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Cell Stress and Chaperones

, Volume 22, Issue 5, pp 729–742 | Cite as

Hsp90 inhibitors radicicol and geldanamycin have opposing effects on Leishmania Aha1-dependent proliferation

  • Katharina Bartsch
  • Antje Hombach-Barrigah
  • Joachim ClosEmail author
Original Paper

Abstract

Hsp90 and its co-chaperones are essential for the medically important parasite Leishmania donovani, facilitating life cycle control and intracellular survival. Activity of Hsp90 is regulated by co-chaperones of the Aha1 and P23 families. In this paper, we studied the expression of L. donovani Aha1 in two life cycle stages, its interaction with Hsp90 and the phenotype of Aha1 null mutants during the insect stage and inside infected macrophages. This study provides a detailed in vitro analysis of the function of Aha1 in Leishmania parasites and the first instance of a reverse genetic analysis of Aha1 in a protozoan parasite. While Aha1 is non-essential under standard growth conditions and at elevated temperature, Aha1 protects against ethanol stress. However, both overexpression and lack of Aha1 affected parasite growth in the presence of the Hsp90 inhibitors radicicol (RAD) and geldanamycin (GA). Under RAD pressure, P23 and Aha1 act in an antagonistic way. By contrast, expression levels of both co-chaperones have similar effects under GA treatment, indicating different inhibition mechanisms by the two compounds. Aha1 is also secreted in virulence-enhancing exosomes. This may explain why the loss of Aha1 reduces the infectivity of L. donovani in ex vivo mouse macrophages, indicating a role during the intracellular mammalian stage.

Keywords

Leishmania Aha1 Hsp90 Co-chaperone Inhibitors Exosomes 

Notes

Acknowledgements

We thank Eugenia Bifeld and Julia Eick for providing bone marrow-derived macrophages. The work described here was funded by the Deutsche Forschungsgemeinschaft, Grant CL 120/8-1.

Supplementary material

12192_2017_800_MOESM1_ESM.pdf (62 kb)
Fig. S1 Anti-Aha1 antibody testing. (His)10: Aha1 was used to raise antibodies in a chicken. Western blot analysis showed that Aha1 antibodies recognise the natural protein in the lysates of L. major, L. donovani, the recombinant protein (His)10::Aha1, but not the orthologues in T. cruzi Y and T. cruzi Tulahuen lysates. Positions of size markers [kD] are shown on the left. (PDF 61 kb)
12192_2017_800_MOESM2_ESM.pdf (78 kb)
Fig. S2 Schematic representation of gene constructs. A. Plasmids constructed for Aha1 gene replacement. B. Schematic representation of homologous recombination between the SwaI-linearised replacement constructs and the Aha1 gene alleles. (PDF 77 kb)
12192_2017_800_MOESM3_ESM.pdf (97 kb)
Fig. S3 Cell body and flagellum length analysis of Aha1−/− null mutants and control strains. L. donovani cells of various genetic background as indicated were seeded into 10 mL of supplemented M199 (1 × 105 mL−1) and grown for 3 days at 25 °C and pH 7.0. Cells were then fixed, stained with mouse anti-α-tubulin and DAPI and visualised by fluorescence microscopy using the EVOS FL Auto epifluorescence microscope. Cell body length measurements (A) and flagellum length measurements (B) were then performed using the measuring tool of the EVOS FL Auto software. n = 2; *p ≤ 0.05,**p ≤ 0.01 (Mann-Whitney U-test) (PDF 96 kb)

