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UniversidadedeLisboa
FaculdadedeMedicinaInstitutodeMedicinaMolecular
INTERACTIONSOFPLASMODIUMBLOODANDLIVERSTAGESWITHINASINGLEHOST
SlviaVilarPortugal
AdissertationforthedegreeofDoctorofPhilosophyinBiomedicalSciences
SpecializationinBiopathologicalSciences
SupervisedbyMariaManuelMota,M.Sc,Ph.D
PrincipalInvestigatorofUnidadedeMalriainInstitutodeMedicinaMolecular
AuxiliaryProfessoratFaculdadedeMedicinadaUniversidadedeLisboa
2010
II
A impresso desta dissertao foi aprovada pela ComissoCoordenadora do Conselho Cientifico da Faculdade de Medicina daUniversidadedeLisboaemreuniode13deAbrilde2010.As opinies expressas nesta publicao so da responsabilidade doseuautor.
III
IV
TheresearchdescribedinthisthesiswasperformedattheInstitutode Medicina Molecular, Lisboa, Portugal, and was financiallysupported by Fundao para a Cincia e Tecnologia, Portugal(SFRH/BD/31523/2006).O trabalho de investigao descrito nesta tese foi realizado noInstituto de Medicina Molecular, Lisboa, Portugal, e foi financiadopela Fundao para a Cincia e Tecnologia, Portugal(SFRH/BD/31523/2006).
V
Preface
This dissertation assembles data obtained duringmyPh.D researchproject, developed at Faculdade de Medicina da Universidade deLisboa, Instituto deMedicinaMolecular, Unidade deMalaria, underthe supervision of Prof.MariaM.Mota, fromOctober 2006 toApril2010.Thisthesisisstructuresin5chapters,precededbyasummarybothinPortugueseandEnglish,outlining theaims, resultsandoutcomesofthisproject.Thefirstchapterprovidesaninsightonpreviousmalariaknowledge,andtheaimsofthiswork.The second chapter contains the description of the methods andmaterialsemployedtocarryoutthepresentwork.Thethirdchaptercontainstheoriginaldataregardingthisproject.Chapter four encloses an overall discussion and conclusions of thestudiesperformed,togetherwithasectionwherefutureperspectivesoftheworkdevelopedaredescribed.In the Appendix is included a table with the information obtainedwithamicroarrayanalysisperformedduringthecourseofthiswork.
Thedatadescribedinthisdissertationistheresultofmyownwork.Thisworkhasneverbeenpreviouslysubmittedforanydegreeatthisoranyotheruniversity
VI
acknowledgements
VII
Acknowledgements
The oneswho got to knowme duringmytime in Lisbon, know that I like a lot towork,alwaysasmuchasIcan,andhaveitdone as soon as possible. Butmost of thetimes, in order to be able to doalmost asmuchaswhatIplannedfor,Icountedwithmany important people without whom itwouldhavebeen impossible toachieveallthis. ThankyouJeremieforthephoto.
Maria,poracharquevaliaapena termeno laboratriodesdeodiaemque me propus vir. Pela responsabilidade que me foi gradualmenteatribuindoequemepermitiucrescernolaboratrioathoje.Epelapressoboa, que sempre me fez sentir, para estar altura do que precisava otrabalho.
Obrigada pelo projecto que deu origem a esta tese, e obrigada por tantasvezes teresacreditadoquea soluoestava jalinaprximaexperincia, eobrigada ainda pormuitas emuitas vezes teresminimizado o facto de quenotnhamosencontradosoluonenhuma...
Mar, porque me ensinou a mexer na bancada, me deixou participaractivamente no seu trabalho, e no final me delegou responsabilidadesefectivasnafinalizaodeprojectosseus.Eporterfeitoistotudocomumcarinhosemprepresenteesempremuitovisvel.
AnaGomes,Mosqeao Johny,por tantasvezesseremcorreiodecaixascomgelosecoeamostras,oupormelevaremetrazeremaolabshoraquetenhoquelestar,poraceitaremosmeushorrioseaindameouviremfalardasexperinciasduranteo jantarsempreatrasado.EaAnaGomes fez istovezessemconta!!
Sem vocs seria efectivamente muito mais difcil ou incrivelmente maisaborrecido.
To Unidade de Malria, to everyone i met in the several UMAs I crossedsince the summer of 2005. All of you, Im sure, at a certain momentcontributedtotheworkpresentedhere,andtothefunIhadproducingit.
ackowledgements
VIII
In particular I thank Cline for the contribution with the analysis of themicroarray results and for helping me interpret them with so muchpatience.
Depois emparticular querodizermuito obrigada Vanessapelo trabalhoquederamasminhasamostrasemMuniqueepelaimensadisponibilidadeparadiscutirtrabalhoouajudarcomumcasacodeEltonJohnouumgorrode Nikita; Carina pelas discusses cientficas ou as conversas da vidaquandoficmosatrabalharatsmil;aoMiguelpelascrticaseficienteseporexemplo,poratravessarmeiacidadecomigodemota,parareporantesdas10hotremosquepartidaUBD;S,PamplonaeMargaridaVigriopor ajudarem nas minhas dvidas de imunologia; S por me ensinar aesplectomizarratinhos,Fernandapormepermitirpedidosdemosquitosforadetempoeporencurtarasminhasausnciasdacasadosanimaiscomumainjecoaquioualienquantoviajo;eElianapelaprontidocomquemeaceitounasuacasaquemepermitiuexperinciasrelmpagoemParis.
To Mario Recker for the great help in producing the model that allowedfitting the results produced in the lab during this thesis, with fieldobservationinmalariaendemicsettings.
ToChrisNewbold,HalDrakesmithandAndyArmitagefortheinputonthehepcidinexperiments,andforthehelppreparingthefinalarticle.
Lgia pela contribuio com os hepatcitos primrios, e por tornaragradveisdiasaproduzirmuitomenosclulasdoqueasquegostaramos.
AoCludioMarinhoporgostardefalarcomigosobreoprojectoquelevouaestatese,epelastantasperguntasquefezsempre.
AoJooFerreiraeaoBrunoSilvaSantosqueenquantoComitdestatesemequestionaram e me incentivaram a perseguir os objectivos. E porperguntaremsemprecomoqueest?!omistrio.
AtodoostaffdascasasdosanimaisdoIMMedoIGC,eAlina,Doloreseao Manel em particular por me deixarem abusar, atender os pedidos deratinhos em cima da hora, usar a cmara de fluxo sem marcao, poracreditaremqueeraimportanteeupodertrabalhartudooquequisesse.
acknowledgements
IX
RosaMaria pela prontido com que produziu os vrios anticorpos queuseiaolongodestetrabalho.
To all of you that read parts of this thesis_ Miguel, Pat, Teresa, Bruno,Margarida,Vanessa,Pamplona,andAndy
Thankyouforyourtime,yourcomments,corrections,andsuggestions.IhopeIdiditright!
AoRuben,aoDanieleaoPedroobrigadaportrataremdomeucomputador.
CatieaoseuamigoManelquemeajudaramaidentificaronomedoqueescolhiparaacapadestatese.EaoCsarqueaceitouimprimir.
primaPaulaquenoinciomedeuasuacasapormaisdeumano.
DepoisAnapornoseimportardepartilharcasacomumaquasesempreausente,porsepreocuparquedurma,quecoma,quedescanse.
Aos meus pais por participarem activamente agora como antes, desde otempoemqueomeupaivendiaoscalendriosdosescuteirosquedeveriavendereu,atatemposmaisrecentesemqueaminhameforraasgaiolasdosmosquitoscomredeenastroquenosservematodosno lab.EQuelquetantassegundasfeiramelevouaocomboioahorasindecentesparaquepudesse comear a trabalhar a horas decentes. E Di porme perdoar ostantosratinhossacrificados.
Obrigadapormedeixaremcontinuarasercuidadaporvocs.
EaoRicardopelompetoquemedoseuamor,emtudoemaisnotrabalho.Obrigadapormeouviresjdemasiadotardeeaindasobretrabalho,porvirescomigoao lab emnoites oumadrugadas emquedevamos snamorar, porleres as coisas que escrevo ainda que tarde, e por perguntares e se fosseassim...?porajudaresnonomedateseeporarranjaresosartigosacabadosdesairdoprelooudaarcadavelha,aosquaisnotenhoacesso.
X
resumo
XI
Resumo A infeco pelo agente causador da malria, o parasita Plasmodium,
encontrase ainda hoje disseminada pelas populaes de 108 pases no
mundo.Amalriaproduzsintomasquevariamentrefebres ligeirasatao
coma,anemiasevera, sndromerespiratriaagudaoumalriacerebral.S
em 2008 esta infeco foi responsvel por mais de 800000 mortes, das
quais a maioria se ficou a dever ao parasita Plasmodium falciparum.
Adicionalmenteamalriaaindaresponsvelporumareduode1.3%no
crescimentoeconmicodospasesdemaiorendemicidade.
O parasita Plasmodium, pertence ao filo Apicomplexa e partilha com o
Homem a presena na Terra desde o prprio aparecimento da espcie
humana,tendoaadaptaoparasitahospedeirovindoaevoluiraolongodo
tempo, procurando um balano entre a transmisso do parasita e a
sobrevivnciadohospedeiro.
comapicadadeumafmeademosquitoAnophelesqueoparasitachega
ao hospedeiro mamfero. Depois de uma passagem pela pele, os
esporozotosmigramvia corrente sanguneaatao fgadoondedepoisde
atravessar vrios hepatcitos, invademumltimo coma formaode um
vacolo parasitrio. No hepatcito dse um processo de crescimento e
replicao que no homem demora vrios dias at formao de vrios
milhares de merozotos que sero libertados de novo na corrente
sangunea. Uma vez no sangue osmerozotos entram numa nova fase de
reproduo assexuada, com sucessivos ciclos de invaso de eritrcitos,
replicao e libertao para a corrente sangunea e nova invaso de
eritrcitos. nesta fase da infeco que todos os sintomas associados
malriasurgem.
resumo
XII
Ocasionalmente, o ciclo de reproduo assexuada dentro dos eritrcitos
para formao de novos merozotos substitudo pela formao de
gametcitos femininos oumasculinos que podero ser aspirados durante
umanovapicadademosquito.nohemocliodestevectorquesedafase
sexualdoparasitadamalriacomaformaodeumzigotoesubsequente
oocinetoquemigraparaalminabasalondesetransformanumoocisto.A
partir da esquizogonia dos oocistos surgem novos esporozotos que uma
vezchegadossglndulassalivaresdomosquitosestoprontosparaquese
inicieumanovainfeco.
Em zonas de alta transmisso de Plasmodium provvel a ocorrncia de
sobreposiodas faseshepticaesanguneanumshospedeiro,bastando
para isso que uma picada infecciosa ocorra num indivduo que alberga j
uma infeco circulante proveniente de uma picada anterior. Apesar de
potencialmente importantes, as possveis interaces entre as diferentes
fasesdedesenvolvimentodoparasitaeohospedeiroqueosacolhenunca
foramobjectodeestudo.
