Transcriptional Responses of Praziquantel Exposure in Schistosomes Identifies a Functional Role for Calcium Signalling Pathway Member CamKII

English version České info

Treatment for clinical schistosomiasis has relied centrally on the broad spectrum anthelmintic praziquantel; however, there is limited information on its mode of action or the molecular response of the parasite. This paper presents a transcriptional and functional approach to defining the molecular responses of schistosomes to praziquantel. Differential gene expression in Schistosoma japonicum was investigated by transcriptome-wide microarray analysis of adult worms perfused from infected mice after 0.5 to 24 hours after oral administration of sub-lethal doses of praziquantel. Genes up-regulated initially in male parasites were associated with “Tegument/Muscle Repair” and “Lipid/Ion Regulation” functions and were followed by “Drug Resistance” and “Ion Regulation” associated genes. Prominent responses induced in female worms included up-regulation of “Ca²⁺ Regulation” and “Drug Resistance” genes and later by transcripts of “Detoxification” and “Pathogen Defense” mechanisms. A subset of highly over-expressed genes, with putative drug resistance/detoxification roles or Ca²⁺-dependant/modulatory functions, were validated by qPCR. The leading candidate among these was CamKII, a putative calcium/calmodulin-dependent protein kinase type II delta chain. RNA interference was employed to knockdown CamKII in S. japonicum to determine the role of CamKII in the response to praziquantel. After partial-knockdown, schistosomes were analysed using IC₅₀ concentrations (50% worm motility) and quantitative monitoring of parasite movement. When CamKII transcription was reduced by 50–69% in S. japonicum, the subsequent effect of an IC₅₀ dosage of praziquantel was exacerbated, reducing motility from 47% to 27% in female worms and from 61% to 23% in males. These observations indicated that CamKII mitigates the effects of praziquantel, probably through stabilising Ca²⁺ fluxes within parasite muscles and tegument. Together, these studies comprehensively charted transcriptional changes upon exposure to praziquantel and, notably, identified CamKII as potentially central to the, as yet undefined, mode of action of praziquantel.

Vyšlo v časopise: Transcriptional Responses of Praziquantel Exposure in Schistosomes Identifies a Functional Role for Calcium Signalling Pathway Member CamKII. PLoS Pathog 9(3): e32767. doi:10.1371/journal.ppat.1003254
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1003254

Souhrn

Zdroje

1. KingCH, DickmanK, TischDJ (2005) Reassessment of the cost of chronic helminthic infection: a meta-analysis of disability-related outcomes in endemic schistosomiasis. Lancet 365: 1561–1569.

2. DoenhoffMJ, CioliD, UtzingerJ (2008) Praziquantel: mechanisms of action, resistance and new derivatives for schistosomiasis. Current Opinion in Infectious Diseases 21: 659–667.

3. Pica-MattocciaL, CioliD (2004) Sex- and stage-related sensitivity of Schistosoma mansoni to in vivo and in vitro praziquantel treatment. Int J Parasitol 34: 527–533.

4. CioliD, Pica-MattocciaL (2003) Praziquantel. Parasitol Res 90 Supp 1: S3–9.

5. Pica-MattocciaL, ValleC, BassoA, TroianiAR, VigorosiF, et al. (2007) Cytochalasin D abolishes the schistosomicidal activity of praziquantel. Exp Parasitol 115: 344–351.

6. KasinathanRS, MorganWM, GreenbergRM (2010) Schistosoma mansoni express higher levels of multidrug resistance-associated protein 1 (SmMRP1) in juvenile worms and in response to praziquantel. Mol Biochem Parasitol 173: 25–31.

7. MesserliSM, KasinathanRS, MorganW, SprangerS, GreenbergRM (2009) Schistosoma mansoni P-glycoprotein levels increase in response to praziquantel exposure and correlate with reduced praziquantel susceptibility. Mol Biochem Parasitol 167: 54–59.

8. Pica-MattocciaL, OrsiniT, BassoA, FestucciA, LibertiP, et al. (2008) Schistosoma mansoni: lack of correlation between praziquantel-induced intra-worm calcium influx and parasite death. Exp Parasitol 119: 332–335.

9. Pica-MattocciaL, DoenhoffMJ, ValleC, BassoA, TroianiAR, et al. (2009) Genetic analysis of decreased praziquantel sensitivity in a laboratory strain of Schistosoma mansoni. Acta Trop 111: 82–85.

10. SatoH, KuselJR, ThornhillJ (2004) Excretion of fluorescent substrates of mammalian multidrug resistance-associated protein (MRP) in the Schistosoma mansoni excretory system. Parasitology 128: 43–52.