References

  1. Barak E, Amin-Spector S, Gerliak E, Goyard S, Holland N, Zilberstein D (2005) Differentiation of Leishmania donovani in host-free system: analysis of signal perception and response. Mol Biochem Parasitol 141:99–108CrossRefPubMedGoogle Scholar
  2. Bates PA (1993) Axenic amastigote culture of Leishmania amastigotes. Parasitol Today 9:143–146CrossRefPubMedGoogle Scholar
  3. Bates PA (1994) Complete developmental cycle of Leishmania mexicana in axenic culture. Parasitology 108:1–9CrossRefPubMedGoogle Scholar
  4. Batista FA, Almeida GS, Seraphim TV, Silva KP, Murta SM, Barbosa LR, Borges JC (2015) Identification of two p23 co-chaperone isoforms in Leishmania braziliensis exhibiting similar structures and Hsp90 interaction properties despite divergent stabilities. FEBS J 282:388–406CrossRefPubMedGoogle Scholar
  5. Bifeld E, Chrobak M, Zander D, Schleicher U, Schönian G, Clos J (2015) Geographical sequence variation in the Leishmania major virulence factor P46. Infect Genet Evol 30:195–205CrossRefPubMedGoogle Scholar
  6. Bifeld E, Tejera Nevado P, Bartsch J, Eick J, Clos J (2016) A versatile qPCR assay to quantify trypanosomatidic infections of host cells and tissues. Med Microbiol Immunol 205:449–458CrossRefPubMedGoogle Scholar
  7. Brandau S, Dresel A, Clos J (1995) High constitutive levels of heat-shock proteins in human-pathogenic parasites of the genus Leishmania. Biochem J 310(Pt 1):225–232CrossRefPubMedPubMedCentralGoogle Scholar
  8. Buchner J (1999) Hsp90 & Co.—a holding for folding. Trends Biochem Sci 24:136–141CrossRefPubMedGoogle Scholar
  9. Cartwright CP, Juroszek JR, Beavan MJ, Ruby FMS, Demorais SMF, Rose AH (1986) Ethanol dissipates the proton-motive force across the plasma-membrane of Saccharomyces cerevisiae. J Gen Microbiol 132:369–377Google Scholar
  10. Choudhury K, Zander D, Kube M, Reinhardt R, Clos J (2008) Identification of a Leishmania infantum gene mediating resistance to miltefosine and SbIII. Int J Parasitol 38:1411–1423CrossRefPubMedGoogle Scholar
  11. Chrobak M, Förster S, Meisel S, Pfefferkorn R, Förster F, Clos J (2012) Leishmania donovani HslV does not interact stably with HslU proteins. Int J Parasitol 42:329–339CrossRefPubMedGoogle Scholar
  12. Chua CS, Low H, Lehming N, Sim TS (2012) Molecular analysis of Plasmodium falciparum co-chaperone Aha1 supports its interaction with and regulation of Hsp90 in the malaria parasite. Int J Biochem Cell Biol 44:233–245CrossRefPubMedGoogle Scholar
  13. Clos J, Brandau S (1994) pJC20 and pJC40—two high-copy-number vectors for T7 RNA polymerase-dependent expression of recombinant genes in Escherichia coli. Prot Expression Purif 5:133–137CrossRefGoogle Scholar
  14. Clos, J., and Hombach, A. (2015) Heat shock proteins of Leishmania: chaperones in the driver's seat. Leishmania: current biology and control 17–36Google Scholar
  15. Forafonov F, Toogun OA, Grad I, Suslova E, Freeman BC, Picard D (2008) p23/Sba1p protects against Hsp90 inhibitors independently of its intrinsic chaperone activity. Mol Cell Biol 28:3446–3456CrossRefPubMedPubMedCentralGoogle Scholar
  16. Ghosh S, Shinogle HE, Garg G, Vielhauer GA, Holzbeierlein JM, Dobrowsky RT, Blagg BS (2015) Hsp90 C-terminal inhibitors exhibit antimigratory activity by disrupting the Hsp90alpha/Aha1 complex in PC3-MM2 cells. ACS Chem Biol 10:577–590CrossRefPubMedGoogle Scholar
  17. Gu X, Xue W, Yin Y, Liu H, Li S, Sun X (2016) The Hsp90 co-chaperones Sti1, Aha1, and P23 regulate adaptive responses to antifungal azoles. Front Microbiol 7:1571PubMedPubMedCentralGoogle Scholar
