Cryptococcus neoformans is an opportunistic fungal pathogen that causes serious human disease in immunocompromised populations. Its polysaccharide capsule is a key virulence factor which is regulated in response to growth conditions, becoming enlarged in the context of infection. We used microarray analysis of cells stimulated to form capsule over a range of growth conditions to identify a transcriptional signature associated with capsule enlargement. The signature contains 880 genes, is enriched for genes encoding known capsule regulators, and includes many uncharacterized sequences. One uncharacterized sequence encodes a novel regulator of capsule and of fungal virulence. This factor is a homolog of the yeast protein Ada2, a member of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex that regulates transcription of stress response genes via histone acetylation. Consistent with this homology, the C. neoformans null mutant exhibits reduced histone H3 lysine 9 acetylation. It is also defective in response to a variety of stress conditions, demonstrating phenotypes that overlap with, but are not identical to, those of other fungi with altered SAGA complexes. The mutant also exhibits significant defects in sexual development and virulence. To establish the role of Ada2 in the broader network of capsule regulation we performed RNA-Seq on strains lacking either Ada2 or one of two other capsule regulators: Cir1 and Nrg1. Analysis of the results suggested that Ada2 functions downstream of both Cir1 and Nrg1 via components of the high osmolarity glycerol (HOG) pathway. To identify direct targets of Ada2, we performed ChIP-Seq analysis of histone acetylation in the Ada2 null mutant. These studies supported the role of Ada2 in the direct regulation of capsule and mating responses and suggested that it may also play a direct role in regulating capsule-independent antiphagocytic virulence factors. These results validate our experimental approach to dissecting capsule regulation and provide multiple targets for future investigation.
Vyšlo v časopise: Toward an Integrated Model of Capsule Regulation in. PLoS Pathog 7(12): e32767. doi:10.1371/journal.ppat.1002411 Kategorie: Research Article prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1002411
1. HeitmanJKozelTRKwon-ChungJPerfectJCasadevallA 2011 Cryptococcus, from human pathogen to model yeast. Washington, D.C. ASM Press 620
2. GilesSSDagenaisTRBottsMRKellerNPHullCM 2009 Elucidating the pathogenesis of spores from the human fungal pathogen Cryptococcus neoformans. Infect Immun 77 3491 3500 doi: 10.1128/IAI.00334-09
3. Garcia-HermosoDJanbonGDromerF 1999 Epidemiological evidence for dormant Cryptococcus neoformans infection. J Clin Microbiol 37 3204 9
4. ParkBJWannemuehlerKAMarstonBJGovenderNPappasPG 2009 Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23 525 30
5. GomezBLNosanchukJD 2003 Melanin and fungi. Curr Opin Infect Dis 16 91 96
6. CoxGMMukherjeeJColeGTCasadevallAPerfectJR 2000 Urease as a virulence factor in experimental cryptococcosis. Infect Immun 68 443 8
7. CoxGMMcDadeHCChenSCTuckerSCGottfredssonM 2001 Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans. Mol Microbiol 39 166 175
8. OkagakiLHStrainAKNielsenJNCharlierCBaltesNJ 2010 Cryptococcal cell morphology affects host cell interactions and pathogenicity. PLoS Pathog 6 e1000953
9. ZaragozaOGarcía-RodasRNosanchukJDCuenca-EstrellaMRodríguez-TudelaJL 2010 Fungal cell gigantism during mammalian infection. PLoS Pathog 6 e1000945
10. DoeringTL 2009 How sweet it is! Capsule formation and cell wall biogenesis in Cryptococcus neoformans. Annu Rev Microbiol 63 223 247
11. RiveraJFeldmesserMCammerMCasadevallA 1998 Organ-dependent variation of capsule thickness in Cryptococcus neoformans during experimental murine infection. Infect Immun 66 5027 30
12. VartivarianSEAnaissieEJCowartRESpriggHATinglerMJ 1993 Regulation of cryptococcal capsular polysaccharide by iron. J Infect Dis 167 186 90
13. LittmanML 1958 Capsule synthesis by Cryptococcus neoformans. Trans N Y Acad Sci 20 623 48
14. GrangerDLPerfectJRDurackDT 1985 Virulence of Cryptococcus neoformans. Regulation of capsule synthesis by carbon dioxide. J Clin Invest 76 508 16
15. ClancyCJNguyenMHAlandoerfferRChengSIczkowskiK 2006 Cryptococcus neoformans var. grubii isolates recovered from persons with AIDS demonstrate a wide range of virulence during murine meningoencephalitis that correlates with the expression of certain virulence factors. Microbiology 152 2247 55
