Sterol Biosynthesis Is Required for Heat Resistance but Not Extracellular Survival in

English version České info

Leishmania parasites are transmitted through the bite of sandflies causing a spectrum of serious diseases in humans. Current drugs are inadequate and no safe vaccine is available. These parasites produce different types of sterols from humans, making the sterol synthesis pathway a valuable target of selective inhibitors. However, functions of sterols and sterol synthesis in protozoa are poorly understood, which hinders the development of new and improved treatments. In this study, we investigated the role of sterol C14α-demethylase, a key enzyme in sterol metabolism and the primary target of azole drugs. Loss of sterol C14α-demethylase completely altered the sterol composition in Leishmania, leading to increased membrane fluidity, failure to maintain lipid rafts, and hypersensitivity to heat stress. Despite these defects, null mutants of sterol C14α-demethylase were viable during the promastigote stage (found in sandflies) and could still cause disease in mice (although at a reduced capacity). Our findings provide direct evidence to support the role of specific sterols in membrane stability and stress response. The new knowledge may also help the development of new treatments or improve the efficacy of current drugs against pathogenic protozoa.

Vyšlo v časopise: Sterol Biosynthesis Is Required for Heat Resistance but Not Extracellular Survival in. PLoS Pathog 10(10): e32767. doi:10.1371/journal.ppat.1004427
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1004427

Souhrn

Zdroje

1. BernC, MaguireJH, AlvarJ (2008) Complexities of assessing the disease burden attributable to leishmaniasis. PLoS Negl Trop Dis 2: e313.

2. MurrayHW, BermanJD, DaviesCR, SaraviaNG (2005) Advances in leishmaniasis. Lancet 366 : 1561–1577.

3. DesjeuxP (2004) Leishmaniasis: current situation and new perspectives. Comp Immunol Microbiol Infect Dis 27 : 305–318.

4. CroftSL, OlliaroP (2011) Leishmaniasis chemotherapy–challenges and opportunities. Clin Microbiol Infect 17 : 1478–1483.

5. GingerML, PrescottMC, ReynoldsDG, ChanceML, GoadLJ (2000) Utilization of leucine and acetate as carbon sources for sterol and fatty acid biosynthesis by Old and New World Leishmania species, Endotrypanum monterogeii and Trypanosoma cruzi. Eur J Biochem 267 : 2555–2566.

6. GoldsteinJL, BrownMS (1990) Regulation of the mevalonate pathway. Nature 343 : 425–430.

7. GaylorJL (2002) Membrane-bound enzymes of cholesterol synthesis from lanosterol. Biochem Biophys Res Commun 292 : 1139–1146.

8. BrownDA, LondonE (1998) Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol 14 : 111–136.

9. SchroederRJ, AhmedSN, ZhuY, LondonE, BrownDA (1998) Cholesterol and sphingolipid enhance the Triton X-100 insolubility of glycosylphosphatidylinositol-anchored proteins by promoting the formation of detergent-insoluble ordered membrane domains. J Biol Chem 273 : 1150–1157.

10. DaumG, LeesND, BardM, DicksonR (1998) Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae. Yeast 14 : 1471–1510.

11. SwanTM, WatsonK (1998) Stress tolerance in a yeast sterol auxotroph: role of ergosterol, heat shock proteins and trehalose. FEMS Microbiol Lett 169 : 191–197.

12. DahlC, BiemannHP, DahlJ (1987) A protein kinase antigenically related to pp60v-src possibly involved in yeast cell cycle control: positive in vivo regulation by sterol. Proc Natl Acad Sci U S A 84 : 4012–4016.

13. PayneAH, HalesDB (2004) Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocr Rev 25 : 947–970.

14. GoadLJ, HolzGGJr, BeachDH (1984) Sterols of Leishmania species. Implications for biosynthesis. Mol Biochem Parasitol 10 : 161–170.