  18. Holmes JL, Sharp SY, Hobbs S, Workman P (2008) Silencing of HSP90 cochaperone AHA1 expression decreases client protein activation and increases cellular sensitivity to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin. Cancer Res 68:1187–1196Google Scholar
  19. Hombach A, Ommen G, Chrobak M, Clos J (2013) The Hsp90-Sti1 interaction is critical for Leishmania donovani proliferation in both life cycle stages. Cell Microbiol 15:585–600CrossRefPubMedGoogle Scholar
  20. Hombach A, Ommen G, MacDonald A, Clos J (2014) A small heat shock protein is essential for thermotolerance and intracellular survival of Leishmania donovani. J Cell Sci 127:4762–4773CrossRefPubMedPubMedCentralGoogle Scholar
  21. Hombach A, Ommen G, Sattler V, Clos J (2015) Leishmania donovani P23 protects parasites against HSP90 inhibitor-mediated growth arrest. Cell Stress Chaperones 20:673–685CrossRefPubMedPubMedCentralGoogle Scholar
  22. Hübel A, Brandau S, Dresel A, Clos J (1995) A member of the ClpB family of stress proteins is expressed during heat shock in Leishmania spp. Mol Biochem Parasitol 70:107–118CrossRefPubMedGoogle Scholar
  23. Johnson JL, Brown C (2009) Plasticity of the Hsp90 chaperone machine in divergent eukaryotic organisms. Cell Stress Chaperones 14:83–94CrossRefPubMedGoogle Scholar
  24. Kapler GM, Coburn CM, Beverley SM (1990) Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression. Mol Cell Biol 10:1084–1094CrossRefPubMedPubMedCentralGoogle Scholar
  25. Krobitsch S, Brandau S, Hoyer C, Schmetz C, Hübel A, Clos J (1998) Leishmania donovani heat shock protein 100. Characterization and function in amastigote stage differentiation. J Biol Chem 273:6488–6494CrossRefPubMedGoogle Scholar
  26. Laban A, Wirth DF (1989) Transfection of Leishmania enriettii and expression of chloramphenicol acetyltransferase gene. Proc Natl Acad Sci U S A 86:9119–9123CrossRefPubMedPubMedCentralGoogle Scholar
  27. Li J, Richter K, Buchner J (2011) Mixed Hsp90-cochaperone complexes are important for the progression of the reaction cycle. Nat Struct Mol Biol 18:61–66CrossRefPubMedGoogle Scholar
  28. Li J, Soroka J, Buchner J (2012) The Hsp90 chaperone machinery: conformational dynamics and regulation by co-chaperones. Biochim Biophys Acta 1823:624–635CrossRefPubMedGoogle Scholar
  29. Lotz GP, Lin H, Harst A, Obermann WM (2003) Aha1 binds to the middle domain of Hsp90, contributes to client protein activation, and stimulates the ATPase activity of the molecular chaperone. J Biol Chem 278:17228–17235CrossRefPubMedGoogle Scholar
  30. Mann HB, Whitney DR (1947) On a test of whether one of 2 random variables is stochastically larger than the other. Ann Math Stat 18:50–60CrossRefGoogle Scholar
  31. Mollapour M, Neckers L (2012) Post-translational modifications of Hsp90 and their contributions to chaperone regulation. Biochim Biophys Acta 1823:648–655CrossRefPubMedGoogle Scholar
  32. Morales MA, Watanabe R, Dacher M, Chafey P, Osorio y Fortea J, Scott DA, Beverley SM, Ommen G, Clos J, Hem S et al (2010) Phosphoproteome dynamics reveal heat-shock protein complexes specific to the Leishmania donovani infectious stage. Proc Natl Acad Sci U S A 107:8381–8386CrossRefPubMedPubMedCentralGoogle Scholar
  33. Ommen G, Clos J (2010) Heat shock proteins in protozoan parasites—Leishmania spp. Heat Shock Proteins 4:135–151CrossRefGoogle Scholar
  34. Ommen G, Lorenz S, Clos J (2009) One-step generation of double-allele gene replacement mutants in Leishmania donovani. Int J Parasitol 39:541–546CrossRefPubMedGoogle Scholar
  35. Ommen G, Chrobak M, Clos J (2010) The co-chaperone SGT of Leishmania donovani is essential for the parasite's viability. Cell Stress Chaperones 15:443–455CrossRefPubMedGoogle Scholar
