Ras1 Acts through Duplicated Cdc42 and Rac Proteins to Regulate Morphogenesis and Pathogenesis in the Human Fungal Pathogen

English version České info

Proliferation and morphogenesis in eukaryotic cells depend on the concerted activity of Rho-type GTPases, including Ras, Cdc42, and Rac. The sexually dimorphic fungus Cryptococcus neoformans, which encodes paralogous, non-essential copies of all three, provides a unique model in which to examine the interactions of these conserved proteins. Previously, we demonstrated that RAS1 mediates C. neoformans virulence by acting as a central regulator of both thermotolerance and mating. We report here that ras1Δ mutants accumulate defects in polarized growth, cytokinesis, and cell cycle progression. We demonstrate that the ras1Δ defects in thermotolerance and mating can be largely explained by the compromised activity of four downstream Rho-GTPases: the Cdc42 paralogs, Cdc42 and Cdc420; and the Rac paralogs, Rac1 and Rac2. Further, we demonstrate that the separate GTPase classes play distinct Ras-dependent roles in C. neoformans morphogenesis and pathogenesis. Cdc42 paralogs primarily control septin localization and cytokinesis, while Rac paralogs play a primary role in polarized cell growth. Together, these duplicate, related signaling proteins provide a robust system to allow microbial proliferation in the presence of host-derived cell stresses.

Vyšlo v časopise: Ras1 Acts through Duplicated Cdc42 and Rac Proteins to Regulate Morphogenesis and Pathogenesis in the Human Fungal Pathogen. PLoS Genet 9(8): e32767. doi:10.1371/journal.pgen.1003687
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003687

Souhrn

Zdroje

1. Pylayeva-GuptaY, GrabockaE, Bar-SagiD (2011) RAS oncogenes: weaving a tumorigenic web. Nature Reviews Cancer 11: 761–774.

2. AlspaughJA, CavalloLM, PerfectJR, HeitmanJ (2000) RAS1 regulates filamentation, mating and growth at high temperature of Cryptococcus neoformans. Molecular Microbiology 36: 352–365.

3. FengQ, SummersE, GuoB, FinkG (1999) Ras signaling is required for serum-induced hyphal differentiation in Candida albicans. Journal of bacteriology 181: 6339–6346.

4. FortwendelJR, FullerKK, StephensTJ, BaconWC, AskewDS, et al. (2008) Aspergillus fumigatus RasA regulates asexual development and cell wall integrity. Eukaryotic Cell 7: 1530–1539.

5. GimenoCJ, LjungdahlPO, StylesCA, FinkGR (1992) Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell 68: 1077–1090.

6. ShinI, KimS, SongH, KimH-RC, MoonA (2005) H-Ras-specific activation of Rac-MKK3/6-p38 pathway: its critical role in invasion and migration of breast epithelial cells. The Journal of Biological Chemistry 280: 14675–14683.

7. ChengC-M, LiH, GasmanS, HuangJ, SchiffR, et al. (2011) Compartmentalized Ras proteins transform NIH 3T3 cells with different efficiencies. Molecular and Cellular Biology 31: 983–997.

8. QiuRG, AboA, McCormickF, SymonsM (1997) Cdc42 regulates anchorage-independent growth and is necessary for Ras transformation. Molecular and Cellular Biology 17: 3449–3458.

9. QiuRG, ChenJ, KirnD, McCormickF, SymonsM (1995) An essential role for Rac in Ras transformation. Nature 374: 457–459.

10. WangY, XuHP, RiggsM, RodgersL, WiglerMH (1991) byr2, a Schizosaccharomyces pombe gene encoding a protein kinase capable of partial suppression of the ras1 mutant phenotype. Molecular and Cellular Biology 11: 3554–3563.

11. OnkenB, WienerH, PhilipsMR, ChangEC (2006) Compartmentalized signaling of Ras in fission yeast. Proceedings of the National Academy of Sciences of the United States of America 103: 9045–9050.

12. NicholsCB, FerreyraJ, BallouER, AlspaughJA (2009) Subcellular localization directs signaling specificity of the Cryptococcus neoformans Ras1 protein. Eukaryotic Cell 8: 181–189.

13. PanX, HeitmanJ (1999) Cyclic AMP-dependent protein kinase regulates pseudohyphal differentiation in Saccharomyces cerevisiae. Molecular and Cellular Biology 19: 4874–4887.

14. MöschHU, RobertsRL, FinkGR (1996) Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the United States of America 93: 5352–5356.

15. ZhuY, FangH-M, WangY-M, ZengG-S, ZhengX-D, et al. (2009) Ras1 and Ras2 play antagonistic roles in regulating cellular cAMP level, stationary-phase entry and stress response in Candida albicans. Molecular Microbiology 74: 862–875.

