Peptide gene fused to minor coat protein gene Phage coat proteins displaying foreign proteins Peptide gene fused to majorcoat protein gene Foreign proteins are displayed on some coat proteins Cell ID: 238099
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REVIEWOpenAccessBacteriophagesandtheirimplicationsonfuturebiotechnology:areviewIrshadUlHaq,WaqasNasirChaudhry,MahaNadeemAkhtar,SaadiaAndleebandIshtiaqQadri *Correspondence:irshadulhaq85@gmail.comNUSTCenterofVirology&Immunology(NCVI),NationalUniversityofSciences&Technology(NUST),H-12,Islamabad44000,PakistanetalVirologyJournalhttp://www.virologyj.com/content/9/1/9 ©2012Haqetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited. (Mumbai)[6].Inwesttheconceptofphagetherapydiedoutin1940duetotheemergenceofantibiotics,butinformerSovietUnionitwasusedandisstillinpractice.TheEliavainstituteinTbilisiGeorgiaisconsideredthepioneerinthisregardwherephagetherapyisextensivelystudiedandapplied[7].Westhasremainedreluctanttousephagetherapyduetotheunreliableearlytrialsofphagetherapy.ButstillphagetherapygotattentioninUSA.Williamsmithandhisfellowsreportedthesuccessfuluseofphagesagainstinmice[8].Oneofthereasonsoftheavoidanceofthephagether-apyinmostofthewesterncountrieswasunreliableandinconsistentresultsofmanyphagetherapytrials.Buttodayitisacceptedthatthemainreasonbehindthefail-urewaspoorunderstandingofphagebiologyandsomeotherissueslikequalitycontrolduringpreparationoftherapeuticstocks[9].Phagetherapyhasbeenusedinanimals,plants,andhumanswithdifferentdegreeofsuccess.Phageshaveseveralpotentialadvantagesoverantibioticsbutatthesametimeitdoeshavedisadvan-tagesaswell.Themainadvantageofphagesistheirspecificityfortargetbacteriawhichreducesthedamagetonormalfloraofthehostgreatly.Thebacteriatobetargetedmustbeidentifiedfirstorotherwiseacocktailofphagesshouldbeused.Bacteriophagesareself-limitingi.e.theyrequiretheirhoststobeconstantlygrowing;ifthebac-terialpathogenstheyarespecificforareabsenttheywillnotpersistforlongenough[1].Replicationatthesiteofinfectionisanotheradvantageofphages.Theyaresafewithnoorlesssideeffects[10,11].Ifbacteriabecomeresistanttophagesthenphagesdoevolvenaturallytoinfecttheaforementionedresistantbacteria,hencemini-mizingthechancesofbacterialescape,whichscoresanotheradvantageofphageoverantibiotics[10].Aftertheiradministrationphagescandissipateveryquicklythroughoutthebodyreachingalmosteveryorgan;buttheimmunesystemswiftlyclearssystemicphageswhichposeyetanotherproblemtotheiraccep-tanceastherapeuticagent[12,13].Oneoftheseriousconcernsabouttheuseofphageinvivoisastrongantibodyresponsewhichwouldclearthephagesmorequicklyandthustheuseofphagesforextendedperiodoftimewouldnotbepos-sible[1].Otherdrawbacksofphagesastherapeuticagentsaretheirnarrowhostranges,andthefactthatphagesarenotalwayslyticundercertainphysiologicalconditions.Duringthepreparationofphagestocksitmustbeensuredthatphagepreparationsarefreeofbac-teriaandbacterialtoxinsinordertoavoidsecondaryinfections.Butsterilizingphagescouldinactivatethem.Phagesmayimparttoxicpropertiestothebacteriaresultinginvirulence[5].Onewayaroundistheuseofphagelyticenzymeendolysin,ratherthanadministeringthewholevirion[14-16].Similarlygeneticallymodifiedphagescanbeused,whichwillonlydelivertheDNAessentialformak-ingantibacterialsthatwouldbespecificforthetargetbacteria[17].Atthemomentitseemsabitfarthatphagetherapywillreplaceantibioticsexclusively,butthereisthehopethatitwillbeusedcomplementarytoantibioticsespe-ciallyforantibioticresistantstrains[1].Phageswillbemuchmorereliablewhenusedexternallyandwheretheimmunesystemgivesitachancebyfavoringittoper-sistwithinthebodyforalittlewhile[1].PhagedisplayTheconceptofphagedisplaywasfirstintroducedin1985[18](Figure1).Phagedisplayisamoleculartech-niqueusedforsynthesizingpolypeptideswithnovelcharacteristics.TheDNAthatencodesthepolypeptideisfusedwithphagecoatproteingenes,andthedesiredproteinisexpressedonthesurfaceofthephageparticle[18,19].ForphagedisplayfilamentousphageM13ofisextensivelyused,otherphageslikelambdaandT7arealsousedinphagedisplaysystem[20,21].Phagedisplaylibrariescanbeusedforthescreeningandisola-tionofpeptidesthatarehighlyspecificandwhichhaveaffinityfortargetproteins.Thesepeptidescanbeusedindrugdesignasreagentsforunderstandingmolecularrecognitionanditalsominimizemimicsforreceptors[19].Thesepeptidescanbeusedastherapeuticagentsbyinhibitingreceptor-ligandinteractionoractingasMoreovertheseproteinscanbeusedforthedetectionofpathogensandagentsthatareconsideredtobeapotentialthreattotheenvironment[22].Directedevolu-tionofproteinscanbeusedtoenhancetheenzymaticactivityandbindingproperties[23].Theactivesiteoftheenzymeisrandomlyalteredandtheactivityoftheenzymeisincreased[1].PhagedisplaycanalsobevariedbyusingphagestodisplaytheFabantibodyfragmentslibrarymostlyonfilamentousphagesurfaces[24].Theselibrarieshavemanyusesinresearchbutoneofthemostimportantusesofitisinthetreatmentagainstcocaineaddictionwherephagesareadministerednasallyandthenultimatelytheymaketheirwaytocentralner-voussystem(CNS).Incentralnervoussystemthedis-playedantibodybindstococainemoleculeandinhibitsitsactiononbrain[25].Intensiveandstateoftheartworkdonebymanyresearchershavemadephagedis-playaphenomenalpartofbiotechnology.Amongstotherapplicationsphageantibodieshaverevolutionizedtheconceptoftherapeuticdrugsanddrugdesign[19].Molecularevolutionandprotein-ligandinteractionhasbeenexplainedbyphagedisplayunambiguously[21].etalVirologyJournalhttp://www.virologyj.com/content/9/1/9Page2of8 Phage particle Peptide gene fused to minor- coat protein gene Phage coat proteins displaying foreign proteins Peptide gene fused to major-coat protein gene Foreign proteins are displayed on some coat proteins Cellscontaining phagemid expressing coat protein fusion gene are infected Phage particle Phage particle Some coat proteins displaying foreign proteins Phage DNA Plasmid DN A Minor coat protein and Gene Major coat protein and Gene Foreign peptide and gene Origin of replication Figure1 Somemethodsthatareusedtofuseforeignpeptidestothesurfaceofphage .Foreignpeptidescanbedisplayedonmorethan onephagecoatproteins.Smallerforeignpeptidesaredisplayedinmorenumbersbutitalsodependsonthetypeofantigen,coatproteinand thephage.