ContentslistsavailableatScienceDirect
Drug
Resistance
Updates
j ou rn a l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / d r u p
Cryptic
prophages
as
targets
for
drug
development
Xiaoxue
Wang
a,∗,
Thomas
K.
Wood
b,c,∗∗aKeyLaboratoryofTropicalMarineBio-resourcesandEcology,GuangdongKeyLaboratoryofMarineMateriaMedica,SouthChinaSeaInstituteof
Oceanology,ChineseAcademyofSciences,Guangzhou510301,PRChina
bDepartmentofChemicalEngineering,PennsylvaniaStateUniversity,UniversityPark,PA16802-4400,UnitedStates
cDepartmentofBiochemistryandMolecularBiology,PennsylvaniaStateUniversity,UniversityPark,PA16802-4400,UnitedStates
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received27March2016
Receivedinrevisedform30May2016 Accepted30May2016
Keywords: Crypticprophage Antibioticresistance Antibiotictolerance Horizontalgenetransfer Toxin–antitoxinsystem
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Bacterialchromosomesmaycontainupto20%phageDNAthatencodesdiverseproteinsrangingfrom thoseforphotosynthesistothoseforautoimmunity;hence,phagescontributegreatlytothemetabolic potentialofpathogens.Activeprophagescarryinggenesencodingvirulencefactorsandantibiotic resis-tancecanbeexcisedfromthehostchromosometoformactivephagesandaretransmissibleamong differentbacterialhostsuponSOSresponses.Crypticprophagesareartifactsofmutagenesisinwhich lysogenicphagearecapturedinthebacterialchromosome:theymayexcisebuttheydonotformactive phageparticlesorlysetheircaptors.Hence,crypticprophagesarerelativelypermanentreservoirsof genes,manyofwhichbenefitpathogens,inwayswearejustbeginningtodiscern.Hereweexplorethe roleofactiveprophage-andcrypticprophage-derivedproteinsintermsof(i)virulence,(ii)antibiotic resistance,and(iii)antibiotictolerance;antibiotictoleranceoccursasaresultofthenon-heritable phe-notypeofdormancywhichisaresultofactivationoftoxinsoftoxin/antitoxinlocithatarefrequently encodedincrypticprophages.Therefore,crypticprophagesarepromisingtargetsfordrugdevelopment. ©2016TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Contents
1. Introduction...30
2. Prophagesarereservoirsofvirulencegenes...31
3. Prophagesarereservoirsofantibioticresistancegenes ... 32
4. Prophagesarereservoirsofantibiotictolerancegenes...34
5. Antibioticstriggerprophageexcision...34
6. Drugsandphage...35
7. Perspectives...35
Conflictofintereststatement...36
Acknowledgements ... 36
References...36
1. Introduction
Bacteriophagesandbacteriaarethemostabundantlifeforms onEarth. They also interact frequently, and each phage infec-tionhasthepotentialtointroducenewgeneticmaterialintothe
∗ Correspondingauthorat:KeyLaboratoryofTropicalMarineBio-resourcesand Ecology,SouthChinaSeaInstituteofOceanology,ChineseAcademyofSciences, Guangzhou510301,PRChina.
∗∗ Correspondingauthorat:DepartmentofChemicalEngineering,Pennsylvania StateUniversity,UniversityPark,PA16802-4400,UnitedStates.
E-mailaddresses:[email protected](X.Wang),[email protected](T.K.Wood).
bacterialhost,therebydrivingtheevolutionofbacteria.The intro-ductionofnovelgenesbyphagesintothebacterialhostcanconfer beneficialphenotypesthatenabletheexploitationofcompetitive environments(Canchayaetal.,2003;LawrenceandOchman,1998; Penadésetal.,2015).Forexample,marinebacteriophageencode photosynthesisgeneswhichmayproviderelieffromintense sun-lightinoceansforthephageandhost(Mannetal.,2003),aswellas encodeadaptivebacterialimmunesystemstoprovideimmunity fromcompetingphageknownasclusteredregularlyinterspaced shortpalindromicrepeats(CRISPR)/CRISPR-associated(Cas) sys-tems(Bellasetal.,2015).
http://dx.doi.org/10.1016/j.drup.2016.06.001
1368-7646/©2016TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4. 0/).
Fig.1.Differenttypesofphagesandprophages.Genesincrypticprophagesareabbreviatedasint:integrase,xis:excisionase,RM:restriction–modificationsystems,andTA: toxin–antitoxinsystems.
Amongthebeneficialgenes,phagesprovideDNAforvirulence, resistance,andtolerancetoantibioticsasthreemajorfactorsfor pathogensduringinfection.Resistanceinvolvesgeneticmutations thatallowforgrowthinthepresenceofantibioticswhereas toler-anceinvolvesmetabolicdormancyfromtheactivationoftoxins of toxin/antitoxin (TA) systems that allows pathogens tosleep throughacourseofantibiotictreatment(Wood,2016).
Treatingpathogenicbacteriathatevadeantibioticshasbecome aglobalissue.Thisreviewwillfocusonthevirulence,resistance, andtolerancegenescarriedbyprophages,theantibioticsorother chemicalsthatcantriggerthespreadofthesegenesbyprophage excision/integration,andalsosomepreventativestrategiestotreat pathogens bytargeting prophages.We alsoemphasize ways to avoidtheadverseeffectsoftriggeringvirulencedisseminationby prophages.
