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1、The structural unit of melanin in the cell wall of the fungalpathogenCryptococcusneoformansReceivedforpublication,April1,2019,andinrevisedform,May19,2019Published,PapersinPress,May22,2019,DOI10.1074/jbc.RA119.008684X Emma Camacho1,2,X Raghav Vij1,X Christine Chrissian3,X Rafael Prados-Rosales?*4X Da
2、vid Gil*,Robert N.OMeally,X Radames J.B.Cordero,X Robert N.Cole,J.Michael McCaffery5,X Ruth E.Stark?,and X Arturo Casadevall6FromtheDepartmentofMolecularMicrobiologyandImmunology,JohnsHopkinsBloombergSchoolofPublicHealth,TheJohnsHopkinsUniversity,Baltimore,Maryland21205,theDepartmentofChemistryandBi
3、ochemistry,CityCollegeofNewYorkandCUNYInstituteforMacromolecularAssemblies,NewYork,NewYork10031,theCityUniversityofNewYorkPh.D.ProgramsinBiochemistryand?Chemistry,NewYork,NewYork10016,the?DepartmentofMicrobiologyandImmunology,AlbertEinsteinCollegeofMedicine,YeshivaUniversity,Bronx,NewYork10461,the*C
4、ICbioGUNE,48160Derio,Vizcaya,Spain,theDepartmentofPreventiveMedicineandPublicHealthandMicrobiology,AutonomaUniversityofMadrid,28049Madrid,Spain,theJohnsHopkinsMassSpectrometryandProteomicFacility,TheJohnsHopkinsUniversity,Baltimore,Maryland21205,andtheIntegratedImagingCenter,DepartmentofBiology,Engi
5、neeringinOncologyCenter,andInstituteforNanoBioTechnology,TheJohnsHopkinsUniversity,Baltimore,Maryland 21218Edited by Joseph M.JezMelanins are synthesized macromolecules that are found inall biological kingdoms.These pigments have a myriad of rolesthat range from microbial virulence to key components
6、 of theinnateimmuneresponseininvertebrates.Melaninsalsoexhibitunique properties with potential applications in physics andmaterial sciences,ranging from electrical batteries to noveltherapeutics.In the fungi,melanins,such as eumelanins,arecomponents of the cell wall that provide protection againstbi
7、otic and abiotic elements.Elucidation of the smallest fungalcell wall-associated melanin unit that serves as a building blockis critical to understand the architecture of these polymers,itsinteraction with surrounding components,and their functionalversatility.In this study,we used isopycnic gradien
8、t sedimenta-tion,NMR,EPR,high-resolutionmicroscopy,andproteomicstoanalyze the melanin in the cell wall of the human pathogenicfungus Cryptococcus neoformans.We observed that melanin isassembledintothecryptococcalcellwallinsphericalstructures?200 nm in diameter,termed melanin granules,which are intur
9、n composed of nanospheres?30 nm in diameter,termedfungal melanosomes.We noted that melanin granules areclosely associated with proteins that may play critical roles inthe fungal melanogenesis and the supramolecular structure ofthis polymer.Using this structural information,we propose amodelforC.neof
10、ormansmelanizationthatissimilartothepro-cessusedinanimalmelanizationandisconsistentwiththephy-logenetic relatedness of the fungal and animal kingdoms.Melanins(Greekmelano,meaningdarkorblack)areperhapsthe most enigmatic biopolymers in the biosphere despite beingubiquitous in nature(1).These pigments
11、carry out a vast arrayof functions in all biological kingdoms,including thermoregu-lation,radical scavenging,energy transduction,camouflage,invertebrate immunity,and sexual display(24).In the micro-bial world,melanins contribute to the virulence of many path-ogenicmicrobes.Melaninsareproducedthrough
12、theoxidationand polymerization of phenolic/indolic precursors;notably,these polymers are characterized by a strong negative charge,high molecular weight,and hydrophobic nature(5).In the fun-gal field,black-to-brown insoluble pigments designated aseumelanins confer greater fitness to melanotic specie
