47. BardarovS, BardarovSJr, Jr, PavelkaMSJr, Jr, SambandamurthyV, LarsenM, et al. (2002) Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology 148: 3007–3017.

48. VilchezeC, BaughnAD, TufarielloJ, LeungLW, KuoM, et al. (2011) Novel Inhibitors of InhA Efficiently Kill Mycobacterium tuberculosis under Aerobic and Anaerobic Conditions. Antimicrob Agents Chemother 55: 3889–3898.

49. LiL, MahanCS, PalaciM, HorterL, LoeffelholzL, et al. (2010) Sputum Mycobacterium tuberculosis mRNA as a marker of bacteriologic clearance in response to antituberculosis therapy. J Clin Microbiol 48: 46–51.

50. MenendezMC, GarciaMJ, NavarroMC, Gonzalez-y-MerchandJA, Rivera-GutierrezS, et al. (2002) Characterization of an rRNA operon (rrnB) of Mycobacterium fortuitum and other mycobacterial species: implications for the classification of mycobacteria. J Bacteriol 184: 1078–1088.

51. NunezMC, MenendezMC, RebolloMJ, GarciaMJ (2008) Transcriptional analysis of Mycobacterium fortuitum cultures upon hydrogen peroxide treatment using the novel standard rrnA-P1. BMC Microbiol 8: 100.

52. VilchezeC, WeinrickB, WongKW, ChenB, JacobsWRJr (2010) NAD+ auxotrophy is bacteriocidal for the tubercle bacilli. Mol Microbiol 76: 365–377.

53. MolleV, KremerL, Girard-BlancC, BesraGS, CozzoneAJ, et al. (2003) An FHA phosphoprotein recognition domain mediates protein EmbR phosphorylation by PknH, a Ser/Thr protein kinase from Mycobacterium tuberculosis. Biochemistry 42: 15300–15309.

54. KremerL, GuerardelY, GurchaSS, LochtC, BesraGS (2002) Temperature-induced changes in the cell-wall components of Mycobacterium thermoresistibile. Microbiology 148: 3145–3154.

55. PawelczykJ, BrzostekA, KremerL, DziadekB, Rumijowska-GalewiczA, et al. (2011) AccD6, a key carboxyltransferase essential for mycolic acid synthesis in Mycobacterium tuberculosis, is dispensable in a nonpathogenic strain. J Bacteriol 193: 6960–6972.

56. KremerL, DoverLG, CarrereS, NampoothiriKM, LesjeanS, et al. (2002) Mycolic acid biosynthesis and enzymic characterization of the beta-ketoacyl-ACP synthase A-condensing enzyme from Mycobacterium tuberculosis. Biochem J 364: 423–430.

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