2. Prophagesarereservoirsofvirulencegenes
Bacteriophagesprovideoneofthemostefficientvehiclesfor movingDNA sequences(theirown andthehost’s DNAby mis-take),viatransduction,betweenbacterialcells.Horizontaltransfer ofgeneticinformationbyphagesismuchmoreprevalentthan pre-viouslythought,andtheenvironmentplaysacrucialroleinthe phage-mediatedtransferofvirulencegenes(Penadésetal.,2015). Therelativelyhighnumbersofphage(1030phageandapproximate
ratioof10 phagetoeach bacterium)leadstofrequentlyticand lysogenicphageinfections(1025infections/s)(Chibani-Chennoufi etal.,2004).Unlikelyticphages,temperatephagesareintegrated intothebacterialgenomeandmaintainalong-termlysogenic rela-tionshipwiththeirhosts(Fig.1).Lysogenyhasauniquerolewithin
thebacterium-phagearmsraceinthatitfavorsthedevelopment ofasymbioticrelationshipbyprovidinganecologicalwindowfor theevolutionofmutually-beneficialfunctions(Feineretal.,2015). Hence,thesefrequentphageinfectionsprovideampleopportunity toaffectvirulence.
Therearemajordifferencesbothbetweenandwithindifferent bacterialspecies in theirability tocauseinfection. Opportunis-ticandpathogenicbacterialspeciesincludeEscherichiacoli,Vibrio cholerae,Pseudomonasaeruginosa,Listeriaspp.,Salmonellaenterica, Enterococcusfaecalis,Streptococcusspp.,andStaphylococcusspp.A majordrivingforceintheemergenceandevolutionofpathogenic isolates is the horizontal transfer and acquisition of virulence factors. Several mobile genetic elements (insertion sequences, plasmids, bacteriophages,and pathogenicity islands)have been implicatedinthehorizontaltransferofvirulencegenes;oneofthe mostsignificantgroupsisthebacteriophages(reviewedinHastings etal.(2004)).
Lysogenicconversionbyprophagesencodingtoxinsandother virulencedeterminantsisthemostostensiblecontributionto bac-terialpathogenesis(Brussowetal.,2004).Manydiseasesarecaused bytoxinsthatareencodedbyphagessuchasdiphtheria,cholera, dysentery,botulism,foodpoisoning,scaldedskinsyndrome, necro-tizingpneumoniaorscarletfever(HackerandKaper,2000).Among these,exotoxinproductionsuchasscarlatinaltoxin,choleratoxin andShigatoxinistheamongthebestdocumentedvirulence fac-tors(WagnerandWaldor,2002).Bacteriophagescanalsoalterhost physiology toincreasevirulence atdifferentstagesof infection, includingbacterialadhesion,colonization,invasion,resistanceto hostimmunedefenses,andtransmissibilityamonghumanhost(as reviewedbyWagnerandWaldor(2002)).
Thecontributionofphagestopathogenicitywasfirst discov-eredin streptococciin 1927when it wasshown that nontoxic streptococci acquired the ability to produce scarlatinal toxin throughthephagesreleasedbythetoxicstreptococci(Frobisher andBrown,1927).In the1950s,toxin-encoding bacteriophages of Corynebacterium diphtheria further linked phages to bacte-rialpathogenicity (Barksdaleand Arden, 1974; Freeman, 1951; Groman,1953).
ThecholeratoxinofV.choleraeillustrateswellacaseofhow multiplephagescontributetobacterialpathogenicity(Brüssowand Hendrix,2002;Davisetal.,2000;Karaolisetal.,1998,1999;Waldor andMekalanos,1996).Shigatoxin(Stx)ispresentinShigella dysen-teriaetype1andshigatoxinproducingE.coli(STEC).Twomajor classes of Stx are foundin STEC, Stx1 and Stx2. The stxgenes inE.colistrainsarethecentralcontributorstothevirulence of enterohemorrhagicE.coli(EHEC),andEHECinfectioncancause bloodydiarrhea andcan leadtohemolyticanemia, thrombocy-topenia,renalfailure,andeventodeath(Kaperetal.,2004).Stx inE.coliO157:H7isencodedasalategeneproductbytemperate bacteriophageintegratedintothechromosome.Phagelategenes, includingstx,aresilentinthelysogenicstate,andShigatoxin(Stx) productiondependsontheactivationoftheStxprophage.Stress signals,includingsomeinducedbyantibiotics,triggerthephage toenterthelyticcycle,andphagereplicationandStxproduction occurconcurrently(Kaperetal.,2004).
ProphagealsoplaysaroleinthevirulenceofP.aeruginosa.P. aeruginosaisanimportantopportunisticpathogenwithabroad hostrange(plants,invertebrates,andvertebrates)(Palleroni,1984), andP.aeruginosaisthemostcommoncauseofchroniclung infec-tionsincysticfibrosis(CF)patients(Lyczaketal.,2002).ThePf4 prophageisessentialforseveralstagesoftheP.aeruginosabiofilm lifecycle,anditsignificantlycontributes toitsvirulenceinvivo (Mai-Prochnowetal.,2004;Riceetal.,2009).
S. enterica serovar Typhimurium harbors two functional prophages, Gifsy-1 and Gifsy-2 that contain virulence genes (Figueroa-BossiandBossi,1999).ProphageGifsy-2carriesthesodC geneforaperiplasmic[Cu,Zn]-superoxidedismutaseinvolvedwith thedefenseagainstkillingbymacrophages(Farrantetal.,1997). Theremovalofbothprophagesleadstoasignificantattenuationof virulence,andtheremovalofprophageGifsy-2significantlyreduces theabilityofS.entericaserovarTyphimuriumtoestablishasystemic infectioninmice(Figueroa-BossiandBossi,1999).