11. KwaMS, VeenstraJG, Van DijkM, RoosMH (1995) Beta-tubulin genes from the parasitic nematode Haemonchus contortus modulate drug resistance in Caenorhabditis elegans. J Mol Biol 246: 500–510.

12. JamesCE, HudsonAL, DaveyMW (2009) Drug resistance mechanisms in helminths: is it survival of the fittest? Trends Parasitol 25: 328–335.

13. KoppSR, ColemanGT, TraubRJ, McCarthyJS, KotzeAC (2009) Acetylcholine receptor subunit genes from Ancylostoma caninum: altered transcription patterns associated with pyrantel resistance. Int J Parasitol 39: 435–441.

14. AragonAD, ImaniRA, BlackburnVR, CupitPM, MelmanSD, et al. (2009) Towards an understanding of the mechanism of action of praziquantel. Mol Biochem Parasitol 164: 57–65.

15. SabahAA, FletcherC, WebbeG, DoenhoffMJ (1986) Schistosoma mansoni: chemotherapy of infections of different ages. Exp Parasitol 61: 294–303.

16. Hines-KayJ, CupitPM, SanchezMC, RosenbergGH, HaneltB, et al. (2012) Transcriptional analysis of Schistosoma mansoni treated with praziquantel in vitro. Mol Biochem Parasitol 186: 87–94.

17. BrindleyPJ, SherA (1987) The chemotherapeutic effect of praziquantel against Schistosoma mansoni is dependent on host antibody response. J Immunol 139: 215–220.

18. RibeiroF, MelloRT, TavaresCA, KuselJR, CoelhoPM (2004) Synergistic action of praziquantel and host specific immune response against Schistosoma mansoni at different phases of infection. Rev Inst Med Trop Sao Paulo 46: 231–233.

19. GunasekeraAM, PatankarS, SchugJ, EisenG, WirthDF (2003) Drug-induced alterations in gene expression of the asexual blood forms of Plasmodium falciparum. Mol Microbiol 50: 1229–1239.

20. NatalangO, BischoffE, DeplaineG, ProuxC, DilliesMA, et al. (2008) Dynamic RNA profiling in Plasmodium falciparum synchronized blood stages exposed to lethal doses of artesunate. BMC Genomics 9: 388.

21. DenningerV, FigarellaK, SchonfeldC, BremsS, BusoldC, et al. (2007) Troglitazone induces differentiation in Trypanosoma brucei. Exp Cell Res 313: 1805–1819.

22. Vande WaaEA, CampbellCK, O'LearyKA, TracyJW (1993) Induction of Schistosoma mansoni glutathione S-transferase by xenobiotics. Arch Biochem Biophys 303: 15–21.

23. RedmanCA, RobertsonA, FallonPG, ModhaJ, KuselJR, et al. (1996) Praziquantel: an urgent and exciting challenge. Parasitol Today 12: 14–20.

24. XiaoSH, FriedmanPA, CattoBA, WebsterLTJr (1984) Praziquantel-induced vesicle formation in the tegument of male Schistosoma mansoni is calcium dependent. J Parasitol 70: 177–179.

25. XiaoSH, YouJQ, ZhangRQ (1985) [Scanning electron microscope observation on the tegumental surface alterations of Schistosoma japonicum induced by praziquantel at different developmental stages]. Yao Xue Xue Bao 20: 577–583.

26. XiaoSH (2005) Development of antischistosomal drugs in China, with particular consideration to praziquantel and the artemisinins. Acta Trop 96: 153–167.

27. XiaoSH, MeiJY, JiaoPY (2009) The in vitro effect of mefloquine and praziquantel against juvenile and adult Schistosoma japonicum. Parasitol Res 106: 237–246.

28. WuX, DeweyTG (2006) From microarray to biological networks: Analysis of gene expression profiles. Methods Mol Biol 316: 35–48.

29. IskarM, CampillosM, KuhnM, JensenLJ, van NoortV, et al. (2010) Drug-induced regulation of target expression. PLoS Comput Biol 6: pii.

30. BraschiS, WilsonRA (2006) Proteins exposed at the adult schistosome surface revealed by biotinylation. Mol Cell Proteomics 5: 347–356.

31. MulvennaJ, MoertelL, JonesMK, NawaratnaS, LovasEM, et al. (2009) Exposed proteins of the Schistosoma japonicum tegument. Int J Parasitol 40: 543–54.

