Regulation of Antigenic Variation in Giardia lamblia

20 Pages·2011·0.58 MB·English

MI65CH30-Lujan ARI 27July2011 9:40
Regulation of Antigenic
Variation in Giardia lamblia
Ce´sar G. Prucca, Fernando D. Rivero,
org and Hugo D. Luja´n
ualreviews.al use only. LUanbioverarstiotryyooffCBoiordcohbema,iCstPryXa5n0d0M4AoSleKcuClaorrBdoioblao,gAy,rgSecnhtoinoal;oefmMaeild:ihcliunjea,[email protected]
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5:611-setts - Annu.Rev.Microbiol.2011.65:611–30 Keywords
ol. 2011.6Massachu FJTuihrlyset6Ap,nu2nb0ul1ias1lhRedevoienwlinofeMasicaroRbeiovlioegwyiisnoAndlivnaenacteon pexaprarseistsei,oinm,mvaucncieneevasion,RNAinterference,surfaceantigen,gene
ev. Microbiniversity of mT10hi.c1irs1oa4.ra6tn/icnalnuena’slurdreeovvii:-emwsic.ororg-090110-102940 AAcrnbotosigtrgreaanncicitsmvasr,iaintivoonl,veasctlhoenpalerpmhaenneontytpsiwcitvcahriinatgioonfhdoemveololopgeodubs,yanmtii--
nu. Rby U CAlolpryigrhigthstre(cid:2)cser2v0e1d1byAnnualReviews. genically different cell surface molecules. In pathogenic microorgan-
An isms,antigenicvariationisoftendescribedasamechanismtoevadethe
0066-4227/11/1013-0611$20.00 hostimmunesystemandthereforeisresponsibleforthegenerationof
chronicand/orrecurrentinfections.However,antigenicvariationhas
alsobeeninvolvedinexpandinghostdiversityanddifferentialcourses
of the diseases. The intestinal protozoan parasite Giardia lamblia un-
dergoes antigenic variation through the continuous exchange of ap-
proximately200variant-specificsurfaceproteins.Herewereviewthe
principalissuesregardingthesignificanceofantigenicvariationduring
Giardiainfections,theparticularfeaturesofthevariant-specificsurface
proteins,andthecurrentknowledgeonthemechanismsthatregulate
thisprocess,aswellastherelevanceofdisruptingantigenicvariationas
anovelapproachtodesigneffectiveantiparasiticvaccines.
611
MI65CH30-Lujan ARI 27July2011 9:40
Surface antigenic variation is a major
Contents evasion system developed by pathogenic
microorganismstomaintainchronicinfections
INTRODUCTION.................. 612
underthecontinuousimmunepressuresgener-
ANTIGENICVARIATIONIN
atedbytheirhosts(34,35,64,136).Studiesof
GIARDIALAMBLIA............... 613
antigenicvariationinseveralorganismsfound
VARIANT-SPECIFICSURFACE
that they use different mechanisms to switch
PROTEINS....................... 614
theexpressionoftheirvariablesurfaceantigens
MOLECULARMECHANISMS
(34).However,regardlessofthediversemech-
INVOLVEDINANTIGENIC
anismsofantigenicvariation,thisphenomenon
VARIATION..................... 616
GIARDIA always requires (a) a family of homologous
POSTTRANSCRIPTIONAL genes present in the genomes of these organ-
GENESILENCING ismsencodingimmunodominant,antigenically
org MACHINERY.................... 619 differentsurfacemolecules;(b)amechanism(s)
ualreviews.al use only. MANESTCWIHGIATENCNIHISCMINVSGAOR.F.IA.A.T.N.I.TO.IN.G..IE.N.N.V.IC.I.V..O. 620 tsmhioeancthgoaufnaiorsamnnlytfeoeorsnretehvaeenrmstiibguletyunaswlaltyitacehxtciinlmugseti;vheaeneedxxpp(rrcee)ssa--
annson ANDHOSTIMMUNE sionofthesemoleculesinindividualcells(34).
Downloaded from www.erst on 01/24/12. For per DISRVMREAOUSRDPPIAOTETLINOIOSONEFNSOIIN.FN.F.A.AEN.NC.T.T.AI.IGN.O.EI.NMN...AI..C.L........... 662221 AvTNCtoiarvranergeynitisap,iedsgnayetieynrdiesinaotamos,ir,ocfaesmnavpBmsadaaoo,rrirGngiarAaahseitinlbatitioraaliaecdnp,l,ilsamathoPhssaminaupcsetnareubmo,cdmeoiePaeerornslngcgayy(aoss3dmnot4oiaitsos)nch,d.muteiIiumrMstgm,ueiesyinn,nncsiiotBcccppeleealduvalcbaldseuuimrinslliniaaaaaga-r-,,
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63A tion,assuggestedbyrecentreports(14,37)and
5:611-setts - INTRODUCTION b(3y8t)h,ewsheeqrueetnhceinpgreosfemncaenyofmhiocmrooblioagloguensogmenees
6u
11.ach Oneofthemostremarkablecharacteristicsof families encoding potential surface molecules
0s
ev. Microbiol. 2niversity of Mas mi(egt1xeyi3cnce7ertp)oo.toeiUxoarpgdnnaraaiencpllseitysslimltocuoanlsapircasphbortalorhengfiegaoilrneefssiersxamtitpnorsia,ddtolihiynrfedfapeidnreaeanarnptrviytticiiruancotlagnaeprmta,ihbneaeintrlioe-rt htGaona.zsdoTbiabnheneteeesnstGfltiikiandangaereolndilwistla)ia,nfiteleaopddmna,(reb2bal4isianio,tu2fe(cs6slyt,eohn5afe.t7eh,Gdum7.m8po)daas.urtnaosdscieatoninmcadlimpsdroooonr--
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nu. by evasive or resistant forms to survive extreme mestic animals (2), also undergoes antigenic
n
A conditions(137). variation (2, 90–92). In addition, because of
Pathogenic bacterial (107), fungal (58), or its particular biological characteristics (29),
protozoan (39, 83, 113, 123) microorganisms Giardia has intrigued microbiologists since
are exposed to hostile conditions during their AntonyvanLeeuwenhoekdiscoveredthispar-
passagebetweentheenvironment,vectors,and asiteinhisownfeces300yearsago(2).
