Differential role of gpaB and sidA gene expressions in relation to virulence in Aspergillus species from patients with invasive aspergillosis

h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Bacterial

and

Fungal

Pathogenesis

Differential

role

of

gpaB

and

sidA

gene

expressions

in

relation

to

virulence

in

Aspergillus

species

from

patients

with

invasive

aspergillosis

Nayereh

Ghods

_,

_Mehraban

_Falahati

_,

_Maryam

_Roudbary

_,

_Shirin

_Farahyar

_,

Masoud

Shamaei

_,

_Mahin

_Pourabdollah

_,

_Farhad

_Seif

a_{IranUniversityofMedicalSciences,SchoolofMedicine,DepartmentofMedicalMycologyandParasitology,Tehran,Iran}

b_{ClinicalTuberculosisandEpidemiologyResearchCenter,NationalResearchInstituteofTuberculosisandLungDiseases(NRITLD),}

ShahidBeheshtiUniversityofMedicalSciences,Tehran,Iran

c_{ChronicRespiratoryDiseasesResearchCenter,NationalResearchInstituteofTuberculosisandLungDiseases(NRITLD),Shahid}

BeheshtiUniversityofMedicalSciences,Tehran,Iran

d_{IranUniversityofMedicalSciences,SchoolofMedicine,DepartmentofImmunology,Tehran,Iran}

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Articlehistory:

Received15April2017 Accepted4October2017 Availableonline3February2018 AssociateEditor:RosanaPuccia

Keywords: A.flavus A.fumigatus sidAgene gpaBgene Bronchoalveolarlavage

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Thevirulencegenesininvasiveaspergillosis(IA)havenotbeenanalyzedadequately.The presentstudywasdesignedtoevaluatetheexpressionofgpaBandsidAgenes,whichare importantvirulencegenesinAspergillusspp.frombronchoalveolarlavage(BAL)samples. DirectexaminationandcultureonCzapekAgarandSabouraudDextroseAgarmediawere performedfor600BALspecimensisolatedfrompatientswithpossibleaspergillosis.A Galac-tomannanELISAassaywasalsocarriedout.TheexpressionlevelsofthegpaBandsidA genesinisolateswereanalyzedusingquantitativereal-timePCR(qRT-PCR).Weidentified 2species,includingAspergillusflavus(A.flavus)andAspergillusfumigatus(A.fumigatus)in25 positivesamplesforinvasiveaspergillosisasvalidatedusingGM-ELISA.A.flavusisthemain pathogenthreateningtransplantrecipientsandcancerpatientsworldwide.Inthisstudy,A. flavushadlowlevelsofthegpaBgeneexpressioncomparedtoA.fumigatus(p=0.006).The highestsidAexpressionwasdetectedintransplantrecipients(p=0.05).Therewasno sig-nificantcorrelationbetweensidAexpressionandunderlyingdisease(p=0.15).ThesidAand gpaBgeneexpressionpatternsmayprovideevidencethatthesevirulencegenesplay impor-tantrolesinthepathogenicityofAspergillusisolates;however,thereareseveralregulatory genesresponsiblefortheunexpressedsidAandgpaBgenesintheisolates.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthorsat:DepartmentofMedicalMycologyandParasitology,SchoolofMedicine,IranUniversityofMedicalSciences,}

Tehran,Iran.

E-mails:mehrabanfalahati@yahoo.com(M.Falahati),mroudbary@yahoo.com,Roudbari.mr@iums.ac.ir(M.Roudbary).

https://doi.org/10.1016/j.bjm.2017.10.003

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Invasiveaspergillosis(IA)isaseverediseaseandoneofthe primary causes of mortality among immunocompromised individuals,includingcysticfibrosis andcancerpatients, as wellastransplantrecipients.1_{AvarietyofAspergillusspecies}

actascausativeagentsofIA,butA.fumigatusandA.flavusare themajorcausesofthedisease.Infact,thesmallsizeoftheir conidiafacilitatestheentryoffungiintothealveolarcavity.2–4

Ifwewanttomapthegenenetwork,whichisassociatedwith theprogressionofIAinimmunocompromisedindividuals,we willneeddetailedinformationrelatedtothevirulencegene profilesofAspergillusspp.

