The Genome of Highlights a Fish Pathogen Adapted to Fluctuating Environments


Spironucleus salmonicida causes systemic infections in salmonid fish. It belongs to the group diplomonads, binucleated heterotrophic flagellates adapted to micro-aerobic environments. Recently we identified energy-producing hydrogenosomes in S. salmonicida. Here we present a genome analysis of the fish parasite with a focus on the comparison to the more studied diplomonad Giardia intestinalis. We annotated 8067 protein coding genes in the ∼12.9 Mbp S. salmonicida genome. Unlike G. intestinalis, promoter-like motifs were found upstream of genes which are correlated with gene expression, suggesting a more elaborate transcriptional regulation. S. salmonicida can utilise more carbohydrates as energy sources, has an extended amino acid and sulfur metabolism, and more enzymes involved in scavenging of reactive oxygen species compared to G. intestinalis. Both genomes have large families of cysteine-rich membrane proteins. A cluster analysis indicated large divergence of these families in the two diplomonads. Nevertheless, one of S. salmonicida cysteine-rich proteins was localised to the plasma membrane similar to G. intestinalis variant-surface proteins. We identified S. salmonicida homologs to cyst wall proteins and showed that one of these is functional when expressed in Giardia. This suggests that the fish parasite is transmitted as a cyst between hosts. The extended metabolic repertoire and more extensive gene regulation compared to G. intestinalis suggest that the fish parasite is more adapted to cope with environmental fluctuations. Our genome analyses indicate that S. salmonicida is a well-adapted pathogen that can colonize different sites in the host.


Vyšlo v časopise: The Genome of Highlights a Fish Pathogen Adapted to Fluctuating Environments. PLoS Genet 10(2): e32767. doi:10.1371/journal.pgen.1004053
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004053

Souhrn

Spironucleus salmonicida causes systemic infections in salmonid fish. It belongs to the group diplomonads, binucleated heterotrophic flagellates adapted to micro-aerobic environments. Recently we identified energy-producing hydrogenosomes in S. salmonicida. Here we present a genome analysis of the fish parasite with a focus on the comparison to the more studied diplomonad Giardia intestinalis. We annotated 8067 protein coding genes in the ∼12.9 Mbp S. salmonicida genome. Unlike G. intestinalis, promoter-like motifs were found upstream of genes which are correlated with gene expression, suggesting a more elaborate transcriptional regulation. S. salmonicida can utilise more carbohydrates as energy sources, has an extended amino acid and sulfur metabolism, and more enzymes involved in scavenging of reactive oxygen species compared to G. intestinalis. Both genomes have large families of cysteine-rich membrane proteins. A cluster analysis indicated large divergence of these families in the two diplomonads. Nevertheless, one of S. salmonicida cysteine-rich proteins was localised to the plasma membrane similar to G. intestinalis variant-surface proteins. We identified S. salmonicida homologs to cyst wall proteins and showed that one of these is functional when expressed in Giardia. This suggests that the fish parasite is transmitted as a cyst between hosts. The extended metabolic repertoire and more extensive gene regulation compared to G. intestinalis suggest that the fish parasite is more adapted to cope with environmental fluctuations. Our genome analyses indicate that S. salmonicida is a well-adapted pathogen that can colonize different sites in the host.


Zdroje

1. AdlSM, SimpsonAG, LaneCE, LukesJ, BassD, et al. (2012) The revised classification of eukaryotes. J Eukaryot Microbiol 59: 429–514.

2. Brugerolle G, Lee JJ (2002) Order Diplomonadida. In: Lee JJ, Leedale GF, Bradbury P, editors. An Illustrated Guide to the Protozoa, 2nd edn. Lawrence, Kansas: Society of Protozoologists. pp. 1125–1135.

3. TovarJ, León-AvilaG, SánchezLB, SutakR, TachezyJ, et al. (2003) Mitochondrial remnant organelles of Giardia function in iron-sulphur protein maturation. Nature 426: 172–176.