O objectivo desta tese prendese precisamente com o estudo das
interacesquesurgirodestasituaoaquedamosonomedereinfeco.
Quo efectivos sero os esporozotos na infeco do fgado de indivduos
comPlasmodiumnosangue,quandocomparadoscoma infecodo fgado
deindivduossemqualquerparasitademalriapresente?
Fazendo uso de modelos animais previamente estabelecidos e diferentes
clonesdeparasitas, quenospermitiramdistinguir as infecesheptica e
sangunea nos animais reinfectados, verificmos uma reduo fortssima
na infeco no fgado de animais reinfectados. A capacidade dos
esporozotosparainfectarhepatcitosderatinhoscominfecosanguneaa
decorrer apareceu altamente limitada quando comparada com a mesma
capacidadeparainfectarhepatcitosderatinhosnaive.
resumo
XIII
O estudodetalhadodeste fenmenopermitiunos relacionar a reduoda
infeco heptica com a diminuio tanto do nmero como do
desenvolvimento das formas exoeritrocticas (EEF no original) no fgado.
Estareduoverificouseindependentedonveldaparasitmiadainfeco
primria,desdequeestaseencontrasseacimadeumvalorquesemostrou
ser baixo e rapidamente atingido. Mais ainda, verificouse que a
administraodeumtratamentoantimalricoaosratinhosinfectadoscom
Plasmodium no sangue antes da reinfeco, resulta na perda deste efeito
protector.
Vriashipteses,baseadasemtrabalhosanterioresaesteenumestudode
expressogenticaquerealizmosparamelhorcompreenderasalteraes
hepticas em resposta presena de Plasmodium no sangue, foram
colocadas na tentativa de entender o mecanismo pelo qual se observa
tamanhareduona infecode fgadoderatinhosreinfectados.Diversas
molculas associadasao sistema imunitrio, inflamaoe apoptoseno
fgado foram testadas pormeio de ratinhos transgnicos, ou pelo uso de
anticorpos depletantes ou drogas bloqueantes sem que nenhum dos
factorestestadosindicasseserrelevante.
A infeco de hepatcitos cocultivados com eritrcitos infectados com
Plasmodium, no mostrou ser eficiente em produzir o mesmo tipo de
reduo,afastandoahiptesedeumfactorsolvellibertadopeloeritrcito
infectado.
Ao avaliar alteraes no fgado relacionas com a disponibilidade de
nutrientes verificmos que o gene codificante da hormona reguladora do
ferro, hepcidina se encontrava sobreexpresso no fgado de animais com
parasitasnosangue.Sabendoqueadisponibilidadedeferropodelimitaro
crescimentodevriospatogniosincluindooPlasmodium,perguntmonos
seseriaesseocernedareduonainfecoverificada.
resumo
XIV
A expresso do gene codificante da hepcidina provou estar intimamente
ligadapresenadeeritrcitos infectadosnosanguederatinhos, subindo
rapidamente com poucos ciclos replicativos de Plasmodium no sangue, e
voltando rapidamente a valores basais com o tratamento antimalrico, o
que rapidamente reduz a parasitemia para zero permitindo ento que a
infeco heptica se processe como em ratinhos naive. Alm disso, pde
verificarse uma redistribuio de ferro no fgado, perdendose parte do
contedo nos hepatcitos para incrementar o contedo em macrfagos
residenteseinfiltradosnofgado.
Adicionalmente,mostrousequeahepcidinapersepodereduzirainfeco
heptica.Aadministraoaratinhosdeumadenovrusexpressandoogene
codificante desta hormona reguladora de ferro confirmou que este
componente promovido pelo parasita no sangue,mesmona sua ausncia,
podeactuarlimitandoainfecoporesporozotos.
Ao tentar perceber a implicao destes resultados na malria humana,
deparmonos com dados epidemiolgicos h muito conhecidos mas
parcamente explicados. Em reas altamente endmicas conhecido
consistentementequeaincidnciadainfecoaumentainicialmentecoma
idade das crianas para depois decrescer, possivelmente pela aco da
imunidade adquirida. Ao mesmo tempo, a complexidade da infeco, em
nmerodeclonesdeparasitasdiferentesnosanguedeindivduos,aumenta
medidaqueascrianascrescememidade.
Divisando um modelo baseado unicamente na existncia de um valor
mnimodeparasitmiaqueinibisseoestabelecimentodainfecoheptica,
tal como foi observado no decurso desta tese, e assumindo o h muito
estabelecido,queadensidadedeparasitasnosanguedecrescecomaidade
dos indivduos. Instituise ento que a probabilidade de um picada
infecciosaproduzir infecodependedonveldeparasitmianomomento
resumo
XV
dainfecoedahistriaclnicadohospedeironoqueconcerneaepisdios
prvios. Sob estas assumpes mnimas o modelo criado prev
correctamente um aumento inicial de infeces nas crianas seguindose
um declnio medida que os indivduos adquirem imunidade devido a
repetidasinfeces.Queristodizerqueumadensidademnimadeparasitas
nosangue,daqualdependaainibiodenovasinfecesdePlasmodiumno
fgado, pode por si justificar o aumento do risco de infeco e crescente
complexidadedasmesmasemcrianasnovas.
A aplicao do modelo com os resultados esperados mais
proeminentementeobservadaquandotestadosegundoreascomtaxasde
transmisso moderadas ou elevadas, e permite explicar ainda algumas
diferenasdeincidnciadadoenaaolongodaidadedascrianasemreas
comdiferentestaxasdetransmisso.
O ciclo de vida do Plasmodium tem vindo a desenvolverse ao longo de
milhes de anos de coevoluo das interaces hospedeiroparasita, com
implicaes importantes para a sade humana. A infeco de eritrcitos
acimadeumadensidademnimaelevaaproduodahormonareguladora
doferro,hepcidina,queredistribuindooferroprotegeonichodoparasita
existente, inibindo o estabelecimento de uma infeco secundria,
prevenindo assim a superinfeco. Este fenmeno actua
independentementeedeformacomplementarimunidadeadquiridaevem
aclarar observaes epidemiolgicas prvias, podendo ainda ter
implicaesemfuturasintervenesnalutacontraamalria.
Palavraschave:malria,reinfeco,superinfeco,hepcidina,ferro
XVI
summary
XVII
Summary
Inregionsofhighmalariatransmission,infectedindividualsareconstantly
exposed to potential reinfection. Mosquito bites transmit livertropic
sporozoitesintosubjectswhoalreadyhavebloodstageparasitaemia.How
these two stages of thePlasmodium life cycle interactwithin their host is
unknown. Here, using a rodent model, we show ongoing blood stage
infections impair the growth of subsequently inoculated sporozoites.
Secondary infectionsarearrested in liverhepatocytesand fail to compete
forcolonizationofredbloodcells.Thisprotectionoftheerythrocyteniche
onlyoccursbeyondacertainthresholdofbloodparasitedensity,andsois
phenotypicallyakintoquorumsensing.WeeliminatePlasmodiumsecreted
factors,hostcellsurvivalandinnateoradaptiveimmunityasexplanations
for this observation. Instead, we find parasitized erythrocytes induce
expression of the host iron regulatory hormone hepcidin, which by
divertingironfromhepatocytestomacrophages,limitsPlasmodiumgrowth
intheliver.Presumingasimilarinteractionbetweenmalariaandthehuman
host we demonstrate how parasite thresholddensity dependent growth
inhibitionalonecanexplaintheepidemiologicalpatternsofagerelatedrisk
and complexity of infections in young children. Our findings thus have
broad implications for malaria and have general relevance for
understandinghostpathogeninteractions.
XVIII
XIX
Top 10 Abreviations
CQ ChloroquineEEF ExoErythocyticFormsEIR EntomologicalInoculationRategfp GreenFluorescentProteinhamp Hepcidinhprt HypoxanthineGuaninePhosphoribosyltransferaseiRBC InfectedRedBloodCellPbA P.bergheiANKAPbNK P.bergheiNK65iRBCsRBC RedBloodCell
XX
XXI
Table of contents
Preface VAcknowledgements VIIResumo XISummary XVIITop10Abreviations XIXTableofcontents XXIIntroduction 1Malaria 3Plasmodiumphylogenyanditscoevolutionwithman 4ThePlasmodiumlifecycleinmammals 8Malariatransmissionandnaturallyacquiredimmunity 22Aims 28
MaterialsandMethods 31Results 47BloodstagePlasmodiumparasitessuppresscoinfectionintheliver 49AdditionalResults 87
Discussion 99Generaldiscussionandconclusions 101Thenextinline 123
Appendix 127Bibliography 153
XXII
Introduction
2
introduction
3
Fromthebeginning
Malaria
Descriptions of malaria appear in history prior to its name in several
ancestralcivilizations.AncientChinesemanuscriptsdatingasearlyas2700
BChavedescriptionsofpatientswithfeveraccompaniedbyenlargedspleen
(reviewed in 1). Later, Indian and Egyptian manuscripts also recorded
indications of malaria infection. Furthermore, DNA of one of the species
causing malaria, Plasmodium falciparum was recently isolated from an
approximately 4000 year old Egyptian mummy 2 and Plasmodium
falciparum DNA was also found in the mummy of Egyptian King
Tutankhamunandthreemembersofhisfamily3.ReportsbyHippocratesin
400BCdescribed for the first time thevariousperiodicmalaria fevers. In
theCorpusHippocraticum,hedistinguishedthe intermittentmalarial fever
from the continuous feverofother infectiousdiseases, andalsonoted the
daily, everyotherday, and everythirdday increase in body temperature
(reviewedin4).
Fig. I1.LaMal'aria (18501851), oil over canvas by the French artist AntoineAuguste Ernest Hbert (18171908). Reproduction rights requested to Muse dOrsay andMuseHbert.
introduction
4
ThenameMalaria appears later from the Italianmal'aria, translating into
badair,asinRome,wherethediseaseragedforcenturies,itwascommonly
believed until the 20th century that swamp and marshes produced the
illness(reviewedin5).
Inworldhistory, thedisentanglementof theGreekEmpire isattributedto
malariaandthediseaseisalsobelievedtohavestoppedarmiesofEuropean
andAsianempiresondifferentoccasions1.Nowasthen,malariastilllimits
civilizations. Indeed, in the secondhalf of the20th century, countrieswith
intensive malaria showed an average increase of gross domestic product
(GDP)5timeslowerthanthatobservedfornonmalarialcountries6.