Antigenicvariation: hostsanddevelopextraordinarymechanismsto Giardia belongs to the earliest diverging
aclonalphenotypic adapttosuchconditions(47,141).Duringan branch of the Eukaryota (82, 126). For this
variationinvolvingcell infection, however, survival of pathogenic or- reason, Giardia is considered an excellent
surfaceantigenic
ganismsdependsnotonlyontheircapacityto model system to study evolutionary aspects
determinants
adapttoanewhostbutalsoontheircapability of basic cellular processes (29). In addition
Giardialamblia: an
to neutralize the innate and adaptive immune to its biological relevance, Giardia is one
intestinalparasiteof
defenses generated by the infected individual of the leading causes of human intestinal
humans
(64,89,149). diseaseworldwide;themostfrequentcauseof
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defined waterborne outbreaks of diarrhea in clinical signs. Children and the elderly are
developed countries; and a common cause of particularly susceptible to Giardia infections
diarrhea in day care centers, institutionalized (23).Symptomsinhumanstypicallyoccurone
individuals,backpackers,andtravelers(130).In totwoweeksafterinfectionandlasttwotofive
Asia,Africa,andLatinAmericaapproximately days (91). Giardiasis seems to be self-limiting
200 million people have symptomatic giar- iftheimmunesystemofthehostisfullydevel-
diasis, with some 500,000 new cases reported oped,indicatingthateffectivehostdefensesare
eachyear(2).Inregionswherebasicsanitation abletocontrolthedisease.Insomecases,how-
is deficient, Giardia infections are almost ever, chronic infections occur in the absence
universalinmembersofthepopulationbythe ofanyapparentimmunodeficiency(91).
ageoftwo(76).Giardiahasbeenrecognizedas
a reemergent infection and has been recently
ANTIGENICVARIATION
includedinthe“NeglectedDiseasesInitiative”
org bytheWorldHealthOrganization(122). INGIARDIALAMBLIA
ualreviews.al use only. iGso.llPaatmheysbllohiagaveaesnseaetlmilcobwslaetugddetsihe(esAiodtofenGntui)fim;AcearatoinoudnsBoGfairsaeervcdeaina- Aosytnmhteipgrteonpmiactshvoaogrfieanttiiohcnemwdiacissreoianosrietgiaalncloyisrmdreisslcaotinevderwewhdiictihnh
nnon pableofinfectinghumans(81,92,102).Assem- peaks in parasitemia and differential surface
as
om www.2. For per bt(1hla2egn9e).ssTeArhveeanGadsiaBrredsiianerfhevucomtirmsanfaoanrsysehmmuambmlaamngeainlAsf,e(rwcethpioircneh-s avanaprthiiageteninoonmexienpnrGoesnisaiorodcnicau(3wrr5ai)ns.gBoriynigcivonintartlorlya.sTot,bhaseenrtfviagecedtonaricss
Downloaded frerst on 01/24/1 swsineehnmfeitccbehtdlsacgbhaeyunmtBbheae(nriWenspfaerBncedtsieseodmnltnaiectadeet)ub(dr8yao1lte,lhys9ebn2yG,o1tGS0i/.n2Mmf)e,ucbirtsuiomst.laiAtcthesee-), lGtceeuairladntruisdnrioega,vtwveoaretrrhtieiaemdbdiieflifiseticynroeivtnnercorseeypschoirnfoeaztsonourtirtiyfgea/seecnxemci-clraaevibntaoertlraiiayinntpgieordpnoadiitnn--
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63A recent report of the draft genome sequencing uctspresentintheculturemediumofdifferent
5:611-setts - obefthcleasGsiSfi/eMdiassolaatdeisffuegrgenesttssptheactietshiwshgreonucpocman- pthoepudleavteiolonpsmofetnhteopfarsapseitceifi(3c,m6,o9n0o,c9l4o,n9a8l)a,nantid-
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11.ach paredwiththegenomeoftheWBisolate(46). bodies(mAbs)thatrecognizethesurfaceofonly
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ev. Microbiol. 2niversity of Mas aIwtannhtfieiGiocncnhitfaieoaricndrnteiiavtihpesheatcasryussaespandtpssaemiinnrmidttspthmaeleedavflleelibfgcyieeenstcitaney(tsg7citvle0ieens)t.ecitAoo,ronnffltpsaeoihgrsfoteeilzcnxloyacgtisytetosesd-f,. sToGocihmcaSeurerudresicbafesoinlearlqsentu,whtieeagintneahttnbiiengiscxeeapnnvpeciaroceripimovautafeliraoanitnantistyoiaondilmnes(om9mi3noo)unc(nGcFseutiiragrpasrurtdeedridsaeusrut1sihrtaniea)lgt.l.