13、s in vivo,therebycontributingtotheirvirulence(6).InCryptococcusneo-formans,a facultative intracellular pathogen,melanized cellsare able to modulate the immune response of the host throughmultiple mechanisms,e.g.altering cytokine profiles(7),reduc-ing antibody-mediated phagocytosis,and decreasing the
14、 toxic-ity of microbial peptides,reactive oxygen species(ROS),7andantifungal drugs(8,9).Recent synthesis of a soluble melaninThisworkwassupportedbyNationalInstitutesofHealthGrantR01-AI052733.The authors declare that they have no conflicts of interest with the con-tents of this article.The content is
15、 solely the responsibility of the authorsand does not necessarily represent the official views of the National Insti-tutes of Health.This article contains Figs.S1S3 and Table S1.1Both authors contributed equally to this work.2Recipient of a Johns Hopkins Malaria Research Institute fellowship.3Recipi
16、ent of a fellowship award from the United States Department of Edu-cationGraduateAssistanceinAreasofNationalNeed(GAANN)PrograminBiochemistry,Biophysics,and Biodesign at the City College of New York.4Supported in part by National Institutes of Health Grant AI115091 fromNIAID,SpanishMinistryofEconomya
17、ndCompetitivenessGrantSAF2016-77433-R,CICbioGUNEthroughtheSeveroOchoaExcellenceAccreditationSEV-2016-0644,and is a“Ramon y Cajal”fellow from the Spanish Ministryof Economy and Competitiveness.5SupportedbyHopkinsIntegratedImagingCenterandNationalInstitutesofHealth Grant 1S10OD012342 from NCRR for the
18、 acquisition of the TalosTEM.6Towhomcorrespondenceshouldbeaddressed:Dept.ofMicrobiologyandMolecular Immunology,Bloomberg School of Public Health,The JohnsHopkinsUniversity,615N.WolfeSt.,Rm.E5132,Baltimore,MD21205.Tel.:410-955-3457;Fax:410-955-0105;E-mail:acasade1jhu.edu.7Theabbreviationsusedare:ROS,
19、reactiveoxygenspecies;FDR,falsediscov-ery rate;L-DOPA,dihydroxyphenylalanine;CPMAS,cross-polarizationmagic-angle spinning;DARR,dipolar assisted rotational resonance;GXM,glucoronoxylomannan;SEM,scanning electron microscopy;TEM,trans-mission electron microscopy;DLS,dynamic light scattering;ssNMR,solid
20、-state NMR;MAS,magic-angle spinning;GPI,glycosylphosphatidylinositol;DHI,5,6-dihydroxyindole;DHICA,5,6-dihydroxyindole-2-carboxylic acid;MVB,multivesicular body;MCP,membrane compartment of Pma1.croARTICLEJ.Biol.Chem.(2019)294(27)104711048910471 2019 Camacho et al.Published under exclusive license by
21、 The American Society for Biochemistry and Molecular Biology,Inc.at SHANGHAI JIAOTONG UNIVERSITY on August 15,2019http:/www.jbc.org/Downloaded from at SHANGHAI JIAOTONG UNIVERSITY on August 15,2019http:/www.jbc.org/Downloaded from at SHANGHAI JIAOTONG UNIVERSITY on August 15,2019http:/www.jbc.org/Do
22、wnloaded from at SHANGHAI JIAOTONG UNIVERSITY on August 15,2019http:/www.jbc.org/Downloaded from at SHANGHAI JIAOTONG UNIVERSITY on August 15,2019http:/www.jbc.org/Downloaded from at SHANGHAI JIAOTONG UNIVERSITY on August 15,2019http:/www.jbc.org/Downloaded from(homogentesic acid andL-DOPA at a 1:1
23、ratio)demonstratedthat it suppresses the production of cytokines and ROS understimulationoffungalcomponentsthusverifyingmelaninsabil-ity to modulate the immune system(10).C.neoformans is a unique system for the study of melaninbiology because,unlike other melanotic fungi,only exoge-nously provided c
24、atecholamines or diphenolic substrates,suchas dihydroxyphenylalanine(L-DOPA),are utilized for melaninsynthesisandnotasanenergysource(1113).Consequently,itis possible to selectively label the melanin polymer with NMRor radioactive isotopes,a property that has been exploited formelanin structural stud
25、ies(1419).Melanization of C.neofor-mans results in the deposition of the polymer at the innermostsurfaceof the cell wall,which becomes filled throughout overtime(20).The pigment is composed of closely packed spher-ical particles ranging from 40 to 130 nm in diameter,whichare found arranged in concen
26、tric layers in the cell wall(21).Early ultrastructural observations in Agaricus bisporus(22),Fonsecaea pedrosoi(23),Cladosporium carrionii,and Hor-moconis resinae(24)suggested compartmentation of fungalmelanins into organelles akin to mammalian melanosomes(25).Evidence for trans-cellwall vesicular t
27、ransport infungi associated with virulence(26)supported the hypothe-sis that fungal melanization may occur within specializedvesicles(2730).Studies using isotopically-labeled precursors in conjunctionwith high-resolution solid-state NMR(ssNMR)revealed thatthe melanin polymer is likely to be covalent
28、ly bonded to cell-wall chitin and was also found strongly associated with othernonpigment cellular moieties,including polysaccharides suchaschitosanandvesicleand/orplasmamembrane-derivedlipids(16,19).These components serve as the scaffold for melaninsynthesis.Indeed,C.neoformans strains with aberran
29、t chitinand/or chitosan biosynthesis are unable to retain the pigmentwithin the cell wall and display a leaky-melanin phenotype(3134),whereas boosting cell-wall chitin/chitosan contentincreases melanin deposition and assembly(14).The associa-tionbetweenfungalmelaninandchitinwasinitiallyreportedinAsp
30、ergillus nidulans(35),followed by work done on Exophiliadermatitides(36)and Candida albicans(29).The concept of afibrillary matrix upon which melanin is deposited extends tootherspecies,includinghumaneumelaninwhereitisknowntoalso play a critical role in absorbing toxic oxidative melaninintermediates
31、 and to provide the elliptical shape to melano-somes(37).In addition to the relevance of melanin to all biota and itsgreat impact on fungal pathogenesis,this polymer has numer-ous potential applications in biophysics,material sciences,andeven in the cosmetics and healthcare industries(3840).Theremar
32、kablepotentialofmelaninderivesbothfromitschemicalcomposition on the molecular level and its versatility as a com-ponentofthesupramolecularstructurewithinacellularmilieu.Investigationofthemelaninstructureisanotoriouslychalleng-ing task due to the insoluble,amorphous,and heterogeneousnature of this ar
33、chitecturally complex polymer(41).Despitethese challenges,over the past 2 decades a wealth of molecularlevelstructuralinformationhasbeenuncoveredusingmultiplenondestructive methodologies,among them are the following:X-ray scattering,ssNMR,EM,and atomic force microscopy(16,4246).Current views of eume
34、lanin molecular architec-ture posit that these pigments are composed of a commonchemical motif and arranged as graphite-like?-stacked planarsheets of locally ordered monomers,mainly derivatives of 5,6-dihydroxyindole(DHI)(47,48),which give rise to subunitnanoparticlesthatthenundergospontaneousaggreg
35、ationuntilsubmicron particles are formed(45,46).Nonetheless,naturaleumelaninsdifferinstructurefromsyntheticmelaninandneu-romelanin(44,45),suggesting that the mode of synthesis canaffect the final structure.A graphical schematic highlightingthe current views on eumelanin synthesis and the relationshi
36、pof melanin to the fungal cell wall,capsule,and cell body showsthat melanin resides in the cell wall(Fig.1).Previous studies from our group have shown that the bud-dingofmelanizedC.neoformanscellsinvolvesadecreaseinthemelanin thickness of the pigmented cell wall near the bud-site(49,50).We have hypo
37、thesized that the pigmented nanopar-ticles released into the extracellular space as part of the normalcell-wallremodeling process are the smallest structural unitsofmelaninfoundanchoredtothecellwall.Hence,wehavenowinvestigated the physical properties,supramolecular structure,and protein content of t