15. UrbinaJA, ConcepcionJL, RangelS, VisbalG, LiraR (2002) Squalene synthase as a chemotherapeutic target in Trypanosoma cruzi and Leishmania mexicana. Mol Biochem Parasitol 125 : 35–45.

16. ZakaiHA, ZimmoS, FouadMA (2003) Effect of itraconazole and terbinafine on Leishmania promastigotes. J Egypt Soc Parasitol 33 : 97–107.

17. ZakaiHA, ZimmoSK (2000) Effects of itraconazole and terbinafine on Leishmania major lesions in BALB/c mice. Ann Trop Med Parasitol 94 : 787–791.

18. BucknerF, YokoyamaK, LockmanJ, AikenheadK, OhkandaJ, et al. (2003) A class of sterol 14-demethylase inhibitors as anti-Trypanosoma cruzi agents. Proc Natl Acad Sci U S A 100 : 15149–15153.

19. ConsigliJ, DanieloC, GalleranoV, PapaM, GuidiA (2006) Cutaneous leishmaniasis: successful treatment with itraconazole. Int J Dermatol 45 : 46–49.

20. BeachDH, GoadLJ, HolzGGJr (1988) Effects of antimycotic azoles on growth and sterol biosynthesis of Leishmania promastigotes. Mol Biochem Parasitol 31 : 149–162.

21. HaughanPA, ChanceML, GoadLJ (1995) Effects of an azasterol inhibitor of sterol 24-transmethylation on sterol biosynthesis and growth of Leishmania donovani promastigotes. Biochem J 308 (Pt 1): 31–38.

22. RodriguesJC, AttiasM, RodriguezC, UrbinaJA, SouzaW (2002) Ultrastructural and biochemical alterations induced by 22,26-azasterol, a delta(24(25))-sterol methyltransferase inhibitor, on promastigote and amastigote forms of Leishmania amazonensis. Antimicrob Agents Chemother 46 : 487–499.

23. BaginskiM, ResatH, BorowskiE (2002) Comparative molecular dynamics simulations of amphotericin B-cholesterol/ergosterol membrane channels. Biochim Biophys Acta 1567 : 63–78.

24. LemkeA, KiderlenAF, KayserO (2005) Amphotericin B. Appl Microbiol Biotechnol. 68 : 151–162.

25. JhaTK, GiriYN, SinghTK, JhaS (1995) Use of amphotericin B in drug-resistant cases of visceral leishmaniasis in north Bihar, India. Am J Trop Med Hyg 52 : 536–538.

26. de SouzaW, RodriguesJC (2009) Sterol Biosynthesis Pathway as Target for Anti-trypanosomatid Drugs. Interdiscip Perspect Infect Dis 2009 : 642502.

27. UrbinaJA (1997) Lipid biosynthesis pathways as chemotherapeutic targets in kinetoplastid parasites. Parasitology 114 Suppl: S91–99

28. Gebre-HiwotA, FrommelD (1993) The in-vitro anti-leishmanial activity of inhibitors of ergosterol biosynthesis. J Antimicrob Chemother 32 : 837–842.

29. DebeljakN, FinkM, RozmanD (2003) Many facets of mammalian lanosterol 14alpha-demethylase from the evolutionarily conserved cytochrome P450 family CYP51. Arch Biochem Biophys 409 : 159–171.

30. GuengerichFP, SohlCD, ChowdhuryG (2011) Multi-step oxidations catalyzed by cytochrome P450 enzymes: Processive vs. distributive kinetics and the issue of carbonyl oxidation in chemical mechanisms. Arch Biochem Biophys 507 : 126–134.

31. KeberR, MotalnH, WagnerKD, DebeljakN, RassoulzadeganM, et al. (2011) Mouse knockout of the cholesterogenic cytochrome P450 lanosterol 14alpha-demethylase (Cyp51) resembles Antley-Bixler syndrome. J Biol Chem 286 : 29086–29097.