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nu. by trophozoites are released (49). Trophozoites infectionsofhumansandlaboratoryanimals(7,
n
A arenotinvasiveandproliferateattachedtothe 8,96,97).Inthecourseofinfection,prolifer-
surfaceoftheintestinalepithelialcellsbymeans ating trophozoites give rise to subpopulations
ofasuckingdisk(2).Trophozoitesarerespon- thathaveswitchedtodifferentsurfaceantigens
sible for the clinical manifestations associated andthereforeareabletoeludehostimmunere-
with the disease, which vary from asymp- sponses.Thesesubpopulationscanbethenrec-
tomatic infections to acute or chronic severe ognizedbytheimmunesystem,butcontinuous
diarrhea(23).Someindividualsarechronically switching to novel surface molecules prevents
infected without presenting symptoms of di- clearance of the infection (41, 87, 88, 90–92,
arrheabutsuffermalabsorptionandlong-term 94).
detrimentaleffectsongrowthanddevelopment Initialexperimentsinwhichantibodieswere
(thelastofwhichoccursprimarilyinchildren) addedtothecultureresultedinaggregationand
(131). The host immune status influences the death of cells expressing a given surface pro-
susceptibility to infection and the severity of teinspecificallyrecognizedbyamAb(93,94).
www.annualreviews.org • RegulationofAntigenicVariationinG.lamblia 613
MI65CH30-Lujan ARI 27July2011 9:40
a b VARIANT-SPECIFIC
SURFACEPROTEINS
AntigenicvariationinGiardiainvolvesvariant-
specificsurfaceproteins(VSPs)(86).VSPsare
key players in Giardia antigenic variation and
are the focus of great controversy in the field
Extracellular domain
20–200 kDa rich in CXXC motifs becausetheircharacteristicsmayberelatedto
regulationoftheswitchingprocess(29).VSPs
are cysteine-rich integral membrane proteins
thatpossessavariableextracellularN-terminal
c
regionandaconservedC-terminaldomainthat
includesauniquetransmembraneregionanda
Transmembrane short, 5-amino-acid-long cytoplasmic tail (85,
g domain
or 101)(Figure1b).VSPsformathickcoatonthe
ualreviews.al use only. memPblarsamnea pfmaaceremas(bi1tre1a1nth)e.aTtgerheneperdreaestneesnsittasyttohhfeicVhkSosPsuts-rpfianacretahscietoepailtnatsthmeraa-t
nn
no
as might act as a physical barrier to prevent the
w.er CRGKA cytoplasmic tail
wwor p immune system from accessing other compo-
om 2. F Figure1 nentsoftheplasmamembrane(91,92,111).
65:611-630. Downloaded frusetts - Amherst on 01/24/1 (tdMtapaAbhrnnionolfeadtupftpiegeivbuhagr)naoleoeaticrdnfiontzioaiytocinolbcaViadnstltbveeeiiSraooer,sPevrlfncicieystaG(tdh4soteltii0eadncaoeb0yrlnintyen×dtouloieiteca.np-ahdl(relteGbpiarwiic)asoriohmiSarptnftrchehiiudthccxohictuteahztelrlmaeoaailipisacrilatma.aeateVrrl(insblcaceSut)lsiiiosPlniaCbattf.eoarc(ot(siduhgrnan.yelr)eefAtge,oduIeslimmct)nrto,ahre)namluionanl,icauunmyadtbgnudisoemhcroiltcaneeflhutotgdhiouneunlerfooewngsraflsttieaueatruGshrnrarocesfniteaartaiseaegncrmmxiseedcnpcnieeooeaermindneafcVssocsbwvssoeScraiailnmaPyratonghis,neaosestaaDanhidlyooAaonuwmP.siiInaniggn, tofitwg(8iiegrami2stnlnhtT)eomi,czmhtcaoeRheasdmeselNslefiepbmcfArdlyroesobtmstTmmleeaVpqSAsgGlueASeebqePi4tanuBi61orcewA7dnenc,i7alacoooswe(afnf3adsoeVts)sefh;esSGraemctePnwrSpsboi-o/eboAltMqaerh6ygtudee,-eeerHdawunAVrshc7(siS4icinncl(8lPago4hg)t,6n.eaaio)eNrst,f,pWrtaottheanhhwcraBdee--t,
011.sach monoclonalantibodiesdirectedtotwodifferentVSPsontrophozoitesinwhich assemblage E clone P15 (59), the sequences
ev. Microbiol. 2niversity of Mas wAtshubhepbiecRrrheiNmvaiAnapttioiiopgsinaettnisho:icwnRvaNinayrAhyiaeia,tlsliRoobwnNe)ehAcnaaisndnbitbseerereufnopebrtdeesedenrr.ceveDge;uduVlaaiSnltePlaad,b.pveMaalirirnaiaggonnf(tir-dfiesidcpvaieadtcniiiondfingcg6rtsre3uoe0rnpf×,ahc.oezporitoetseiinn. oatgireffiencpoaartmveiavoeiinloawuboslifletyh.aiIpdtnpyerpnsoiitlcxiiaficimloedfasettaaeuntldyudire2ems0sl0aeondfvysttpnohoigstvehenpelerVisodtSepePnein-rs
RU By using mAbs as powerful tools, it was pos- family (variable, cysteine-rich N-terminal re-
nu. by sible to clone trophozoites expressing a given gion,aconservedtransmembranedomain,and
n
A
antigen,demonstratingthatGiardiaspeciesex- theCRGKAcytoplasmictail)(85).Aprevious
press only one antigen on their surface at a reportestimatedthattheVSPrepertoirepos-
time,butthatswitchingtoanotherantigenically sesses about 133 to 151 VSP variants (101).
