微生物外文文獻(xiàn)

微生物外文文獻(xiàn)

TheresistanceoftheyeastSaccharomycescerevisiaetothebiocidepolyhexamethylenebiguanide:involvementofcellwallintegritypathwayandemergingroleforYAP1。

CarolinaElsztein1,RodrigoMdeLucena1andMarcosAdeMoraisJr1,2*

Background

Previousworkhasshownthatpolyhexamethylenebigua-nide(PHMB)waseffectiveinselectivelykillingtheyeaststhatareregardedascontaminantsoftheethanolfermentationprocess,withspecialattentionbeingpaidtoDekkerabruxellensis[1].PHMBhasbeenusedasanantisepticanddisinfectantinmedicineandindustry,anditscurrentapplicationsincludethefollowing:impregnationoffabricstoinhibitmicrobialgrowth[2,3],watertreatment[4],disinfectionofawiderangeofsolidsurfacessuchascontactlenses[5],。

InterdepartmentalResearchGroupinMetabolicEngineering.Av.MoraesRego,1235,CidadeUniversitária,50670-901,Recife,PE,Brazil。

Fulllistofauthorinformationisavailableattheendofthearticle

Elszteinetal.BMCMolecularBiology2011,12:38

surfacessuchasbacterialcellwallsanditsbiocidalmechanismmayincludedamagetothecellmembrane[16].Atlowerconcentrations,ithasbeensuggestedthatthebacteriostaticeffectsofPHMBmaybepartlyduetoapowerfulcooperativeinteractionwith(polya-nionic)nucleicacids[17].ItwassuggestedthatPHMBshouldinteractwithphospholipidsoftheAcantha-moebacellmembraneandthuscausechangesincellpermeability[18,19],andthiswasfurthersupportedbyworkonE.coli[14].TheexternallayerofSaccharo-mycescerevisiaeandotheryeastspeciesplasmamem-braneisenhancedbyphosphatidylcholine,ergosterolandsphingolipids[20].OwingtothecationicnatureofPHMB,itstoxiceffectmaybemediatedbyitslinkstothenegativephospholipidsontheyeastcellsurface.InthepresenceofPHMB,thehomogeneousdistributionofphospholipidsthatareusuallylinkedtobiologicalmembranes,istransformedintoamosaicofindividualphospholipiddomainsthatproducefluidandliquidcrystallineregionsinthecellmembrane[15].Bymeansofthewhole-genometranscriptionalprofile,Allenetal[14]showthatE.colirespondstobacteriostaticlevelsofPHMBbyalteringtheexpressionofmanyofthegenesinvolvedatalllevelsofthecellultrastructure,i.

e.theoutermembrane,periplasm,innermembraneandcytoplasmicdomains,butnotthelipolysaccharidelayer.Therewasalsoanalterationintheexpressionofgenesassociatedwithstressessuchasacidresistance,alkaliresistance,osmoticshockandcell-envelopeperturbation.

AswellasthefungicideactivityinD.bruxellensis,PichiagaleiformesandCandidatropicalis,theyeastspe-ciesisolatedPHMBasacontaminantthroughethanolfermentation,andthisalsodifferentiallyaffectedSac-charomycescerevisiaeindustrialstrains,forexamplethePE-2strainwasmoreaffectedthanJP1[1].ThisresultmightimposeconstraintsontheuseofPHMBincon-trollingincidentsofyeastcontaminationinindustrialfermentations.Thus,thefurtherstepstowardsdevelop-ingindustrialformulationsofPHMB,mustrelyondeterminingtheidentificationofbiologicalactivityandtargetsofPHMBontheyeastcells,andidentifyingtheyeastmechanismsthatrespondtothedamagecausedbythisbiocide.

Inthisstudy,wehavesoughttounderstandtheaspectsofPHMB-cellinteractiondiscussedabove,byadoptingtwostrategies:1)testingthebiocideeffectsofPHMBontheexpressionofyeastgenesinvolvedinthecellwallintegritymechanism(CWI)and2)bytestingyeaststrainswithmutationsingenesinvolvedinCWIandtheiroxidativestressresponse.TheresultsstronglyindicatedthatPHMBmayactbydestabilizingtheyeastcellwall,thusinducingthecellwallintegritypathwayresponse.Moreover,anaccountisgivenofthepotentialregulatoryroleofYap1pontheexpressionofCWIgenes.

ResultsandDiscussion

PHMBtreatmentinducesgenesoftheyeastCellWallIntegrity(CWI)sensingmechanism

OurpreviousresultsshowedthatthelethaleffectofPHMBonyeastcellswaspartlymitigatedbytrehalose[1],whichisknowntobeaprotectiveagentofthecellenvelope[20].Moreover,previousglobalgeneexpres-sionanalysisrevealedthatexposureofE.colicellstoPHMBinducedtheexpressionofgenesinvolvedincellwallmaintenance[14].ThisledustosearchforCWIgenesintheyeastgenome(Additionalfile1).Moreover,recentstudieshaveshownthatsomegenesinvolvedintrehalosemetabolismareregulatedbystressresponseelements(STRE)intheirpromoterregion;[21],inviewofthiswehaveincludedsomeyeastgenescontainingSTREintheirpromoterinouranalysis.Fromalistofselectedgenes(Additionalfile1),weconductedaquan-titativePCRanalysisaftercellexposuretoPHMBandtheresultsrevealedthattheCHS1,FKS1,GAS1,HSP150,KRE6,MSN2,MSN4,PKH1andYLR194cgeneswereup-regulatedinthePHMB-resistantJPIstrain,whileremainingunchangedinthePHMB-sensi-tivePE-2strain(Figure1).Moreover,CRZ1andRLM1geneswereinducedinJP1andrepressedinPE-2,whileCIN5andMNN9didnotchangeinJP1,butwererepressedinPE-2.TheexpressionoftheSLT2Figure1Relativeexpressionsoftheyeastgenesinvolvedinthecellwallintegritymechanismandinthegeneralstressresponse.RelativeQuantityrepresentstheexpressionofthe。

studiedgenesinthePHMB-treatedcells(0.01%)overnon-treatedcellsofthetwoindustrialstrainsJP1(redcolumn)andPE-2(bluecolumn).

generemainedunchangedinboththeindustrialstrains(Figure1).

Crz1pisacalcineurin-dependentzincfinger-typetranscriptionfactorthatbindstothepromotersoftheFKS1andCHS1genesinresponsetocalcofluorandcal-cium,respectively[23].Bothgeneswerefoundtobeco-expressed,withapeakinG1,whentheisotropiccellwallsynthesisalloweddaughter-cellexpansion[23]andourresults(Figure1)corroboratethat