16. ChenC, DickmanMB (2004) Dominant active Rac and dominant negative Rac revert the dominant active Ras phenotype in Colletotrichum trifolii by distinct signalling pathways. Molecular Microbiology 51: 1493–1507.

17. NicholsCB, PerfectZH, AlspaughJA (2007) A Ras1-Cdc24 signal transduction pathway mediates thermotolerance in the fungal pathogen Cryptococcus neoformans. Molecular Microbiology 63: 1118–1130.

18. BoyceKJ, HynesMJ, AndrianopoulosA (2005) The Ras and Rho GTPases genetically interact to co-ordinately regulate cell polarity during development in Penicillium marneffei. Molecular Microbiology 55: 1487–1501.

19. BallouER, NicholsCB, MigliaKJ, KozubowskiL, AlspaughJA (2010) Two CDC42 paralogues modulate Cryptococcus neoformans thermotolerance and morphogenesis under host physiological conditions. Molecular Microbiology 75: 763–780.

20. BassilanaM, ArkowitzRA (2006) Rac1 and Cdc42 have different roles in Candida albicans development. Eukaryotic Cell 5: 321–329.

21. WaughMS, NicholsCB, DeCesareCM, CoxGM, HeitmanJ, et al. (2002) Ras1 and Ras2 contribute shared and unique roles in physiology and virulence of Cryptococcus neoformans. Microbiology (Reading, England) 148: 191–201.

22. VallimMA, NicholsCB, FernandesL, CramerKL, AlspaughJA (2005) A Rac homolog functions downstream of Ras1 to control hyphal differentiation and high-temperature growth in the pathogenic fungus Cryptococcus neoformans. Eukaryotic Cell 4: 1066–1078.

23. BallouER, SelvigK, NarlochJL, NicholsC, AlspaughA (2013) Two Rac paralogs regulate polarized growth in the human fungal pathogen Cryptococcus neoformans. Fungal Genetics and Biology DOI:10.1016/j.fgb.2013.05.006

24. LebererE, HarcusD, DignardD, JohnsonL, UshinskySC, et al. (2001) Ras links cellular morphogenesis to virulence by regulation of the MAP kinase and cAMP signalling pathways in the pathogenic fungus Candida albicans. Molecular Microbiology 42: 673–687.

25. LeeN, KronstadJW (2002) ras2 Controls morphogenesis, pheromone response, and pathogenicity in the fungal pathogen Ustilago maydis. Eukaryotic Cell 1: 954–966.

26. FortwendelJR, ZhaoW, BhabhraR, ParkS, PerlinDS, et al. (2005) A fungus-specific ras homolog contributes to the hyphal growth and virulence of Aspergillus fumigatus. Eukaryotic Cell 4: 1982–1989.

27. PiispanenAE, BonnefoiO, CardenS, DeveauA, BassilanaM, et al. (2011) Roles of Ras1 membrane localization during Candida albicans hyphal growth and farnesol response. Eukaryotic Cell 10: 1473–1484.

28. WaughMS, VallimMA, HeitmanJ, AlspaughJA (2003) Ras1 controls pheromone expression and response during mating in Cryptococcus neoformans. Fungal Genetics and Biology 38: 110–121.

29. LoftusBJ, FungE, RoncagliaP, RowleyD, AmedeoP, et al. (2005) The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans. Science 307: 1321–1324.

30. ShenG, ZhouE, AlspaughJA, WangP (2012) Wsp1 is downstream of Cin1 and regulates vesicle transport and actin cytoskeleton as an effector of Cdc42 and Rac1 in Cryptococcus neoformans. Eukaryotic Cell 11: 471–481.

31. GaoY, XingJ, StreuliM, LetoTL, ZhengY (2001) Trp(56) of rac1 specifies interaction with a subset of guanine nucleotide exchange factors. The Journal of Biological Chemistry 276: 47530–47541.

32. RittingerK, WalkerPA, EcclestonJF, NurmahomedK, OwenD, et al. (1997) Crystal structure of a small G protein in complex with the GTPase-activating protein rhoGAP. Nature 388: 693–697.

33. EkenaJL, StantonBC, Schiebe-OwensJA, HullCM (2008) Sexual development in Cryptococcus neoformans requires CLP1, a target of the homeodomain transcription factors Sxi1alpha and Sxi2a. Eukaryotic Cell 7: 49–57.

34. BenardV, BohlBP, BokochGM (1999) Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. The Journal of Biological Chemistry 274: 13198–13204.

35. KimJ-S, DieboldBA, BabiorBM, KnausUG, BokochGM (2007) Regulation of Nox1 activity via protein kinase A-mediated phosphorylation of NoxA1 and 14-3-3 binding. The Journal of Biological Chemistry 282: 34787–34800.