32. ShawMK, ErasmusDA (1983) Schistosoma mansoni: the effects of a subcurative dose of praziquantel on the ultrastructure of worms in vivo. Z Parasitenkd 69: 73–90.

33. GobertGN, StenzelDJ, McManusDP, JonesMK (2003) The ultrastructural architecture of the adult Schistosoma japonicum tegument. Int J Parasitol 33: 1561–1575.

34. BernatchezPN, SharmaA, KodamanP, SessaWC (2009) Myoferlin is critical for endocytosis in endothelial cells. Am J Physiol Cell Physiol 297: C484–492.

35. KirkhamM, PartonRG (2005) Clathrin-independent endocytosis: new insights into caveolae and non-caveolar lipid raft carriers. Biochim Biophys Acta 1746: 349–363.

36. ShifmanJM, ChoiMH, MihalasS, MayoSL, KennedyMB (2006) Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by calmodulin with two bound calciums. Proc Natl Acad Sci U S A 103: 13968–13973.

37. RogerE, GourbalB, GrunauC, PierceRJ, GalinierR, et al. (2008) Expression analysis of highly polymorphic mucin proteins (Sm PoMuc) from the parasite Schistosoma mansoni. Mol Biochem Parasitol 157: 217–227.

38. GobertGN, MoertelL, BrindleyPJ, McManusDP (2009) Developmental gene expression profiles of the human pathogen Schistosoma japonicum. BMC Genomics 10: 128.

39. SempleF, DorinJR (2012) beta-Defensins: multifunctional modulators of infection, inflammation and more? J Innate Immun 4: 337–348.

40. TarrDE (2012) Distribution and characteristics of ABFs, cecropins, nemapores, and lysozymes in nematodes. Dev Comp Immunol 36: 502–520.

41. KumagaiT, OsadaY, OhtaN, KanazawaT (2009) Peroxiredoxin-1 from Schistosoma japonicum functions as a scavenger against hydrogen peroxide but not nitric oxide. Mol Biochem Parasitol 164: 26–31.

42. SilbereisenA, TrittenL, KeiserJ (2011) Exploration of novel in vitro assays to study drugs against Trichuris spp. J Microbiol Methods 87: 169–175.

43. SmithRA, PontiggiaL, WatermanC, LichtenwalnerM, WassermanJ (2009) Comparison of motility, recovery, and methyl-thiazolyl-tetrazolium reduction assays for use in screening plant products for anthelmintic activity. Parasitol Res 105: 1339–1343.

44. SmoutMJ, KotzeAC, McCarthyJS, LoukasA (2010) A novel high throughput assay for anthelmintic drug screening and resistance diagnosis by real-time monitoring of parasite motility. PLoS Negl Trop Dis 4: e885.

45. PaxR, BennettJL, FettererR (1978) A benzodiazepine derivative and praziquantel: effects on musculature of Schistosoma mansoni and Schistosoma japonicum. Naunyn Schmiedebergs Arch Pharmacol 304: 309–315.

46. KimI, JeHD, GallantC, ZhanQ, RiperDV, et al. (2000) Ca2+-calmodulin-dependent protein kinase II-dependent activation of contractility in ferret aorta. J Physiol 526 Pt 2: 367–374.

47. DayTA, BennettJL, PaxRA (1994) Serotonin and its requirement for maintenance of contractility in muscle fibres isolated from Schistosoma mansoni. Parasitology 108(Pt 4): 425–432.

48. MaierLS, BersDM (2007) Role of Ca2+/calmodulin-dependent protein kinase (CaMK) in excitation-contraction coupling in the heart. Cardiovasc Res 73: 631–640.

49. GrueterCE, AbiriaSA, WuY, AndersonME, ColbranRJ (2008) Differential regulated interactions of calcium/calmodulin-dependent protein kinase II with isoforms of voltage-gated calcium channel beta subunits. Biochemistry 47: 1760–1767.

50. KohnAB, Roberts-MisterlyJM, AndersonPA, GreenbergRM (2003) Creation by mutagenesis of a mammalian Ca(2+) channel beta subunit that confers praziquantel sensitivity to a mammalian Ca(2+) channel. Int J Parasitol 33: 1303–1308.

51. KohnAD, MoonRT (2005) Wnt and calcium signaling: beta-catenin-independent pathways. Cell Calcium 38: 439–446.

52. KampTJ, HellJW (2000) Regulation of cardiac L-type calcium channels by protein kinase A and protein kinase C. Circ Res 87: 1095–1102.