InStaphylococcusaureus,expressionofthephageencodedsea, seg2,sek2andsaktoxinsisgreatlyincreasedfollowingprophage induction(Sumbyand Waldor,2003).In S.aureus,tst,thegene thatencodes toxicshock syndrometoxin,is carried bya 15kb longpathogenicityisland(HochhutandWaldor,1999).Prophage andprophage-likeelementsarealsothemajorsourcesofvariation betweenthegenomesfromStreptococcuspyogenesstrainsinvolved intwodistinctpathologies,woundinfectionsandrheumaticfever. Theprophagesencodeseveralsecretedproteinsinvolvedinthe human–bacteriuminteraction,includingthescarletfevertoxin.
The mammalian intestine is home to a dense community of bacteria and its associated bacteriophage, which influence virulence. The Gram-positive bacterium E. faecalis is a natural inhabitantof themammaliangastrointestinaltractand is com-monlyfoundinsoil,sewage,water,andfood,frequentlythrough fecalcontamination(Matoset al.,2013).E.faecalisis an oppor-tunisticpathogenthatisamajorcauseofurinarytractinfections, bacteremiaand infectiveendocarditis,and E.faecalisV583 har-borsacompositephagederivedfromtwodistinctchromosomally encoded prophage elements. One prophage encodesthe struc-turalgenesnecessaryforphageparticleproductionandtheother prophageisrequiredforphageinfectionofsusceptiblehost bacte-ria.E.faecalisV583usesphageparticlestoestablishandmaintain dominanceofitsintestinalnicheinthepresenceofclosely-related
competingstrains.Recentstudiesofthehumanfecalviromeshow thattemperateratherthanlyticphagesarelong-term contribu-torstothemicrobialhostphenotypethroughprovisionofadaptive genes(Reyesetal.,2010).
3. Prophagesarereservoirsofantibioticresistancegenes Many microorganisms produce secondary metabolites with antimicrobialactivitiesandreleasethemintotheirnatural habi-tats.Theseantibiotic-producingmicroorganismsareresistantto theantibioticstheyproduce, butfor thenon-resistantbacteria, theyneedtodevelopresistancemechanismstoensuresurvival in these environments (Muniesa et al., 2013). The presence of antibioticsintheenvironmentmayexertlong-termselective pres-surefortheemergenceandhorizontaltransmissionofresistance mechanisms in the non-producing microorganisms. In recent years,theexplosivespreadofantibiotic-resistancedeterminants amongpathogenic,commensal,and environmentalbacteria has reached a global dimension. Prophages not only encode toxin genesforhumanpathogensbutalsocarrygenesthatenhancethe fitnessofthebacterialcellinecologicalniches(Hendrix,2003).For example,prophagescontaingenesthat provideprotectionfrom attackbyotherphages,suchasrestriction-modificationsystems (Vasu and Nagaraja, 2013) and CRISPR/Cas systems (Deveau et al., 2010). Growing evidence shows that phages also carry ortransfer genesthatparticipate inothercellularprocess such as inactivating antibiotics (Muniesa et al., 2013).Erythromycin resistancemethylases(Erm)conferresistancetothreeclassesof clinically-importantantibiotics(themacrolides,thelincosamides, andthestreptograminsB),andarewidespreadinStaphylococcus and otherbacterial species (Seppälä etal., 1998).For example, in Staphylococcus xylosus isolated from bovine mastitis milk, a novel macrolide–lincosamide–streptogramin B resistance gene islocatedona53-kbprophagethatissite-specificallyintegrated intotheS.xylosuschromosome(Wipfetal., 2014).In Staphylo-coccusfleurettii,this gene is locatedin a genomic island which is site-specifically integrated into thehousekeepinggene guaA, andexhibitstheabilitytocircularize(Wipfetal.,2015).Transfer oferythromycinresistanceviaprophagesofclinicallyisolatedS. pyogeneswassuggested toberesponsible fortheemergenceof streptococciwithmultipleresistancesintheclinicalenvironment (HyderandStreifeld,1978).P1bacteriophageslysogenizebacteria asindependentplasmid-likeelements,andarecentreportshows that P1-like bacteriophage carrying SHV-2 extended-spectrum -lactamaseispresentinclinicalstrainsofE.coli(Billard-Pomares etal.,2014).Acquiredresistanceto-lactamantibioticsis con-ferredprincipallyby-lactamasesandpenicillin-bindingproteins (LivermoreandWoodford,2006).Two-lactamasegenesandone gene encoding a penicillin-binding protein have been detected in the bacteriophage DNA fraction of sewage, river water, and fecalwastefromfarmedanimals,suggestingthatbacteriophages can be environmental vectors for the horizontal transfer of antibioticresistancegenes (Colomer-Lluch and Muniesa,2011). Quinoloneantibioticresistancegenes(qnrAandqnrS) havealso beenfoundin phageDNAisolated fromurbanwastewater and animal wastewater, suggesting that spreading genetic informa-tionviabacteriophageshasgainedimportanceintheresistance disseminationinenvironments(Colomer-Lluchetal.,2014).