  36. Panaretou B, Siligardi G, Meyer P, Maloney A, Sullivan JK, Singh S, Millson SH, Clarke PA, Naaby-Hansen S, Stein R et al (2002) Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone aha1. Mol Cell 10:1307–1318CrossRefPubMedGoogle Scholar
  37. Park JE, Tan HS, Datta A, Lai RC, Zhang H, Meng W, Lim SK, Sze SK (2010) Hypoxic tumor cell modulates its microenvironment to enhance angiogenic and metastatic potential by secretion of proteins and exosomes. Mol Cell Proteomics 9:1085–1099CrossRefPubMedPubMedCentralGoogle Scholar
  38. Racoosin EL, Beverley SM (1997) Leishmania major: promastigotes induce expression of a subset of chemokine genes in murine macrophages. Exp Parasitol 85:283–295CrossRefPubMedGoogle Scholar
  39. Racoosin EL, Swanson JA (1989) Macrophage colony-stimulating factor (rM-CSF) stimulates pinocytosis in bone marrow-derived macrophages. J Exp Med 170:1635–1648CrossRefPubMedGoogle Scholar
  40. Rehn AB, Buchner J (2015) p23 and Aha1. Subcell Biochem 78:113–131CrossRefPubMedGoogle Scholar
  41. Rey-Ladino JA, Joshi PB, Singh B, Gupta R, Reiner NE (1997) Leishmania major: molecular cloning, sequencing, and expression of the heat shock protein 60 gene reveals unique carboxy terminal peptide sequences. Exp Parasitol 85:249–263CrossRefPubMedGoogle Scholar
  42. Roe SM, Prodromou C, O'Brien R, Ladbury JE, Piper PW, Pearl LH (1999) Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin. J Med Chem 42:260–266CrossRefPubMedGoogle Scholar
  43. Rogers MB, Hilley JD, Dickens NJ, Wilkes J, Bates PA, Depledge DP, Harris D, Her Y, Herzyk P, Imamura H et al (2011) Chromosome and gene copy number variation allow major structural change between species and strains of Leishmania. Genome Res 21:2129–2142CrossRefPubMedPubMedCentralGoogle Scholar
  44. Rosenzweig D, Smith D, Opperdoes F, Stern S, Olafson RW, Zilberstein D (2008) Retooling Leishmania metabolism: from sand fly gut to human macrophage. FASEB J 22:590–602CrossRefPubMedGoogle Scholar
  45. Rutherford SL, Zuker CS (1994) Protein folding and the regulation of signaling pathways. Cell 79:1129–1132CrossRefPubMedGoogle Scholar
  46. Sambrook J, Russell DW (2001) Molecular cloning, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  47. Schäfer C, Tejera Nevado P, Zander D, Clos J (2014) ARM58 overexpression reduces intracellular antimony concentration in Leishmania infantum. Antimicrob Agents Chemother 58:1565–1574CrossRefPubMedPubMedCentralGoogle Scholar
  48. Schlüter A, Wiesgigl M, Hoyer C, Fleischer S, Klaholz L, Schmetz C, Clos J (2000) Expression and subcellular localization of cpn60 protein family members in Leishmania donovani. Biochim Biophys Acta 1491:65–74CrossRefPubMedGoogle Scholar
  49. Seraphim TV, Alves MM, Silva IM, Gomes FE, Silva KP, Murta SM, Barbosa LR, Borges JC (2013) Low resolution structural studies indicate that the activator of Hsp90 ATPase 1 (Aha1) of Leishmania braziliensis has an elongated shape which allows its interaction with both N- and M-domains of Hsp90. PLoS One 8:e66822CrossRefPubMedPubMedCentralGoogle Scholar
  50. Silva KP, Seraphim TV, Borges JC (2013) Structural and functional studies of Leishmania braziliensis Hsp90. Biochim Biophys Acta 1834:351–361CrossRefPubMedGoogle Scholar
  51. Silverman JM, Chan SK, Robinson DP, Dwyer DM, Nandan D, Foster LJ, Reiner NE (2008) Proteomic analysis of the secretome of Leishmania donovani. Genome Biol 9:R35CrossRefPubMedPubMedCentralGoogle Scholar
  52. Silverman JM, Clos J, de’Oliveira CC, Shirvani O, Fang Y, Wang C, Foster LJ, Reiner NE (2010a) An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages. J Cell Sci 123:842–852CrossRefPubMedGoogle Scholar