different surface antigen occurred once every However,thereportofthefirstGiardiagenome
6 to 16 generations, depending on the isolate (82)indicatesthatGiardiaencodesarepertoire
andtheantigen(95).Nevertheless,simultane- of235to275vspgenes,whichwasconfirmedby
Variant-specific ous expression of two antigens on the surface arecentanalysis(4).Inthelatterstudy,303vsp
surfaceprotein ofindividualtrophozoiteswasobservedduring geneswereidentifiedinthecurrentassemblyof
(VSP): anantigen
switching, when dual labeling of the tropho- thegenomeoftheWBisolate.Ofthe303vsp
involvedinantigenic
zoitesurfacewithdifferentmAbslastedabout genes,228werecomplete,whereas75werepar-
variationinGiardia
15h(92),andduringdifferentiationtoandfrom tialorincompletesequences.Thesegenesare
vsp: VSP-codinggene
cysts(28,79). clustered in groups of two to nine distributed
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alloverthefivechromosomes.Becauseofthe anti-HA-specific antibodies showed that the
similaritythatexistsbetweenvspupstreamse- completesurfaceofthetrophozoitesbecomes
quences,ithasbeenproposedthatthesereper- labeled,confirmingthattheN-terminalregion
PV: peripheral
toiresresultedfromdivergenceandsubsequent is extracellular and VSPs are type 1a integral
vacuole
recombination events (146). This assumption membrane proteins (65). On the other hand,
is supported by the fact that of the 228 vsp the C-terminal portion of the VSPs exhibits
genesestimatedintherecentanalysis,10were high conservation among the different family
identical inverted gene pairs (4). In addition, members,showingalmost90%identityinthe
someVSPssharesomedegreeofhomologyand aminoacidsofthetransmembranedomainand
canberecognizedevenbythesamemAb(28). a totally conserved 5-amino-acid cytoplasmic
Moreover,thestudyofantigenicvariationus- tail(CRGKA)(85).
ing polyclonal antibodies yields confusing re- Severalposttranslationalmodificationshave
sultsbecauseseveralVSPsmaysharecommon been reported in VSPs; however, due to the
org epitopes(113). conflictingresultsthathavebeeninformed(re-
ualreviews.al use only. gperonAmessoawtreeirtshbeamlrieeovsretedloatttoihvheeralyvGes.nhoolarimtntbarlnoiadnsghaeannvdeest,lhivmesipr- vnmieeocwdeiesfisdacrianytiRotonesfdeaernetendrcmtehi1en1ier3)tp,hopesrsoeibxfoilseutnerndelcesetvuaodnficetesheaforseer
nnon itedsequenceconservation.The3(cid:2)untranslated thebiologyofG.lamblia.
as
www.or per raelsgoiotnenodfsGtoiabredisahogretn,etys,piicnacllluydninoglovnsgpegretnheasn, theTplhaesmtraanmsepmorbtroafnneeiswsliymsiylnarthtoestihzaetdoVfhSiPgshteor
om 2. F 30nucleotides(nt)(1,2,92,101).However,the eukaryotes. The presence of a recognizable
Downloaded frerst on 01/24/1 pctThorheenisssienisniinttciiineatigtaootforofarcPocedyaleAnomndait,edgwnaTtateTsisri,pnewcriteehisnaeettrnoleytrasietnulgegamgrbeeoepsnutretteds4(eI0(nn4%rt)s). sptphiolgaernstmdaioliarnpemecopteiftmoidntbhearela(nm1pe0or,bov7ytee1mti)hneeinnictsotonhefsenttoihatumuegtiihVnveSot-Psotesecrertmnoestioutnhrrayeel
0. mh
63A oftheidentifiedvspgenesoftheWBgenome, pathway. Two reports present contradictory
5:611-setts - sintrdeiacmatirneggitohnesloafctkhoefvshpogmenoleosg.Iynoafdtdhiteio5n(cid:2),uvpsp- rwehsuenltscorengsaidrderininggtthheetcroannssperovretdofcytthoeplaVsSmPics
6u
11.ach genesarenotnecessarilytelomeric,andrecom- tail.Asreported,eliminationormodificationof
0s
ev. Microbiol. 2niversity of Mas bgitnhiionactnTaotsunhionoterndfaeatashrmtsgestiooongcoaewinan-htttoeeaimgrdtmewewniahiincstaahfsvloarnuperoilnoaotrdtctbiaioieontneinoon(nt1ohd,ftoeo2rct,houp4tema)hr.eeVanrsSitrtPeeed-ss tvtochhoetheennetsceereodnranvdutsehotedheprvolecaerxysdsitmtofaooipmcuflannirnsdeemowttiihalccycuaittsldauytsimhnloetdhfe(oet7lsehi5misez).einVndoCaSVttPoiShontPanvavesioelrffspraeotrnlhemyye-,
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nu. by presentsahighdegreeofvariationamongthe effectonnormalVSPtransporttotheplasma
n
A different members of the family (ranging in membrane (133). There is no satisfactory
size from 20 to 200 kDa), with variability in explanationfortheseconflictingresults.
thenumberofCXXCmotifs(whereCiscys- On the basis of the results of immunogold
teineandXrepresentsanyaminoacid)(2).The transmission electron microscopy (77, 111),
extracellular N terminus forms the interface VSPsarepresentonthesurfaceoftheparasite,
between the parasite and the host and is the formingacontinuouscoatthatcoverstheen-
portionofthemoleculerecognizedbyspecific tirecellsurface,includingtheflagellaandthe
antibodiesgeneratedduringGiardiainfections ventraldisk,aswellastheroughendoplasmic
(85,93).Moreover,theconstitutiveexpression reticulum(siteofsynthesis)andtheperipheral
ofamino-terminalhemagglutinin(HA)-tagged vacuoles (PVs), which seems to represent a
VSPs and the posterior identification of the primitive endosomal/lysosomal system that is
localization of the chimeric protein using located underneath the plasma membrane of
www.annualreviews.org • RegulationofAntigenicVariationinG.lamblia 615
MI65CH30-Lujan ARI 27July2011 9:40
the parasite (67). What is the reason for the MOLECULARMECHANISMS
presenceofVSPsinthePVs?ArethePVsthe INVOLVEDINANTIGENIC
siteofVSPdegradation? VARIATION
RNAinterference
Animportantissueinneedoffurtherinves- For several years, many research groups
(RNAi): amechanism
fordegradingdsRNA tigation is how a VSP is replaced by another have focused on elucidating the regulatory
inasequence-specific VSPduringantigenicswitching.Thisinforma- mechanisms that are responsible for anti-
manner tionisrelevantnotonlyforGiardia,butalsofor genic switching in Giardia (92). However,
otherorganismsthatundergoantigenicvaria- these investigations were unable to clarify
tion.AretheVSPstransportedfromtheplasma whetherregulationtakesplaceatthegenomic,
membranetothePVsforrecycling,orarethe transcriptional,orposttranscriptionallevel(2).