4. Jerlström-HultqvistJ, EinarssonE, XuF, HjortK, EkB, et al. (2013) Hydrogenosomes in the diplomonad Spironucleus salmonicida. Nat Commun 4: 2493.

5. RameshMA, MalikSB, LogsdonJMJr (2005) A phylogenomic inventory of meiotic genes; evidence for sex in Giardia and an early eukaryotic origin of meiosis. Curr Biol 15: 185–191.

6. CooperMA, AdamRD, WorobeyM, SterlingCR (2007) Population genetics provides evidence for recombination in Giardia. Curr Biol 17: 1984–1988.

7. AnderssonJO (2012) Double peaks reveal rare diplomonad sex. Trends Parasitol 28: 46–52.

8. AnderssonJO, SjögrenÅM, HornerDS, MurphyCA, DyalPL, et al. (2007) A genomic survey of the fish parasite Spironucleus salmonicida indicates genomic plasticity among diplomonads and significant lateral gene transfer in eukaryote genome evolution. BMC Genomics 8: 51.

9. AnderssonJO, SjögrenÅM, DavisLAM, EmbleyTM, RogerAJ (2003) Phylogenetic analyses of diplomonad genes reveal frequent lateral gene transfers affecting eukaryotes. Curr Biol 13: 94–104.

10. MorrisonHG, McArthurAG, GillinFD, AleySB, AdamRD, et al. (2007) Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science 317: 1921–1926.

11. UpcroftJ, UpcroftP (1998) My favorite cell: Giardia. Bioessays 20: 256–263.

12. AdamRD (2001) Biology of Giardia lamblia. Clin Microbiol Rev 14: 447–475.

13. AnkarklevJ, Jerlström-HultqvistJ, RingqvistE, TroellK, SvärdSG (2010) Behind the smile: cell biology and disease mechanisms of Giardia species. Nat Rev Microbiol 8: 413–422.

14. WilliamsCF, LloydD, PoyntonSL, JorgensenA, MilletCOM, et al. (2011) Spironucleus species: economically-important fish pathogens and enigmatic single-celled eukaryotes. J Aquac Res Development S2.

15. KoliskoM, CepickaI, HamplV, LeighJ, RogerAJ, et al. (2008) Molecular phylogeny of diplomonads and enteromonads based on SSU rRNA, alpha-tubulin and HSP90 genes: implications for the evolutionary history of the double karyomastigont of diplomonads. BMC Evol Biol 8: 205.

16. KentML, EllisJ, FournieJW, DaweSC, BagshawJW, et al. (1992) Systemic hexamitid (Protozoa, Diplomonadida) infection in seawater pen-reared Chinook salmon Oncorhynchus tshawytscha. Dis Aquat Organ 14: 81–89.

17. JørgensenA, SterudE (2006) The marine pathogenic genotype of Spironucleus barkhanus from farmed salmonids redescribed as Spironucleus salmonicida n. sp. J Eukaryot Microbiol 53: 531–541.

18. Roxström-LindquistK, Jerlström-HultqvistJ, JørgensenA, TroellK, SvärdSG, et al. (2010) Large genomic differences between the morphologically indistinguishable diplomonads Spironucleus barkhanus and Spironucleus salmonicida. BMC Genomics 11: 258.

19. JørgensenA, TorpK, BjorlandMA, PoppeTT (2011) Wild arctic char Salvelinus alpinus and trout Salmo trutta: hosts and reservoir of the salmonid pathogen Spironucleus salmonicida (Diplomonadida; Hexamitidae). Dis Aquat Organ 97: 57–63.

20. Jerlström-HultqvistJ, EinarssonE, SvärdSG (2012) Stable transfection of the diplomonad parasite Spironucleus salmonicida. Eukaryot Cell 11: 1353–1361.

21. FranzénO, Jerlström-HultqvistJ, CastroE, SherwoodE, AnkarklevJ, et al. (2009) Draft genome sequencing of Giardia intestinalis assemblage B isolate GS: are human giardiasis caused by two different species? PLoS Pathog 5 (8) e1000560.

