Functional Analysis of Host Factors that Mediate the Intracellular Lifestyle of
Cryptococcus neoformans (Cn), the major causative agent of human fungal meningoencephalitis, replicates within phagolysosomes of infected host cells. Despite more than a half-century of investigation into host-Cn interactions, host factors that mediate infection by this fungal pathogen remain obscure. Here, we describe the development of a system that employs Drosophila S2 cells and RNA interference (RNAi) to define and characterize Cn host factors. The system recapitulated salient aspects of fungal interactions with mammalian cells, including phagocytosis, intracellular trafficking, replication, cell-to-cell spread and escape of the pathogen from host cells. Fifty-seven evolutionarily conserved host factors were identified using this system, including 29 factors that had not been previously implicated in mediating fungal pathogenesis. Subsequent analysis indicated that Cn exploits host actin cytoskeletal elements, cell surface signaling molecules, and vesicle-mediated transport proteins to establish a replicative niche. Several host molecules known to be associated with autophagy (Atg), including Atg2, Atg5, Atg9 and Pi3K59F (a class III PI3-kinase) were also uncovered in our screen. Small interfering RNA (siRNA) mediated depletion of these autophagy proteins in murine RAW264.7 macrophages demonstrated their requirement during Cn infection, thereby validating findings obtained using the Drosophila S2 cell system. Immunofluorescence confocal microscopy analyses demonstrated that Atg5, LC3, Atg9a were recruited to the vicinity of Cn containing vacuoles (CnCvs) in the early stages of Cn infection. Pharmacological inhibition of autophagy and/or PI3-kinase activity further demonstrated a requirement for autophagy associated host proteins in supporting infection of mammalian cells by Cn. Finally, systematic trafficking studies indicated that CnCVs associated with Atg proteins, including Atg5, Atg9a and LC3, during trafficking to a terminal intracellular compartment that was decorated with the lysosomal markers LAMP-1 and cathepsin D. Our findings validate the utility of the Drosophila S2 cell system as a functional genomic platform for identifying and characterizing host factors that mediate fungal intracellular replication. Our results also support a model in which host Atg proteins mediate Cn intracellular trafficking and replication.
Vyšlo v časopise:
Functional Analysis of Host Factors that Mediate the Intracellular Lifestyle of. PLoS Pathog 7(6): e32767. doi:10.1371/journal.ppat.1002078
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.ppat.1002078
Souhrn
Cryptococcus neoformans (Cn), the major causative agent of human fungal meningoencephalitis, replicates within phagolysosomes of infected host cells. Despite more than a half-century of investigation into host-Cn interactions, host factors that mediate infection by this fungal pathogen remain obscure. Here, we describe the development of a system that employs Drosophila S2 cells and RNA interference (RNAi) to define and characterize Cn host factors. The system recapitulated salient aspects of fungal interactions with mammalian cells, including phagocytosis, intracellular trafficking, replication, cell-to-cell spread and escape of the pathogen from host cells. Fifty-seven evolutionarily conserved host factors were identified using this system, including 29 factors that had not been previously implicated in mediating fungal pathogenesis. Subsequent analysis indicated that Cn exploits host actin cytoskeletal elements, cell surface signaling molecules, and vesicle-mediated transport proteins to establish a replicative niche. Several host molecules known to be associated with autophagy (Atg), including Atg2, Atg5, Atg9 and Pi3K59F (a class III PI3-kinase) were also uncovered in our screen. Small interfering RNA (siRNA) mediated depletion of these autophagy proteins in murine RAW264.7 macrophages demonstrated their requirement during Cn infection, thereby validating findings obtained using the Drosophila S2 cell system. Immunofluorescence confocal microscopy analyses demonstrated that Atg5, LC3, Atg9a were recruited to the vicinity of Cn containing vacuoles (CnCvs) in the early stages of Cn infection. Pharmacological inhibition of autophagy and/or PI3-kinase activity further demonstrated a requirement for autophagy associated host proteins in supporting infection of mammalian cells by Cn. Finally, systematic trafficking studies indicated that CnCVs associated with Atg proteins, including Atg5, Atg9a and LC3, during trafficking to a terminal intracellular compartment that was decorated with the lysosomal markers LAMP-1 and cathepsin D. Our findings validate the utility of the Drosophila S2 cell system as a functional genomic platform for identifying and characterizing host factors that mediate fungal intracellular replication. Our results also support a model in which host Atg proteins mediate Cn intracellular trafficking and replication.