VSPsreleasedbyspecificcleavage,assuggested InclonesexpressingonlyoneVSPontheir
(108)?Iftheyareendocytosed,howisthefor- surface, it was found that the only transcript
merVSPdiscriminatedfromthenovelVSPif (mRNA)detectedbyNorthernblottingwasthe
org they have the same cytoplasmic tails? Are the onecorrespondingtotheVSPthatisexpressed
ualreviews.al use only. stVauSiglPgasess(ti1eg3dn2pa,ol1sft3ot4rra)?dnisIslfcartsiioom,nianislamttihooendriefibceaatwtsiioegnennsaolbftothhtahet (ts5reoe).lm,Tebhdeictsoafiubnseedtirnoafgnstschuregibg2ee0dst0wshvtrseaprnegsacesrntihpesetiooontnhalelyrcsoornnee--
nnon marks the former VSP before the newly syn- mainedsilent.Itwaslaterdemonstratedthatall
as
www.or per tmheemsizberdanVe?SPOrisariencthoerpVoSraPtsedcleinavtoedtbhyesppleacsimfiac vsislpengecendesbayreatnraRnsNcrAibiendtebruftertehnatceall(RbuNtAoni)e-laikree
om 2. F proteasesfacingthecellexteriorforreleaseinto mechanism. This mRNA always corresponds
Downloaded frerst on 01/24/1 ttchhoeenIsetcinduvielsitrrueordwneemlmsleeenckdrtne?iutoomwryn/(7etx1hc,arte9t8oV)rySaPndsprhoapadvpueecatbsreeoinnf t(oa5onr,ictgeh8ine5oa,fvlaa8tr6driaa)i.snntstAincefrctxitepprVtredSVsiPsSseaPmdppRosewnNairttAhscehw(3ipin,tagh8r5atos,hic1tece4ua4srp,usp1,ref4taa7hcr)ee-.
0. mh
63A G. lamblia that could be involved in divert- IthasbeenproposedthatGiardiaisatetraploid
5:611-setts - isnwgitcthheediimtsmournigeinsaylstVemSPw(9h1il)e. Tthheenp,agraivsietne oofrgeaancihsmallealnedotfhaevrespfogreenpeo(1ss7e)s.sIetshfaosubreceonpriee-s
6u
11.ach that the cleavage sequence is the same for all portedthatonlythevspgenesthatpossesstan-
0s
ev. Microbiol. 2niversity of Mas VtcadhosiSsfenfaPmefyorsse,cnah(p1tloe0Vrw8fmSo)c?PriacmsnrAcoeaoldstclhpooworcpouiayttlgheihzaesamdeniAmdbdboimfstfflheusronppeworenfloctiituafiecoticyenrtesotasomtmcoeeotntwtnhcrogyee dasaleetelrcmeaveerfgrtdrioaevtimpenhneaatntthtiuosmemncVelabynS(eo5Prbn1-eeo,Af1ar6l4erlce4geolp)ee.gneFonaetfoi,sztrw,ehdehseixensbanecmyteeipaxsillptleeerx,wlepeosarssnseewesdosiaebtalhd--t
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nu. by surface of individual cells (99, 134), whether thelevelofprotein,waslostinclonesthatdo
n
A differentVSPsarelocatedindifferentregions not express that variant (5). The presence of
of the plasma membrane (132, 134) remains allele-specificexpressionofvspgenessuggests
unclear. But again, whether the CRGKA tail anepigeneticmechanismregulatessurfaceanti-
is important for VSP switching, transport, or genexpressioninGiardia,butnoexperimental
both,andhowoneVSPisreplacedbyanother evidencesupportsthisidea(144–147).
antigenically different VSP remain a mystery. During the process of trophozoite differ-
Nevertheless, either the natural half-lives of entiation into cysts (encystation) or from cyst
the VSPs (involving degradation, release, or to trophozoites (excystation), VSP switching
both) linked to differences in level of their becomes evident in the isolate WB (79).
