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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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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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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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

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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

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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).

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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

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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

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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

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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

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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.

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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

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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

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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

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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

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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