Prophagesmayalsobecometrappedinthehostgenomedue to mutation (Canchaya et al., 2003); these inactive prophage elementsarereferredtoascrypticprophages(Fig.1),andtheyalso playaroleinantibioticresistanceandtolerance.Forexample,the E.coliK-12genomehasgained1600kbofnovelDNA(18%)since itsdivergencefromSalmonellasp.100millionyearsago(Lawrence and Ochman,1998), and contains one activeLambda prophage
Table1
SummaryofgenesthatparticipateinantibioticresistanceortoleranceinthecrypticprophagesofE.coliK-12.
Genename Prophage Function Antibioticstested References
ydaC rac Putativedouble-strandbreak
reductionprotein
Erythromycin Sooetal.(2011)
ralR rac DNase,toxinoftypeITApairRalR/RalA Fosfomycin Guoetal.(2014)
kilR rac Toxin,FtsZinhibitor Novobiocin,bicyclomycin,azlocillin Peteretal.(2004),Sabinaetal.(2003) relE Qin ToxinofTApairRelE/RelB,
sequence-specificendoribonuclease
Cefotaxime,ofloxacin,tobramycin, ciprofloxacin,ampicillin
GotfredsenandGerdes(1998),Keren etal.(2004),Maisonneuveetal.(2011)
dicB Qin Controlofcelldivision Azlocillin Wangetal.(2010)
hokD Qin Smalltoxicmembranepolypeptide Kanamycin,novobiocin Kohanskietal.(2007),Peteretal. (2004)
emrE DLP12 Multidrugresistancepump Methylviologen,tetracycline, ethidium;tetraphenylphosphonium
Morimyoetal.(1992),Yerushalmi etal.(1995)
ompT DLP12 Outermembraneprotease Streptomycin,chlortetracycline Huietal.(2010),Lietal.(2008)
yfdO CPS-53 Uncharacterizedprotein Lidocaine,nalidixicacid Sooetal.(2011)
rnlA CP4-57 ToxinofRnlA/RnlBTApair Gentamicin Kogaetal.(2011)
yfjZ CP4-57 AntitoxinofputativeTApairYpjF-YfjZ Novobiocin Peteretal.(2004)
ypjF CP4-57 ToxinofputativeTApairYpjF-YfjZ Novobiocin Peteretal.(2004)
cbtA CP4-44 AntitoxinofTApairCbtA/CbeA, cytoskeletonbundling-enhancing factorA
Norfloxacin,novobiocin Masudaetal.(2012),Sabinaetal. (2003)
cbeA CP4-44 ToxinofTApairCbtA/CbeA, cytoskeletonbindingtoxin
Norfloxacin,spectinomycin Masudaetal.(2012)
ykfI CP4-6 ToxinofputativeTApairYkfI-YafW ColicinE3 Walkeretal.(2004)
yafW CP4-6 AntitoxinofputativeTApairYkfI-YafW ColicinE3,kasugamycin Walkeretal.(2004) yagE CP4-6 2-Keto-3-deoxygluconate(KDG)
aldolase
Novobiocin,norfloxacin,ampicillin, streptomycin
Bhaskaretal.(2011),Peteretal.(2004)
andninecrypticprophages(Blattneretal.,1997).Atleastcryptic prophageracisaphagefossilthatispresentinotherE.colistrains havingbeenacquiredover4.5millionyearsago,whichappears moreancientthantheLambdaprophage(Pernaetal.,2001).These cryptic prophage are not inactive DNA remnants generated in thecourseof host evolution but are importantfor host fitness in terms of both antibiotic and stress resistance (Wang et al., 2010).For example,by deletingall thecrypticprophage genes inE.coli(166kb),ithasbeenshownthatthatcrypticprophages contributesignificantlytoresistancetosub-lethalconcentrations ofquinoloneand-lactamantibioticsandthattheprophagesare beneficialfor withstandingosmotic,oxidative,and acidstresses (Wangetal.,2010).
Specifically,the nine crypticprophages in E. coli K-12 con-tain165putativegenes,and50ofthemarerelatedtoantibiotic resistanceeither bytranscriptome studies orin wholegenome screeningtests(Kohanskietal.,2007;Peteretal.,2004;Walker etal.,2004).AsshowninTable1,17crypticprophagegenesaffect antibioticresistancebysurvivaltestsormetabolicactivityassays usingdeletionstrainsorusingplasmidstoexpresstheseprophage genes.Inparticular,theproductsofkilRinracprophageanddicB inQin prophage areresponsiblefor inhibiting celldivision and areimportant forresistancetonalidixic acid(aquinolone)and azlocillin(a-lactam)(Wangetal.,2010).YdaCencodedbyrac wasidentifiedinthescreenforantibioticresistanceusingpooled plasmidsfromtheASKAlibrarythatshowedincreasedresistanceto erythromycin(Sooetal.,2011),andwehaveconfirmedthis phen-totypeinsurivialassaysusingtwodifferentconstructstoexpress ydaC(unpulisheddata).Rac-likeprophageistransmissibletoother E.colistrains(Asadulghanietal.,2009),thusenablingittospread theseresistancegenes.
InGram-negativebacteria,antimicrobialagentsmusttraverse both theoutermembraneand plasmamembrane togain entry intothecell.Oneofmanyeffectivecellularresistancestrategies involvestheextrusionoftheantimicrobialfromthecellby trans-porters,whichmaybeanchoredintheinnermembraneorresidein thetrans-membranespaceandwhicharealsoencodedbycryptic prophage.Forexample,thetransporterethidiummultidrug resis-tanceproteinE(EmrE)inE.coliisaproton-dependentsecondary transporterfromcrypticprophageDLP12(Yerushalmietal.,1995).