Several putative virulence factors contributing to the pathogenesisofaspergillosis,5_{havebeenidentified,including}

pigmentproduction,adhesionmoleculesonthecellsurface, hydrolyticenzymesandtoxinsecretion.Additionally,it has beenproventhatanetworkofgenesisrequiredtosupport thevirulenceoftheAspergillusspp.6

G-proteins,involvedinsignaltransductionpathways,and siderophoresareencodedbyspecificgenesintheAspergillus

genome7 _{and they are essential for A. fumigatus to}

repro-gramitsphysiologyinresponsetotheenvironmentandiron uptake.8,9 G protein signaling and the way by which it is regulatedhavebeen studiedinanumber ofmodelsinthe pastdecade.Studies on the pathogenicfungi haveopened anewavenuetotheconceptoftheregulatorymechanisms ofdevelopment,virulence/pathogenicityestablishment, mat-ing, and generation of secondary metabolites in different fungi.Certainly,A.fumigatusG␤andG␥subunitsareinevitable for viability and vegetative growth, because gene deleted mutants are extremely defective in germination and veg-etative growth.10 _{gpaB genes encode G-proteins that have}

a central role in the integrity, morphogenesis, and viru-lenceofA.fumigatus,aswellaselicitingthehost’simmune response.ApreviousstudydemonstratedthatthegpaBgene mutantstrainofAspergillusisavirulentinanimal-infectedIA models.11

InastudyperformedbyGehrkeetal.it wasshownthat deletionof twoputative G-protein-coupled receptors, GprC andGprD,causeddevastatinggrowthdefects. Furthermore, hyphal extension and germination were decreased. They showedthatthemutantsstraindisplayedanattenuationof virulenceinamurineinfectionmodel.12

Askew and colleagues showed the gene networks with probablerolesintheintegrityofthecellwalland environmen-talchange-relatedsignalingpathways.13_{Siderophoreproteins}

areencodedbysidAgenesandplayimportantrolesinseveral vitalprocesses,includinggermination,oxidativestress resis-tance,andironstorageinAspergillusspp.9,14 _{Interestingly,it}

hasbeenshownthatA.fumigatusexploitssidAgene expres-sionforvirulenceandpathogenicity.15,16

HissenindicatedthatarescuedstrainofAspergillus(Delta sidA+sidA)exhibitedsiderophoresandhadsimilargrowthto thewildtypeonIron-limitedmedia.Incontrasttothe wild-typeandotherrescuedstrains,itwasreportedthattheDelta sidAstrainwasvirulentinamousemodelofIA.It demon-stratesthatsidAplaysapivotalrole inthepathogenesisof

A. fumigatus.15 _{Previousstudies havealready described the}

roleofgenesinthepathogenesisofAspergillus.Notonlyitis notwell-definedwhichgeneisexactlyinvolvedinAspergillus

pathogenesis but alsotheexact function ofeach genehas remainedunknown.6_{TheexpressionofsidAandgpaBgenes}

inAspergillusspp.wasnotcompletelydefinedinBAL speci-mensisolatedfromimmunosuppressivesubjects.Therefore, regardingtheimportanceofgpaBandsidAgenesinvirulence ofAspergillusspp.eitherinvitroorinvivo.Thisstudyfocusedon gpaBandsidAgeneexpressionsinA.fumigatusandA.flavus

forthefirsttime,astheprimarygenesinvolved(incriminated) inthepathogenesisofAspergillusspp.inBALsamplesisolated fromimmunocompromisedandhospitalizedsubjects predis-posedtodevelopingIA.