synthesis or an active mechanism of exchange This mechanism is not universal because
may be playing a role in this process. Further after completion of the life cycle in the GS
experimentsarenecessarytoclarifythisissue. isolatetheoriginalVSPremainsunchangedby
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616 Prucca Rivero Luja´n
MI65CH30-Lujan ARI 27July2011 9:40
other immunologically different VSPs (128). (PTGS)wereinitiallydescribedashostdefense
However, it is also unclear whether antigenic responses,inwhichsiRNAsworkasmediators
switching occurs duringdifferentiation or cell of a surveillance mechanism in charge of de-
Transcription: the
division, events intrinsically linked to both stroyingmRNAsofduplicatedoraberrantge-
processofgenerating
encystationandexcystationprocesses(2). neticelements(112).Ontheotherhand,adif- messengerRNAsfrom
Transcriptionalcontrolofconstitutivelyex- ferentclassofsmallRNAs,calledmicroRNAs aDNAtemplate
pressedandregulatedgenesinGiardiaisdriven (miRNAs), encoded within the genome of dsRNA: double-
by small promoter sequences identified up- certain organisms as short hairpin structures strandedRNA
stream of the translational start sites (148). and active during normal development, reg- Dicer(Dcr): an
Studiesofthesesequencessuggestthatthepres- ulate expression of developmental genes by endoribonuclease
enceofAT-richfragmentscanallowtranscrip- repressing the synthesis of target proteins RNAseIIIfamily
enzymespecificfor
tion initiation in G. lamblia (2). However, no (13, 52). Later, a variety of small RNAs were
processingdsRNA
VSPpromotersequencehasbeenidentifiedto described and linked to different aspects of
into23-to25-nt-long
org date,andtheupstreamregionsofthevspgenes gene expression (27, 33, 44, 60, 66, 69). The smallRNAs
ualreviews.al use only. dtaroofrnlatocrttaisnohsnocwroifpavtnisoypnpg.ueAtnadetaisvmceosenetqtaauli.en(n4tc)heienthidnaiicttaciataetnodcrtohsnea--t amRrNotilfiAecciiualmlesaicnhitnrintooedruyccetilsolpsnectihofiafctatlphlyoosssseuepssspmraeansllseasRctNtihvAee PpgeoTnstGetrsSail:nesnccriinpgtional
nnon quence PyAatgTT (see above). Nevertheless, expression of their homologous endogenous
as
www.or per nteormexipneeriwmheenthtaelrdoantalyhathveisbfereacntpiornovoidfethdetoVdSeP- gperenseesn(c4e3,o5f3d–s5R5N,1A0s4)i.sInanalelstsheensteiarlepaosprtesc,tthoef
om 2. F repertoire containing initiator element motifs thePTGSmechanism.
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0. mh
63A IsVSPexpressionregulatedatthetranscrip- family of endogenous genes during the entire
5:611-setts - trieoansoaln,otrhepeoxsptterrainmscernitpstiroenqaulirleedvetol?anFsowresrotmhies lGifiearcdyicalePoTfGthSismmeicchraonoirsgmanriessmem.Ablltehsoguegnhetshie-
6u
11.ach question,suchasnuclearrun-onassaysandsen- lencingphenomenathatcontroltheexpression
0s
ev. Microbiol. 2niversity of Mas saeMwixthpboiverrreteoehsoaResdvirTneorgr-,anPnlafCyogRseroinnsuogeesfvliVeneVrgSVaSPlpPSriPyismte,rtaarewranrsenssrcsittecrhirpawinbttoasecstdaonnrfneroapotmcomlcroptlnlteeihaefdyers. otbaabefrlrfgeIoenorsteeftsithgvseasnnoptdfgsgopteuegrndineemynes,,seottruwhhsseeatghsGeeaxeintpavesrraisedml.suiasuaiPtoletnTadnoGebfoSyaumlusalseryigcnahegmannspuuilmsiimfty--
RU
nu. by nucleioftheparasiteorwhethermanyvspgenes mostofthevspgenerepertoire.Theseprimers
n
A aretranscribedsimultaneouslyandthenallbut allowed the identification of novel vsp genes
onearesilencedbeforetranslation,assuggested and were useful to determine the presence of
tooccurforvargenesinPlasmodium(18). senseandantisenseVSPtranscriptsindifferent
Severalgene-silencingphenomena,includ- Giardia clones by RT-PCR. These results in-
ing transgene silencing and co-suppression in dicate that several sense vsp mRNAs, includ-
plants,quellinginfungi,andRNAinterference ing the one that is translated, were amplified
(RNAi) in metazoans, have many common whenthereverseprimerswereusedintheRT
features.MessengerRNAs(mRNAs)fromthe reaction. Subsequent nuclear run-on experi-
targetgenearedegradedintosmallinterfering ments supported the simultaneous transcrip-
RNAs (siRNAs) by double-stranded RNA tionofmanyvspgenesandthepossibilityofam-
(dsRNA)-specificribonucleaseDicer(Dcr)ina plifyingantisensevspRNAs,excludingtheone
sequence-specificmanner(15,110,150).These thatisfoundbyNorthernblotandexpressedin
forms of posttranscriptional gene silencing thetrophozoitesurface,whenthesenseprimers
www.annualreviews.org • RegulationofAntigenicVariationinG.lamblia 617
MI65CH30-Lujan ARI 27July2011 9:40
a RNAi mechanism in antigenic variation b Disruption of antigenic variation
Key
Dicer
Ago (RISC)
RdRP
Ribosomes
VSP A mRNA/
protein
VSP B mRNA
VSP C mRNA
g
or
ualreviews.al use only.
nn
no
as
w.er
wwor p
om 2. F
0. Downloaded frmherst on 01/24/1 FSncuighculeemrieoat2ficthoefpthareamsiteecahnadnitsrmancsopnotrrtoeldlintogtahneticgyetnoipclavsamri.atTiohnerien,GRidaRrdPiag.e(an)erInatewsiladn-ttiyspenesceeVllsS,PmmanRyNVASsP,pmrRodNuAcisnagreVgSePndersRatNedAinthe
63A complexes.GiardiaDicer(andpossibleAgo)detectsthesedsRNAs,cleavingtheminto25-to27-nt-longsmallRNAs.OnlyoneVSP
5:611-setts - tVraSnPscthriapttwbiylplabseseesxpthreesssieldenocnintgheprtorocepshsobzyoiatestsiullrufancken.o(bw)nInmceecllhsainniswmh,icahcctuhmeuRlNateAsiipnatthhewcayythoapslabseme,nadnidsriuspttheedn(tfroarnesxlaatmedplien,tDoitcheer’s
011.6sachu eVxSpPressosinonthheassubrfeaecnekonfoinckdeivdidduoawlntr),omphaonzyoViteSsP.Atrbabnrsecvriiapttisoancsh:iAegveo,aAsrtegaodnya-ustteatperloetveeilnt;hdastRaNlloAw,sdothueblseim-sturlatanndeeoduRsNexAp;ressionofmany
ol. 2Mas mRNA,messengerRNA;RdRP,RNA-dependentRNApolymerase;RNAi,RNAinterference;RISC,RNA-inducedsilencing
ev. Microbiniversity of complex;VSP,variant-specaaisrfisecayusussresfdahcoeinwpertodhteetihnRa.Tt mreaancyt,ioifn.noNtuacllle,avrsprugne-noens sbuegggeesnteinragtetdhaptotshttorsaensacnritpisteionnseallRy.NBAustcwouhladt
RU
nu. by aretranscribedunderconditionsinwhichonly isthetranscriptionalmachineryofG.lamblia?