EmrEconfersresistancetopositivelychargedhydrophobic antibi-oticssuchastetracyclinebyactivelyexpellingthedrug(Viveiros etal.,2005).OutermembraneproteaseOmpTfromprophageDLP12 hasbeenshowntoincreaseresistancetostreptomycinand chlorte-tracycline(Lietal.,2008).TheimpactofOmpTonE.coliresistance tourinarycationicpeptideswasinvestigatedbytestinganompT knockoutstrain,andOmpTmayhelpthehostpersistlongerinthe urinarytractbyenablingittoresisttheantimicrobialactivityof urinarycationicpeptides(Huietal.,2010).
Inotherspecies,manyresistancegenesarelocatedongenomic islands,andthereareseveralcommonfeatures sharedby cryp-tic prophage and genomic islands. Both of them harbor phage integraseorexcisionasethatdirectlyregulatetheintegrationor excisionofthesemobileelements.Anothercommonfeatureisthe presenceoftwoperfectornear-perfectrepeatsatthebordersof thesemobilegeneticelements,andtheyareusedassite-specific recombinationeventsduringexcision.P4orP4-likeintegrasegenes arenormallyadjacenttothetRNAsortRNAmodificationgenes, whichserve asthephageattachments (Williams,2002).Mobile geneticelementsthatcarry P4-likeintegrases aretermed cryp-tic prophages in E. coli (e.g. CP4-6, CP4-44, CP4-57) but often are referred to as genomic islands or pathogenicity islands in other speciessuch asSalmonella and Shigella. For example, the genomicislandthatcarriesresistancetoampicillin, chlorampheni-col, streptomycin, sulfonamides, and tetracycline in S. enterica TyphymuriumphagetypeDT104isflankedbyanearperfect18-bp repeatandinsertedinthetrmEgeneencodingatRNAmodification enzyme(Cabedoetal.,1999).Theresistancelocuspathogenicity islandinShigellaspp.mediatesresistancetostreptomycin, ampi-cillin, chloramphenicol, and tetracycline, and it can be excised fromthechromosome viasite-specific recombinationmediated bytheP4-likeintegrase(Turneretal.,2004).Wehaveshownin E.coli that excisionaseAlpAin CP4-57 can lead to a complete removalof CP4-57prophage (Wangetal., 2009)and that exci-sionaseXisRinracandamodifiedhostproteinH-NScanleadto acompleteremovalofracprophage(Hongetal.,2010;Liuetal., 2015).Therefore,theglobaldisseminationofmultipleantibiotic resistancesharboredbymobilegeneticislandsinpathogenic bacte-riaseemstobecloselyrelatedtothesite-specificrecombination events.
4. Prophagesarereservoirsofantibiotictolerancegenes Inmanycases,TAlociarecloselylinkedtomobilegenetic ele-ments.Forexample, V.cholera has13TA pairsand allof them areclusteredinthemegaintegrononChrII,thesmallerofitstwo chromosomes(Buddeetal.,2007;Gerdesetal.,2005).Also,typeI, typeIIandtypeIVTAlocihavebeenidentifiedintheninecryptic prophagesofE.coli(Table1),indicatingTAsareoverrepresentedin E.coliprophages(∼8%).Thepresenceofthesetoxinsisimportant inthatactivationofalltoxinstodateleadstoadramaticincrease inpersistercells(Chowdhuryetal.,2016a;Wood,2016).Sothese crypticprophagesnotonlyprovidethemeansforantibiotic resis-tance,buttheyalsoprovidethemeanstomakethecellsdormant andmorepersistent.
ThetypeItoxin/antitoxinpairRalR/RalAinE.coliraccryptic prophageincreasesresistancetobroad-spectrumfosfomycin(Guo etal.,2014),andtheunderlyingmechanismremainstobe deter-mined.RelEtoxinofTypeIITARelB/RelEinE.coliprophageQinis asequence-specificendoribonuleasewhichblockstranslationby cleavageofmRNAs(Christensenetal.,2001;Neubaueretal.,2009). Critically,RelEleadstohighpersistercellformationinthepresence ofhighconcentraionsofciprofloxacin,ampicillin,andtobramycin (Kerenetal.,2004;Kogaetal.,2011).Also,thetoxinofthetypeII TAsystemRnlA/RnlBoftheE.colicrypticprophageCP4-57causes inhibitionofcellgrowthandrapiddegradationofcellularmRNAs. ThetoxinoffirstrecognizedtypeIVTApair,CbtA/CbeAinE.coli crypticprophageCP4-44,notonlyinhibitscellgrowth,butalters cellshape byinhibiting thepolymerizationofcytoskeletal pro-teinsFtsZandMreBviadirectprotein-proteininteraction(Masuda etal.,2012).Moreover,thisTApairhasrelatedtoresistanceto nor-floxacin,novobiocin,andspectinomycin(Tanetal.,2011;Kohanski etal.,2007;Masudaetal.,2012).Theonlyothertwohomologous TAlociofCbtA/CbeAalsoresideinprophages,YkfI/YafWonE.coli crypticprophageCP4-6andYpjF/YfjZonE.colicrypticprophage CP4-57(BrownandShaw,2003).Interestingly,YkfI/YafWisrelated totheresistancetobacteriocincolicinE3(Walkeretal.,2004),and YpjF/YfjZisrelatedtoresistancetonovobiocin(Peteretal.,2004). OneofthemoststrikingfeaturesoftheseP4-likecrypticprophages inE.coliisthattheyarepervasivelymosaic,withdifferent seg-mentsseemtohavedistinctevolutionaryhistories(Brussowetal., 2004).ThepresenceofthreehomologousTAlociinthreeP4-like prophages(CP4-6,CP4-44,andCP4-57)suggeststhathorizontal geneticexchangeplaysadominantroleinshapingthesegenome architectures.