  53. Silverman JM, Clos J, Horakova E, Wang AY, Wiesgigl M, Kelly I, Lynn MA, McMaster WR, Foster LJ, Levings MK et al (2010b) Leishmania exosomes modulate innate and adaptive immune responses through effects on monocytes and dendritic cells. J Immunol 185:5011–5022CrossRefPubMedGoogle Scholar
  54. Singh M, Shah V, Tatu U (2014) A novel C-terminal homologue of Aha1 co-chaperone binds to heat shock protein 90 and stimulates its ATPase activity in Entamoeba histolytica. J Mol Biol 426:1786–1798CrossRefPubMedGoogle Scholar
  55. Soroka J, Wandinger SK, Mausbacher N, Schreiber T, Richter K, Daub H, Buchner J (2012) Conformational switching of the molecular chaperone Hsp90 via regulated phosphorylation. Mol Cell 45:517–528CrossRefPubMedGoogle Scholar
  56. Student (1908) The probable error of a mean. Biometrika 6:1–25CrossRefGoogle Scholar
  57. Tejera Nevado P, Bifeld E, Höhn K, Clos J (2016) Clustering of drug resistance and fitness-related genes in Leishmania infantum. Antimicrob Agents Chemother 60:5262–5275CrossRefPubMedPubMedCentralGoogle Scholar
  58. Tripathi V, Darnauer S, Hartwig NR, Obermann WM (2014) Aha1 can act as an autonomous chaperone to prevent aggregation of stressed proteins. J Biol Chem 289:36220–36228CrossRefPubMedPubMedCentralGoogle Scholar
  59. Twu O, de Miguel N, Lustig G, Stevens GC, Vashisht AA, Wohlschlegel JA, Johnson PJ (2013) Trichomonas vaginalis exosomes deliver cargo to host cells and mediate hostratioparasite interactions. PLoS Pathog 9:e1003482CrossRefPubMedPubMedCentralGoogle Scholar
  60. Ubeda JM, Raymond F, Mukherjee A, Plourde M, Gingras H, Roy G, Lapointe A, Leprohon P, Papadopoulou B, Corbeil J et al (2014) Genome-wide stochastic adaptive DNA amplification at direct and inverted DNA repeats in the parasite Leishmania. PLoS Biol 12:e1001868CrossRefPubMedPubMedCentralGoogle Scholar
  61. Vergnes B, Gourbal B, Girard I, Sundar S, Drummelsmith J, Ouellette M (2007) A proteomics screen implicates HSP83 and a small kinetoplastid calpain-related protein in drug resistance in Leishmania donovani clinical field isolates by modulating drug-induced programmed cell death. Mol Cell Proteomics 6:88–101CrossRefPubMedGoogle Scholar
  62. Wandinger SK, Richter K, Buchner J (2008) The Hsp90 chaperone machinery. J Biol Chem 283:18473–18477CrossRefPubMedGoogle Scholar
  63. Wiesgigl M, Clos J (2001) Heat shock protein 90 homeostasis controls stage differentiation in Leishmania donovani. Mol Biol Cell 12:3307–3316CrossRefPubMedPubMedCentralGoogle Scholar
  64. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119CrossRefPubMedGoogle Scholar
  65. Yau W-L, Pescher P, Macdonald A, Zander D, Retzlaff S, Blisnick T, Rotureau B, Bastin P, Clos J, Späth G (2014) Cyclophilin 40-deficient Leishmania donovani fail to undergo stress-induced development of the infectious metacyclic stage. Cell Microbiol 93:80–97Google Scholar
  66. Yau WL, Lambertz U, Colineau L, Pescher P, MacDonald A, Zander D, Retzlaff S, Eick J, Reiner NE, Clos J et al (2016) Phenotypic characterization of a Leishmania donovani cyclophilin 40 null mutant. J Eukaryot Microbiol 63:823–833CrossRefPubMedGoogle Scholar
  67. Zamora-Veyl FB, Kroemer M, Zander D, Clos J (2005) Stage-specific expression of the mitochondrial co-chaperonin of Leishmania donovani, CPN10. Kinetoplastid Biol Dis 4:3CrossRefPubMedPubMedCentralGoogle Scholar
  68. Zilberstein D, Shapira M (1994) The role of pH and temperature in the development of Leishmania parasites. Annu Rev Microbiol 48:449–470CrossRefPubMedGoogle Scholar

Copyright information

© Cell Stress Society International 2017

Authors and Affiliations

  1. 1.Bernhard Nocht Institute for Tropical MedicineHamburgGermany

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