22. Jerlström-HultqvistJ, FranzénO, AnkarklevJ, XuF, NohynkovaE, et al. (2010) Genome analysis and comparative genomics of a Giardia intestinalis assemblage E isolate. BMC Genomics 11: 543.

23. KeelingPJ, DoolittleWF (1997) Widespread and ancient distribution of a noncanonical genetic code in diplomonads. Mol Biol Evol 14: 895–901.

24. ManningG, ReinerDS, LauwaetT, DacreM, SmithA, et al. (2011) The minimal kinome of Giardia lamblia illuminates early kinase evolution and unique parasite biology. Genome Biol 12: R66.

25. O'ConnellMJ, KrienMJ, HunterT (2003) Never say never. The NIMA-related protein kinases in mitotic control. Trends Cell Biol 13: 221–228.

26. CollinsL, PennyD (2005) Complex spliceosomal organization ancestral to extant eukaryotes. Mol Biol Evol 22: 1053–1066.

27. FranzenO, Jerlström-HultqvistJ, EinarssonE, AnkarklevJ, FerellaM, et al. (2013) Transcriptome profiling of Giardia intestinalis using strand-specific RNA-seq. PLoS Comput Biol 9: e1003000.

28. KamikawaR, InagakiY, TokoroM, RogerAJ, HashimotoT (2011) Split introns in the genome of Giardia intestinalis are excised by spliceosome-mediated trans-splicing. Curr Biol 21: 311–315.

29. CarltonJM, HirtRP, SilvaJC, DelcherAL, SchatzM, et al. (2007) Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis. Science 315: 207–212.

30. PetersenTN, BrunakS, von HeijneG, NielsenH (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8: 785–786.

31. AkopianD, ShenK, ZhangX, ShanSO (2013) Signal recognition particle: an essential protein-targeting machine. Annu Rev Biochem 82: 693–721.

32. TeodorovicS, WallsCD, ElmendorfHG (2007) Bidirectional transcription is an inherent feature of Giardia lamblia promoters and contributes to an abundance of sterile antisense transcripts throughout the genome. Nucleic Acids Res 35: 2544–2553.

33. BestAA, MorrisonHG, McArthurAG, SoginML, OlsenGJ (2004) Evolution of eukaryotic transcription: insights from the genome of Giardia lamblia. Genome Res 14: 1537–1547.

34. IyerLM, AnantharamanV, WolfMY, AravindL (2008) Comparative genomics of transcription factors and chromatin proteins in parasitic protists and other eukaryotes. Int J Parasitol 38: 1–31.

35. FuentesV, BarreraG, SanchezJ, HernandezR, Lopez-VillasenorI (2012) Functional analysis of sequence motifs involved in the polyadenylation of Trichomonas vaginalis mRNAs. Eukaryot Cell 11: 725–734.

36. WilliamsCF, VaccaAR, LloydD, SchelkleB, CableJ (2013) Non-invasive investigation of Spironucleus vortens transmission in freshwater angelfish Pterophyllum scalare. Dis Aquat Organ 105: 211–223.

37. JanuschkaMM, ErlandsenSL, BemrickWJ, SchuppDG, FeelyDE (1988) A comparison of Giardia microti and Spironucleus muris cysts in the vole: an immunocytochemical, light, and electron microscopic study. J Parasitol 74: 452–458.

38. WoodAM, SmithHV (2005) Spironucleosis (Hexamitiasis, Hexamitosis) in the ring-necked pheasant (Phasianus colchicus): detection of cysts and description of Spironucleus meleagridis in stained smears. Avian Dis 49: 138–143.

39. MorfL, SpycherC, RehrauerH, FournierCA, MorrisonHG, et al. (2010) The transcriptional response to encystation stimuli in Giardia lamblia is restricted to a small set of genes. Eukaryot Cell 9: 1566–1576.

40. KonradC, SpycherC, HehlAB (2010) Selective condensation drives partitioning and sequential secretion of cyst wall proteins in differentiating Giardia lamblia. PLoS Pathog 6: e1000835.