32. BardM, LeesND, TuriT, CraftD, CofrinL, et al. (1993) Sterol synthesis and viability of erg11 (cytochrome P450 lanosterol demethylase) mutations in Saccharomyces cerevisiae and Candida albicans. Lipids 28 : 963–967.

33. KalbVF, WoodsCW, TuriTG, DeyCR, SutterTR, et al. (1987) Primary structure of the P450 lanosterol demethylase gene from Saccharomyces cerevisiae. DNA 6 : 529–537.

34. WatsonPF, RoseME, EllisSW, EnglandH, KellySL (1989) Defective sterol C5–6 desaturation and azole resistance: a new hypothesis for the mode of action of azole antifungals. Biochem Biophys Res Commun 164 : 1170–1175.

35. LambDC, KellyDE, WatermanMR, StromstedtM, RozmanD, et al. (1999) Characteristics of the heterologously expressed human lanosterol 14alpha-demethylase (other names: P45014DM, CYP51, P45051) and inhibition of the purified human and Candida albicans CYP51 with azole antifungal agents. Yeast 15 : 755–763.

36. BucknerFS, JoubertBM, BoyleSM, EastmanRT, VerlindeCL, et al. (2003) Cloning and analysis of Trypanosoma cruzi lanosterol 14alpha-demethylase. Mol Biochem Parasitol 132 : 75–81.

37. LepeshevaGI, NesWD, ZhouW, HillGC, WatermanMR (2004) CYP51 from Trypanosoma brucei is obtusifoliol-specific. Biochemistry 43 : 10789–10799.

38. HargroveTY, WawrzakZ, LiuJ, NesWD, WatermanMR, et al. (2011) Substrate preferences and catalytic parameters determined by structural characteristics of sterol 14alpha-demethylase (CYP51) from Leishmania infantum. J Biol Chem 286 : 26838–26848.

39. BucknerFS (2008) Sterol 14-demethylase inhibitors for Trypanosoma cruzi infections. Adv Exp Med Biol 625 : 61–80.

40. SuryadevaraPK, RacherlaKK, OlepuS, NorcrossNR, TatipakaHB, et al. (2013) Dialkylimidazole inhibitors of Trypanosoma cruzi sterol 14alpha-demethylase as anti-Chagas disease agents. Bioorg Med Chem Lett 23 : 6492–6499.

41. Soeiro MdeN, de SouzaEM, da SilvaCF, Batista DdaG, BatistaMM, et al. (2013) In vitro and in vivo studies of the antiparasitic activity of sterol 14alpha-demethylase (CYP51) inhibitor VNI against drug-resistant strains of Trypanosoma cruzi. Antimicrob Agents Chemother 57 : 4151–4163.

42. BakS, KahnRA, OlsenCE, HalkierBA (1997) Cloning and expression in Escherichia coli of the obtusifoliol 14 alpha-demethylase of Sorghum bicolor (L.) Moench, a cytochrome P450 orthologous to the sterol 14 alpha-demethylases (CYP51) from fungi and mammals. Plant J 11 : 191–201.

43. LepeshevaGI, ZaitsevaNG, NesWD, ZhouW, AraseM, et al. (2006) CYP51 from Trypanosoma cruzi: a phyla-specific residue in the B' helix defines substrate preferences of sterol 14alpha-demethylase. J Biol Chem 281 : 3577–3585.

44. LepeshevaGI, WatermanMR (2011) Structural basis for conservation in the CYP51 family. Biochim Biophys Acta 1814 : 88–93.

45. CruzA, BeverleySM (1990) Gene replacement in parasitic protozoa. Nature 348 : 171–173.

46. BangsJD, UyetakeL, BrickmanMJ, BalberAE, BoothroydJC (1993) Molecular cloning and cellular localization of a BiP homologue in Trypanosoma brucei. Divergent ER retention signals in a lower eukaryote. J Cell Sci 105 (Pt 4): 1101–1113.