36. GladfelterAS, PringleJR, LewDJ (2001) The septin cortex at the yeast mother-bud neck. Current opinion in microbiology 4: 681–689.

37. KozubowskiL, HeitmanJ (2010) Septins enforce morphogenetic events during sexual reproduction and contribute to virulence of Cryptococcus neoformans. Molecular Microbiology 75: 658–675.

38. LewDJ, ReedSI (1995) A cell cycle checkpoint monitors cell morphogenesis in budding yeast. The Journal of Cell Biology 129: 739–749.

39. LinX, PatelS, LitvintsevaAP, FloydA, MitchellTG, et al. (2009) Diploids in the Cryptococcus neoformans serotype A population homozygous for the alpha mating type originate via unisexual mating. PLoS Pathogens 5: e1000283.

40. BallouER, AlspaughJ, NicholsCB (2012) Morphogenesis of Cryptococcus neoformans. Morphogenesis and Pathogenicity in Fungi, Topics in Current Genetics 22: 197–223.

41. JohnsonJM, JinM, LewDJ (2011) Symmetry breaking and the establishment of cell polarity in budding yeast. Current Opinion in Genetics and Development 21: 740–746.

42. BadalyanSM, PolakE, HermannR, AebiM, KüesU (2004) Role of peg formation in clamp cell fusion of homobasidiomycete fungi. Journal of Basic Microbiology 44: 167–177.

43. Kwon-ChungKJ, PopkinTJ (1976) Ultrastructure of septal complex in Filobasidiella neoformans (Cryptococcus neoformans). Journal of Bacteriology 126: 524–528.

44. WickesBL, MayorgaME, EdmanU, EdmanJC (1996) Dimorphism and haploid fruiting in Cryptococcus neoformans: association with the alpha-mating type. Proceedings of the National Academy of Sciences of the United States of America 93: 7327–7331.

45. ZhengY, BenderA, CerioneRA (1995) Interactions among proteins involved in bud-site selection and bud-site assembly in Saccharomyces cerevisiae. The Journal of Biological Chemistry 270: 626–630.

46. MöschHU, KüblerE, KrappmannS, FinkGR, BrausGH (1999) Crosstalk between the Ras2p-controlled mitogen-activated protein kinase and cAMP pathways during invasive growth of Saccharomyces cerevisiae. Molecular Biology of the Cell 10: 1325–1335.

47. HoffmanCS, WinstonF (1991) Glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene occurs by a cAMP signaling pathway. Genes & Development 5: 561–571.

48. MillerPJ, JohnsonDI (1994) Cdc42p GTPase Is Involved in Controlling Polarized Cell Growth in Schizosaccharomyces pombe. Molecular and Cellular Biology 14: 1075–1083.

49. LiYC, ChenCR, ChangEC (2000) Fission yeast Ras1 effector Scd1 interacts with the spindle and affects its proper formation. Genetics 156: 995–1004.

50. HopeH, SchmauchC, ArkowitzRA, BassilanaM (2010) The Candida albicans ELMO homologue functions together with Rac1 and Dck1, upstream of the MAP Kinase Cek1, in invasive filamentous growth. Molecular Microbiology 76: 1572–1590.

51. GowNAR, van de VeerdonkFL, BrownAJP, NeteaMG (2012) Candida albicans morphogenesis and host defence: discriminating invasion from colonization. Nature Reviews Microbiology 10: 112–122.

52. HudsonDA, SciasciaQL, SandersRJ, NorrisGE, EdwardsPJB, et al. (2004) Identification of the dialysable serum inducer of germ-tube formation in Candida albicans. Microbiology (Reading, England) 150: 3041–3049.

53. VandenBergAL, IbrahimAS, EdwardsJE, ToenjesKA, JohnsonDI (2004) Cdc42p GTPase regulates the budded-to-hyphal-form transition and expression of hypha-specific transcripts in Candida albicans. Eukaryotic Cell 3: 724–734.

54. MartinR, WaltherA, WendlandJ (2005) Ras1-induced hyphal development in Candida albicans requires the formin Bni1. Eukaryotic Cell 4: 1712–1724.

55. UshinskySC, HarcusD, AshJ, DignardD, MarcilA (2002) CDC42 Is Required for Polarized Growth in Human Pathogen Candida albicans. Eukaryotic Cell 1: 95–104.

56. MahlertM, LevelekiL, HlubekA, SandrockB, BölkerM (2006) Rac1 and Cdc42 regulate hyphal growth and cytokinesis in the dimorphic fungus Ustilago maydis. Molecular Microbiology 59: 567–578.

57. ViragA, LeeMP, SiH, HarrisSD (2007) Regulation of hyphal morphogenesis by cdc42 and rac1 homologues in Aspergillus nidulans. Molecular Microbiology 66: 1579–1596.