(a)Thegeneforaforeignpeptideisdirectlyfusedtotheminorcoat-proteingene.Theforeignantigenisdisplayedbyallminorcoat proteins.(b)Foreignpeptidegeneisattachedtomajorcoatproteingenewhileanothercopyofthegene(majorcoatproteins)isalsopresent. Foreignproteinisdisplayedonsomemajorcoatproteins.(c)Cellscontainingaphagemid(plasmidthathavebothplasmidandbacteriophage originofreplication)areinfectedwithunchangedhelperphagewhichthenexpressestheforeignpeptideorprotein.Foreignantigensare displayedbysomecoatproteins. Haq etal . VirologyJournal 2012, 9 :9 http://www.virologyj.com/content/9/1/9 Page3of8 PhagetypingThespecificityofphagesforbacterialcellsenablesthemtobeusedforthetypingofbacterialstrainsandthedetectionofpathogenicbacteria[1].Phagetypingisalsoknownastheuseofsensitivitypatternstospecificphagesforpreciselyidentifyingthemicrobialstrains.Thesensitivityofthedetectionwouldbeincreasedifthephagesboundtobacteriaaredetectedbyspecificantibodies[26].Forthedetectionofunknownbacterialstrainitslawnisprovidedwithdifferentphages,andiftheplaque(clearzones)appearsthenitmeansthatthephagehasgrownandlysedthebacterialcell,makingiteasytoidentifythespecificbacterialstrain[1].Therearecertainothermethodswhichcanbeemployedtodetectpathogenicbacteriasuchastheuseofphagesthatcandeliverreportergenes{e.g.lux}specifically[27]orusinggreenfluorescentprotein,[28]thatwouldexpressafterinfectionofbacteria.Similarlyphageshav-ingafluorescentdyecovalentlyattachedtotheircoatscanbeusedtodetectspecificadsorption[29,30].Thedetectionofsomeofthereleasedcomponentssuchasadenylatekinase[31]afterthespecificlysisofbacteriaandtheuseofantibodiesandpeptidesthataredisplayedbyphagescanalsobeused,thatwillbindtotoxinsandbacterialpathogensspecifically[22].Dualphagetechnol-ogyisanotherapplicationofphagesindetectionofbac-teria,inwhichphagesareusedtodetectthebindingofantibodiestospecificantigens[7].Phageamplificationassaycanalsobeusedtodetectpathogenicbacteria[32].ThetechniquehasmostextensivelybeenusedforthedetectionofMycobacteriumtuberculosisPseudomonas,Salmonella,Listeria,andCampylobacterspecies[33].TargetedgenedeliverythroughPhagesPhagesarethepotentialtherapeuticgenedeliveryvehi-cles[33,34].TherationaleofusingphagesfortargetedgenedeliveryissimilartothatofusingphagesforDNAvaccinesdeliveryinwhichthephagecoatprotectstheDNAinsidefromdegradationafterithasbeeninjected.Butconceptuallybotharedifferent.Phagesabilitytodisplayforeignproteinsontheirsurfacesenablethemtotargetspecificcelltypeswhichisaprerequisiteforsuc-cessfulgenetherapy[1].Phagedisplayandartificialcovalentconjugationareusedtodisplaytargetingandprocessingmoleculesonthesurfacesofphages[35,36].Forthedeliveryofphages,targetingsequencessuchasfibroblastgrowthfactorhavebeenusedtothecellshav-ingtheappropriatereceptors[37,38].Enhancingtheuptakeandendosomalreleaseofphages,proteinssequencessuchaspentonbaseofadenoviruswhichmediatesentry,attachmentandendosomalreleaseareused[39].Theproteintransductiondomainofhumanimmunodeficiencyvirus(HIV)tatproteinandthesimianvirus40(SV40)Tantigennuclearlocalizationsignalhavealsobeenusedtoenhancetheuptakeandnucleartargetingofphageslikelambdathathavebeenmodified[40].Otherdisplayedpeptidesthatcanfacili-tategenedeliveryviaphagesincludeintegrinbindingpeptideswhichenhancebindinganduptake[37]andDNAdegradationreducingDNaseIIinhibitor[38].Toscreentheabilityofphagesfortargetingspecificcellsandtissues,phagedisplaylibrarieshavebeenusedinmicemanytimesandeverytimephageswerefoundinspecifictissues[41].Forinstanceisolatingphagesthattargetliver,micewereinoculatedwithphagedisplaylibrariesandphageswereisolatedafterextractingthelivers[1].Similarinvitrostrategyisusedfortheisola-tionofphagedisplayedpeptidesthatenhancedcytoplas-micuptakeintomammaliancells[42].Soagainphagesprovedthemselvestobeversatilebymakingitpossibletotargetspecifictissueseitherbyscreeningphagedis-playlibrariesrandomlyorbyrationaldesign[1].PhagesasvehiclesforvaccinesdeliveryPhageshavebeenusedasvehiclesforthedeliveryofvaccines(Figure2).Phageparticlescanbeuseddirectlycarryingthevaccineantigensexpressedontheirsur-faces.ButincaseofDNAvaccinesthesequencesthatareessentialforthevaccineantigensynthesisareincor-poratedintothephagegenomeandthephagewouldthenactasvehicleforthedeliveryofDNAvaccine[13].Phagedisplaycanbeusedtoconstructphagesthatwoulddisplaythespecificantigenicpeptideontheirsur-faces[1].Phagedisplaylibrariescanbescreenedwithspecificantiserumtodetectnovelantigensandmime-topes.Mimetopesarethepeptidesthatmimictheanti-genicpropertiesandsecondarystructuresofprotectiveprotein,lipidorcarbohydrate,althoughhavingdifferentprimarystructure[43,44].Phagedisplaylibrariescanalsobescreenedagainsttheserumofconvalescentsfortheidentificationofpotentialvaccinesagainstspecificdiseases[45].Therearesomecasesinwhichwholephageparticlesthatdisplayedantigenicpeptideshavebeenusedasvaccinesinanimalmodels[46,47].Ratherthantranscriptionalfusiontoacoatprotein,somesubstancescanbeartificiallyconjugatedtothephagesurfaceaftergrowth,whichwillincreasetherangeofdisplayedantigens[48].Phagesareconsideredtobenaturalimmunostimulators[13,49]thereforeanantigenthatispresentedonthephagecoatproteinwouldcomereadyconjugatedwithanaturaladjuvantactivity,needingnoseparateproteinpurificationandsubsequentconjugationtoacarrierproteinbeforeimmunization.RecentlyithasbeenshownthatunchangedphagescanbeusedtodeliverDNAvaccinesetalVirologyJournalhttp://www.virologyj.com/content/9/1/9Page4of8 WZ}P v WZP ]oÇ]vP ](]]ooÇ }viµP } ]À v]P v }v ] µ( W} ]À v]P v }viµP ](]]ooÇ } ZP } } ]v /}o P v v}]vP (} } ]À v]P v W} ]À v]P v P v (µ } ZP } } ]v P v W} ]À v]P v ]oÇ }v µ( }lÇ}] } } ]v (µ]}v ~ ~ µlÇ}] Æ ]}v À } µlÇ}] Æ ]}v À } o}v ]v} ZP P v}u WZP }v]v]vP À]v P v ]v µlÇ}] Æ ]}v À } v ]oÇ]vP } ]À v]P v s]v P v ~ ~ ~ µlÇ}] Æ ]}v À } }v]v]vP À]v P v Figure2 Someexamplesofmethodsforvaccinesdeliveryviaphages .(a)Hostcouldbeinoculatedforphage-deliveredproteinvaccine.(b) Asin(a)hostisinoculatedforphage-deliveredproteinvaccinebuttheprotectiveantigenisexpressedasprokaryoticcoatproteinfusion.(c) InoculationofhostforphagemediatedDNAvaccination.(d)Hostcanbeinoculatedforhybridphagevaccination,whereinoneconstruct proteinandDNAvaccinesaredeliveredthroughphage.