The clinical features of malaria infection, which made it so recognisable
throughout centuries, occur only during the asexual cycles ofPlasmodium
parasite inside red blood cells (RBCs). In humans, symptoms range from
mildfevertocoma,severeanaemia,respiratorydistressorcerebralmalaria7.The2009WorldMalariaReportoftheWorldHealthOrganization(WHO)
states that 108 countries were endemic for malaria in 2009, and that
863000deathswereattributabletomalariain2008,themajorityofwhich
were African children under 5 years of age 8. Moreover, the disease is
estimatedtoberesponsibleforanannualaveragereductionof1.3%inthe
economicgrowthforthosecountrieswiththehighestburden9.
Fromtheverybeginning
Plasmodiumphylogenyanditscoevolutionwithman
Plasmodium is the causative agent of malaria and belongs to the
Apicomplexaphylum.Apicomplexanshaveprobablybeenpresentonearth
since the earliest animals appeared and modern Plasmodium species
descend from apicomplexans that interacted and adapted to increasingly
sophisticated hosts over hundreds of millions of years 10. Species of the
genus Plasmodium have coexisted with humankind throughout its
introduction
5
evolution.P.falciparum,P.vivax,P.ovaleandP.malariaearethespeciesthat
systematically infecthumans. Inaddition,a fifthone,P.knowlesi,anatural
parasite ofmacaquemonkeys in southeast Asia,was recently reported to
infect considerable numbers of humans 11, besides several reports on
isolated cases in severalAsian regions 12,13. Likewise, recentwork reports
that P. falciparum infections are not rare in gorillas 14. These species
represent only a glimpse of the systematic and ecological diversity of
PlasmodiumandrelatedgeneraofApicomplexaparasites.Severalhundreds
of malaria parasite species use squamat reptiles, birds, and mammals as
vertebrate hosts along with many genera of dipteran vectors 15, but the
evolutionary and ecological events that led to this diversification and
successremainpoorlyresolved.Recentgeneticdatachallengedtheclassical
systematic classification ofmalaria parasites, bothwithin thePlasmodium
genus, and in its relations among the currently recognized genera of the
orderHaemosporidiaeofApicomplexaparasites.Astudyrecoveringmulti
gene phylogeny ofmalaria parasites, including the analysis of sequencing
datafrom4genes,indicatedthatparasitesidentifiedasPlasmodiumspecies
fall in twomajor clades, each cladebeingassociatedwithauniquevector
family,onecladecontainingparasitesofmammals,andotheroflizardsand
birds. Within the mammalian parasite clade, there are 4 monophyletic
lineages: the human parasite, P. falciparum, a primate lineage that
containsP. vivax andP. knowlesi, a lineage of parasites that infectAfrican
rodents,andthethreesamplesofHepatocystisisolatedfrombats16.
Evolutionarily,Plasmodiumemergencewasaccompaniedbyamajorchange
in parasites life cycle that differed from other genera. In addition to an
initial roundofasexual replication in fixed tissues thatwasgeneral forall
parasites, thePlasmodium life cycle includes additional rounds of asexual
reproduction in blood cells, revealing increasing complexity when
compared to ancestral parasites. There was also a shift to the use of
introduction
6
mosquitovectors,whichseems tohavemade theexploitationofagreater
varietyofvertebratetaxaashostspossible16.
Fig.I2.PhylogenetictreeofmalariacausingparasitesAdaptedfromamultygene analysis study performed by Ellen S.Martinsen and collaborators, using 11mammalian parasite species, 7 species that infect lizards, and 39 lineages fromavianhosts.
It has been established that Plasmodium entered mammals only once,
coincident with a switch from Culicine to Anopheline mosquitoes 17. All
knownvectorsofmammalianinfectingPlasmodiabelong to theAnopheles
genus 18, indicating that the shift of Plasmodium into mammals was
associatedwithspecializationtoanophelinevectors.Itiswellacceptedthat
the closest identified sister taxon of P. falciparum is P. reichenowi whose
hostisthechimpanzee19.ItwasrecentlyproposedthatextantP.falciparum
populationsoriginated fromP. reichenowi as late as1.5millionyears ago,
introduction
7
likelybyasinglehost transfer 20,anevent thatappears tohavehappened
frequentlybetweenprimates, includinghumans 19.Othersbelieve that the
appearance of P. falciparum occurred even later during the initiation of
agriculture, ten thousand years ago 21. Still, there is some controversy
around theseproposalsand the relationshipbetweenP. falciparumandP.
reichenowiisnotyetcompletelyclear.
Nevertheless, it isclear thatPlasmodium (or itsancestors)and theirhosts
had millions of years to coevolve and coadapt, leading to a balance
between parasite replication and host survival enabling transmission to
otherhosts.ApossibleexplanationforP.falciparumbeingthemostvirulent
speciesinfectinghumansisthefactthat,beingmuchmorerecentthanother
Plasmodium species, it hasnot yet had thenecessary time to finetune its
effectsonthehostpopulation.
Despite the significant burden of malariarelated mortality only a small
proportion of infections lead to severe disease or death. Plasmodium
infections in malaria endemic regions most often go unnoticed even in
children, who are themost affected sector of the population. In children,
around half of all infectious bites produce no infection, about a quarter
leads to asymptomatic infections, roughly the other quarter gives rise to
uncomplicated malaria with mild symptoms, and only a very small
proportion of the infective bites will induce severe malaria with
manifestations such as coma, cerebral malaria, respiratory distress, or
severeanaemia22thatcanultimatelyleadtodeath.Still,theprevalenceofP.
falciparum is high enough to kill more than half amillion children every
yearinsubSaharanAfrica.
EvidencefortheadaptationofPlasmodiumtoman,andmansstrategiesto
circumvent malaria infection can be seen in how the infection has
modulated certain human genes in affected populations, selecting for
resistance.AsHaldanehypothesisedandAllisondemonstrated23,malariais
introduction
8
knowntobeastrongevolutionaryforceofselectionintherecenthistoryof
the human genome. Sicklesell disease, thalassemia, and glucose6
phosphatase deficiency, among other erythrocyte defects that together
comprise the most common Mendelian human diseases, are under
Plasmodiuminfectionselectionforce,andcanaidunderstandingthegenetic
basis of resistance shownby some exposed populations. Indeed, different
resistant alleles have been identified for sickle cell trait in different areas
andatdifferenttimes,suggestingconvergentevolution(reviewedin24).
Plasmodiummodusoperandi
ThePlasmodiumlifecycleinmammals
Alfonse Laveran identified the exflagelating malaria parasite in human
blood in 1880 25. Eight years later, using an avian model, Ronald Ross
attributed the transmissionofmalariadisease to theanophelinemosquito26.Thefirstobservationofthepreerythrocyticstageparasitewasdescribed
laterin1948byH.E.ShortandP.C.Garnhamintheliverofasimianmodel27.Thethreestagesofmalariainfectionwerethendescribed,andfromthere
onresearchtriestofillinthegaps,andunderstandtheexactdetailsofthe
entire life cycle, what the parasites needs are, and how host and vector
moleculescontributetoorfightinfection.
Duringitslifecyclethemalariaparasitetakesmanyforms,shiftingbetween
invasive and replicative stages both in the vertebrate host and in the
anophelinevector.
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9
Fig.I3.Plasmodiumlifecycle(A)Duringabloodmealananophelinemosquitoinjects Plasmodium sporozoites into the host dermis. (B) After reaching a bloodvessel, sporozoites will travel to the liver where after traversing severalhepatocytes, sporozoites invade a final one. (C) After asexual replication anddevelopment inside a hepatocyte,merozoites are released into the blood stream.(D) Merozoites infect red blood cells during cycles of asexual replication. (E)Occasionally replication cycles will originate female and male gametocytes. (F)Throughanotherbloodmeal, amosquito ingestsgametocytes into itsmidgut. (G)Fertilization of gametes occurs in the mosquito midgut with the formation ofookinetesand latertheoocysts. (H)Sporozoitesreleasedfromtheoocystmigratetothesalivaryglandofthemosquitoawaitingthenextbloodmeal.Depositionintheskin.Allnaturalhumanmalariainfectionsstartwiththe
depositionofPlasmodium sporozoitesbya femaleanophelinemosquito in
thehostsdermis,duringabloodmeal.Eachsporozoiteisapproximately10
mlongand1mwide,andonceinthedermis,oneoffourpossiblefates
willdetermineitsfuture:(i)itcaninvadeabloodvesselandreachtheliver27,(ii)itcaninvadealymphaticvesselandaccumulateinthedraininglymph
node28,(iii)itcanremaininthedermis29,or(iv)itcanbereingestedinto
the mosquito gut during the blood meal 30. Only the first of these fates
allowsinfectiontoproceed.
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10
Intheavasculartissuesofthedermis,sporozoitesactivelyglideforwardin
random directions. This movement however, seems to be specific to its
substrate, as it differs from the one observed in salivary gland ducts and
from that exhibited in vitro 28. Like other apicomplexan parasites,
Plasmodium sporozoites display a unique kind of locomotion, known as
glidingmotility,definedby the lackofobviousmodification in themoving
cell shape, and theneed for a supporting substrate 31. Theparasite glides
both to achievemotility and to invade cells, and the twomechanisms are
madepossiblebythesameactomyosinmotorlocatedinthecorticalspace
inbetweentheplasmamembraneandtheinnermembranecomplex(IMC),
a continuous layerof flattenedvesiclesunderneath theplasmamembrane3134. In this space, transmembrane proteins displaying adhesive
extracellular domains and anchored to the motor trigger either forward
locomotionor penetration into thehost cells 31,35,36. One suchmolecule of
particular importance is the thrombospondinrelated anonymous protein
(TRAP),whoseexpressionisrestrictedtoPlasmodiumsporozoites37,38.
Mostsporozoitesremaininthedermisforatleast5minutes,astheremoval
of the biting site 5 minutes after the bite greatly reduces the number of
infections in mice. However, a similar removal 15 minutes after the
mosquito bite does not cause any significant alteration in the outcome of
liverinfection39,showingthatduringthistimeframefollowinginoculationa
significantandsufficientproportionofthesporozoitesleavethebitingsite
to find a blood vessel, which will later lead to infection of hepatocytes.
Nevertheless, it takes one hour for half of the inoculated sporozoites to
leave the dermis, either into blood or lymphatic vessels, and sporozoites
havebeenfoundinthedermisupto7hoursafter injection40.Duringthis
period in the skin, sporozoites might trigger the first line of immune
response.
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11
To leave thedermis sporozoitesmaymakeuseof their ability to traverse
cells41,disruptingmembranesofendothelialcellsandpenetratingvessels.
Severalproteinshavebeenidentifiedasessentialforcelltraversal,suchas
the sporozoite microneme protein essential for cell traversal (SPECT) 42,
SPECT243, cell traversalprotein forookinetesandsporozoites (CelTOS) 44
orphospholipase (PL) 45. Indeed, sporozoites lackingSPECTareunable to
reachbloodvessels41.