Materials

and

methods

Patientcharacteristics

Bronchoalveolar lavage (BAL) samples were collected by a pulmonologist via bronchoscopy from 600 patients with underlyingdisease andsusceptibletoIAbetween2014and 2015atMasihDaneshvariHospital,Tehran,Iran.Thepatient populationsincludecancerpatients(leukemia,liver), trans-plant recipients (bone marrow, lung, and kidney), chronic obstructive pulmonary disease (COPD), hemoptysis (with animmunosuppressivecondition),andallergic bronchopul-monaryaspergillosis(ABPA)patientstakingcorticosteroid.17

Alloftheparticipantsfilledinformedconsent.Thepatients’ medicalrecords andother data,including age,gender, and typeofdisease were collected and recorded inadatabase. These data were kept fully anonymous. The study proto-colwasapprovedbytheResearchEthicsCommitteeofIran UniversityofMedicalSciences(IUMS).AllBALsampleswere collectedtwiceperpatient.

Specimenculture

TheBALsampleswere immediately transferredtoa medi-calmycologylaboratory.Thespecimenswereusedinadirect smearwithKOH10%andthenculturedonCzapekAgarand Sabouraud Dextrose Agar media (2% glucose, 1% peptone, 0.05%chloramphenicol,2%agar)(Merck,Germany)for7days at25◦C.ThegrowthrateofAspergilluswascheckedandthe specieswereidentifiedusingconventionalphenotyping meth-odssuchascolonycolorsandslidecultures.

ConfirmationofaspergillosisusingGM-ELISA

The GM-ELISA (Platelia Aspergillus; Bio-Rad, Edmonds, WA) was performed on positive BAL samples, according to the manufacturer’s recommendations.18 _{Briefly, 100␮L of the}

Platelia treatmentsolutionwas addedto300␮LoftheBAL specimen and heated at 100◦C for 3min before undergo-ing centrifugation. The supernatant and the horseradish peroxidase-labeledmonoclonalantibody(EBA-2)were incu-batedat37◦Cinantibody-precoatedmicroplates.Theplates werewashedandthenincubatedwithasubstratechromogen reaction solution. Thereaction was stopped by the use of 100␮Lofsulfuricacidand after30min,theoptical density

Table1–Sequenceofprimersinquantitativereal-timePCR.

Genename Sequenceofprimers Productsize(bp) Tm(◦C) Primerefficiency(%)

Forward(5_→3_{) Reverse(5}_→3₎

sidA GCGACGATAGCCCATTTGTC ATTAGCAGGATTAGGATCAAGG 155 59 98

gpaB CCCGACTTCCTATGACGAGC CAACGCCTATGCATTCAGCC 140 60 98

ˇ-tubulin CGACAACGAGGCTCTGTACG CAACTTGCGCAGATCAGAGTTGAG 200 58 99

(ODvalue)ofeachwellwasreadusingamicroplatereaderat 450nm.Onewellforthenegativecontrol,twowellsforthe cut-offcontrol,andonewellforthepositivecontrolwereused.The resultsofGalactomannanELISAassayareexpressedas “galac-tomannanindex”(GMI=ODsample/meanODcut-offcontrol), bycomparisontothe“cutoff”control.AGMIindexof≥1.0in twoconsecutivesampleswasconsideredpositive.

CultureofAspergillusspp.

ForRNAextraction,theAspergillusspp.wasculturedinaYeast SucroseBroth (YSB)medium (Merck,Germany) at25◦C for 48–72h. Theoptimalgrowthratewasdeterminedbasedon theexpansionofmyceliumonthesurfaceofthemedium.

RNAextraction,cDNAsynthesis,andquantitative real-timePCR

RNAcontents withinall the samples were extractedusing theRNA-XPLUSsolution(Cinaclon,cat=RN7713C-s).After dis-rupting the Aspergillus spp. cell wallusing liquid nitrogen, theRNA-XPLUSsolutionwasaddedtothesample. Chloro-formwasaddedaftercentrifugationofliquidat2000rpmfor 5min,theaqueousphasewastransferredtoanewtube.The equalvolumeofisopropanoladded,thenthetubewas cen-trifugedat2000rpmfor5minandwashedwith75%ethanol, thesemidriedpellet ofRNAwasdissolvedinDEPC treated water.TheRNAwastreated withDNase(DNaseI) (Invitro-gen,CatNo18068015)toremovegenomicDNAcontamination. Thequalityand quantityofthe extractedRNA, beforeand aftertreatment,were examinedusingagel electrophoresis andBioPhotometerplus(Germany),respectively.

First-strandcDNAwassynthesizedusingahigh-capacity cDNA reverse transcription kit (Hyper script reverse trans-criptase, Gene All, Cat No 601-100), according to the manufacturer’s protocol. The cDNA samples were kept at −80◦_{Cuntilbeingused.TheexpressionofthegpaBandsidA} geneswasquantifiedbyreal-timePCRanalysis,aspreviously described.19_{Thereactionscontained2␮LoftemplatecDNA,}

0.6␮Lofeachspecificprimer(10pmol)forthesidAandgpaB genes,10␮LofSYBRGreenmastermix,and6.8␮LofDEPC waterinafinalvolume of20␮L.Theresultswere normal-izedusing␤-tubulinamplificationsrunonthesameplateas thehousekeepinggene.Thereal-timePCRsystemwas pro-grammedtoaninitialdenaturationandpolymeraseactivation for2minat95◦C,denaturationfor15sat95◦C,andannealing andelongationfor1minat62◦Cover35cyclesusingaCorbet thermocycle(RG-6000,Australia).Specificprimersfor quan-titativereal-timeRT-PCRweredesignedwithonlineprimer designsoftware(Genscript,Piscataway,NJ).Fordetermination ofprimersefficiencyvalues,wepaperedtheserialdilutions

(25ng,5ng,1ng,0.2ng,0.04ngpermicroliter)ofthepositive controlforsidAandgapBgenes.Then,theprimersefficiency valuesweredeterminedusingaCorbetthermocycle(RG-6000, Australia)(Table1).Thespecificityoftheprimerswasassessed with genomic DNA from different Aspergillus species. The standardspeciesofA.fumigatusandA.flavus(ascontrolgroup) wereusedinrelativequantitativemethodusingreal-timePCR. ThelevelofsidAandgapBexpressionswasdividedintoeither down-regulation(≤meanofgeneexpression)orup-regulation (>meanofgeneexpression),asdescribedpreviously.20

Statisticalanalysis

The mean of gene expression was used as the cut-off for categorization inlow and high expressiongroups. Relative expression was expected using the formula 2−Ct. The expression ofthe sidA and gpaBgenes wasnormalized to ␤-tubulin expression. The data were analyzed using SPSS software, version 20 (SPSS,Chicago, IL, USA). The associa-tion between the level of gpaB and sidA expressions and patientcharacteristics wereexplored usingeitherPearson’s chi-squareorFisher’sexacttest.Variationsintheexpression ofsidAandgpaBbetweenimmunosuppressivediseaseswere assessedusingtheMann–WhitneyUtest.Ap-valueof≤0.05 wasconsideredtobestatisticallysignificant.

Results

Populationstudy

Twenty-five positive cases were detected through the screeningof600BALsamplesreceivedfromstudiedpatients. The A.flavus isolateswere foundin 17 (68%)ofthe cases, andA.fumigatusisolateswerealsodetectedin8(32%)ofthe cases. Twelve(48%) patientswere male, and 13(52%) were female (M/Fratio=0.92); they rangedin age from 40 to 83 years(mean=59andmedian=60years).Theobtainedresults frompatient’sdatashowedthat9(36%)ofthepatientshad COPD,whiletransplantrecipientsandcancerpatientswere foundinsimilarfrequencies(24%).Inaddition, hemoptysis and ABPAweredetectedinonly2cases(8%)(Table2).The resultsofGM-ELISAtestindicatedthatGMantigenwas suc-cessfullydetectedintheserumofthepatientswithpositive

Aspergillusspp.inBALsamples,theresultanalyzedaccording toGMindex.

AnalysisofgpaBgeneexpressioninpatientsandits correlationwithpatientcharacteristics

For analysisofgpaBgeneexpression,firstly,theexpression level of gpaB gene in each case was normalized with

␤-Table2–Patients’characteristicsandcorrelationbetweenthelevelsofsidAandgpaBgeneexpressions(boldvaluesare significant).

Patientdata Totalno. sidAexpression(mean=2.4) p-Value gpaBexpression(mean=33) p-Value

Low High Low High

Age(mean=59) ≤59 14(56) 10(71) 4(29) 0.54 13(93) 1(7) 0.17 >59 11(44) 9(82) 2(18) 8(73) 3(27) Gender Male 12(48) 9(75) 3(25) 0.9 10(83) 2(17) 0.93 Female 13(52) 10(77) 3(23) 11(85) 2(15) Disease Transplantrecipient 6(24) 3(50) 3(50) 0.15 6(100) 0(0) 0.07 Cancer 6(24) 6(100) 0(0) 6(100) 0(0) COPDa _{9(36) 8(89) 1(14) 6(67) 3(33)} Hemoptysis 2(8) 1(50) 1(50) 1(50) 1(50) ABPAb _{2(8) 1(50) 1(50) 2(100) 0(0)} Aspergillusspp. flavus 17(68) 12(71) 5(29) 0.28 17(100) 0(0) 0.006 fumigatus 8(32) 7(88) 1(12) 4(50) 4(50)

a _{Chronicobstructivepulmonarydisease.} b _{Allergicbroncopulmonaryaspergillosis.}

tubulin,ashousekeepinggeneandthefinalgeneexpression levelhasbeencalculated(2−Ct).Then,meanvalueofgpaB geneexpression wasselected asa cut-off valueto catego-rizethelevelofgeneexpression.ThemeangpaBexpression (mean=33)waschosenasacut-offvaluetoclassifythe sam-plesintotwocategories:eachcasewithlowerthanofmean genelevelwasconsideredasthedown-regulationexpression, andeachcasewithhigherthanofmeangenelevelwas consid-eredastheup-regulationexpression.Down-regulationgpaB expressionwasfoundmainlyincancerpatientsand trans-plantrecipients;thesesamepatientsshowednoinstancesof up-regulationgpaBexpression(Table2).

Correlations between the level of gpaB expression and patientcharacteristicswere exploredusingeitherPearson’s chi-squareorFisher’sexacttest.Asignificantassociationwas observedbetweengpaBexpressionandAspergillusspp.using fisherexacttest,indicatingthatA.flavusexpressedlower lev-els ofgpaBthan A. fumigatus(p=0.006, Table2). Moreover, atrendbecameevidentbetweendecreasedgpaBexpression andunderlyingdiseaseusingChi-squaretest(p=0.07,Table2). NocorrelationwasfoundbetweengpaBexpressionandother patientcharacteristics,includingageandgender(p=0.17and

p=0.93,respectively).Amongcases,acomparisonofthe dif-ferencesbetweenexpressionusingtheMann–WhitneyUtest demonstratedamarginaltrendbetweencancerpatientsand bothhemoptysis patientsand transplantrecipients(p=0.08 andp=0.09,respectively).Inaddition,amarginaltrendwas foundbetweentransplantrecipientsandhemoptysispatients. (p=0.08)(Fig.1).