(e)HostinoculationforstandardDNAvaccination. Haq etal . VirologyJournal 2012, 9 :9 http://www.virologyj.com/content/9/1/9 Page5of8 moreefficientlythanstandardplasmidDNAvaccination[13,50-52].Thegeneforvaccineisclonedunderthecontrolofeukaryoticexpressioncassetteinalambdabacteriophageandpurifiedphageparticlesareinjectedintothehost.ThecoatprotectsDNAfromdegradationandasitactsasavirus-likeparticleitwouldtargetthevaccinetotheantigenpresentingcells[1].WhenitwascomparedwiththestandardDNAvaccination,theanti-bodyresponsewasverymuchsuperiorinmice[52]andrabbits[50].Recentlythepossibilityofproducingahybridphagehasbeenproposed,aDNAvaccinecon-tainedinphageparticleundertheeukaryoticpromoterandaphagedisplayvariantofthesameantigenispre-sentonthephagesurface[1].Suchavaccinewouldeffi-cientlytargetbothhumoralandcellularimmunesystems[13].Itcanalsobeextendedtothemodificationofsurfaceofthephagevaccinebyincorporatingspecificproteinsequencestotargetparticularimmunecellstypeslikegalactoseresiduesthatwilltargetgalactoserecognizinghepaticreceptorsintheliver[48].Similarlydendritic[53]andlangerhanscellscouldbetargetedbyisolatingpeptidefromthephagedisplaylibraries[54].PhagesasbiocontrolandbacteriophagebioprocessingPhagescouldbeusedaspredatorsofpests(bacteria)foundinassociationwithplants,fungiortheirproducts[55,56].PhagemediatedbiocontrolofplantpathogenshassuccessfullybeenattemptedagainstXanthomonaspruniassociatedbacterialspotofpeachestocontrolinfectionsofpeaches,cabbageandpeppers.PhageshavealsobeenusedtocontrolRalstoniasolanacearumtobacco.TheyhavebeensuccessfullyemployedagainstXanthomonascampestriswhichcausespotsontomatoes.Similarlybacterialblotchofmushroomscausedbydomonastolaasiicanbetreatedwithphages[57].Phageshavealsobeenconsideredasameansofcontrollingthebiofoulingofthermalpowerplantscondensertubes[58].Bacteriophagesinbioprocessingareusedtoreducethebacterialloadinfoodsusuallyintheminimallyprocessedfoodstoavoidcookingassociatedflavorortexture[59].Controllingpathogensoffruitsandvegetablesisofmuchconcernasthesefoodscannotbefurtherprocessedthatwouldkillanypathogenpresent.Controlofpathogensviaphagesisanon-thermalinterventionbywhichgrowthCampylobacteronchickenskin[60]Salmonellaenteritidisincheese[61]Listeriamonocyto-onmeat[62]andfreshcutfruit[63]isreduced.Extendingtheshelflifeofanimalproducts,phagebiopro-cessingcouldbeused[64].ConclusionDetailsgivenabovegiveaglimpseofthelargerangeofapplicationsofphagesinthefieldofbiotechnologyandmedicalscience.Theapplicationsofphagesrangefromthediagnosisofthedisease,throughphagetyping,anditsprevention(phagevaccine),tothetreatment(phagetherapy).Thereisthehopethatphagescouldbeusefultohumansinmanyways.Bymakingacocktailofphagesitwouldbecomeeasytotreatawidevarietyofbacterialinfectionsthatareotherwiseresistanttothelatestgenerationsofantibiotics.Aphagecanbeusedindividuallytotreatabacterialinfectionbylysingthebacterialcellasitishavingthelyticpotential.Atthesametimetheversatilityofphageswouldallowustousetheantibodiesagainstthebacteriathathavebeendisplayedonthephagesurface.SimilarlyaprotectiveantigencouldbedeliveredasaDNAorphagedisplayvaccine.Soamixtureofphagesthataremodifiedgeneticallywouldbemorehelpfulinaddressingalltheseproblems.Phageshavealsobeengoodtocopewiththefoodspoilageproblem,andtotreatthebacterialinfec-tionofplantsandfruits.Therearesomeconcernsabouttheuseofphages.Itincludesthesafetyandefficacyissues,aswellasimmuneresponsetotheadministeredphages.Growthoptimizationandpurificationstrategiesofphagesarealsosomeissuesneededtobeaddressed.Duetotherapidprogressinthefieldsofbiotechnologyandmole-cularbiologyitishopedthattheseentities(phages)whicharepresentabundantlyinthebiospherecouldanswermanyquestionshumanbeingsarehaving.HIV:Humanimmunodeficiencyvirus;CNS:Centralnervoussystem;SV40:Simianvirus40.AcknowledgementsWeacknowledgeNationalUniversityofSciences&Technology(NUST)Islamabadforprovidingfinancialassistance.IUHsurveyedtheliterature,collectedthereferencesconcernedwiththisreviewanddraftedthemanuscript.MNAdesignedthefigures.WNC,SAandIQcontributedinrevisingandeditingthemanuscript.Alltheauthorsreadandapprovedthefinalmanuscript.CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.Received:11June2011Accepted:10January2012Published:10January20121.ClarkJR,MarchJB:Bacteriophagesandbiotechnology:vaccines,genetherapyandantibacterials.TrendsBiotechnol2.AckermanHW:Tailedbacteriophages:theCaudovirales.AdvVirusRes3.InalJM:Phagetherapy:areappraisalofbacteriophagesasantibiotics.ArchImmunolTherExp4.SummersWC:Bacteriophagediscovered.FelixdHerelleandtheOriginsofMolecularBiologyYaleUniversityPress;1999,47-59.5.HermosoJA,GarciaJL,GarciaP:Takingaimonbacterialpathogens:fromphagetherapytoenzybiotics.CurrOpinMicrobioletalVirologyJournalhttp://www.virologyj.com/content/9/1/9Page6of8 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58.SakaguchiI,ShinshimaK,KawarataniK,SugaiO:Controlofmicrobiofoulingusingbacteriophage2.Detectionofphagesandfundamentalstudyoftheirlyticeffectonfoulingbacteria.ChuoKenkyushoHokoku1989,1-32.59.GarcíaP,RodríguezL,RodríguezA,MartínezB:Foodbiopreservation:promisingstrategiesusingbacteriocins,bacteriophagesandendolysins.TrendsFoodSciTechnol60.GoodeD,AllenVM,BarrowPA:ReductionofexperimentalSalmonellaandCampylobactercontaminationofchickenskinbyapplicationoflyticApplEnvironMicrobiol61.ModiR,HirviY,HillA,GriffithsMW:EffectofphageonsurvivalofSalmonellaenteritidisduringmanufactureandstorageofcheddarcheesemadefromrawandpasteurizedmilk.JFoodProt62.DykesGA,MoorheadSM:CombinedantimicrobialeffectofnisinandalisteriophageagainstListeriamonocytogenesinbrothbutnotinbufferoronrawbeef.IntJFoodMicrobiol63.LeverentzB,ConwayWS,CampMJ,JanisiewiczWJ,AbuladzeT,YangM,SaftnerR,SulakvelidzeA:BiocontrolofListeriamonocytogenesonfresh-cutproducebytreatmentwithlyticbacteriophagesandabacteriocin.ApplEnvironMicrobiol64.GreerGG,DiltsBD:ControlofBrochothrixthermosphactaspoilageofporkadiposetissueusingbacteriophages.JFoodProtdoi:10.1186/1743-422X-9-9Citethisarticleas:etalBacteriophagesandtheirimplicationsonfuturebiotechnology:areview.VirologyJournal Thorough peer review No space constraints or color Þgure chargesResearch which is freely available for redistribution www.biomedcentral.com/submit etalVirologyJournalhttp://www.virologyj.com/content/9/1/9Page8of8