47. ReinhartMP, BillheimerJT, FaustJR, GaylorJL (1987) Subcellular localization of the enzymes of cholesterol biosynthesis and metabolism in rat liver. J Biol Chem 262 : 9649–9655.

48. HommaK, YoshidaY, NakanoA (2000) Evidence for recycling of cytochrome P450 sterol 14-demethylase from the cis-Golgi compartment to the endoplasmic reticulum (ER) upon saturation of the ER-retention mechanism. J Biochem 127 : 747–754.

49. SacksDL, PerkinsPV (1984) Identification of an infective stage of Leishmania promastigotes. Science 223 : 1417–1419.

50. ZotchevSB (2003) Polyene macrolide antibiotics and their applications in human therapy. Curr Med Chem 10 : 211–223.

51. BaginskiM, CzubJ, SternalK (2006) Interaction of amphotericin B and its selected derivatives with membranes: molecular modeling studies. Chem Rec 6 : 320–332.

52. BrownDA, LondonE (1998) Structure and origin of ordered lipid domains in biological membranes. J Membr Biol 164 : 103–114.

53. Zhang K, Showalter M, Revollo J, Hsu FF, Turk J, et al. (2003) Sphingolipids are essential for differentiation but not growth in Leishmania. EMBO J 22 : 6016–6026. PMCID: 275442.

54. DennyPW, FieldMC, SmithDF (2001) GPI-anchored proteins and glycoconjugates segregate into lipid rafts in Kinetoplastida. FEBS Lett 491 : 148–153.

55. ShapiraM, PinelliE (1989) Heat-shock protein 83 of Leishmania mexicana amazonensis is an abundant cytoplasmic protein with a tandemly repeated genomic arrangement. Eur J Biochem 185 : 231–236.

56. SpathGF, GarrawayLA, TurcoSJ, BeverleySM (2003) The role(s) of lipophosphoglycan (LPG) in the establishment of Leishmania major infections in mammalian hosts. Proc Natl Acad Sci USA 100 : 9536–9541.

57. GoadLJ, HolzGGJr, BeachDH (1985) Sterols of ketoconazole-inhibited Leishmania mexicana mexicana promastigotes. Mol Biochem Parasitol 15 : 257–279.

58. XuW, XinL, SoongL, ZhangK (2011) Sphingolipid degradation by Leishmania major is required for its resistance to acidic pH in the mammalian host. Infect Immun 79 : 3377–3387.

59. DufourcEJ (2008) Sterols and membrane dynamics. J Chem Biol 1 : 63–77.

60. HarringtonJM, ScelsiC, HartelA, JonesNG, EngstlerM, et al. (2012) Novel African trypanocidal agents: membrane rigidifying peptides. PLoS One 7: e44384.

61. BermanJD, HolzGGJr, BeachDH (1984) Effects of ketoconazole on growth and sterol biosynthesis of Leishmania mexicana promastigotes in culture. Mol Biochem Parasitol 12 : 1–13.

62. RogT, Pasenkiewicz-GierulaM (2001) Cholesterol effects on the phosphatidylcholine bilayer nonpolar region: a molecular simulation study. Biophys J 81 : 2190–2202.

63. RogT, Pasenkiewicz-GierulaM (2004) Non-polar interactions between cholesterol and phospholipids: a molecular dynamics simulation study. Biophys Chem 107 : 151–164.

64. BlochKE (1983) Sterol structure and membrane function. CRC Crit Rev Biochem 14 : 47–92.

65. PoyryS, RogT, KarttunenM, VattulainenI (2008) Significance of cholesterol methyl groups. J Phys Chem B 112 : 2922–2929.

66. RogT, Pasenkiewicz-GierulaM, VattulainenI, KarttunenM (2007) What happens if cholesterol is made smoother: importance of methyl substituents in cholesterol ring structure on phosphatidylcholine-sterol interaction. Biophys J 92 : 3346–3357.