AnalysisofsidAgeneexpressioninpatientsandits correlationwithpatientcharacteristics

ForanalysisofsidAgeneexpression,firstly,the expression level of sidA gene in each case was normalized with

␤-300 250 200 150 100 50 0 Transplant recipient

Cancer COPD Hemoptysis ABPA

Underlying diseases *7

GpaB e

xpression

o5

Fig.1–AnalysisofgpaBexpressionlevelsinpatientsusing Mann–WhitneyUtest.Onthebasisofthestandard definitions,Box-plotshowsmedian(boldline), interquartileline(box),outliers(circle),andextreme observations(star).COPD,chronicobstructivepulmonary disease;ABPA,allergicbroncopulmonaryaspergillosis.

tubulin,ashousekeepinggeneandthefinalgeneexpression levelhasbeencalculated(2−Ct).Then,meanvalueofsidA geneexpressionwasselectedasacut-offvaluetocategorize the levelofgene expression.Themean ofsidAexpression (mean=2.4)waschosenasacut-offvaluetoclassifythe sam-plesintotwocategories:Eachcasewithlowerthanofmean genelevelwasconsideredasthedown-regulationexpression, andeachcasewithhigherthanofmeangenelevelwas consid-eredastheup-regulationexpression.LowsidAexpressionwas frequentlyobservedinCOPDandcancercases(Table2). Corre-lationsbetweenthelevelofsidAexpressionandpatient char-acteristicswereexploredusingeitherPearson’schi-squareor

* * * 8 4 14 25 20 15 10 5 0 Transplant recipients

Cancer COPD Hemoptysis ABPA

Underlying diseases

SidA e

x

pression

p = 0.05

Fig.2–AnalysisofsidAexpressionlevelsinpatientsusing Mann–WhitneyUtest.Onthebasisofthestandard definitions,Box-plotshowsmedian(boldline), interquartileline(box),outliers(circle),andextreme observations(star).COPD,chronicobstructivepulmonary disease;ABPA,allergicbroncopulmonaryaspergillosis.

Fisher’sexacttest.Down-regulationofsidAexpressionwas mainlyfoundinA.flavus,butnosignificantassociationwas foundbetweensidAexpressionandAspergillusspp.(p=0.28,

Table2).Althoughdown-regulation ofsidAexpressionwas

detectedincancerandCOPDpatients,nosignificant corre-lation was foundbetween sidA expressionand underlying disease(p=0.15).Amongthepatients,acomparisonofthe dif-ferencesbetweensidAexpressionusingtheMann–Whitney

Utestdemonstratedasignificantdifferencebetweencancer patientsandtransplantrecipients (p=0.05),and amarginal trendbetweencancerpatientsandbothhemoptysisandABPA patients(p=0.08andp=0.08,respectively)(Table2andFig.2).

CorrelationbetweenAspergillusspp.andunderlying diseases

A.flavus wasthe mainpathogen incancer patients(100%) andtransplantrecipients(83%).A.flavusandA.fumigatusplay similarrolesinothercases,includingCOPD,hemoptysis,and ABPA.

Discussion

TheincidenceofIAhasincreasedinrecentyearsinpatients with predisposing factors.21 _{A. fumigatus and A. flavus are}

knownasimportantagentsthatcancauseIAwithenhanced morbidity and mortalityin immunocompromised patients, suchaschronicobstructive pulmonarydisease (COPD)and cancerpatients,patientstakingcorticosteroidsand antimi-crobialprophylacticdrugs,andtransplantrecipients.22,23_Due

tothesuppressedimmunesystemsofsuchindividuals,the

Aspergillus conidia can pass through the alveolar epithe-lial cells byconidiationand germination, thereby inducing pathogenicityinpatients.24,25_{Furthermore,theexpressionof}

someAspergillusgeneshasbeenobservedtobechangedwith extensiveprophylaxiswithfluconazole,whichincreasesthe virulencepotency.26

Thefunctionsandcharacteroftheputativevirulence fac-tors of A. fumigatus are still under investigation.Although severalgeneshavebeendiscovered,therearealotofother genes whichareresponsible forthe regulationofvirulence gene expression. Itoftenmakes it impossible todesignate withcertaintywhichofthemcontributetothemechanism ofpathogenesis.12 _{Regardingthedominantroleofvirulence}

genes in the pathogenesis of IA, there is no comprehen-sive informationconcerninggeneexpressioninAspergillus,

althoughafewstudieshaveinvestigatedthisissueinrecent years.