41. RawlingsND, MortonFR, KokCY, KongJ, BarrettAJ (2008) MEROPS: the peptidase database. Nucleic Acids Res 36: D320–325.

42. SajidM, McKerrowJH (2002) Cysteine proteases of parasitic organ. Mol Biochem Parasitol 120: 1–21.

43. AdamRD, NigamA, SeshadriV, MartensCA, FarnethGA, et al. (2010) The Giardia lamblia vsp gene repertoire: characteristics, genomic organization, and evolution. BMC Genomics 11: 424.

44. NashTE, BanksSM, AllingDW, MerrittJWJr, ConradJT (1990) Frequency of variant antigens in Giardia lamblia. Exp Parasitol 71: 415–421.

45. PagnyS, LerougeP, FayeL, GomordV (1999) Signals and mechanisms for protein retention in the endoplasmic reticulum. J Exp Bot 50: 157–164.

46. DavidsBJ, ReinerDS, BirkelandSR, PreheimSP, CiprianoMJ, et al. (2006) A new family of giardial cysteine-rich non-VSP protein genes and a novel cyst protein. PLoS ONE 1: e44.

47. PruccaCG, SlavinI, QuirogaR, EliasEV, RiveroFD, et al. (2008) Antigenic variation in Giardia lamblia is regulated by RNA interference. Nature 456: 750–754.

48. NixonJEJ, WangA, FieldJ, MorrisonHG, McArthurAG, et al. (2002) Evidence for lateral transfer of genes encoding ferredoxins, nitroreductases, NADH oxidase, and alcohol dehydrogenase 3 from anaerobic prokaryotes to Giardia lamblia and Entamoeba histolytica. Eukaryot Cell 1: 181–190.

49. LawCJ, MaloneyPC, WangDN (2008) Ins and outs of major facilitator superfamily antiporters. Annu Rev Microbiol 62: 289–305.

50. DavidsonAL, DassaE, OrelleC, ChenJ (2008) Structure, function, and evolution of bacterial ATP-binding cassette systems. Microbiol Mol Biol Rev 72: 317–364.

51. YoungGB, JackDL, SmithDW, SaierMHJr (1999) The amino acid/auxin: proton symport permease family. Biochim Biophys Acta 1415: 306–322.

52. SchofieldPJ, CostelloM, EdwardsMR, O'SullivanWJ (1990) The arginine dihydrolase pathway is present in Giardia intestinalis. Int J Parasitol 20: 697–699.

53. YarlettN, MartinezMP, MoharramiMA, TachezyJ (1996) The contribution of the arginine dihydrolase pathway to energy metabolism by Trichomonas vaginalis. Mol Biochem Parasitol 78: 117–125.

54. LacourciereGM, MiharaH, KuriharaT, EsakiN, StadtmanTC (2000) Escherichia coli NifS-like proteins provide selenium in the pathway for the biosynthesis of selenophosphate. J Biol Chem 275: 23769–23773.

55. LacourciereGM (2002) Selenium is mobilized in vivo from free selenocysteine and is incorporated specifically into formate dehydrogenase H and tRNA nucleosides. J Bacteriol 184: 1940–1946.

56. HaftDH, SelfWT (2008) Orphan SelD proteins and selenium-dependent molybdenum hydroxylases. Biol Direct 3: 4.

57. ZhangY, TuranovAA, HatfieldDL, GladyshevVN (2008) In silico identification of genes involved in selenium metabolism: evidence for a third selenium utilization trait. BMC Genomics 9: 251.

58. Kopriva S, Patron NJ, Keeling P, Leustek T (2008) Phylogenetic analysis of sulfate assimilation and cysteine biosynthesis in phototrophic organisms. In: Hell R, Dahl C, Knaff DB, Leustek T, editors. Sulfur Metabolism in Phototrophic Organisms: Springer Netherlands. pp. 31–58.

59. TakahashiH, KoprivaS, GiordanoM, SaitoK, HellR (2011) Sulfur assimilation in photosynthetic organisms: molecular functions and regulations of transporters and assimilatory enzymes. Annu Rev Plant Biol 62: 157–184.