38、hese secreted melanin nanoparticles inthe context of current knowledge of the cell-wallassociatedfungal melanin.ResultsProlongedacidhydrolysisofmelaninghostsyieldsmelaninnanoparticlesPriorstudieshaveshownthatthesolidyeast-shapedparticleslacking all internal structures,designated as“melanin ghosts,”a
39、re composed of smaller spherical structures ranging in diam-eter from 40 to 130 nm(21,49).We hypothesized that theseparticles were held together by nonpigment cellular compo-nents,a supposition supported by NMR studies showing thatC.neoformans ghosts contain tightly associated lipid moietiesand cell
40、-wall polysaccharides such as chitin(16,19).Becausepolysaccharides are typically susceptible to acid hydrolysis,wereasoned that prolonging the hydrochloric acid(HCl)incuba-tion conducted during the melanin ghost isolation protocolwould free the smaller spherical melanin particles for furtheranalysis
41、.Consistent with these expectations,increasing theincubation time of the HCl treatment led to significant mor-phological changes in the melanin ghosts samples.Scanningelectronmicrographsshowedthatghosts,subjectedtothepro-longedacidincubation,initiallyappearedtobedeformedastherelatively larger melani
42、n particles,located close to the externalsurface,are released;over time,they took on a“hollowed out”appearance as the smaller melanin nanoparticles were subse-quentlyliberated(Fig.2,AC).Inaddition,theroughnessoftheghostsurfaceincreasedwiththeprolongedacidtreatment,sug-gesting that the removal of aci
43、d-labile nonpigment materialexposes more of the underlying melanin particles.SEM visual-ization of material released during 4 h of HCl boiling revealedthat melanin aggregates between 60 and 160 nm in diametersize,with the majority measuring around 6080 nm,were sur-rounded by smaller granule-like mel
44、anin particles with diame-CellwallmelanizationinC.neoformans10472J.Biol.Chem.(2019)294(27)1047110489 at SHANGHAI JIAOTONG UNIVERSITY on August 15,2019http:/www.jbc.org/Downloaded from terslessthan60nm(Fig.2,DandE).Cross-sectionalimagingofthissuspendedmaterialshowedamixofgranularnanoparticles?30 nm i
45、n diameter with variable electron density and degreesof consolidation(Fig.2F).Further TEM analyses of these parti-cles using negative staining,which involves coating the samplewithaheavymetalstainthatprotectsitsstructurefromcollaps-ing during drying and thus provides contrast for the detectionof str
46、uctural features,revealed images consistent with thenotion that these nanospheres form tightly packed melaninaggregates(Fig.2,GI).Altogether,these results establishedthat melanin ghosts of C.neoformans are composed of aggre-gated melanin nanoparticles arranged as small to large sizedspherical partic
47、les,which are found embedded within an acid-labile matrix that produces a smooth appearance for these cell-sized structures.C.neoformans-derivedmelaningranulesdifferfromauto-polymerizedL-DOPAL-DOPA auto-polymerization occurs both in the presence ofC.neoformans cells or in cell-free conditions(17).Ho
48、wever,inthe various cellular systems where melanogenesis has beenstudied,melanin synthesis occurs within specialized vesiclesknown as melanosomes(24,2729,51).During the buddingprocess of melanized fungal yeasts,melanosomes in the cellwall are either degraded and/or laterally displaced so that thedau
49、ghter cell can emerge(49,50,52).However,in syntheticsystems,melanin formation is due to the auto-polymerizationof precursor molecules,such asL-DOPA.Consequently,wehypothesizedthatthereleasedmelanosomeswouldbefoundinthesupernatantsofC.neoformansmelanizedcultures,possiblyFigure1.Currentviewsaboutthesy
50、nthesisoffungaleumelanins.A,catecholamineprecursorslikeL-DOPAareoxidizedbythepolyphenoloxidaselaccasetoformdopaquinone,whichundergoesfurtheroxidationtoformDHIorDHICA.DHIandDHICAformlocally-orderedoligomersthatinturnformplanarstacks,presumablywithstackingdistancesofabout4.5(44).Thehigherorderstructur