16. ChangYCKwon-ChungKJ 1994 Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence. Mol Cell Biol 14 4912 9
17. JanbonGHimmelreichUMoyrandFImprovisiLDromerF 2001 Cas1p is a membrane protein necessary for the O-acetylation of the Cryptococcus neoformans capsular polysaccharide. Mol Microbiol 42 453 67
18. Pukkila-WorleyRAlspaughJA 2004 Cyclic AMP signaling in Cryptococcus neoformans. FEMS Yeast Res 4 361 7
19. D`SouzaCAAlspaughJAYueCHarashimaTCoxGM 2001 Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans. Mol Cell Biol 21 3179 91
20. CramerKLGerraldQDNicholsCBPriceMSAlspaughJA 2006 Transcription factor Nrg1 mediates capsule formation, stress response, and pathogenesis in Cryptococcus neoformans. Eukaryot Cell 5 1147 56
21. ÒMearaTRNortonDPriceMSHayCClementsMF 2010 Interaction of Cryptococcus neoformans Rim101 and protein kinase A regulates capsule. PLoS Pathog 6 e1000776
22. JungWHSaikiaSHuGWangJFungCK-Y 2010 HapX positively and negatively regulates the transcriptional response to iron deprivation in Cryptococcus neoformans. PLoS Pathog 6 e1001209
23. JungWHShamAWhiteRKronstadJW 2006 Iron regulation of the major virulence factors in the AIDS-associated pathogen Cryptococcus neoformans. PLoS Biol 4 e410
24. ChunCDBrownJCSMadhaniHD 2011 A major role for capsule-independent phagocytosis-inhibitory mechanisms in mammalian infection by Cryptococcus neoformans. Cell Host Microbe 9 243 51
25. LiuOWChunCDChowEDChenCMadhaniHD 2008 Systematic genetic analysis of virulence in the human fungal pathogen Cryptococcus neoformans. Cell 135 174 88
26. BahnY-SKojimaKCoxGMHeitmanJ 2005 Specialization of the HOG pathway and its impact on differentiation and virulence of Cryptococcus neoformans. Mol Biol Cell 16 2285 300
27. ZhangSHachamMPanepintoJHuGShinS 2006 The Hsp70 member, Ssa1, acts as a DNA-binding transcriptional co-activator of laccase in Cryptococcus neoformans. Mol Microbiol 62 1090 101
28. ChangYCMillerGFKwon-ChungKJ 2003 Importance of a developmentally regulated pheromone receptor of Cryptococcus neoformans for virulence. Infect Immun 71 4953 60
29. GerikKJDonlinMJSotoCEBanksAMBanksIR 2005 Cell wall integrity is dependent on the PKC1 signal transduction pathway in Cryptococcus neoformans. Mol Microbiol 58 393 408
30. GerikKJBhimireddySRRyerseJSSpechtCALodgeJK 2008 PKC1 is essential for protection against both oxidative and nitrosative stresses, cell integrity, and normal manifestation of virulence factors in the pathogenic fungus Cryptococcus neoformans. Eukaryot Cell 7 1685 1698
31. ÒMearaTRHayCPriceMSGilesSAlspaughJA 2010 Cryptococcus neoformans histone acetyltransferase Gcn5 regulates fungal adaptation to the host. Eukaryot Cell 9 1193 202
32. KoutelouEHirschCLDentSYR 2010 Multiple faces of the SAGA complex. Curr Opin Cell Biol 22 374 82
33. WangPNicholsCBLengelerKBCardenasMECoxGM 2002 Mating-type-specific and nonspecific PAK kinases play shared and divergent roles in Cryptococcus neoformans. Eukaryot Cell 1 257 72
34. HicksJKBahnY-SHeitmanJ 2005 Pde1 phosphodiesterase modulates cyclic AMP levels through a protein kinase A-mediated negative feedback loop in Cryptococcus neoformans. Eukaryot Cell 4 1971 81
35. GrantPADugganLCôtéJRobertsSMBrownellJE 1997 Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex. Genes Dev 11 1640 50
36. BalasubramanianRPray-GrantMGSelleckWGrantPATanS 2002 Role of the Ada2 and Ada3 transcriptional coactivators in histone acetylation. J Biol Chem 277 7989 95
37. NielsenKCoxGMWangPToffalettiDLPerfectJR 2003 Sexual cycle of Cryptococcus neoformans var. grubii and virulence of congenic a and alpha isolates. Infect Immun 71 4831 41
38. GrantPAEberharterAJohnSCookRGTurnerBM 1999 Expanded lysine acetylation specificity of Gcn5 in native complexes. J Biol Chem 274 5895 900
39. HuisingaKLPughBF 2004 A genome-wide housekeeping role for TFIID and a highly regulated stress-related role for SAGA in Saccharomyces cerevisiae. Mol Cell 13 573 85
40. SellamAAskewCEppELavoieHWhitewayM 2009 Genome-wide mapping of the coactivator Ada2p yields insight into the functional roles of SAGA/ADA complex in Candida albicans. Mol Biol Cell 20 2389 400
41. JohnssonAXue-FranzénYLundinMWrightAPH 2006 Stress-specific role of fission yeast Gcn5 histone acetyltransferase in programming a subset of stress response genes. Eukaryot Cell 5 1337 46