53. KeefKD, HumeJR, ZhongJ (2001) Regulation of cardiac and smooth muscle Ca(2+) channels (Ca(V)1.2a,b) by protein kinases. Am J Physiol Cell Physiol 281: C1743–1756.

54. TokumitsuH, InuzukaH, IshikawaY, IkedaM, SajiI, et al. (2002) STO-609, a specific inhibitor of the Ca(2+)/calmodulin-dependent protein kinase kinase. J Biol Chem 277: 15813–15818.

55. AltschulSF, GishW, MillerW, MyersEW, LipmanDJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410.

56. Krautz-PetersonG, BhardwajR, FaghiriZ, TararamCA, SkellyPJ (2010) RNA interference in schistosomes: machinery and methodology. Parasitology 137: 485–495.

57. FreitasTC, JungE, PearceEJ (2007) TGF-beta signaling controls embryo development in the parasitic flatworm Schistosoma mansoni. PLoS Pathog 3: e52.

58. DuvoisinR, AyukMA, RinaldiG, SuttiprapaS, MannVH, et al. (2012) Human U6 promoter drives stronger shRNA activity than its schistosome orthologue in Schistosoma mansoni and human fibrosarcoma cells. Transgenic Res 21: 511–521.

59. RinaldiG, EckertSE, TsaiIJ, SuttiprapaS, KinesKJ, et al. (2012) Germline transgenesis and insertional mutagenesis in Schistosoma mansoni mediated by murine leukemia virus. PLoS Pathog 8: e1002820.

60. YouH, ZhangW, MoertelL, McManusDP, GobertGN (2009) Transcriptional profiles of adult male and female Schistosoma japonicum in response to insulin reveal increased expression of genes involved in growth and development. Int J Parasitol 39: 1551–1559.

61. LeopoldG, UngethumW, GrollE, DiekmannHW, NowakH, et al. (1978) Clinical pharmacology in normal volunteers of praziquantel, a new drug against schistosomes and cestodes. An example of a complex study covering both tolerance and pharmacokinetics. Eur J Clin Pharmacol 14: 281–291.

62. GobertGN, McInnesR, MoertelL, NelsonC, JonesMK, et al. (2006) Transcriptomics tool for the human Schistosoma blood flukes using microarray gene expression profiling. Exp Parasitol 114: 160–172.

63. LiuF, ChenP, CuiSJ, WangZQ, HanZG (2008) SjTPdb: integrated transcriptome and proteome database and analysis platform for Schistosoma japonicum. BMC Genomics 9: 304.

64. PengJ, HanH, GobertGN, HongY, JiangW, et al. (2011) Differential gene expression in Schistosoma japonicum schistosomula from Wistar rats and BALB/c mice. Parasit Vectors 4: 155.

65. LinD, ShkedyZ, YekutieliD, BurzykowskiT, GohlmannHW, et al. (2007) Testing for trends in dose-response microarray experiments: a comparison of several testing procedures, multiplicity and resampling-based inference. Stat Appl Genet Mol Biol 6: Article26.

66. PoundsSB (2006) Estimation and control of multiple testing error rates for microarray studies. Brief Bioinform 7: 25–36.

67. OgataH, GotoS, SatoK, FujibuchiW, BonoH, et al. (1999) KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res 27: 29–34.

68. The Schistosoma japonicum genome reveals features of host-parasite interplay. Nature 460: 345–351.

69. GobertGN, McManusDP, NawaratnaS, MoertelL, MulvennaJ, et al. (2009) Tissue specific profiling of females of Schistosoma japonicum by integrated laser microdissection microscopy and microarray analysis. PLoS Negl Trop Dis 3: e469.

70. MoralesME, RinaldiG, GobertGN, KinesKJ, TortJF, et al. (2008) RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade. Mol Biochem Parasitol 157: 160–168.

71. RinaldiG, MoralesME, AlrefaeiYN, CancelaM, CastilloE, et al. (2009) RNA interference targeting leucine aminopeptidase blocks hatching of Schistosoma mansoni eggs. Mol Biochem Parasitol 167: 118–126.

72. YouH, ZhangW, JonesMK, GobertGN, MulvennaJ, et al. (2010) Cloning and characterisation of Schistosoma japonicum insulin receptors. PLoS One 5: e9868.

73. ZaldPB, CotterMA2nd, RobertsonES (2000) Improved transfection efficiency of 293 cells by radio frequency electroporation. Biotechniques 28: 418, 420.

74. NdegwaD, Krautz-PetersonG, SkellyPJ (2007) Protocols for gene silencing in schistosomes. Exp Parasitol 117: 284–291.