Zdroje
1. BlancoJLGarciaME 2008 Immune response to fungal infections. Vet Immunol Immunopathol 125 47 70
2. LinXHeitmanJ 2006 The biology of the Cryptococcus neoformans species complex. Annu Rev Microbiol 60 69 105
3. TournuHSerneelsJVan DijckP 2005 Fungal pathogens research: novel and improved molecular approaches for the discovery of antifungal drug targets. Curr Drug Targets 6 909 922
4. FromtlingRAShadomyHJJacobsonES 1982 Decreased virulence in stable, acapsular mutants of Cryptococcus neoformans. Mycopathologia 79 23 29
5. ChangYCKwon-ChungKJ 1994 Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence. Mol Cell Biol 14 4912 4919
6. WangYAisenPCasadevallA 1996 Melanin, melanin “ghosts,” and melanin composition in Cryptococcus neoformans. Infect Immun 64 2420 2424
7. Kwon-ChungKJPolacheckIPopkinTJ 1982 Melanin-lacking mutants of Cryptococcus neoformans and their virulence for mice. J Bacteriol 150 1414 1421
8. MaHMayRC 2009 Virulence in Cryptococcus species. Adv Appl Microbiol 67 131 190
9. LiuOWChunCDChowEDChenCMadhaniHD 2008 Systematic genetic analysis of virulence in the human fungal pathogen Cryptococcus neoformans. Cell 135 174 188
10. NeteaMGFerwerdaGvan der GraafCAVan der MeerJWKullbergBJ 2006 Recognition of fungal pathogens by toll-like receptors. Curr Pharm Des 12 4195 4201
11. NakamuraKMiyagiKKoguchiYKinjoYUezuK 2006 Limited contribution of Toll-like receptor 2 and 4 to the host response to a fungal infectious pathogen, Cryptococcus neoformans. FEMS Immunol Med Microbiol 47 148 154
12. SiegemundSAlberG 2008 Cryptococcus neoformans activates bone marrow-derived conventional dendritic cells rather than plasmacytoid dendritic cells and down-regulates macrophages. FEMS Immunol Med Microbiol 52 417 427
13. VecchiarelliA 2000 Immunoregulation by capsular components of Cryptococcus neoformans. Med Mycol 38 407 417
14. RiveraJZaragozaOCasadevallA 2005 Antibody-mediated protection against Cryptococcus neoformans pulmonary infection is dependent on B cells. Infect Immun 73 1141 1150
15. KawakamiK 2004 Regulation by innate immune T lymphocytes in the host defense against pulmonary infection with Cryptococcus neoformans. Jpn J Infect Dis 57 137 145
16. WozniakKLLevitzSM 2008 Cryptococcus neoformans enters the endolysosomal pathway of dendritic cells and is killed by lysosomal components. Infect Immun 76 4764 4771
17. OsterholzerJJMilamJEChenGHToewsGBHuffnagleGB 2009 Role of dendritic cells and alveolar macrophages in regulating early host defense against pulmonary infection with Cryptococcus neoformans. Infect Immun 77 3749 3758
18. GuillotLCarrollSFBadawyMQureshiST 2008 Cryptococcus neoformans induces IL-8 secretion and CXCL1 expression by human bronchial epithelial cells. Respir Res 9 9
19. Artavanis-TsakonasKLoveJCPloeghHLVyasJM 2006 Recruitment of CD63 to Cryptococcus neoformans phagosomes requires acidification. Proc Natl Acad Sci U S A 103 15945 15950
20. LevitzSMNongSHSeetooKFHarrisonTSSpeizerRA 1999 Cryptococcus neoformans resides in an acidic phagolysosome of human macrophages. Infect Immun 67 885 890
21. AlvarezMCasadevallA 2006 Phagosome extrusion and host-cell survival after Cryptococcus neoformans phagocytosis by macrophages. Curr Biol 16 2161 2165
22. MaHCroudaceJELammasDAMayRC 2006 Expulsion of live pathogenic yeast by macrophages. Curr Biol 16 2156 2160
23. MaHCroudaceJELammasDAMayRC 2007 Direct cell-to-cell spread of a pathogenic yeast. BMC Immunol 8 15
24. LevineBDereticV 2007 Unveiling the roles of autophagy in innate and adaptive immunity. Nat Rev Immunol 7 767 777