42. Xue-FranzénYJohnssonABrodinDHenrikssonJBürglinTR 2010 Genome-wide characterisation of the Gcn5 histone acetyltransferase in budding yeast during stress adaptation reveals evolutionarily conserved and diverged roles. BMC Genomics 11 200
43. MissallTAPusateriMEDonlinMJChambersKTCorbettJA 2006 Posttranslational, translational, and transcriptional responses to nitric oxide stress in Cryptococcus neoformans: implications for virulence. Eukaryot Cell 5 518 529
44. BrownSMCampbellLTLodgeJK 2007 Cryptococcus neoformans, a fungus under stress. Curr Opin Microbiol 10 320 325
45. Cryptococcus neoformans var. grubii H99 Sequencing Project, (n.d.) Broad Institute of Harvard and MIT. Available: http://www.broadinstitute.org/
46. LeeHChangYCVarmaAKwon-ChungKJ 2009 Regulatory diversity of TUP1 in Cryptococcus neoformans. Eukaryot Cell 8 1901 8
47. KoY-JYuYMKimG-BLeeG-WMaengPJ 2009 Remodeling of global transcription patterns of Cryptococcus neoformans genes mediated by the stress-activated HOG signaling pathways. Eukaryot Cell 8 1197 217
48. T-youngRohNgauWCCuiKLandsmanDZhaoK 2004 High-resolution genome-wide mapping of histone modifications. Nat Biotechnol 22 1013 6
49. LiuCLKaplanTKimMBuratowskiSSchreiberSL 2005 Single-nucleosome mapping of histone modifications in S. cerevisiae. PLoS Biol 3 e328
50. MaPWeraSVan DijckPTheveleinJM 1999 The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signaling. Mol Biol Cell 10 91 104
51. SerranoRBernalDSimónEAriñoJ 2004 Copper and iron are the limiting factors for growth of the yeast Saccharomyces cerevisiae in an alkaline environment. J Biol Chem 279 19698 704
52. HelmlingerDMargueratSVillénJGygiSPBählerJ 2008 The S. pombe SAGA complex controls the switch from proliferation to sexual differentiation through the opposing roles of its subunits Gcn5 and Spt8. Genes Dev 22 3184 95
53. JacobsonSPillusL 2009 The SAGA subunit Ada2 functions in transcriptional silencing. Mol Cell Biol 29 6033 45 doi:10.1128/MCB.00542-09
54. RobertFPokholokDKHannettNMRinaldiNJChandyM 2004 Global position and recruitment of HATs and HDACs in the yeast genome. Mol Cell 16 199 209
55. PokholokDKHarbisonCTLevineSColeMHannettNM 2005 Genome-wide map of nucleosome acetylation and methylation in yeast. Cell 122 517 27
56. ChikamoriMFukushimaK 2005 A new hexose transporter from Cryptococcus neoformans: molecular cloning and structural and functional characterization. Fungal Genet Biol 42 646 55
57. MoyrandFFontaineTJanbonG 2007 Systematic capsule gene disruption reveals the central role of galactose metabolism on Cryptococcus neoformans virulence. Mol Microbiol 64 771 81
58. DromerFMathoulinSDupontBBrugiereOLetenneurL 1996 Comparison of the efficacy of amphotericin B and fluconazole in the treatment of cryptococcosis in human immunodeficiency virus-negative patients: retrospective analysis of 83 cases. French Cryptococcosis Study Group. Clin Infect Dis 22 Suppl 2 S154 60
59. CottrellTRGriffithCLLiuHNenningerAADoeringTL 2007 The pathogenic fungus Cryptococcus neoformans expresses two functional GDP-mannose transporters with distinct expression patterns and roles in capsule synthesis. Eukaryot Cell 6 776 785
60. SmythGK 2004 Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3 Article3
61. TusherVGTibshiraniRChuG 2001 Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A 98 5116 21
62. NelsonRTHuaJPryorBLodgeJK 2001 Identification of virulence mutants of the fungal pathogen Cryptococcus neoformans using signature-tagged mutagenesis. Genetics 157 935 947
63. FuJHettlerEWickesBL 2006 Split marker transformation increases homologous integration frequency in Cryptococcus neoformans. Fungal Genet Biol 43 200 212 doi:10.1016/j.fgb.2005.09.007
64. TrapnellCPachterLSalzbergSL 2009 TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25 1105 11
65. TrapnellCWilliamsBAPerteaGMortazaviAKwanG 2010 Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28 511 5
66. LoKGottardoR 2007 Flexible empirical Bayes models for differential gene expression. Bioinformatics 23 328 35
67. LangmeadBTrapnellCPopMSalzbergSL 2009 Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10 R25
68. ZhangYLiuTMeyerCAEeckhouteJJohnsonDS 2008 Model-based analysis of ChIP-Seq (MACS). Genome Biol 9 R137
Zvýšte si kvalifikáciu online z pohodlia domova
Nemáte účet? Registrujte sa
Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.