Closetoonethirdofsporozoitesinoculatedintomicethroughmosquitobite
are later found in lymphaticvessels 28.Fromthere,onlya fewsporozoites
canberescuedbacktobloodcirculation,whiletherestaredrainedtothe
closest lymph node. Once there, some sporozoites have been observed to
developintoyoungandsmallExoErythocyticForms(EEFs)duringthefirst
hours. This initial development is not continued and, instead, these small
EEFs are then cleared, which seems to contribute to the initiation of an
immuneresponse46.
Themechanismofinvasionofthebloodvesselsisstillnotfullyunderstood.
Ithasbeendescribedthatglidingspeedisdecreasedwhensporozoitesare
incontactwiththevesselsanditisknownthattheentryprocesslastsless
thanaminute47.
Theliverstage.Onceinsidethevessel,sporozoitesarecarriedintheblood
flowatthesamespeedaserythrocytes40,andwill laterbearrestedinthe
liverbynotcompletelyunderstoodmechanisms.Circumsporozoiteprotein
(CSP),aproteinencodedbyasinglecopygene,andthemajorcoatprotein
ofPlasmodiumsporozoites48,hasbeenshowntobeinvolvedinthisprocess.
IndeedrecombinantCSPbindsspecificallytosulfatedglycoconjugates49.It
has beenproposed that heparan sulfate proteoglycans (HSPGs) of stellate
cells extending through endothelial fenestrations to the sinusoidal lumen,
canattractandarrestsporozoites.Thesecellssynthesizeproteoglycansthat
areeight timesmoresulphatedthanthoseofhepatocytesand incorporate
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12
twice the amount sulphate into heparan sulphate, known to be
preferentially attractive to sporozoites 50. Being unable to pass through
fenestrationsof the liversinusoid,sporozoitesmustmigratethroughcells,
mostly Kupffer cells, using their capacity to traverse barriers and finally
access the liver parenchyma 51. Still, this is a matter of controversy and
someauthorshaveproposedthatthepassagethroughKupffercellsoccurs
byamechanismakintophagocytosis52.Oncehavinggainingfreeaccessto
the liverparenchyma,sporozoitestraverseseveralhepatocytes,disrupting
theirplasmamembranes toget in andoutof cells 53, until theyultimately
invadeafinalhepatocytewithformationofaparasitophorousvacuole(PV).
Differentstudieshaveshownhepatocytetraversalmakessporozoitesprone
forfinalhepatocyteinfection,eitherbyactivatingsporozoitestoformaPV54,55orbyleadingtothereleaseofhepatocytegrowthfactor,whichsustains
hostcellviability56.Importantly,althoughdemonstratedtobebeneficialfor
infection,traversalabilityofPlasmodiumsporozoiteshasbeenshownnotto
be essential for hepatocyte invasion in vitro, since parasites lacking
essential proteins for cell migration as SPECT, CelTOS or PL still invade
hepatocytes and complete liver stage development similarly to wild type
parasites 4244. Notmuch is known about the signal to stopmigration and
invade,butmousestudiesindicatethatsporozoitesareactivatedtoinitiate
the invasion process in the presence of cells expressing high levels of
sulfatedHSPGs50,54.
To invade hepatocytes, Plasmodium sporozoites, like other apicomplexan
parasites, release proteins from apical organelles termed rhoptries,
micronemesanddensegranules,andattachtohostcellsusingcelladhesive
domains of some of these proteins. TRAP seems to be essential to this
process57.Althoughadhesiontothehostcell is initiallyreversible, it later
becomesirreversible,formingtightjunctionsthatwillallowtheparasiteto
moveforwardpropelledby itsactomyosinmotor,allowing it toenter the
introduction
13
cell.With the completionof the tight junctions, the sporozoite is included
insidethehostcellsurroundedbythePVmembrane58.
Insidehepatocytes,sporozoitesdevelopandreplicateoriginatingthousands
of merozoites that ultimately will proceed to blood infection. The
astonishingreplicationrateobservedmostcertainlyimpliesstrongcellular
andmolecularhostparasiteinteractions.Althoughlittleisknownaboutthe
intrahepatic development of Plasmodium parasites, recent studies have
providedanewandmorecomprehensiveinsightintogeneexpressionand
protein abundance profiles of Plasmodium liver stage 59. Plasmodium
sporozoite and liver stage asparaginerich protein (SLARP) has been
observed to be essential for initiation of EEF development, as parasites
mutant for this protein are arrested in very early development 60. Other
parasiteproteinshavebeenshown tobeessential fordevelopment inside
hepatocytes. Parasites deficient for the microneme protein P36p present
reduced invasion and impaired development 61, and upregulated in
infective sporozoites genes 3 an 4 (UIS3) or (UIS4)deficient parasites
present impaired development 62. Plasmodium Fatty Acid Synthesis II
pathwayenzymeswere found tobemodulatedduring infection, and later
functionalstudiesrevealedthattype2fattyacidsynthesisisimportantfor
liverstagedevelopment63and,morespecifically,essentialforthelatesteps
of liver stage development 64. Another recent study showed that cGMP
dependent protein kinase (PbPKG) disruption in sporozoites leads to an
arrestofliverstages65.
Hostmoleculeshavealsoproventobeimportantforcompletedevelopment
of the Plasmodium parasite inside hepatocytes. The expression of several
host genes is modulated during liver infection, as shown recently in a
microarray study comparing infected and noninfected cells patterns of
geneexpressionover the timecourseof infection 66. In functional studies,
tetraspaninCD81 67 and scavanger receptorB1 68 havebeen shown to be
introduction
14
importantforliverstagedevelopment.Theirabsencereducesliverinfection
toverylowlevelsinrodentmodels.Yetanotherfunctionalscreen,aimedat
the host kinome, has linked an efficient parasite infection with several
humankinases69.
During the liver stage, as will later be shown for the blood stage,
PlasmodiumparasitesseemtoexportproteinsbeyondthePVtothecytosol
of the hepatocyte or even to the hepatocyte nucleus. Up to now this has
been shown for CSP, and mutant parasites incapable of this export
movementshowimpaireddevelopment70.
By theendofexoerythrocyticstage,merozoitesarereleased in theblood
stream within large vesicles known as merosomes 71. These budding
structures full of merozoites squeeze through endothelial cells, and are
initially hidden from the host's innate immune system by being covered
withahepatocytederivedmembrane.
The blood stage. After reaching circulation, merosomes release the
merozoites into the bloodstream. This seems to occur only when
merosomesreachthelungmicrovasculature.Althoughthereasonbehindis
not known, it has been speculated that low macrophage density and
reduced blood velocity with reduced shear forces within the lung
microvasculatureenhancestheabilityofmerozoitestoinvadeerythrocytes,
sinceoncemerozoitesarefreeincirculationtheyquicklyhavetofindared
bloodcelltoinfect.72.
MerozoitesinvadeRBCs,highlydifferentiatedcells,whosemainpurposeis
oxygen distribution throughout the body. However, RBCs lack several
cellular functions as they are anucleated, transcriptionally and
translationally inactive, lack any secretory apparatus, have only a limited
repertoire of solute and iron transporters, and are readily removed from
circulationwhendamaged(reviewedin73).
introduction
15
Invasion of RBCs follows sequential steps: cell recognition, merozoite
binding,reorientationanddeformationoftheRBC, junctionformationand
parasiteentry.IninP.falciparumalone,morethan50surfaceandsecreted
proteins have been identified as playing a role in these events 74. Initial
hostparasite contact is believed to be randombut, once it has happened,
merozoitesurfaceproteins(MSP)immediatelymediateadhesiontothehost
cell 75. Themerozoites surface is covered in glycosylphosphatidylinositol
(GPI)anchoredproteinsandtheirpartners76.Manyoftheseproteinsseem
toactasligandsforRBCs.BesidessharingGPIanchors,surfaceproteinsof
the merozoite also share with sporozoites and gametes the cysteinerich
domains that have been shown to potentiate adherence 77 ofPlasmodium
species.Assuch, it isbelievedtheyplaysimilarrolesinthecytoadherance
during the blood stage of infection. In order to invade RBCs, merozoites
makeuseofcomplexmechanisms,evidencinggreatadaptationtotheirhost.
Someparasitestrainsthatdependonsialicacidreceptors,forexample,can
evenshifttheirinvasionpathwaytoasialicacidindependententryprocess78.
Afterthisinitialbinding,thereisareorientationofthemerozoiteinorderto
position its apical and secretory ornanelles (rhoptries, micronemes and
densegranules)incontactwiththeRBCmembrane.Anindentationisthen
formed in the contact zone.Anumberof proteins of the apical organelles
bindtospecificerythrocytereceptors.Apicalmembraneantigen1(AMA1)
is known to establish the apical interaction through parasite adhesins
initially located at the neck of the rhoptries and in the micronemes 79.
Recently, parasites with green fluorescent protein (GFP)tagged AMA1
were used in live imaging studies that revealed this proteins crucial
function80.
Theactual invasionprocess involves twomajorprotein families, theDuffy
bindinglike (DBL) protein family 81, and the P. falciparum reticulocyte
introduction
16
binding protein homolog (PfRh) family 82. DBL and PfRh proteins are
importantformerozoiteinvasionofRBCs,butarenotconsideredessential,
asgenedisruptionforeachofthemindifferentP.falciparumlinesstillshow
normal bloodstage growth rates 8387. This might be due to
complementationbetweendifferent familymembersor to a great level of
adaptation of Plasmodium to its host cell. When the apical interaction is
formed,themerozoiteestablishesatightjunction,whichisaccompaniedby
the releaseofmoreproteins from themicronemeand rhoptry organelles.
When attachment is finally achieved, the merozoite is hypothesised to
discharge mediators into the RBC, and although these have not yet been
identified, visualization of merozoite invasion by electron migrographs
suggests material transfer 88. Entry is thenmediated by activation of the
actomyosinmotorinthepellicleoftheinvadingmerozoite89.Attheendof
theinvasionprocess,theRBCbilayerenvelopstheinvadingmerozoiteina
newvacuole90,whichlacksnormalproteinsoftheRBCmembrane.Proteins
likeBand3arecleavedbyproteolyticenzymessecretedbytheparasite 91
and are replaced by recruitment of detergentresistantmembrane (DRM)
raftproteins,makingthePVricherinlipidraftsanditsproteins90.
Inside the normally quiescent environment of a RBC, the merozoite
undergoes some rounds of nuclear division, transforming sequentially in
ring,trophozoiteandschizontstages.Iteventuallyegressesandreleases16
to 32 newmerozoites thatwill follow the same sequence of events. InP.
falciparum,theringstagelastsapproximately24h,accountingforhalfofthe
entireerythrocyticcycle92.
Following entry, and throughout all the developmental and replication
processes, theparasitemodifies thepermeability and adhesiveproperties
ofitshostcell,possiblytopromoteitsownsurvival.