An onevsptranscriptaccumulatesinthecytoplasm Giardiagenomesshowthepresenceofseveral,
oftheparasiteandisabletobetranslatedinto butnotall,molecularcomponentsinvolvedin
theproteinthatisthenexpressedonthetropho- thesynthesisandregulationofthisfundamen-
zoitesurface(Figure2a). tal biological process (82, 124). Are all genes
Aconsiderationaboutgenetranscriptionin constitutively transcribed in Giardia? No—
Giardiaisnecessaryatthispoint.Thefinding particularly those genes that are upregulated
of antisense vsp RNAs agrees with a previous during trophozoite differentiation into cysts
reportdocumentingthepresenceoflong,ster- (70). Although several reports indicate the
ileantisensetranscriptsinGiardia,whichwere activity of encystation-specific transcription
suggested to result from a loose mechanism factors, no definitive conclusions have been
controlling transcription (40). Neverthe- drawn(29).IsthesolepresenceofAT-richre-
less, antisense RNAs, including vsp genes, gionslocatedupstreamoftheinitiationcodon
were not detected in nuclear run-on assays, ofallGiardiagenesnecessaryandsufficientto
· ·
618 Prucca Rivero Luja´n
MI65CH30-Lujan ARI 27July2011 9:40
drivetranscriptionofthosegenesinlightofthe that the produced vsp dsRNAs trigger the ac-
reduced transcriptional regulatory machinery tionofanendonucleasecomplexthatprocesses
of the parasite (82)? Nevertheless, exquisite thesemoleculesintoshort25-nt-longdsRNAs
RNA-dependent
metaboliccontrolmustexistgiventheexpen- in an ATP-dependent fashion, a feature of
RNApolymerase
sive energetic cost of synthesizing many tran- RNA-silencingsystems(103,138).Inaddition (RdRP): anenzyme
scriptsthatwillbesilenced,atleastforVSPs. to RdRP, different enzymes involved in pro- thatgenerates
cessingdsRNAsinothereukaryotes,including antisenseRNAusing
aberrantRNAsasa
modules of Dicer (Dcr) (a bidentate RNase
GIARDIA template
III and many ATP-dependent DEAD-box
POSTTRANSCRIPTIONALGENE Ago: Argonaute
RNA-helicases) and Piwi/argonaute (Ago),
SILENCINGMACHINERY protein
were identified in Giardia and were constitu-
AnRNA-dependentRNApolymerase(RdRP) tivelyexpressedintrophozoites(114).Because
homolog that localizes to the rough endo- Giardiabelongstotheearliestdivergingbranch
org plasmic reticulum of trophozoites has been oftheeukaryoticlineofdescent,thepresence
ualreviews.al use only. dhheaosvwceerivabeecrdy,tiom(1pm1la3us,mn1oi1clo4oc)a.rlRinzudactRiloePansrraelorsecualetlsixzpsahetiocotwendt(h9ta)ot; ooalfultstaeaspskaisnra(gatleseditnagshekingienhsse,treineaudwkoahfrioycnoheteegsae)c,nhiesgnpeoenrtefsoucrramprrriiniesg-s
nnon gRdRP is likely associated with ribosomes on ing(Figure3).However,becauseGiardiaDcr
as
www.or per trheteiccuyltuompl.asTmhiecsseidreesoufltthsesuroguggeshteandpoaprtlaicsimpaic- iosbtthaeinsmthaellfiesrtstDtchrreken-odwimnesnosifoarn,ailtswtrauscutuserdetoof
om 2. F tionofRdRPincytoplasmiceventspreceding
Downloaded frerst on 01/24/1 tiadnrniargdnecspltrtaroitnaiconvenossfslovartedmmeptsoeenlcytttrshiobehfmooRsmodmioRnlePotsog,ionwudhgsseeRvrnNesepAasmisls.ecRnrNecTeinAhnge-s DEXDc HELIcDUF2d83oPmAZain RNase III (A/B) DSRHMomo sapiens Dcr1 (1,922 aa)
0. mh
63A phenomena induced by transgenes has been
11.65:611-achusetts - sbohefoethwnenss,uilgaengndecsitdnesgdRpNtrooAcbseespsnr(oe3dc0eu,sc1sea0dr9y)b.iyTntRheedrsmRePerdehisauatlvetess Giardia lamblia Dcr (754 aa)
0s
ev. Microbiol. 2niversity of Mas sotTishufthepicmepsgroueeorflntfotietrpGrhela,eietaiwmrovdhnsoipaadtoetRfrladarpaneRrnsePtctdihrsiiteecphnttasissnttergimunRtcahtNkGuaetrAiaattslhrhdecbiishaypaerrRdneasizdcreyeRtnmeccPrtee-s. doPmAZain Piwi domain Homo sapiens PIWIL1 (861 aa)
RU Divergent
nu. by activeonlyinthepresenceofhomologousVSP PAZ domain
An transcripts (114)? Are antisense transcripts Giardia lamblia Piwi (900 aa)
for many other genes (40) also generated by
thisenzyme?Ifso,howdoesRdRPselectthe RNA recognition RdRP
motif domain
aberrant transcripts for synthesizing antisense
Arabidopsis thaliana RDR6 (1,196 aa)
RNAs?Or,becausetheseantisensetranscripts
as described by others (40) are not evident in
nuclearrun-onassays(114),couldtheybearti-
factsoflibraryconstruction?Thesearerelevant Giardia lamblia RdRP (2,001 aa)
questions that require further experimenta-
Figure3
tion to fully understand gene regulation in
DiagramofthecomponentoftheRNAimachineryofGiardialamblia.