5. Antibioticstriggerprophageexcision
UV irradiationand mitomycinC (MMC)are classical agents thatcanefficientlyinduceprophageexcisioninlysogenicbacteria (Otsujietal.,1959).TheSOSresponseisinducedbyUVradiation orMMC,anditcanalsobeactivatedbyantibioticsthatinhibitDNA replicationorinhibitDNAgyraseactivitiestoproducesingle-strand DNA(ssDNA).DuringthecourseofrepairofDNAdamage,ssDNA isproduced(e.g.,DNAcrosslinksproducedasa resultofMMC). Trimethoprim(dihydrofolatereductaseinhibitor)isanexampleof anSOS-inducingantibioticthatinhibitsDNAreplication(Lewinand Amyes,1991).Fluoroquinolones,broad-spectrumantibioticsthat inhibitbacterialDNAgyraseandtopoisomeraseactivity,alsolead toSOSresponsesbygeneratingDNAdouble-strandbreaks(Drlica andZhao,1997).
ThegenescodingforShigatoxinsaresilentinlysogenicbacteria, and prophage induction is necessary for theirefficient expres-sion and toxin production. Both toxins are usually encoded in thegenomes ofbacteriophages(Stxphages),and theycan lyso-genize E. coli strains, thereby allowing a mechanism for toxin
disseminationviatransferofbacteriophages(Huangetal.,1987; O’Brienetal.,1984).Shigatoxin-producingEHEC(O157Sakai) pos-sesses18prophagesthatencodenumerousgenesrelatedtoEHEC virulence,includingthoseforShigatoxinsandtwootherpotent cytotoxins(Hayashietal.,2001).Nineoutofthe18prophagescan beexcisedtoformacirclebyMMC-mediatedinduction,andthree ofthemaretransferabletothenon-pathogeniccommensalE.coli strainK-12andstablymaintainedinthenewhost(Asadulghani etal.,2009).TheinductionofShigatoxin-convertingprophagesin EHECalsooccursinthepresenceofnorfloxacinandunder oxida-tivestress(Ło´setal.,2010).Hence,thesecrypticprophageshavea highpotentialfordisseminatingvirulence-relatedgenesandother genetictraitstootherbacteriaunderstressconditions,andstress activatespathogenicity.
Increased virulence caused by increased Stx production has beenrelatedtostxprophageinductionbothinvitro(Mühldorfer et al., 1996) and in vivo (Zhang et al., 2000). In particular, clinically-used antibiotics known to trigger the SOS response, includingciprofloxacin,havebeenshowntoenhanceStx produc-tion(Mühldorferetal.,1996).TheSOSresponseinducedbyMMC orfluoroquinolonescausesenhanced intra-intestinaltransferof Stx2prophagesinvivo(Zhangetal.,2000).ProphagesofE. fae-calisV583excisefromthebacterialchromosomeinthepresence ofafluoroquinolone,andareabletoproduceactivephageprogeny (Matosetal.,2013).TheS.typhimuriumfunctionalprophages, Gifsy-1andGifsy-2,canbeinducedbyexposingbacteriatohydrogen peroxide(Figueroa-Bossi and Bossi, 1999).Recent studies have demonstratedthatoxidativestressconditionsmayoccurduring colonizationofthehumanintestine byenteric bacteria(Kumar etal.,2007).Moreover,earlierstudiesonaclinicalisolateofEHEC suggestedthathydrogenperoxideproducedbyhumanneutrophils, mayincreasetheproductionofStx2(Wagneretal.,2001).
Carbadox is a quinoxaline-di-N-oxide, and exposure of Salmonella sp.tocarbadox inducesprophages that cantransfer virulenceandantibioticresistancegenestosusceptiblebacterial hosts(Stantonetal.,2008).Carbadoxfrequentlyinduces general-izedtransducingphagesinmultidrug-resistantphagetypeDT104 andDT120isolates,resultinginthetransferofchromosomaland plasmidDNAthatincludedantibioticresistancegenes(Brunelle, 2014).Metagenomicsapproacheswereusedtoevaluatetheeffect of two antibiotics in feed (carbadox and ASP250 [chlortetracy-cline,sulfamethazine,andpenicillin])onswineintestinalphage metagenomes(viromes),andtheabundanceofphage integrase-encoding genes was significantly increased in the viromes of medicated swine over that in the viromes of non-medicated swine(Allenet al.,2011).Prophage-likeVSH-1wasdetectedin Brachyspira hyodysenteriae cultures treated with mitomycin C, carbadox,metronidazole,andhydrogenperoxide.Carbadox-and metronidazole-inducedVSH-1 particles transmitted tylosin and chloramphenicolresistancedeterminantsbetweenB. hyodysente-riaestrains(Stantonetal.,2008).