The ring stage is followedby the trophozoite stage, duringwhichmost of
the cytoplasm of the RBC is consumed, a process that leads to the
introduction
17
degradationof60to80%ofthehaemoglobinpresentintheRBC93.Thisis
followed by the schizont stage, during which 4 to 5 rounds of binary
replication take place, originating merozoites that will later invade other
RBCs. The most common and concentrated molecule inside RBCs is
haemoglobin that is degraded by Plasmodium. Haemoglobin proteolysis
yieldshaembesidesaminoacidsasendproducts.Haemeisnotmetabolized
orrecycledbytheparasite.Instead,itisstoredashemozoin,apolymerthat
confersthecharacteristicpigmentationtotheorgansofinfectedindividuals94,95.Theaminoacidsofthehaemoglobinpolypeptidechainareusedinthe
synthesisofparasitesproteins96andappeartobeusedasanenergysource95. The recovery of the amino acids is essential for the parasite as it has
limited capacity of de novo synthesis 94. However, the recycling of amino
acids originating from haemoglobin degradation is not sufficient for
parasite maintenance as haeomglobin lacks some essential amino acids,
whichtheparasiteisabletowithdrawfromthehostplasma97.Thefeeding
of theparasite in theRBCoccurs through a cytostome, an invaginationof
thePVmembraneandtheparasiteplasmamembrane,whichingestssmall
packets of haemoglobin from the host cytosol. Budding vesicles full of
haemoglobinaretransportedandfusedtoanacidicdigestivevacuole(DV)
where haemoglobin is degraded and haeme is detoxified by the action of
several proteases 98 acting like haemoglobinases. Data suggest that this
degradation process is ordered and requires, among other reactions, an
initialasparticproteasemediatedcleavage,followedbysecondaryaspartic
proteaseandcysteineproteasecleavages.Theasparticproteaseshavebeen
shown to be Plasmepsin I and II and the cysteine protease has been
identified has Falcipain 99,100. Different patterns of gene expression are
observedduring theerythrocytic cycle, suggesting that their functionsare
not exactly the same. Several other Plasmepsin proteins were later
identifiedbothinsideandoutsidetheDV.Theirfunctionalredundancywas
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18
shown when independent disruption of Plasmepsin I, II, IV or histidine
asparticprotease(HAP)wasobtainedandverylittleeffectontheparasite
growthwasobserved101,102.
Plasmodiumhastheabilitytoexporthundredsof itsownproteinsbeyond
itsplasmamembraneandthePVtothecytosoloftheerythrocyte103.This
represents5%ofitswholegenome.Althoughthefunctionofmanyofthese
proteins remains unknown, some of them have been associated with
virulence,promotingcelladhesionand/orrigidityoftheerythrocyte104,105.
Tobeexported,proteinsmustfirstenterthesecretorypathway106showing
a recessed aminoterminal hydrophobic endoplasmatic reticulum (ER)
signal sequence that allows transport across theplasmamembraneof the
parasite but not the PVmembrane.Most proteins exported across the PV
membrane require an additional sequence element knownasPlasmodium
export element (PEXEL), or a vacuolar transport sign (VTS) that is found
downstream of the ER signal sequence 107. This export movement has
recently been proposed to be performed by a proteinaceous translocon
withinthePVmembrane,anATPpoweredcomplexcontainingheatshock
protein 101, a novel protein PTEX150 and a known parasite protein
identified as exported protein 2 (EXP2) that potentially works as the
commonportalthroughwhichmostorallexportedproteinsmustpass108.
MoreoverPlasmepsinVwasrecentlyshowntobetheproteaseresponsible
forcleavageofthePEXELmotifofproteinstobeexported,therebyallowing
theirtraversalofthePVandtraffickingtotheRBCsurface109,110.Following
thisexportviathistransloconatthePVmembrane,exportedproteinshave
toreachdifferentdestinationsinthehostRBC,suchasthecytoplasmorthe
plasmamembrane. Because RBCs have no secretory system, the parasite
buildsitsowntoallowitsproteinstoreachthehostcellplasmamembrane.
Maurer's cleft is a central structural component of this extracellular
proteinexportsystem111.Thesestacksofflattenedlamellaeoflongslender
introduction
19
membraneswithatranslucent lumenwere identifiedmorethanacentury
agoandarelocatedbelowtheerythrocyteplasmamembrane112,113.Several
parasiteproteinsaresynthesizedintheparasitesERandthentransferred
tothecleft114,115.ItisstillnotcompletelyclearwhethertheMaurer'scleftis
part of a continuousnetwork that connects thePV to the erythrocyte cell
surfacelikesomeimagingreconstructionsofthinsectionsseemtosuggest116, or if the cleft is a welldefined structure continuously supplied with
vesiclesbudding from themembrane lining thePV.Subsequently, vesicles
bud out from the cleft and migrate towards the erythrocyte cell surface,
withsecretionoftheircontentsintothemediumandincorporationofsome
proteinsintheerythrocyteplasmamembrane74.
The very strong remodelling of the host RBC eventually leads to its
distortion,withpositivelychargedknobbyprotrusions.Usingatomic force
microscopy, theknobwasshown toconsistof twosubunits thatmightbe
central to thephenomenonof cytoadherence inP. falciparummalaria 117.
Indeed, inP. falciparum infections,schizontsareknowntobarelycirculate118. Instead, they are sequestered in different organs by adhering to the
endothelial cells of the vessels. Knobs are created by the deposition of
parasite proteins, such as knobassociated histidinerich protein (KAHRP)
andtheadhesionproteinP.falciparumerythrocytemembrane1(PfEMP1),
whichareinsertedintheRBCmembrane.
PfEMP1 is restricted to P. falciparum, and undergoes clonal antigenic
variation switching, changing its antigen type at high frequencies during
intraerythrocyticcycles119,120.PfEMP1belongstoalargepolymorphicgene
familycalledvar,inwhicheachindividualgeneencodesadifferentformof
the protein, and only one is expressed at a time through a mutually
exclusive mechanism. It comprises three regions: the cytoplasmatic or
acidic terminal segment (ATS) that is anchored to the knobs; the
transmembrane regions that are inside the RBC membrane; and the
introduction
20
ectodomain orNterminal segment followed by theDBL domains and the
Cysrich interdomain regions (CIDR) (reviewed in 74) that interact with
endothelial cell receptors, leading to sequestration and thus preventing
destruction of infected RBC (iRBC) in the spleen 121. PfEMP1 is highly
relatedtovirulenceasitisbelievedthattheadherenceitpromotestriggers
much of the associated pathology, including cerebral malaria end
preganancyassociatedmalaria(reviewedin122).
Following this period (whose length varies betweendifferentPlasmodium
species) where Plasmodium replicates with the consumption of the RBC
cytoplasm,parasitesleavetheRBCbyaprocesscalledegressthathappens
veryfastandmustbetightlyregulated.Severalhypotheticalmodelsexplain
egress.AlthoughthesevarybydisagreeingonwhetherthePVmembraneor
theRBCmembranedisintegratesfirst,allthemodelsacceptthatproteases
play a critical role. Protease inhibitors have been shown to prevent P.
falciparumiRBCrupture,andtopromoteaccumulationofmatureschizonts
invitroduetoegressblocking123,whichwaslaterattributedtoprevention
of the proteolytic effect of serine repeat antigen (SERA)5 124. Further
studies showed thatdifferentprotease inhibitorshavedifferent effectson
egress 125. Still, the mechanism by which egress is regulated is only
beginningtobeunderstood.Ithasbeenrecentlyreportedthatjustpriorto
egress essential serine protease PfSUB1 is discharged from Plasmodium
organelles called exonemes, from the merozoite into the PV 126, where it
mediates theproteolyticmaturationofmembersofSERA family thathave
been previously implicated in egress 127. Furthermore PfSUB1 has been
shown to directlymediate primary proteolytic processing ofMSP1,MSP6
andMSP7.PfSUB1seemstopreparenotonlythemerozoiteforreleasefrom
theerythrocytebutalsotoensurethat,once incirculation,merozoitesget
insideanewcellwithoutdelay128,avoidingexposuretothehostresponse.
introduction
21
Shifting tosexualstages.Differentiationandsexual commitmenthappen
prior to schizogeny during asexual replication cycles inside RBCs. All
siblings of a schizont are either asexual, female gametocytes or male
gametocytes 129, but the mechanism that determines which of these
possibilitiesoccursremainsunknown.TranslationalrepressionandmRNA
turnoverhavebeenshowntobekeyplayers indeterminingstagespecific
gene expression in Plasmodium. More specifically, development of zygote
inhibitedRNAhelicase(DOZI)wasidentifiedinthefemalegametocyteand
shown to have a central role in the silencing andmaintenance of steady
statelevelsofapopulationofgametocytespecifictranscripts,allowingthe
coordinated production of essential proteins for the further development
andestablishmentofinfectioninsidethemosquito130.
Inside the mosquito. Mature gametocytes in the blood can later be
engorgedduringamosquitobloodmeal.Theabruptenvironmentalchange
insidethemosquitotriggersgametocytestoroundupandemergefromthe
RBCwithinminutes 131.Thepresenceof xanthurenicacidallows themale
gametocytetotransformintoeightmotilemicrogametesafterthreerounds
of exceedingly fast genome replication followed by nuclear division and
axoneme assembly, and the female emerges from the erythrocyte as a
roundshaped nonmotile gamete 132. Exflagelation of themale gametocyte
and the vibratory movements in waves of its microgametes allow it to
penetrateintothefemalegamete,andfertilizationtooccur133.Theresulting
zygotewillbe theonlyparasitesurvivor in theaggressiveenvironmentof
themosquitogut,andfurtherdevelopsintoatetraploid134motileookinete
that is the only invasive formof thewhole cycle that is not originatedby
replication.Thereisagreatlossofookinetesduringtraversalofthemidgut
epithelialcellstoreachthebasallamina,duetohostprotectivemechanisms135.Onceinthebasallamina,ookinetesbecomesessileand,aftermeiosis136,
transformintooocysts thatare theonlyextracellulardevelopmentalstage
introduction
22
of thewhole cycle. Oocystsmake use of their capsule to recruit nutrients
from the hemolymph 137 to grow, 50 to 60m in diameter, and originate
sporozoites.CSP, theprotein thatwillcoverallofsporozoites,starts tobe
expressed and accumulate in the oocyst plasmamembrane even prior to
sporozoite formation 138. With the retraction on the oocyst plasma
membrane, several lobes called sporoblastsappear in this form 139.CSP is
essential for this formation 140, and also seems to be essential for the
organization of the microtubule organizing centres (MTOCs) 138 that will
later lead to the formationof theapical complexandnucleipositioning in
thedaughtersporozoites.
Sporozoitereleaseintothehemolymphoccursasynchronously141andwas
shown to be dependent on proteinase activity, including that of egress
cysteineprotease1(ECP1)142.Oncereleasedinthehemocoel,sporozoites
canbespreadinthewholemosquitobodybutonlyrecognizespecifichost
receptorsinthebasallaminaofthesalivaryglands139.Invasionoccursonly
inthisorganandseveralparasite ligands, likeCSP143,TRAP144orMAEBL145, have been identified as being important for recognition and invasion.
Afterattaching,sporozoitesbreachthebasallaminaandinvadethroughthe
basalmembraneofsalivarysecretoryacinarcellswiththebrief formation
of a vacuole thatwill allow them to emerge from the apical side of these
samecellsintothesalivaryglandduct146.
Sporozoites are now ready to infect a new mammalian host when the
infectedmosquitotakesanotherbloodmeal.
Malariaoutcome
Malariatransmissionandnaturallyacquiredimmunity
MeasuringmalariaendemicityorPlasmodiumprevalenceinageographical
area has been the subject of an active debate for decades. Surveys of
introduction
23
splenomegaly across apopulation 147, examinationofperipheral blood for
asexual malaria parasites 148, stability/instability of transmission,
entomologicalinoculationrates(EIR)149orhaemoglobinmeasurements150
were, and are, some of the metrics to establish classes of endemicity inwhichagivenpopulationshouldfall.ConsideringAfricaalone,theexisting
endemicpopulationsshowratesoftransmissionthatcandifferinintensity
by100fold.
Fig.I4.Global distribution of malaria transmission risk. Adapdted fromWHOWorldMalariaReport2005.
Clinical presentations ofP. falciparummalaria vary according to different
transmission intensities, but the biological interactions promoting these
differentoutcomesarenot easilyunderstood. Studies across fifty yearsof
field research in areas with different rates of transmission, in several
communitieswithdifferentaccesstohealthsystemsortreatment,andwith
variations in the prevalence of infection occurring from year to year in a
givenplace,allowallsortsofinterpretationsconcerninghowmortalityand
morbidityvarywithtransmissionsrates.Thereis,however,aconsensusin
introduction
24
accepting that the mean age of severe malaria disease decreases as P.
falciparum transmission increases. The frequency of cerebral malaria
declineswith increasing transmission rateswhile anaemia takes over the
clinical burden (reviewed in 151). It is generally accepted that in low to
moderatetransmissionsettings,theincidenceofseverediseasegrowsafter
the first year of age, while in high transmission areas the peak in severe
disease happens between the fifth and the seventh month of age, after
which it declines significantly before the first birthday (reviewed 152).
Common to all settings is the fact that for the first 3 months of age the
incidenceofseveredisease isvery low,most likelydue to thepresenceof
maternalantibodies 153. Inaddition, themuch lowerriskofseveredisease
after5yearsofageindicatesthatsomedegreeofimmunityisacquiredearly
in life. However, under very low transmission regions the risk of clinical
malariaisextendeduntiladulthood154.
Anextensivestudyinanareawithdecreasingparasiteprevalenceover16
years showed that although transmissions decreased significantly in the
first ten years, therewere nomajor alterations in clinical cases reported.
Onlylater,whenparasiteprevalencefellbelowacertainthreshold,coulda
drop in hospital admissions be observed. At the same time, as parasite
prevalencedropped, themean age of slidepositive children increased 155.
Moreover in crosssectional studieswith increasingmalaria transmission,
there isan initial increase in therateofhospitaladmissionswithmalaria,
but thereafter, the risk of hospitalization rises either more slowly or
plateausatintermediaterangesoftransmissionintensityandmaydecrease
slightlyinareasofveryhightransmission156.
Very strong evidence that protection against malaria increases with age
more thanwith any other factors comes from a study performed inMali
where itwas observed that older children treatedwith chloroquinewere
better able to clear chloroquineresistant P. falciparum parasites than
introduction
25
younger children treated in a similar way 157, again pointing towards an
effectiveacquiredimmuneresponse.
However, although in endemic settings older children and adults are
resistant to severe morbidity and death, they are still susceptible to
infection158,showingthatonlyacertainlevelofimmunitycanbedeveloped
againstmalaria.Thisnatural immunity isacquiredat thecostofveryhigh
early mortality and is still defective, as multiple infections are required
before clinical protection is achieved, and persistent infection is typical.
Indeed,backin1900,RobertKochcomparedareasofdifferentendemicities
anddeducedthattherewastheneedforheavyandcontinuedexposureto
theparasite inorder toacquireprotectionagainstmalaria.Later, in1920,
the essential features of naturally acquired immunity againstPlasmodium
weredescribed: itwasaccepted thatnatural immunitywas(i)effective in
adultsafteruninterruptedlifelongheavyexposure,(ii) lostuponcessation
of exposure, (iii) species specific, (iv) somewhat stage specific, and (v)
acquired at a rate which was dependent upon the degree of exposure
(reviewedin159).
In spite of these premises, very little is known about the mechanisms
through which immunity against malaria is acquired. Early studies with
humans, showed that antibodies can protect againstmalaria infection 160,
andseveralantigenshave laterbeenshown tobeassociatedwithmalaria
protection.Antibodies againstMSP1 161,MSP2 162 orMSP3 163were found
significantly elevated in the sera of protected individuals in different
endemic areas and are thought to correlate with protection. These
associations vary between studies, and antigens encountered until now
show considerable polymorphisms through the existence of alternative
allelic forms 164 and through antigenic variation 165, limiting their use as
possibletargetsforvaccinationandtreatment.Also,theantibodyresponse
to themost commonmerozoite antigens is believed to be shortlived 166,
introduction
26
peaking oneweek after themalaria episode and rapidly decaying to very
lowlevelswithin6to8weeks.Thereasonforsuchashorthalflife isstill
notunderstood.Itmightbethatthecatabolichalflifeoftheseantibodiesis
justshorter,butotherhypothesishavebeenraisedsuchasthepredilection
towardsIgG3whichisknowntobeshortlived167,orthepoordevelopment
of memory or longlived plasma cells 168. Independent of the mechanism
behind it, thisshorthalflifeofantimalariaantibodiescontinues tocreate
problems when planning an intervention strategy toward an effective
vaccineagainstmalaria.
Liverstagenaturallyacquiredimmunityisnotbelievedtobestrongenough
or acquired fast enough to provide any kind of protection in endemic
populations. Still, it cannot be said that natural immunity against
Plasmodium preerythrocytic stage is not involved in the final outcome
observed. Indeed, infection of both humans and mice with Plasmodium
sporozoites followed by chloroquine treatment that abrogates the
establishment of blood stages protects individuals from subsequent
infections169,170.Itistrue,however,thatearlystudiesperformedinhumans,
bypassing the hepatic phase of the infection through direct blood stage
infection, showed that immunity could be maintained (reviewed in 162).
However,vaccinedevelopmenteffortshave investeda significanteffort in
preerythrocyticimmunizationandprovenefficientinrenderingprotection
forvariableperiodsoftime,probablybecauseoftheveryhighnumbersof
attenuatedsporozoitesused171,whichareverydifferentfromtheverylow
numbersofsporozoitesthatmosquitoesinjectduringabloodmeal172.Both
attenuatedformsoftheparasite173andlivesporozoites170cancompletely
protect humans, making them resistant to infection. However, so far the
mostadvancedvaccinecandidateagainstPlasmodiumfalciparumisRTS,S.It
is a recombinant, yeastexpressed subunit vaccine using hepatitis B virus
introduction
27
surfaceantigenscarryingepitopesfromP.falciparumCSP174themajorcoat
proteinofsporozoites.
InareaswithveryhighEIRs,aschildrengetoldermalariaattacksbecome
lessfrequentafteraninitialpeakatyoungage,andparasitelevelsbetween
manifestationssteadilydecrease.Althoughthechildseemstobeacquiring
immunity, they remain parasitized, often with higher parasitaemias than
otherchildrenatgreaterriskofclinicaldiseasefromareaswithlowerEIRs175. This protection against clinical disease is the product of acquired
immunity that is able to control parasitaemia but does not fully abrogate
infection, producing only nonsterilizing immunity 176. Several reports of
Plasmodium superinfection describe single individuals hosting more than
one Plasmodium species, or different genotypes of the same Plasmodium
speciesinfectingRBCs177179.However,theseobservationshavebeenmuch
more frequentamongasymptomaticcarriers than inclinicalcases 180,and
havebeenshowntodependontheageofindividualsinapopulation175,181
183.
Premunition or concomitant immunity is theprotection against infections
andclinicaldisease thatassociateswithpersistenceofmultiple infections,
providingprotectionagainstnewinfectionsbymaintainingalowgradeand
generally asymptomatic parasitaemia and high levels of antibodies
(reviewed in 159,184). A better understanding of mechanisms behind
premunition is clearly central to the comprehensionof naturally acquired
immunity tomalaria. Periodic bloodstage infection presumably serves to
boost a preexisting immune response and maintain high frequencies of
effectorcellsinareasofhightransmission.Exposuretoagreatervarietyor
antigensof familieswithassociatedvariance increases the repertoire that
eachindividualrecognises185.
Antigenic variation creates distinct waves of parasitemia that must be
chased by different antibody responses, on the one hand promoting long
introduction
28
infections thatmightendanger thehost,whileon theotherhandallowing
eventualacquisitionofdiseasecontrollingimmunity.Effectiveantiparasitic
immunityis,ifever,achievedonlyafterverymanyandfrequentinfections149,186.
So, thedoubt remains,whether reducing the risk ofPlasmodium infection
willalwaysleadtoadecreaseinthethreatofseveremalariathroughoutlife.
Thisisaconcernthatwasraised50yearsago,whenwonderingifaltering
thenaturalriskofexposuretoparasite,byreducingvectorortransmission
itself,would change the epidemiology of severe disease 187. Knowing that
increasing exposure to Plasmodium parasites is reflected in increasingly
rapidacquisitionofimmuneresponsesthatlimitslifethreateningeffectsof
malaria, the fullunderstandingof the transmission/immunityassociations
isofmajorimportanceindefiningmalariacontrolinterventions.
Aims
Plasmodium passage through the skin followed by infection of liver
hepatocytes and, later, of blood erythrocytes, are natural and sequential
steps of Plasmodium life cycle in themammalian host and should not be
seen as independent entities. The sophistication and complexity of
Plasmodium life cycle are evidence for coevolution with man, and are
reflected in the complicatednatureof relationshipsbetween transmission
rates, immunity and disease severity in endemic areas. Importantly, liver
and blood stages of Plasmodium infection may frequently occur
simultaneouslyinthesameindividualwheretransmissionsratesallowit.
Indeed, after infecting the hepatocyte for approximately a week, P.
falciparumcaninfectRBCsforweeksorevenmonthsafterthat188.Thus,a
new infection initiated by amosquito bitemay occurwhile the parasites
from a previous one are still replicating inside erythrocytes. Very little is
knownabouttheinteractionsbetweenthetwodifferentstagesofamalaria
introduction
29
infection. So far it has been shown that malaria bloodstage can be
immunosuppressive 189, but it is clear that very close interactions are
establishedbetweenparasiteandhostthroughouttheparasiteslifecycle.
The objective of thework presented here arises from intending to clarify
effectsofonestageonanotherinindividualsduallyinfectedwithbloodand
posterior liver stage malaria, and try to understand how reinfection fits
what is known so far regardingdifferentpatterns of infection indifferent
EIRsettings.
ToaccessPlasmodiumbloodstage/liverstage interactionsnewtoolswere
usedtooutwitpastlimitations,wehavemadeuseofwildtypePlasmodium
parasitestostartbloodstageinfectionsandofgfpexpressingorluciferase
expressing Plasmodium parasites to initiate liver stage infections. Using
transgenic parasites we were able to distinguish the two infections
circumventing the lackof specific liverstagemarkers.Because theGFPor
luciferasegenesareunderthecontrolofaPlasmodiumhousekeepinggene
promoter,theirexpressioncorrelateswithPlasmodiumliverload.
ThestudyofthetemporalcoincidenceofPlasmodiumbloodandliverstages
inasinglehostatagiventimewasmostlyperformedinrodentmodelsof
infectiontoanswer4majorquestions:
What would be the impact of an ongoing blood stage infection on the
establishmentofasecondarysporozoiteinfectionintheliver?
Bywhatmolecularmechanisms do any interactive effects between blood
andliverstagesoccur?
What, if any are the consequences of such interaction for the reinfected
individual?
Howsuch interactions adjust to established relationsbetweenpatternsof
infectionandtransmissionratesindifferentendemicsettings?
30
Materials and Methods
32
materials & methods
33
Mice.C57BL/6,BALB/c,BalbSCIDaswellasmicedeficientinRagII,IL10,
caspase 3, MyD88 or Kit Wsh/Wsh deficient were bred in the specific
pathogenfree facilities of the Instituto de Gulbenkian de Cincia (Oeiras,
Portugal).RAGII/cdeficientmicewerekindlyprovidedbyJamesDiSanto
(CytokinesandLymphoidDevelopmentunit,InstitutPasteur,Paris,France).
NOS2 (B6.129P2Nos2tm1Lau/J), TCRdelta (B6.129P2Tcrdtm1Mom/J),
and C5a (B10.D2Hc0.H2d.H2T18c/oSnJ) deficientmice were purchased
atTheJacksonLaboratoryalongwiththeirrespectivewildtypelittermates.
AllmicewerehousedintheInstitutodeMedicinaMolecular(IMM)facilities
andtheIMMAnimalCareCommitteeapprovedallprotocols.IFNdeficient
mice were kindly provided by Rui Appelberg (Microbiology and
Immunology of Infection laboratory, Instituto de Biologia Molecular e
CelularIBMC,Porto,Portugal).ExperimentswithIFNdeficientmicewere
performed at IBMC, and IBMC Animal Care Committee approved all
protocols.AlltransgenicmiceweregenotypedbytailgenomicDNAPCRto
confirmtheirrespectivemutations.
Plasmodium blood infection. Primary blood stage infection ofmicewas
achieved by 30 minutes exposure of mice to 15 Anopheles mosquitoes
infected with P. berghei ANKA (parasite line GFPcon259cl2), or by intra
peritonealinoculationofthedesignatedquantityofredbloodcellsinfected
with P. berghei ANKA (1.49L), P. berghei NK65, P. yoelii 17X NL or P.
chabaudi chabaudi AS. Peripheral blood parasitaemia was determined by
GiemsastainingfollowedbymicroscopiccountingofiRBCsandresultsare
expressed as percentage ofRBCs.On Fig. 1 C parasitemiawas verified by
realtimeinvivoimagingusingtheinvivoIVISLuminaImagingSystem190
asdescribedbellow.AndonFig. 1Eparasitemiawasdeterminedby flow
cytometry, measuring red blood cells infected with gfpexpressing P.
bergheiANKA.Theresultsareexpressedaspercentageofinfectedredblood
cells,aspreviouslydescribed191.
materials & methods
34
Plasmodiumliverinfection.Greenfluorescentprotein(gfp)expressingP.
berghei ANKA (parasite line GFPcon259cl2) or gfpexpressing P. yoelii
sporozoites 191,192 were obtained by dissection of Anopheles stephensi
infectedmosquitoesbredintheinsectariumofIMM.Micewereinfectedby
intravenous inoculationof thedesignatedquantityofGFPsporozoites,or
by30minutesexposureofmiceto15AnophelesmosquitoesinfectedwithP.
berghei ANKA. Parasite liver load was quantified 40, 48 or 72h post
infection.
RealtimeinvivoimagingluminescentPlasmodium.Navemice(control)
andbloodstage infected(PbAinFig.1AandPbNKinFig.1BandFig.1C),
were intraperitoneally injected with 200l of anaesthesia mixture (80
mg/kgKetamineand10mg/kgXylazine)diluted inPBS inorder toallow
eachmouseexposure to15Anophelesmosquitoes infectedwith luciferase
expressingP.berghei (parasite line354cl4).After30minutesofmosquito
bites,micewereshaved intheabdomen.FortyhpostinfectionforFig.1A
and 1B and 6 days postinfection for Fig. 1C, Dluciferin dissolved in PBS
(150mg/kg;CaliperLifeSciences,USA)wasinjectedsubcutaneouslyinthe
neck. Animals were anesthetized again as described above for the whole
duration of measurements (performed within 5 to 10 minutes after the
injectionofDluciferin).Bioluminescenceimagingwasacquiredwitha12.5
cmfieldofview(FOV),mediumbinningfactorandanexposuretimeof180
seconds. Luciferase activity in animalswas visualized through imaging of
wholebodiesusingtheinvivoIVISLuminaImagingSystem190.
qRealTimePCRquantificationof liver infection.Liverswere collected
andhomogenizedindenaturingsolution(4Mguanidinethiocyanate;25mM
sodiumcitratepH7,0.5%NLauroylsarcosineand,0.7%Mercaptoethanol
in DEPCtreated water). Total RNA was extracted using RNeasy Mini kit
(Qiagen),and thenreverse transcribed intocDNAusingTranscriptorFirst
materials & methods
35
Strand cDNA Synthesis kit (Roche), according to the manufacturers
protocols. Infection load in the liver was determined as previously
describedbyqRTPCRusingclassicPbA18SrRNA193specificprimersorgfp
specificprimers(gfpexpressioncorrelateswithPbA18SrRNA,fig.S1).qRT
PCR reactions used Power SYBR Green PCR Master Mix (Applied
Biosystems) and were performed according to the manufacturers
instructions on anABI Prism7000 system (AppliedBiosystems). Relative
amounts of PbA 18S rRNA and gfp mRNA were calculated against the
Hypoxanthine Guanine Phosphoribosyltransferase (hprt) housekeeping
gene,followingaprimedenaturationof10minutesat95C,then50cycles
of95Cfor15secondsand60Cfor1minute.PbA18SrRNA,gfpandhprt
specific primer sequenceswere: 5CGG CTT AAT TTG ACT CAA CAC G3
and5TTAGCATGCCAGAGTCTCGTTC3forPbA18SrRNA,5GTCAGT
GGAGAGGGTGAAGG3and5ACTTCAGCACGTGTCTTGTAGTTC3
forgfpand5TGCTCGAGATGTGATGAAGG3and5TCCCCTGTT
GAC TGG TCA TT 3 for mouse hprt. External standardization was
performed using plasmids encoding the fulllength genes cDNA cloned in
TOPOTA(Invitrogen).
Chloroquine treatment. Mice infected 4 days previously with P. berghei
NK65 and control mice received 0.8 mg of chloroquine (CQ) by intra
peritonealinjectionfor1or2daysbeforereinfection,aprotocolleadingto
decreaseinperipheralbloodparasitaemiauntilzerobyday2oftreatment.
Liver slice histopathology, morphometric analysis and
immunofluorescence. Liver tissues were harvested from control or re
infected mice 40 h after sporozoite infection. Tissues were fixed in 4%
paraformaldehyde for 15 minutes, washed three times in PBS and then
sliced into 50 m sections using a vibratome (VT1000S, Leica). Sections
were later permeabilized and blocked overnight in 0.3% Triton X100
materials & methods
36
(Calbiochem)and1%BovineSerumAlbumin(Sigma)toavoidnonspecific
reactivity. Sections were then incubated overnight at 4C in the same
solution containing antiGFP IgG Alexa flour 488 congugate antibody
(Invitrogen), Alexa 594 phaloidin (Invitrogen) and 4',6diamidino2
phenylindole(DAPI,Sigma).Aftermounting15to20sectionsonslides,the
areasofthesectionsweremeasuredusingascope,theEEFswerecounted
usingtheLeicaDM5000BWidefieldFluorescenceMicroscope,andthesizes
of 20 randomly chosenEEFs of eachmouseweremeasured using a Zeiss
LSM 510 META Point Scanning Confocal Microscope. All images of
immunofluorescencestainedsectionswereanalysedusingtheImageJ1.42b
software. Areas and numbers of EEF were normalized to the total area
observed.
Cells. Huh7 cells, a human hepatoma cell line, were cultured in RPMI
(Gibco/Invitrogen)mediumsupplementedwith10%fetalcalfserum(FCS,
Gibco/Invitrogen), 1% penicillin/streptomycin (pen/strep,
Gibco/Invitrogen), 1% glutamine (Gibco/Invitrogen) at pH 7 and
maintainedat37Cwith5%CO2.
Isolation of murine primary hepatocytes.Mouse primary hepatocytes
were isolated as previously described 194. Briefly, cells were initially
obtainedbyperfusionofmouse liver lobuleswith liverperfusionmedium
and liver digest medium (Gibco/Invitrogen) at 37C using a peristaltic
pump. Hepatocytes were then purified using a 1.12 g/ml, 1.08 g/ml and
1.06g/ml Percoll gradient. Cells were cultured in Williams E medium
containing 4% FCS, 1% pen/strep in 24well plates coated with 0.2%
GelatineinPBS.Cellsweremaintainedincultureat37Cand5%CO2.
Sporozoite infectionanddevelopment incontactwith infectedblood.
Mouse primary hepatocytes and Huh7 cells, a human hepatoma cell line,
were cultured as described above in complete Williams E or RPMI
materials & methods
37
(Gibco/Invitrogen) in transwell system plates (COSTAR/Corning). In the
lower chamber, liverderived cells were infected with 20,000 P. berghei
ANKA sporozoites, and allowed to share medium with upper chambers
containing serum(CTRL),noninfectedblood (NI)orblood containing6x
105P.bergheiNK65iRBCs(PbNK).After36hofcoculture,hepatomacells
werecollectedandtreatedforFACSanalysisandprimaryhepatocyteswere
fixed in4%paraformaldehydefor10minutes,washedthreetimes inPBS,
andlaterpermeabilizedandblocked1hin0.3%TritonX100(Calbiochem)
and 1% Bovine Serum Albumin (Sigma) to avoid nonspecific reactivity.