25. LevineBKroemerG 2008 Autophagy in the pathogenesis of disease. Cell 132 27 42
26. DereticVLevineB 2009 Autophagy, immunity, and microbial adaptations. Cell Host Microbe 5 527 549
27. YorimitsuTKlionskyDJ 2007 Endoplasmic reticulum stress: a new pathway to induce autophagy. Autophagy 3 160 162
28. KabeyaYMizushimaNUenoTYamamotoAKirisakoT 2000 LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19 5720 5728
29. MizushimaNOhsumiYYoshimoriT 2002 Autophagosome formation in mammalian cells. Cell Struct Funct 27 421 429
30. PyBFLipinskiMMYuanJ 2007 Autophagy limits Listeria monocytogenes intracellular growth in the early phase of primary infection. Autophagy 3 117 125
31. YanoTMitaSOhmoriHOshimaYFujimotoY 2008 Autophagic control of listeria through intracellular innate immune recognition in drosophila. Nat Immunol 9 908 916
32. ZhaoZFuxBGoodwinMDunayIRStrongD 2008 Autophagosome-independent essential function for the autophagy protein Atg5 in cellular immunity to intracellular pathogens. Cell Host Microbe 4 458 469
33. Konen-WaismanSHowardJC 2007 Cell-autonomous immunity to Toxoplasma gondii in mouse and man. Microbes Infect 9 1652 1661
34. SchmidDMunzC 2007 Innate and adaptive immunity through autophagy. Immunity 27 11 21
35. KirkegaardKTaylorMPJacksonWT 2004 Cellular autophagy: surrender, avoidance and subversion by microorganisms. Nat Rev Microbiol 2 301 314
36. JacksonWTGiddingsTHJrTaylorMPMulinyaweSRabinovitchM 2005 Subversion of cellular autophagosomal machinery by RNA viruses. PLoS Biol 3 e156
37. WilemanT 2006 Aggresomes and autophagy generate sites for virus replication. Science 312 875 878
38. ChengLWVialaJPStuurmanNWiedemannUValeRD 2005 Use of RNA interference in Drosophila S2 cells to identify host pathways controlling compartmentalization of an intracellular pathogen. Proc Natl Acad Sci U S A 102 13646 13651
39. Stroschein-StevensonSLFoleyEO'FarrellPHJohnsonAD 2006 Identification of Drosophila gene products required for phagocytosis of Candida albicans. PLoS Biol 4 e4
40. AgaisseHBurrackLSPhilipsJARubinEJPerrimonN 2005 Genome-wide RNAi screen for host factors required for intracellular bacterial infection. Science 309 1248 1251
41. QinQMPeiJAnconaVShawBDFichtTA 2008 RNAi screen of endoplasmic reticulum-associated host factors reveals a role for IRE1alpha in supporting Brucella replication. PLoS Pathog 4 e1000110
42. PhilipsJARubinEJPerrimonN 2005 Drosophila RNAi screen reveals CD36 family member required for mycobacterial infection. Science 309 1251 1253
43. DerreIPypaertMDautry-VarsatAAgaisseH 2007 RNAi screen in Drosophila cells reveals the involvement of the Tom complex in Chlamydia infection. PLoS Pathog 3 1446 1458
44. ElwellCACeesayAKimJHKalmanDEngelJN 2008 RNA interference screen identifies Abl kinase and PDGFR signaling in Chlamydia trachomatis entry. PLoS Pathog 4 e1000021
45. ErginCIlkitMKaftanogluO 2004 Detection of Cryptococcus neoformans var. grubii in honeybee (Apis mellifera) colonies. Mycoses 47 431 434
46. Baroni FdeAPaulaCRSilvaEGVianiFCRiveraIN 2006 Cryptococcus neoformans strains isolated from church towers in Rio de Janeiro City, RJ, Brazil. Rev Inst Med Trop Sao Paulo 48 71 75
47. MylonakisEMorenoREl KhouryJBIdnurmAHeitmanJ 2005 Galleria mellonella as a model system to study Cryptococcus neoformans pathogenesis. Infect Immun 73 3842 3850
48. ApidianakisYRahmeLGHeitmanJAusubelFMCalderwoodSB 2004 Challenge of Drosophila melanogaster with Cryptococcus neoformans and role of the innate immune response. Eukaryot Cell 3 413 419
49. BurgessDSHastingsRW 2000 A comparison of dynamic characteristics of fluconazole, itraconazole, and amphotericin B against Cryptococcus neoformans using time-kill methodology. Diagn Microbiol Infect Dis 38 87 93
50. ZhaiBZhouHYangLZhangJJungK 2010 Polymyxin B, in combination with fluconazole, exerts a potent fungicidal effect. J Antimicrob Chemother 65 931 938
51. LungOBlissardGW 2005 A cellular Drosophila melanogaster protein with similarity to baculovirus F envelope fusion proteins. J Virol 79 7979 7989
52. LinXJacksonJCFeretzakiMXueCHeitmanJ 2010 Transcription factors Mat2 and Znf2 operate cellular circuits orchestrating opposite- and same-sex mating in Cryptococcus neoformans. PLoS Genet 6 e1000953
53. Geunes-BoyerSOliverTNJanbonGLodgeJKHeitmanJ 2009 Surfactant protein D increases phagocytosis of hypocapsular Cryptococcus neoformans by murine macrophages and enhances fungal survival. Infect Immun 77 2783 2794
54. PetiotAOgier-DenisEBlommaartEFMeijerAJCodognoP 2000 Distinct classes of phosphatidylinositol 3′-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J Biol Chem 275 992 998
55. BlommaartEFKrauseUSchellensJPVreeling-SindelarovaHMeijerAJ 1997 The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur J Biochem 243 240 246
56. YamamotoATagawaYYoshimoriTMoriyamaYMasakiR 1998 Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct 23 33 42
57. TuckerSCCasadevallA 2002 Replication of Cryptococcus neoformans in macrophages is accompanied by phagosomal permeabilization and accumulation of vesicles containing polysaccharide in the cytoplasm. Proc Natl Acad Sci U S A 99 3165 3170
58. KnechtEAguadoCCarcelJEstebanIEsteveJM 2009 Intracellular protein degradation in mammalian cells: recent developments. Cell Mol Life Sci 66 2427 2443
59. BottsMRHullCM 2010 Dueling in the lung: how Cryptococcus spores race the host for survival. Curr Opin Microbiol 13 437 442
60. CasadevallA 2010 Cryptococci at the brain gate: break and enter or use a Trojan horse? J Clin Invest 120 1389 1392
61. CharlierCNielsenKDaouSBrigitteMChretienF 2009 Evidence of a role for monocytes in dissemination and brain invasion by Cryptococcus neoformans. Infect Immun 77 120 127
62. ChamilosGLionakisMSLewisREKontoyiannisDP 2007 Role of mini-host models in the study of medically important fungi. Lancet Infect Dis 7 42 55
63. MylonakisECasadevallAAusubelFM 2007 Exploiting amoeboid and non-vertebrate animal model systems to study the virulence of human pathogenic fungi. PLoS Pathog 3 e101
64. CasadevallA 2005 Host as the variable: model hosts approach the immunological asymptote. Infect Immun 73 3829 3832
65. KumaAHatanoMMatsuiMYamamotoANakayaH 2004 The role of autophagy during the early neonatal starvation period. Nature 432 1032 1036
66. KomatsuMWaguriSUenoTIwataJMurataS 2005 Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J Cell Biol 169 425 434
67. MizushimaNYamamotoAHatanoMKobayashiYKabeyaY 2001 Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J Cell Biol 152 657 668
68. SaitohTFujitaNHayashiTTakaharaKSatohT 2009 Atg9a controls dsDNA-driven dynamic translocation of STING and the innate immune response. Proc Natl Acad Sci U S A 106 20842 20846
69. ReggioriFTuckerKAStromhaugPEKlionskyDJ 2004 The Atg1-Atg13 complex regulates Atg9 and Atg23 retrieval transport from the pre-autophagosomal structure. Dev Cell 6 79 90
70. ObaraKSekitoTNiimiKOhsumiY 2008 The Atg18-Atg2 complex is recruited to autophagic membranes via phosphatidylinositol 3-phosphate and exerts an essential function. J Biol Chem 283 23972 23980
71. NishidaYArakawaSFujitaniKYamaguchiHMizutaT 2009 Discovery of Atg5/Atg7-independent alternative macroautophagy. Nature 461 654 658