As previously described for SOS induction by MMC, fluoro-quinolone antibiotics, and trimethoprim (Goerke et al., 2006), -lactamsarealsocapableoftriggeringprophageinductioninS. aureuslysogens.-lactam-mediatedphageinductionalsoresulted inreplicationand high-frequencytransferofthestaphylococcal pathogenicityislands,showingthatsuchantibioticsmayhavethe unintendedconsequenceofpromotingthespreadofbacterial vir-ulence factors (Maiques et al., 2006). -Lactam antibiotics are extracellularstimulioftheSOSresponseinS.aureusaswellasin E.colianddemonstrateanothercaseforhorizontaldissemination ofvirulencefactors.
Integratingconjugativeelements(ICE)canalsocarryantibiotic resistance genes and recruit SOS responses to mobilize them-selvesfromonebacterialgenometoanotherbycell-to-cellcontact (Hastings etal.,2004).Therapeuticagents suchas ciprofloxacin
andMMCpromotethespreadofantibioticresistancegenes car-riedonICEinV.choleraeamongavarietyofGram-negativespecies includingE.coli(Beaberetal.,2004;HochhutandWaldor,1999).
6. Drugsandphage
Since MMCand other antibiotics triggerSOSresponses that maycontributetheaugmentation oftoxinproductionby induc-ing stx prophage induction, the treatment of infections using antibiotics resulting in DNA damage and phageinduction may lead to unexpected adverse consequences. Stimulation of gene transferfollowingbacterialexposuretofluoroquinolonesshould beconsideredanadverseeffect,andclinicaldecisionsregarding antibioticselectionforinfectiousdiseasetherapyshouldinclude thispotentialrisk.Antibioticsthatinhibitproteinsynthesis,such aschloramphenicol,tetracyclineandstreptomycin,donotinduce SOSresponses,andneitherdoagentsthatactupontheouter mem-brane(Hastingsetal.,2004).Theuseof fosfomycinwhichisan inhibitorof cell-wallsynthesis didnotcause theintraintestinal transferof Stx2 prophagetransfer inmice (Zhang et al.,2000), andeffectivelyreducedtherisksofhemolytic-uremicsyndrome (Takeda,1998).Thus,effortsshouldbemadeindevelopingnew compoundswithantimicrobialactivitiestargetingmorespecific cellularfunctions/components rather than DNA replication. For example,lassomycinisanewlyidentifiedantibioticthatexhibits potent bactericidal activity against both growing and dormant mycobacteria.ItbindstoahighlyacidicregionoftheClpC1ATPase complexandmarkedlystimulatesitsATPaseactivitywithout stim-ulatingClpP1P2-catalyzedproteinbreakdown,whichisessential forviabilityofmycobacteria(Gavrishetal.,2014).Anothernewly identifiedantibiotic,teixobactin,fromunculturedbacteria,inhibits bacterialcellwallsynthesisbybindingtoahighlyconservedmotif oflipidII(precursorofpeptidoglycan)andlipidIII(precursorofcell wallteichoicacid)(Lingetal.,2015).
Incontrasttoactiveprophagesthataretriggeredtoexciseby theDNArepair(SOS)response,crypticprophagesusuallystayas stableresidentsonthehostchromosomeunderadversegrowing conditionsincludingduringtheSOSresponse.Forexample,forthe nineprophagesinE.coliK-12,e14wastheonlyinducibleprophage uponMMCtreatment(Wangetal.,2010).Amongtheeight cryp-ticprophagesthatdonotexcisewithMMC,twoprophageswere inducedtoexcise duringE.coli biofilmformation, thus provid-ing benefits for the population by creating a subpopulation of prophage-excisedcellswithdifferentbiofilm-relatedphenotypes (Liuetal.,2015;Wangetal.,2009).Furthermore,Pf4prophage exci-sionhasbeenlinkedtobothcelldeathandlysisforP.aeruginosa cellsinbiofilmsasfilamentous-likeprophageexcisionincreases diversityindispersing cellsaswellasimpactsbiofilm architec-ture and virulence (Rice et al., 2009; Webb et al., 2003). It is wellestablishedthatbiofilmsprovideincreasedtolerancetoward antibiotictreatment(Costertonetal.,1995),thusprophagescan also indirectly contribute to antibiotic tolerance by promoting biofilmformation.
Moreover, cryptic prophages carrying TA systems can be activated during stress (Yamaguchi and Inouye, 2011). During oxidativestressandstarvation,proteasessuchasLonandClpXP degradeunstableantitoxinsandreleasefreetoxins(Christensen etal.,2004;Maisonneuveetal.,2013;Wangetal.,2011;Wang andWood,2011).Ithasbeensuggestedthatactivatingtoxinsby deactivatingantitoxinsofTAsystemswouldbebeneficialinterms fightingpathogens (Chan et al., 2015); however,this approach isshort-sightedinthatthisapproachwillindubitablyleadtoan increaseinthenumbersofpathogenpersistercells(Shapiro,2013) sinceproteinsthatreducegrowthsuchastoxinsincrease persis-tence (Chowdhury et al., 2016a).Perhapsthe bestapproach to
target pathogensthat utilize TAsystems likethose encodedby phagestoformpersistercells,istoutilizeacombinationofdrugs withoneusedtokillgrowingcellsandanotheronetokillpersister cells.Examplesofthisapproachoftargetingbothgrowingand dor-mantcellsincludecombiningrifampicinwiththeacyldepsipeptide ADEP4(Conlonetal.,2013)andbycombiningcefoperazoneand doxycyclinewithdaptomycin(Fengetal.,2015).Otherpossible approachesincludeusingeithermitomycinC(Kwanetal.,2015) (forinfectionswhereitdoesnotleadtoextracellulartoxin produc-tionlikeShigatoxins)orcisplatin(Chowdhuryetal.,2016b)tokill simultaneouslybothactivelygrowingpathogensaswellastheir persistercells;bothcompoundskillactiveandpersistercellsby crosslinkingtheirDNA,bothhavebeenshowntobebroadly effec-tiveagainstpathogenssuchasP.aeruginosa,EHEC,S.aureus,and Borreliaburgdorferi(Chowdhuryetal.,2016b;Kwanetal.,2015; Sharmaetal.,2015),andbothareapprovedbytheFoodandDrug Administrationforhumanuse.