67. CourniaZ, UllmannGM, SmithJC (2007) Differential effects of cholesterol, ergosterol and lanosterol on a dipalmitoyl phosphatidylcholine membrane: a molecular dynamics simulation study. J Phys Chem B 111 : 1786–1801.

68. SabatiniK, MattilaJP, KinnunenPK (2008) Interfacial behavior of cholesterol, ergosterol, and lanosterol in mixtures with DPPC and DMPC. Biophys J 95 : 2340–2355.

69. AbeF, UsuiK, HirakiT (2009) Fluconazole modulates membrane rigidity, heterogeneity, and water penetration into the plasma membrane in Saccharomyces cerevisiae. Biochemistry 48 : 8494–8504.

70. FernandezC, Lobo Md MdelV, Gomez-CoronadoD, LasuncionMA (2004) Cholesterol is essential for mitosis progression and its deficiency induces polyploid cell formation. Exp Cell Res 300 : 109–120.

71. Martinez-BotasJ, SuarezY, FerrueloAJ, Gomez-CoronadoD, LasuncionMA (1999) Cholesterol starvation decreases p34(cdc2) kinase activity and arrests the cell cycle at G2. FASEB J 13 : 1359–1370.

72. RaltonJE, McConvilleMJ (1998) Delineation of three pathways of glycosylphosphatidylinositol biosynthesis in Leishmania mexicana. Precursors from different pathways are assembled on distinct pools of phosphatidylinositol and undergo fatty acid remodeling. J Biol Chem 273 : 4245–4257.

73. NadererT, McConvilleMJ (2002) Characterization of a Leishmania mexicana mutant defective in synthesis of free and protein-linked GPI glycolipids. Mol Biochem Parasitol 125 : 147–161.

74. SpathGF, LyeLF, SegawaH, SacksDL, TurcoSJ, et al. (2003) Persistence without pathology in phosphoglycan-deficient Leishmania major. Science 301 : 1241–1243.

75. MorenoSN, DocampoR (2003) Calcium regulation in protozoan parasites. Curr Opin Microbiol 6 : 359–364.

76. Vannier-SantosMA, UrbinaJA, MartinyA, NevesA, de SouzaW (1995) Alterations induced by the antifungal compounds ketoconazole and terbinafine in Leishmania. J Eukaryot Microbiol 42 : 337–346.

77. Vannier-SantosMA, MartinyA, LinsU, UrbinaJA, BorgesVM, et al. (1999) Impairment of sterol biosynthesis leads to phosphorus and calcium accumulation in Leishmania acidocalcisomes. Microbiology 145 (Pt 11): 3213–3220.

78. SaundersEC, NgWW, KloehnJ, ChambersJM, NgM, et al. (2014) Induction of a stringent metabolic response in intracellular stages of Leishmania mexicana leads to increased dependence on mitochondrial metabolism. PLoS Pathog 10: e1003888.

79. PucadyilTJ, TewaryP, MadhubalaR, ChattopadhyayA (2004) Cholesterol is required for Leishmania donovani infection: implications in leishmaniasis. Mol Biochem Parasitol 133 : 145–152.

80. RubA, DeyR, JadhavM, KamatR, ChakkaramakkilS, et al. (2009) Cholesterol depletion associated with Leishmania major infection alters macrophage CD40 signalosome composition and effector function. Nat Immunol 10 : 273–280.

81. Zhang K, Hsu FF, Scott DA, Docampo R, Turk J, et al. (2005) Leishmania salvage and remodelling of host sphingolipids in amastigote survival and acidocalcisome biogenesis. Mol Microbiol 55 : 1566–1578. PMC Journal –⁠ In Process. PMID: 15720561.

82. AlrajhiAA, IbrahimEA, De VolEB, KhairatM, FarisRM, et al. (2002) Fluconazole for the treatment of cutaneous leishmaniasis caused by Leishmania major. N Engl J Med 346 : 891–895.