60. HérbertA, CasaregolaS, BeckerichJ-M (2011) Biodiversity in sulfurmetabolism in hemiascomycetous yeasts. FEMS Yeast Res 11: 366–378.

61. PayneSH, LoomisWF (2006) Retention and loss of amino acid biosynthetic pathways based on analysis of whole-genome sequences. Eukaryot Cell 5: 272–276.

62. MaK, AdamsMWW (1994) Sulfide dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus: a new multifunctional enzyme involved in the reduction of elemental sulfur. J Bacteriol 176: 6509–6517.

63. HagenWR, SilvaPJ, AmorimMA, HagedoornP-L, WassinkH, et al. (2000) Novel structure and redox chemistry of the prosthetic groups of the iron-sulfur flavoprotein sulfide dehydrogenase from Pyrococcus furiosus; evidence for a [2Fe-2S] cluster with Asp(Cys)3 ligands. J Biol Inorg Chem 5: 527–534.

64. AnderssonJO, RogerAJ (2002) Evolutionary analyses of the small subunit of glutamate synthase: gene order conservation, gene fusions and prokaryote-to-eukaryote lateral gene transfers. Eukaryot Cell 1: 304–310.

65. BridgerSL, ClarksonSM, StirrettK, DeBarryMB, LipscombGL, et al. (2011) Deletion strains reveal metabolic roles for key elemental sulfur-responsive proteins in Pyrococcus furiosus. J Bacteriol 193: 6498–6504.

66. LiuY, BeerLL, WhitmanWB (2012) Sulfur metabolism in archaea reveals novel processes. Environ Microbiol 14: 2632–2644.

67. BrownDM, UpcroftJA, UpcroftP (1995) Free radical detoxification in Giardia duodenalis. Mol Biochem Parasitol 72: 47–56.

68. BrownDM, UpcroftJA, UpcroftP (1996) A H2O-producing NADH oxidase from the protozoan parasite Giardia duodenalis. Eur J Biochem 241: 155–161.

69. TestaF, MastronicolaD, CabelliDE, BordiE, PucilloLP, et al. (2011) The superoxide reductase from the early diverging eukaryote Giardia intestinalis. Free Radic Biol Med 51: 1567–1574.

70. VicenteJB, TestaF, MastronicolaD, ForteE, SartiP, et al. (2009) Redox properties of the oxygen-detoxifying flavodiiron protein from the human parasite Giardia intestinalis. Arch Biochem Biophys 488: 9–13.

71. MilletCO, CableJ, LloydD (2010) The diplomonad fish parasite Spironucleus vortens produces hydrogen. J Eukaryot Microbiol 57: 400–404.

72. BabulaP, MasarikM, AdamV, EckschlagerT, StiborovaM, et al. (2012) Mammalian metallothioneins: properties and functions. Metallomics 4: 739–750.

73. AnderssonJO, HirtRP, FosterPG, RogerAJ (2006) Evolution of four gene families with patchy phylogenetic distribution: influx of genes into protist genomes. BMC Evol Biol 6: 27.

74. MastronicolaD, TestaF, ForteE, BordiE, PucilloLP, et al. (2010) Flavohemoglobin and nitric oxide detoxification in the human protozoan parasite Giardia intestinalis. Biochem Biophys Res Commun 399: 654–658.

75. Jerlström-HultqvistJ, StadelmannB, BirkestedtS, HellmanU, SvärdSG (2012) Plasmid vectors for proteomic analyses in Giardia: purification of virulence factors and analysis of the proteasome. Eukaryot Cell 11: 864–873.

76. MillerJR, DelcherAL, KorenS, VenterE, WalenzBP, et al. (2008) Aggressive assembly of pyrosequencing reads with mates. Bioinformatics 24: 2818–2824.

77. LiH, DurbinR (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25: 1754–1760.

78. GardnerPP, DaubJ, TateJ, MooreBL, OsuchIH, et al. (2011) Rfam: Wikipedia, clans and the “decimal” release. Nucleic Acids Res 39: D141–145.