AsaninterestinguseofTAsystemsfromcrypticprophageas drugs,toxinRelEfromcrypticprophageQincausesapoptosiswhen itisproducedinahumanosteosarcomacellline(Yamamotoetal., 2002).Unfortunately,althoughtoxinsofTAsystemshavemanyof thesametargetsasantibiotics,theyareactiveonlyintracellularly; i.e.,theyarenoteffectivewhenaddedextracellularlybutmustbe translocatedtothecellinterior.Forexample,theHoktoxinofthe Hok/SokTAsystemisnotactivewithGrampositiveorGram neg-ativebacteriaunlessitiselectroporatedintothecell(Pecotaetal., 2003).
Also,othernon-TAcomponentsofprophagehavepotentialas drugs.Forexample,recentprogresshasbeenmadeinHIV-1 ther-apybydirectedevolutionofasitespecificrecombinasethatcan recognizea34-bpsequenceflankingthemajorityoftheintegrated provirusHIV-1;thisevolvedrecombinasecanefficientlyand pre-ciselyremove theintegratedprovirusfrominfectedcells andis efficaciousonclinicalHIV-1isolatesinvitroandinvivo(Karpinski etal.,2016).Thus,targetingtherecombinaseofprophagemaybea promisingapproachfortreatingbothviralandbacterialinfections.
7. Perspectives
Thereisgrowingneedtounderstandphage-hostinteractions andbacterial-hostinteractionsincomplexsystems,suchasamong gutmicrobiota.Phagescanregulatethemicrobiomeusing differ-entstrategies,suchaskillingcompetingbacteriatoallowlysogenic bacteriatothrive innicheswithlimited nutrients,byencoding toxinsorvirulencefactorsthatincreasepathogenicity,by encod-inggenes thatincrease antibiotictolerance, and byfunctioning asvehicles forthehorizontaltransfer ofgenesamong different species of bacteria. Clearly, the spread of antibiotic resistance amongpathogenicbacteriahasbecomeaseriousglobalissuefor publichealth,andtheroleofphageinthisprocessshouldnotbe neglected.Increasingevidencehasshownthatprophagesof com-mensalandenvironmentalbacteriaarealsoreservoirsofantibiotic resistanceand tolerance,andtheirrolesinthedisseminationof resistanceandtolerancetopathogenicbacteriathroughhorizontal genetransfershouldberecognized.Prophagescanalsocarrynew families ofvirulence, resistanceand tolerancegenes.Prophages orprophage-likeelementscanbeidentifiedandannotatedinthe sequencedbacterialgenomesthroughwebserverssuchasPHAST (Zhouetal.,2011),ProphageFinder(BoseandBarber,2006), Island-Viewer (Dhillon et al.,2015), and MobilomerFINDER (Ou etal., 2007).Inaddition,theviromesequencesthatarepresentinpublicly availabledatabases(e.g.,MG-RAST(Keeganetal.,2016))canalsobe minedforthepresenceofvirulence,resistanceandtolerancegenes insidesphagesandprophages.
The human gut also contains large amounts of free viral particles, most of them bacteriophages probably released after spontaneousinductionofprophagesoflysogenicbacteriainthe gut (Breitbart et al., 2003). A recent study by Gordon’s group showsthattemperatephagesareprominentinfecalmicrobiota, andaninvivomicestudydemonstratedtheprophageinductionin afecalcommunityoccursuponexitingthehost(Penadésetal., 2015).Moreover,host-associated bacteriaoftenencounter vari-oushost-relatedstressessuchasnutritionaldeprivation,oxidants, temperatureupshifts,andlowpHwhichcanalsotriggerprophage excision.Therefore, the humanmicrobiome and environmental microbiomeprojectsthathavebeeninitiatedthroughouttheworld (Dubilieretal.,2015),shouldstrivetoidentifyprophagesand func-tionalgenesembeddedintheprophages,giventheprominentrole ofphageinvirulence,antibioticresistance,andantibiotictolerance. Conflictofintereststatement
Nonedeclared. Acknowledgements
This work was supported by the National Basic Research Program of China (Grant No. 2013CB955701 to XW), by the National Natural Science Foundation of China (NFSC31270214, NFSC31290233toXW),andtheArmyResearchOffice (W911NF-14-1-0279toT.W.).We thankforDrs. YunxueGuoandPengxia WangattheSouthChinaSeaInstituteofOceanologyfortheirhelp withthetableandthefigureforthismanuscript.XWisthe recip-ientofthe1000-YouthEliteProgram(theRecruitmentProgramof GlobalExpertsinChina),andTWistheBiotechnologyEndowed ProfessoratthePennsylvaniaStateUniversity.
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