58. WangP, NicholsCB, LengelerKB, CardenasME, CoxGM, et al. (2002) Mating-type-specific and nonspecific PAK kinases play shared and divergent roles in Cryptococcus neoformans. Eukaryotic Cell 1: 257–272.

59. SemighiniCP, HarrisSD (2008) Regulation of apical dominance in Aspergillus nidulans hyphae by reactive oxygen species. Genetics 179: 1919–1932.

60. TanakaA, TakemotoD, HyonG-S, ParkP, ScottB (2008) NoxA activation by the small GTPase RacA is required to maintain a mutualistic symbiotic association between Epichloë festucae and perennial ryegrass. Molecular Microbiology 68: 1165–1178.

61. RolkeY, TudzynskiP (2008) The small GTPase Rac and the p21-activated kinase Cla4 in Claviceps purpurea: interaction and impact on polarity, development and pathogenicity. Molecular Microbiology 68: 405–423.

62. LewDJ, ReedSI (1993) Morphogenesis in the yeast cell cycle: regulation by Cdc28 and cyclins. The Journal of Cell Biology 120: 1305–1320.

63. HowellAS, LewDJ (2012) Morphogenesis and the cell cycle. Genetics 190: 51–77.

64. SionovE, LeeH, ChangYC, Kwon-ChungKJ (2010) Cryptococcus neoformans overcomes stress of azole drugs by formation of disomy in specific multiple chromosomes. PLoS Pathogens 6: e1000848.

65. SemighiniCP, AveretteAF, PerfectJR, HeitmanJ (2011) Deletion of Cryptococcus neoformans AIF ortholog promotes chromosome aneuploidy and fluconazole-resistance in a metacaspase-independent manner. PLoS Pathogens 7: e1002364.

66. OkagakiLH, StrainAK, NielsenJN, CharlierC, BaltesNJ, et al. (2010) Cryptococcal Cell Morphology Affects Host Cell Interactions and Pathogenicity. PLoS Pathogens 6: e1000953.

67. ZaragozaO, Garcia-RiveraJ, NosanchukJD, Cuenca-EstrellaM, Rodriguez-TudelaJ, et al. (2010) Fungal Cell Gigantism during Mammalian Infection. PLoS Pathogens 6: 1–18.

68. ShermanF (1991) Getting started with yeast. Methods in enzymology 194: 3–21.

69. Kwon-ChungKJ, EdmanJ, WickesBL (1992) Genetic Association of Mating Types and Virulence in Cryptococcus neoformans. Infection and Immunity 60: 602–605.

70. MurashigeT, SkoogF (1962) A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiologia Plantarum 15: 473–497.

71. NielsenK, CoxGM, WangP, ToffalettiD, PerfectJR, et al. (2003) Sexual Cycle of Cryptococcus neoformans var. grubii and Virulence of Congenic a and alpha isolates. . Infection and Immunity 71: 4831–4841.

72. ToffalettiDL, RudeTH, JohnstonSA, DurackDT, PerfectJR (1993) Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA. Journal of Bacteriology 175: 1405–1411.

73. FraserJA, SubaranRL, NicholsCB, HeitmanJ (2003) Recapitulation of the sexual cycle of the primary fungal pathogen Cryptococcus neoformans var. gattii: implications for an outbreak on Vancouver Island, Canada. Eukaryotic Cell 2: 1036–1045.

74. McDadeHC, CoxGM (2001) A new dominant selectable marker for use in Cryptococcus neoformans. Medical mycology : official publication of the International Society for Human and Animal Mycology 39: 151–154.

75. DavidsonRC, CruzMC, SiaRA, AllenB, AlspaughJA, et al. (2000) Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans. Fungal Genetics and Biology : FG & B 29: 38–48.

76. PriceMS, NicholsCB, AlspaughJA (2008) The Cryptococcus neoformans Rho-GDP dissociation inhibitor mediates intracellular survival and virulence. Infection and Immunity 76: 5729–5737.

77. KozubowskiL, AboobakarEF, CardenasME, HeitmanJ (2011) Calcineurin colocalizes with P-bodies and stress granules during thermal stress in Cryptococcus neoformans. Eukaryotic Cell 10: 1396–1402.

78. CramerKL, GerraldQD, NicholsCB, PriceMS, AlspaughJA (2006) Transcription factor Nrg1 mediates capsule formation, stress response, and pathogenesis in Cryptococcus neoformans. Eukaryotic Cell 5: 1147–1156.

79. SchmittgenTD, LivakKJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nature Protocols 3: 1101–1108.

80. PerfectJR, LangSD, DurackDT (1980) Chronic cryptococcal meningitis: a new experimental model in rabbits. The American journal of pathology 101: 177–194.