79. NawrockiEP, KolbeDL, EddySR (2009) Infernal 1.0: inference of RNA alignments. Bioinformatics 25: 1335–1337.

80. ConsortiumU (2012) Reorganizing the protein space at the Universal Protein Resource (UniProt). Nucleic Acids Res 40: D71–75.

81. LiH, HandsakerB, WysokerA, FennellT, RuanJ, et al. (2009) The Sequence Alignment/Map format and SAMtools. Bioinformatics 25: 2078–2079.

82. HaasBJ, SalzbergSL, ZhuW, PerteaM, AllenJE, et al. (2008) Automated eukaryotic gene structure annotation using EVidenceModeler and the Program to Assemble Spliced Alignments. Genome Biol 9: R7.

83. MajorosWH, PerteaM, SalzbergSL (2004) TigrScan and GlimmerHMM: two open source ab initio eukaryotic gene-finders. Bioinformatics 20: 2878–2879.

84. HyattD, ChenGL, LocascioPF, LandML, LarimerFW, et al. (2010) Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics 11: 119.

85. DelcherAL, BratkeKA, PowersEC, SalzbergSL (2007) Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23: 673–679.

86. FinnRD, MistryJ, TateJ, CoggillP, HegerA, et al. (2010) The Pfam protein families database. Nucleic Acids Res 38: D211–222.

87. SelengutJD, HaftDH, DavidsenT, GanapathyA, Gwinn-GiglioM, et al. (2007) TIGRFAMs and Genome Properties: tools for the assignment of molecular function and biological process in prokaryotic genomes. Nucleic Acids Res 35: D260–264.

88. UniProtC (2012) Reorganizing the protein space at the Universal Protein Resource (UniProt). Nucleic Acids Res 40: D71–75.

89. RutherfordK, ParkhillJ, CrookJ, HorsnellT, RiceP, et al. (2000) Artemis: sequence visualization and annotation. Bioinformatics 16: 944–945.

90. AurrecoecheaC, BrestelliJ, BrunkBP, CarltonJM, DommerJ, et al. (2009) GiardiaDB and TrichDB: integrated genomic resources for the eukaryotic protist pathogens Giardia lamblia and Trichomonas vaginalis. Nucleic Acids Res 37: D526–530.

91. LiL, StoeckertCJJr, RoosDS (2003) OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res 13: 2178–2189.

92. CarverTJ, RutherfordKM, BerrimanM, RajandreamMA, BarrellBG, et al. (2005) ACT: the Artemis Comparison Tool. Bioinformatics 21: 3422–3423.

93. SchattnerP, BrooksAN, LoweTM (2005) The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res 33: W686–689.

94. LagesenK, HallinP, RodlandEA, StaerfeldtHH, RognesT, et al. (2007) RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res 35: 3100–3108.

95. BaileyTL, ElkanC (1994) Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol 2: 28–36.

96. TrapnellC, WilliamsBA, PerteaG, MortazaviA, KwanG, et al. (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28: 511–515.

97. EdgarRC (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5: 113.

98. Beauregard-RacineJ, BicepC, SchliepK, LopezP, LapointeFJ, et al. (2011) Of woods and webs: possible alternatives to the tree of life for studying genomic fluidity in E. coli. Biol Direct 6: 39.

99. MoriyaY, ItohM, OkudaS, YoshizawaAC, KanehisaM (2007) KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res 35: W182–185.

100. KroghA, LarssonB, von HeijneG, SonnhammerEL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305: 567–580.

101. WaterhouseAM, ProcterJB, MartinDM, ClampM, BartonGJ (2009) Jalview Version 2 - a multiple sequence alignment editor and analysis workbench. Bioinformatics 25: 1189–1191.

Štítky
Genetika Reprodukčná medicína

Článok vyšiel v časopise

PLOS Genetics


2014 Číslo 2
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Eozinofilní granulomatóza s polyangiitidou
nový kurz
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa