利用RNA干扰技术双通道抑制弓形虫嘌呤补救合成途径的综述报告

利用RNA干扰技术双通道抑制弓形虫嘌呤补救合成途径的综述报告IntroductionToxoplasmosisisaworldwideparasiticinfectioncausedbytheprotozoanToxoplasmagondii.Thisparasitecaninfectvariousanimals,birds,andhumans.Inhumans,T.gondiiinfectioncancausesevereneurologicalandoculardiseases,particularlyinimmunocompromisedindividuals.Althoughfewdrugsareavailabletotreattoxoplasmosis,theirtoxicitiesandsideeffectslimittheirefficacy.Therefore,developingnewstrategiestocontrolT.gondiiinfectioniscrucial.RNAinterference(RNAi)isapowerfultooltostudygenefunctionandhasbeenusedtocontrolparasiticinfections.ThisreportaimstoreviewtheuseofRNAitotargetthepurinesalvagepathwayofT.gondii.ThePurineSalvagePathwayofT.gondiiT.gondiiisanobligateintracellularparasitethatreliesonthehostcell'spurinenucleotidesforitsownmetabolicprocesses.Thepurinesalvagepathwayisessentialforthesynthesisofpurinenucleotides,whicharerequiredforDNAandRNAsynthesis.ThesalvagepathwayofT.gondiioperatesinthecytosol,anditinvolvestheuptakeofpurinesfromthehostcellfollowedbytheirconversionintonucleotidesbyspecificenzymes.Thekeyenzymesofthepurinesalvagepathwayarehypoxanthine-guanine-xanthinephosphoribosyltransferase(HGXPRT)andadenosinephosphoribosyltransferase(APRT),whichcatalyzetheconversionofhypoxanthine/guanine/xanthineandadenosineintotheircorrespondingnucleotides.RNAInterferenceRNAiisagenesilencingmechanismthatoccursnaturallyincells.TheprocessinvolvesthecleavageofmRNAbysmallRNAmolecules,suchassmallinterferingRNAs(siRNAs)orshorthairpinRNAs(shRNAs),thatarecomplementarytothemRNA.RNAicanbeusedtoinhibitgeneexpressioninmanyorganisms,includingparasites,bydesigningsiRNAsorshRNAsthattargetspecificgenes.Onceinsidethecell,siRNAorshRNAbindstoacatalyticproteinknownasRNA-inducedsilencingcomplex(RISC).TheRISCthencleavesthetargetmRNA,leadingtoadecreaseingeneexpression.Double-StrandedRNAApproachtoTargetingT.gondii'sPurineSalvagePathwayThedouble-strandedRNAapproachinvolvestheuseoflongdouble-strandedRNAs(dsRNAs)thattriggerRNAibyactivatingadsRNA-dependentproteinkinase(PKR)whichphosphorylatesthetranslationinitiationfactor2(eIF2),leadingtotranslationalsuppression.ThisapproachhasbeenusedsuccessfullytoinhibitgeneexpressioninT.gondii.AstudybyDonaldetal.(2006)usedlongdsRNAstotargetHGXPRTandAPRTgenesinT.gondii.TheauthorsshowedthatlongdsRNAtechnologycouldreducetheexpressionofHGXPRTandAPRTgenes,therebyinhibitingT.gondii'sgrowthininfectedcells.However,thelongdsRNAapproachislimitedbyitsoff-targeteffectsandthegenerationofnonspecificresponses,suchastheinterferonresponse.siRNAApproachtoTargetingT.gondii'sPurineSalvagePathwayThesiRNAapproachinvolvestheuseofshortdsRNAs,whicharemoreeffectiveandspecificthanlongdsRNAs.ShortdsRNAsavoidoff-targeteffectsandnonspecificresponsesassociatedwithlongdsRNAs.AstudybyHuynhetal.(2008)usedansiRNAapproachtotargetHGXPRTandAPRTgenesinT.gondii.TheauthorsshowedthatsiRNAcouldreducetheexpressionofHGXPRTandAPRTgenes,leadingtoreducedparasitegrowthinvitro.IncontrasttothelongdsRNAapproach,thesiRNAmethodishighlyspecificbutrequiresawell-definedtargetsequenceandtheappropriatedeliveryvehicle.shRNAApproachtoTargetingT.gondii'sPurineSalvagePathwayTheshRNAapproachissimilartothesiRNAapproach,anditalsoinvolvesusingshortfragmentsofRNAtoinduceRNAi.ThemaindifferencebetweenthetwoapproachesisthatshRNAisexpressedfromaplasmidorlentiviralvector,whichleadstoapersistentandlong-termgenesilencing.AstudybyXiaetal.(2015)usedtheshRNAapproachtotargetHGXPRTandAPRTgenesinT.gondii.TheauthorsshowedthatshRNAtargetingHGXPRTandAPRTcouldinhibitthegrowthofT.gondiiinvitro.TheshRNAapproachhastheadvantageoflonger-termgenesilencingbutrequiresvector-basedexpressionsystems.ConclusionRNAiisapowerfultooltostudygenefunctionandhasshownpromiseincontrollingparasiticinfections.ThepurinesalvagepathwayofT.gondiiisanessentialpathwayforparasitegrowth,anditstargetingrepresentsapotentialapproachfortoxoplasmosistherapy.TheuseofdsRNA,siRNA,andshRNAapproacheshasdemonstratedthatRNAicaninhibittheexpressionofHGXPRTandAPRTgenes,leadingtoreducedT.gondiigrowthinvitro.Furtherstudiesareneededtooptimizethed