Coverslipswere then incubated1hat4C in the same solution containing
antiGFP IgG Alexa flour 488 congugate antibody (Invitrogen), Alexa 594
phaloidin (Invitrogen) and DAPI (Sigma). After mounting coverslips on
slides,EEFswerecountedusingtheLeicaDM5000BWidefieldFluorescence
Microscope, and the sizes of 20 randomly chosen EEFs of each coverslip
were measured using a Zeiss LSM 510 META Point Scanning Confocal
Microscope. All immunofluorescencestained images were analysed using
theImageJ1.42bsoftware.
Fluorescence activated cell sorting (FACS) analysis of sporozoite
infection.FACSanalysisofsporozoiteinfectedHuh7hepatomacellcultures
at36hpostsporozoiteadditionwasperformedtodeterminethepercentage
ofparasitecontainingcellsandparasiteGFPintensitywithininfectedcells.
CellsamplesforFACSanalysiswereprocessedaspreviouslydescribed195.
Transcription profiling. Total RNA from livers of nave mice (control
group), mice infected with 106 P. berghei NK65 iRBCs for 7 days (Blood
stagegroup,BS),miceinfectedwith5x104P.bergheiANKAGFPsporozoites
for40h(Liverstagegroup,LS),andmiceinfectedwith106P.bergheiNK65
iRBCs for 7 days then reinfected with 5x104 P. berghei ANKAGFP
sporozoitesfor40h(Reinfectiongroup,Reinf)wasextractedusingRNeasy
materials & methods
38
Mini kit (Qiagen), following the manufacturers instructions. Three
biological replicates were analysed for each group. RNA quality and
quantitywasassessedonaBioanalysernanochip(Agilent).TotalRNAwas
reverse transcribed and endlabelled as cRNA using the GeneChip WT
Amplified DoubleStranded cDNA Synthesis Kit and the GeneChip WT
TerminalLabelingKitasrecommendedbyAffymetrix.Hybridisation toan
AffymetrixMouseGene1.0STArraywascarriedoutat45Cfor16hunder
rotation (60 rpm). Arrays were washed on an Affymetrix FS450 and
scanned using an Affymetrix Genechip Scanner 3000 7G. Fluorescence
intensities were background adjusted, quantile normalised and median
polished into expression values using the robust multiarray averaging
programRMA196inR/Bioconductorsuite197(softwarepackage:affy198)To
assess significance and differential expression (DE), each infected group
wascomparedtothenoninfectedgroup,andoutof28815mainprobesets
represented on the array, 6273 transcript clusters were classified as
significant (Ftest, p
materials & methods
39
(ii)IFN.MiceinfectedwithP.bergheiNK65andcontrolmicereceived0.4
mgofIFNspecificmonoclonalantibody(cloneR46A2,RatIgG1)1,2and3
days, after inoculation with infected blood, a protocol leading IFN
depletion at the timeof sporozoite infection, 5 dayspostP. bergheiNK65
infection.
(iii)TNF.MiceinfectedwithP.bergheiNK65andcontrolmicereceived0.4
mgofTNFspecificmonoclonal antibody (cloneXT3.11,Rat IgG1)4and6
days after inoculation of infected blood, a protocol leading to TNF
depletion at the timeof sporozoite infection, 5 dayspostP. bergheiNK65
infection.
NNitroLarginine methyl ester hydrochloride treatment. Mice
infectedwithP. bergheiNK65 and controlmice received50mg/kgofN
NitroLargininemethylesterhydrochloride(LNAME,Sigma)3,4,5and6
daysafterinoculationofinfectedblood,aprotocolleadingtoNOSinhibition
atthetimeofsporozoiteinfection,7dayspostP.bergheiNK65infection.
Smethylisothioureatreatment.Mice infectedwithP.bergheiNK65and
controlmicereceived100gofSmethylisothiourea(SMT,Sigma)3,4,5
and 6 days after inoculation of infected blood, a protocol leading to iNOS
inhibitionatthetimeofsporozoite infection,7dayspostP.bergheiNK65
infection.
C3a receptor antagonist treatment. C5a deficient (B10.D2Hc0.H2d.H2
T18c/oSnJ)naveorP.bergheiNK65infectedmicereceived0.5mgofC3a
receptor antagonist (SB290157, Calbiochem) 4h before sporozoite re
infection,5dayspostP.bergheiNK65infection.
Kupffer cell depletion using clodronate liposomes 201,202.Kupffer cells
wereeliminatedfromnaveandP.bergheiNK65infectedmicebyinjection
of200lclodronateliposomes(liposomeencapsulateddichloromethylene
materials & methods
40
diphosphonate;clodLip)daily,fromdayoneafterP.bergheiNK65infection
untilthedayofsporozoiteinfection(4dayspostbloodstageinfection).As
controls, the same volume of PBS containing liposomeswas injected into
naveorP. bergheiNK65 infectedmice. Clodronatewas a gift fromRoche
Diagnostics GmbH (Mannheim, Germany). It was encapsulated into
liposomes,aspreviouslydescribed203.Kupffercelldepletionwasverifiedby
immunofluorescencemicroscopy.Liverswerecollectedand50msections
obtained and fixed, as described above. Sections were then incubated
overnight at 4C in a solution containing rat antimouse pan tissue fixed
macrophages antibody (Fitzgerald) and Alexa 660 phaloidin (Invitrogen).
After washing twice with PBS, sections were incubated for 6h with
secondary Alexa fluor 595 donkey antirat IgG (Invitrogen) and DAPI
(Sigma).Aftermountingsectionsonslides,sectionswereimagedonaZeiss
LSM 510 META Point Scanning Confocal Microscope. Images of
immunofluorescencestainedsectionswereanalysedusingtheImageJ1.42b
software.
Splenectomy. Intraperitoneal injection of 250 l of anaesthesia mixture
(80mg/kgKetamineand10mg/kgXylazine)wasadministeredtomice10
to 15 min before surgery. After observing the animals for response to
anaesthesia,theanimalswerelaidontheirrightside;andthefurremoved
with a razor. A 1.5 to 2 cm long skin incision was made in the left
hypochondrium, the connective tissue under the skin was loosened and
another incision in the peritoneal wall was made. Gently the spleen was
pulledontotheexteriorsurfaceoftheperitoneum.Theartery,attachedto
thehilumofthespleen,closetothestomachwastiedoffwithasingleknot
using 30 plain CATGUT suture. The efferent venule attached at the other
end of the spleen was tied off similarly by a single knot using 30 plain
CATGUT.Cuttingawaythemesenteryandconnectivetissue,thespleenwas
removed. Peritoneal wall and underlying muscles were closed with plain
materials & methods
41
CATGUT (30) and the skin incisions closed with wound clips. Mice with
accessorial spleen were excluded from the study. The control group
underwent a sham surgery and they were maintained in the same
conditions.
Liver hepcidin (hamp1) quantification.After RNA extraction and cDNA
conversionasdescribedabove,hepcidin(hamp1)mRNAexpression in the
liverwasdeterminedbyTaqManqRTPCRusingTaqManGeneExpression
MasterMix(AppliedBiosystems)andAppliedBiosystemsinventoriedgene
expression assays for hamp1 (ABI TaqMan Gene Expression Assay
probe/primer set Mm00519025_m1) and gapdh (Glyceraldehyde3
phosphate dehydrogenase) (ABI TaqMan Gene Expression Assay
probe/primer set Mm99999915_g1), according to the manufacturers
instructions. qRTPCR was carried out using an ABI Prism 7500 FAST
system (Applied Biosystems). Changes in hepcidin mRNA expression
betweencontrolandreinfectedmouse liverswerecalculatedusing the2
DDCtmethodwithgapdhastheendogenouscontrolgene.
Ferritinstaining.Liverswerecollectedand50msectionswereobtained
andfixedasdescribedabove.Sectionswerethenincubatedovernightat4C
in a solution containing rabbit antimouse heavy and light ferritin chain
antibody(akindgiftbyPaoloArosio,FacultyofMedicine,Brescia,Italy)and
Alexa 660 phaloidin (Invitrogen). After washing twice with PBS, sections
were incubatedfor6hwithsecondaryAlexafluor595donkeyantirat IgG
(Invitrogen) andDAPI (Sigma). Sectionswere imagedonaZeissLSM510
METAPointScanningConfocalMicroscope.Imagesofimmunofluorescence
stainedsectionswereanalysedusingtheImageJ1.42bsoftware.
Totalnonhemeironquantification.Totalnonhemeironwasmeasured
as described previously 204, in brief after drying samples (spleens and
primaryhepatocytes) for48hat45C, tissueswereweighed,anddigested
materials & methods
42
for 48h in 10% trichloroacetic acid/10% HCl at 65C. Two hundred
microlitersof the extractwere thenadded to1mlof chromogen solution
(0.01%bathophenanthrolinedisulfonic acid, 0.1% thioglycolic acid, 2.5M
sodium acetate) and after 15 minutes of incubation, absorbance was
measured at 535 nm. A standard curve performed for known iron
concentrationsbetween10and500g/mlrevealedalinearityofresponse
with a slope of ~1. Sampleswere diluted appropriately to fallwithin the
linearrange.Valueswereexpressedas%ofcontrol,inmicrogramsofiron
pergramofdryweight.
HAMPadenovirus in vivo infection.Micewere infectedwith adenoviral
5x1011 pu of pAd.CMV.GFP (control) or pAd.CMV.Hamp.ires.GFP.Wpre
(HAMP.adV) kindlyprovidedby StefanoRivella for (Weill CornellMedical
College,USA)48hprior tosporozoite infection,hepcidinexpressionwas2
fold upregulated by the Hamp.adv compared to uninfected and GFP.adv
control groups. All mice were then infected with 2x104 P. berghei ANKA
(1.49L)sporozoites,and40h later liverswerecollectedand liver infection
wasdeterminedbyexpressionofPbA18SrRNA.
Iron treatments. (i) In vitro: Ferric Ammonium Citrate (FAC, Sigma)
bathophenanthrolinedisulfonate (BPS, Acros) and Desferrioxamine (DFO,
Sigma)solutionswerepreparedinat thedesignatedconcentrations.Huh7
cells were incubated with different concentrations of FAC, BPS or DFO,
using water as control for FAC and PBS as control for BPS and DFO
respectively, 24h prior to sporozoite infection and for 36h postinfection,
afterwhichcellswerecollectedforFACSanalysis.
(ii) In vivo:DFO (Sigma) solutionwas prepared in PBS at the designated
concentration.Micewere inoculated intraperitonealywith 250mg/Kg of
DFOusingPBSascontrol24hpriortosporozoiteinfection.
materials & methods
43
Tuneldetectionofapoptosis.Livertissueswereharvestedfromwildtype
andcaspase3deficientmiceinfectedwithP.bergheiNK65for8daysalong
with noninfected wild type mice. Tissues were frozen in OCT freezing
medium and then sliced into 16msections using a cryostat. Analysis of
D