Ourfindings demonstrated theup-regulationexpression ofthegpaBgeneinA.fumigatuscomparedtoA.flavus.This mayindicatethatgpaBplaysanessentialroleinthe patho-genesis of A. fumigatus. G-proteins, as a main component of gpaB, have been shown to have a fundamental effect on transduction ofthe signals into the fungal cells.27,28 _In

fact, the activation of G-protein subunits in the cell wall of A. fumigatus increases cyclicadenosine monophosphate (cAMP) signaling, mitogen-activated protein kinase (MAPK) andproteinkinaseA(PKA)pathwaysinthefungi,and dele-tion of A. fumigatus GprC and GprD genes lead to growth destruction in all conditions, and an analysis ofvirulence revealedasignificantdecreaseinvirulenceanddelayed mor-talityinamurinemodelofaspergillosis.12_{Down-regulation}

gpaBexpressionwasdetectedmainlyinCOPDpatients;the patients exhibited noexpressionof this gene.This finding is supported by the activation of other regulatory mech-anisms, such as the MAPK signal pathway, ray proteins, histidinekinases,calciumsignalingorsignaltransductionby othervirulencesubunitsofG-proteinsinthepathogenesisof IA.29

Weobservedthe highestsidA geneexpressionin trans-plantrecipientsandlowexpressionofthisgeneincancerand COPDpatients.Ithasbeenwell-establishedthatsidAplaysa crucialroleinvirulenceduetoitshigh-affinityironuptake sys-temsinfungi,whichtriggerspathogenesisinthepatients.30

Additionally, sidA genes exciteAspergillus growth inlavage fluid,alsosiderophoredeficiencydecreasestissue inflamma-tionandinvasion.31

Consistent with our finding, Gravelat et al. showed MedA accomplishes conidiation, because in medA strain of A. fumigatus, biofilm formation and adherence to pul-monaryepithelial,endothelialcellsandfibronectinhavebeen impaired in vitro. The medA strain alsohad reduced the ability to damage pulmonary epithelial cells. Moreover, A. fumigatusmedAstraindisplayedlowvirulenceinbothan invertebrateandamammalianmodelofIA.32

ThelackofasignificantcorrelationbetweensidA expres-sionandunderlyingdiseasesmayberelatedtotheactivation ofothersystemsforironuptakebytheAspergillusspecies.33

Themetaloreductases(MR)increasedironuptakeupon the initiationofpulmonaryIAinfectionsinanimalmodels.34,35

ItwouldbebeneficialforfuturestudiestoinvestigatesidA and gpaBgene expressions in other immunocompromised patients,suchasHIVandhepatitispatients.Insummary,the sidAandgpaBgeneexpressionpatternsinpatientsindicated

thatthesegenesplayimportantrolesinthepathogenicityof

Aspergillusisolates;nevertheless,severalregulatorygenesand proteinsappearedtoberesponsiblefortheunexpressedsidA andgpaBgenesintheisolates,whichexplaintheroleofthis mainmechanism.

Conclusion

Taken together, our findings indicated that the A. flavus

wasthe majorpathogen incancer patientsand transplant recipients. A high expression of gpaB in COPD samples and an increased expression of sidA in transplant recip-ient may be related to the critical role of predisposing factors in the expression of specific genes in our study population.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgment

ThisstudywassupportedbyagrantfromIranUniversityof MedicalSciences,Tehran,Iran(Grant#25653).

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