Giardia.
SchematicillustrationofthedifferentdomainsofRdRP,Dicer,and
AfterthesynthesisofantisenseVSPmRNA Piwi/argonauteofG.lambliacomparedwithsimilarmoleculesofhigher
transcripts by Giardia RdRP, it was suggested eukaryotes.Abbreviation:RdRP,RNA-dependentRNApolymerase.
www.annualreviews.org • RegulationofAntigenicVariationinG.lamblia 619
MI65CH30-Lujan ARI 27July2011 9:40
thisclassofenzymesandwasfoundtobeactive efficiency,asreportedforotherparasiticorgan-
evenintheabsenceoftheRNAhelicasedomain isms(12,19,80,106).
(72–74). This domain has been reported to After transcription of the entire vsp reper-
conferspecificityandtocontrolactivityofDcr toire,differencesinthelevelofindividualtran-
in higher organisms (72–74). Therefore, this scriptsmightserveastheexclusionfactorthat
findingmustexplainwhyGiardiaDcrisableto allows the vsp mRNA with the highest con-
cleaveanydsRNA(syntheticorendogenous)in centration to evade the action by the Giardia
vitro(74,114).Experimentsinvivoconsisting RdRP and subsequent degradation by the si-
of either the introduction of exogenous small lencingmachinery(114).Becausecomponents
dsRNAs into the cells or the endogenous of the RNAi machinery, including Piwi/Ago
production of small dsRNAs are expected to familymembers,havebeeninvolvedinhistone
induceastrongRNAiresponse.Itwasrecently modificationsandtranscriptionalderepression
reported, however, that the Giardia RNA inotherorganisms,Agomightwellberequired
org pathwayisactiveonlyagainstlongandspecific forasimilarfunctioninGiardia.However,si-
ualreviews.al use only. dstsuRdTNiehAsepsmer(of1ol1er7cmu),eladrwbhcyoicomhtphoecrnoengnrtrotasudpoiscft(sG74ipa,rr1de1iva4io).sui-s ldteiioncnactiininnggGo.tfhlaaAmtgbiotliamre(a1suy1l4the)a,dvi.eein.a,ncuonanvdtirdaobitllileoinncgaelltlhsfeu,niinnc---
nnon lencingmachineryshowhomologytothoseof tegrityofthegenomebyblockingtheactivityof
as
www.or per htiiognheirnotrhgiasnpisrmims,itisvueggceesltlin(g11a4).simDiilraerctfuenvci-- tsroamnsepcoeslolunlsar(1m3e9c)h.aRndiRsmPscoopuelrdatbineginavftoelrvetrdanin-
om 2. F dence for the participation of RdRP and Dcr scriptiontorecognizehighlyhomologousmR-
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0. mh
63A these enzymes resulted in an alteration of the a concentration-dependent sensor of aberrant
5:611-setts - VonSePVeSxPprewsassiopnropdauttceerdnbsyucihndtihvaidtumalotrreopthhaon- mfuRrtNheArsb(y1e5x0p).erTimhiesnhtsypinodthiceastiisnwgatshastupafpfionritteyd-
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11.ach zoites,demonstratingtheessentialroleofthese purified HA-tagged Giardia RdRP generates
0s
ev. Microbiol. 2niversity of Mas MmceosEsleC(cFuHilgeuAsraNetdI1iScffMaenredSnFtOsigtFeuprAseoN2fbtTh).eIGsilEenNciInCgpro- itohwnhfihgeatehhVdn-eimSfmmfPeooirlbxeertenceuiutnrtlVehgara(Se1-nPxw1p,o4eru)inegn.eshAdsetevtdtrsepRtomhtrNerpatctAhnsoestncooitrnrvispoeiltlreveoniixstfcrpaeoprseretesirostsohienonnelngyrt
RU
nu. by SWITCHING promoter,donotinfluenceantigenicvariation,
n
A GiventhatthisendogenousRNAisystemisin- and continuous switching occurs even under
volved in the control of antigenic variant ex- thesecircumstances(114).Similarresultswere
pressioninGiardia,howdoesasinglevsptran- reportedwhenaDNAfragmentcontainingthe
script bypass this silencing process? In some vspH7genetaggedwithanHAepitopewasinte-
organisms,siRNAscorrelatewithmethylation gratedintotheGSgenome(65).Trophozoites
ofhomologousDNAsequences,whichinturn could express only the native vspH7 gene and
modulates transcription (32, 105, 109); how- nottheHA-taggedvspH7,evenwhentheup-
ever, DNA methylation has not been demon- streamanddownstreamregionsaswellasthe
strated in G. lamblia (2, 114). Hence, other coding regions are virtually identical. These
nuclear modifications, such as variations in results indicate that the control of VSP ex-
chromatin structure linked to the cell cy- pressionislikelyindependentofthepromoter
cle,mightproducedifferencesintranscription that drives its expression, and that another
· ·
620 Prucca Rivero Luja´n