83. MomeniAZ, JalayerT, EmamjomehM, BashardostN, GhassemiRL, et al. (1996) Treatment of cutaneous leishmaniasis with itraconazole. Randomized double-blind study. Arch Dermatol 132 : 784–786.

84. WeinrauchL, LivshinR, el-OnJ (1987) Ketoconazole in cutaneous leishmaniasis. Br J Dermatol 117 : 666–668.

85. OlivieriBP, MolinaJT, de CastroSL, PereiraMC, CalvetCM, et al. (2010) A comparative study of posaconazole and benznidazole in the prevention of heart damage and promotion of trypanocidal immune response in a murine model of Chagas disease. Int J Antimicrob Agents 36 : 79–83.

86. PrenticeAG, GlasmacherA (2005) Making sense of itraconazole pharmacokinetics. J Antimicrob Chemother 56 Suppl 1i17–i22.

87. HaDS, SchwarzJK, TurcoSJ, BeverleySM (1996) Use of the green fluorescent protein as a marker in transfected Leishmania. Mol Biochem Parasitol 77 : 57–64.

88. KaplerGM, CoburnCM, BeverleySM (1990) Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression. Mol Cell Biol 10 : 1084–1094.

89. SpathGF, BeverleySM (2001) A lipophosphoglycan-independent method for isolation of infective Leishmania metacyclic promastigotes by density gradient centrifugation. Exp Parasitol 99 : 97–103.

90. BeverleySM (2003) Protozomics: trypanosomatid parasite genetics comes of age. Nat Rev Genet 4 : 11–19.

91. Zhang O, Wilson MC, Xu W, Hsu FF, Turk J, et al. (2009) Degradation of host sphingomyelin is essential for Leishmania virulence. PLoS Pathog 5(12): e1000692. PMCID: 2784226.

92. MoreiraW, LeblancE, OuelletteM (2009) The role of reduced pterins in resistance to reactive oxygen and nitrogen intermediates in the protozoan parasite Leishmania. Free Radic Biol Med 46 : 367–375.

93. de IbarraAA, HowardJG, SnaryD (1982) Monoclonal antibodies to Leishmania tropica major: specificities and antigen location. Parasitology 85 (Pt 3): 523–531.

94. ConnellND, Medina-AcostaE, McMasterWR, BloomBR, RussellDG (1993) Effective immunization against cutaneous leishmaniasis with recombinant bacille Calmette-Guerin expressing the Leishmania surface proteinase gp63. Proc Natl Acad Sci U S A 90 : 11473–11477.

95. MoralesMA, PescherP, SpathGF (2010) Leishmania major MPK7 protein kinase activity inhibits intracellular growth of the pathogenic amastigote stage. Eukaryot Cell 9 : 22–30.

96. SpathGF, EpsteinL, LeaderB, SingerSM, AvilaHA, et al. (2000) Lipophosphoglycan is a virulence factor distinct from related glycoconjugates in the protozoan parasite Leishmania major. Proc Natl Acad Sci USA 97 : 9258–9263.

97. FolchJ, LeesM, Sloane StanleyGH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226 : 497–509.

98. Zhang O, Xu W, Pillai A, Zhang K (2012) Developmentally Regulated Sphingolipid Degradation in Leishmania major. PLoS One 7(1):e31059: PMCID: 3267774.

99. RacoosinEL, BeverleySM (1997) Leishmania major: promastigotes induce expression of a subset of chemokine genes in murine macrophages. Exp Parasitol 85 : 283–295.

100. PillaiAB, XuW, ZhangO, ZhangK (2012) Sphingolipid degradation in Leishmania (Leishmania) amazonensis. PLoS Negl Trop Dis 6: e1944.

101. TitusRG, MarchandM, BoonT, LouisJA (1985) A limiting dilution assay for quantifying Leishmania major in tissues of infected mice. Parasite Immunol 7 : 545–555.