72. ShimizuSKonishiANishidaYMizutaTNishinaH 2010 Involvement of JNK in the regulation of autophagic cell death. Oncogene 29 2070 2082
73. SchnaithAKashkarHLeggioSAAddicksKKronkeM 2007 Staphylococcus aureus subvert autophagy for induction of caspase-independent host cell death. J Biol Chem 282 2695 2706
74. SwansonMSIsbergRR 1995 Association of Legionella pneumophila with the macrophage endoplasmic reticulum. Infect Immun 63 3609 3620
75. RomanoPSGutierrezMGBeronWRabinovitchMColomboMI 2007 The autophagic pathway is actively modulated by phase II Coxiella burnetii to efficiently replicate in the host cell. Cell Microbiol 9 891 909
76. WangYHWuJJLeiHY 2009 The autophagic induction in Helicobacter pylori-infected macrophage. Exp Biol Med (Maywood) 234 171 180
77. WangYHWuJJLeiHY 2009 When Helicobacter pylori invades and replicates in the cells. Autophagy 5 540 542
78. RomanoPSArboitMAVazquezCLColomboMI 2009 The autophagic pathway is a key component in the lysosomal dependent entry of Trypanosoma cruzi into the host cell. Autophagy 5 6 18
79. WangYWeissLMOrlofskyA 2009 Host cell autophagy is induced by Toxoplasma gondii and contributes to parasite growth. J Biol Chem 284 1694 1701
80. OrvedahlAAlexanderDTalloczyZSunQWeiY 2007 HSV-1 ICP34.5 confers neurovirulence by targeting the Beclin 1 autophagy protein. Cell Host Microbe 1 23 35
81. OrvedahlALevineB 2008 Autophagy and viral neurovirulence. Cell Microbiol 10 1747 1756
82. AxeELWalkerSAManifavaMChandraPRoderickHL 2008 Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol 182 685 701
83. HuGHachamMWatermanSRPanepintoJShinS 2008 PI3K signaling of autophagy is required for starvation tolerance and virulenceof Cryptococcus neoformans. J Clin Invest 118 1186 1197
84. KronstadJWAttarianRCadieuxBChoiJD'SouzaCA 2011 Expanding fungal pathogenesis: Cryptococcus breaks out of the opportunistic box. Nat Rev Microbiol 9 193 203
85. ZaragozaOGarcia-RodasRNosanchukJDCuenca-EstrellaMRodriguez-TudelaJL 2010 Fungal cell gigantism during mammalian infection. PLoS Pathog 6 e1000945
86. FeldmesserMKressYCasadevallA 2001 Dynamic changes in the morphology of Cryptococcus neoformans during murine pulmonary infection. Microbiology 147 2355 2365
87. CruickshankJGCavillRJelbertM 1973 Cryptococcus neoformans of unusual morphology. Appl Microbiol 25 309 312
88. KechichianTBSheaJDel PoetaM 2007 Depletion of alveolar macrophages decreases the dissemination of a glucosylceramide-deficient mutant of Cryptococcus neoformans in immunodeficient mice. Infect Immun 75 4792 4798
89. MaHHagenFStekelDJJohnstonSASionovE 2009 The fatal fungal outbreak on Vancouver Island is characterized by enhanced intracellular parasitism driven by mitochondrial regulation. Proc Natl Acad Sci U S A 106 12980 12985
Štítky
Hygiena a epidemiológia Infekčné lekárstvo LaboratóriumČlánok vyšiel v časopise
PLOS Pathogens
2011 Číslo 6
- Parazitičtí červi v terapii Crohnovy choroby a dalších zánětlivých autoimunitních onemocnění
- Očkování proti virové hemoragické horečce Ebola experimentální vakcínou rVSVDG-ZEBOV-GP
- Koronavirus hýbe světem: Víte jak se chránit a jak postupovat v případě podezření?
Najčítanejšie v tomto čísle
- High Affinity Nanobodies against the VSG Are Potent Trypanolytic Agents that Block Endocytosis
- Structural and Mechanistic Studies of Measles Virus Illuminate Paramyxovirus Entry
- Sporangiospore Size Dimorphism Is Linked to Virulence of
- The Binding of Triclosan to SmeT, the Repressor of the Multidrug Efflux Pump SmeDEF, Induces Antibiotic Resistance in