Molecular determinants of SR-B1-dependent Plasmodium … · 88 SR-B1 is a highly glycosylated...

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Molecular determinants of SR-B1-dependent Plasmodium sporozoite entry into1

hepatocyticcells.2

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Anne-Claire Langloisa, Giulia Manzonia, Laetitia Vincensinia, Romain Coppéeb, Carine4

Marinacha, Maryse Guérinc, Thierry Hubyc, Véronique Carrièred, François-Loïc Cossete,5

MarlèneDreuxe,EricRubinsteina,OlivierSilviea6

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aSorbonneUniversite, INSERM,CNRS,Centred’ImmunologieetdesMaladies Infectieuses,8

CIMI-Paris,F-75013,Paris,France.9

bUniversitédeParis,UMR261MERIT,IRD,F-75006Paris,France.10

c SorbonneUniversité, INSERM,Unité de recherche sur lesmaladies cardiovasculaires, le11

métabolismeetlanutrition,ICAN,F-75013Paris,France.12

dSorbonneUniversité,INSERM,CentredeRecherchedeSt-Antoine,F-75012,Paris,France.13

eCIRI – Centre International de Recherche en Infectiologie, Univ Lyon,Université Claude14

BernardLyon1,Inserm,U1111,CNRS,UMR5308,ENSLyon,F-69007,Lyon,France.15

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Correspondingauthor:OlivierSilvie;[email protected] 18

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Runningtitle:SR-B1structureandPlasmodiuminfection20

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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 18, 2020. . https://doi.org/10.1101/2020.03.16.994731doi: bioRxiv preprint

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ABSTRACT22

SporozoiteformsofthemalariaparasitePlasmodiumaretransmittedbymosquitoes23

andfirstinfecttheliverforaninitialroundofreplicationbeforeparasiteproliferationinthe24

blood. The molecular mechanisms involved during sporozoite invasion of hepatocytes25

remain poorly understood.Two receptors of theHepatitis C virus (HCV), the tetraspanin26

CD81andthescavengerreceptorclassBtype1(SR-B1),playanimportantroleduringthe27

entry of Plasmodium sporozoites into hepatocytic cells. In contrast to HCV entry, which28

requiresbothCD81andSR-B1togetherwithadditionalhostfactors,CD81andSR-B1operate29

independently during malaria liver infection. Sporozoites from human-infecting P.30

falciparumandP.vivaxrelyrespectivelyonCD81orSR-B1.Rodent-infectingP.bergheican31

useSR-B1toinfecthostcellsasanalternativepathwaytoCD81,providingatractablemodel32

toinvestigatetheroleofSR-B1duringPlasmodiumliverinfection.Hereweshowthatmouse33

SR-B1islessfunctionalascomparedtohumanSR-B1duringP.bergheiinfection.Wetook34

advantageofthisfunctionaldifferencetoinvestigatethestructuraldeterminantsofSR-B135

requiredforinfection.Usingastructure-guidedstrategyandchimericmouse/humanSR-B136

constructs, we could map the functional region of human SR-B1 within apical loops,37

suggestingthatthisregionoftheproteinmayplayacrucialroleforinteractionofsporozoite38

ligandswithhostcellsandthustheveryfirststepofPlasmodiuminfection.39

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IMPORTANCE45

Malaria is caused by Plasmodium parasites and remains one of the deadliest parasitic46

diseases worldwide. The parasite is transmitted by a blood feeding mosquito and first47

invadestheliverforaninitial,obligatoryandsilentroundofreplication.Theliverinfection48

isanattractivetargetforantimalarialvaccinestrategies,howeverthemolecularmechanisms49

ofparasite invasionofhepatocytesremain tobe fullyelucidated.Twohepatocyte surface50

proteinsareknowntobeimportantforparasiteentryintohepatocytes,thetetraspaninCD8151

andthescavengerreceptorclassBtype1(SR-B1).Thesereceptorsconstituteindependent52

gateways depending on the Plasmodium species. Here, we identified the structural53

determinantsofSR-B1,an importanthepatocyteentry factor forhuman-infectingP.vivax.54

This study paves theway toward a better characterization of themolecular interactions55

underlyingthecrucialearlystagesofinfection,apre-requisiteforthedevelopmentofnovel56

malariavaccinestrategies.57

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INTRODUCTION68

Despiteprogressinmalariacontroloverthelasttwodecades,Plasmodiumparasites69

continuetocausemorethan200millioncaseseveryyear(1).Aftertheirinoculationintothe70

skinbyinfectedAnophelesmosquitoes,Plasmodiumsporozoitesrapidlymigratetotheliver71

using gliding motility and cell traversal activity. Once in the liver, they first traverse72

hepatocytes before invading them and developing into exo-erythrocytic forms (EEFs),73

surroundedbyaparasitophorousvacuole(PV)membrane.InsidethePV,theydifferentiate74

intothousandsofmerozoites,whicharereleasedintothebloodcirculationandinvadered75

bloodcells,provokingthesymptomaticphaseofthedisease.76

Several host and parasite factors implicated in sporozoite invasion have been77

identifiedbuttheunderlyingmolecularinteractionsremainunknown.Humanandmurine78

parasitessharesimilarinvasionroutes,withtwodistinctinvasionpathwaysthatdependon79

thetetraspaninCD81or thescavengerreceptorclassBtype1(SR-B1)(2–5).Thehuman80

parasiteP. falciparumandthemurineparasiteP.yoeliibothrequireCD81(3),whereasP.81

vivaxentershumanhepatocytesusingSR-B1(4).Interestingly,themurineparasiteP.berghei82

caninvadecellsusingeitherCD81or,alternatively,aSR-B1-dependentrouteintheabsence83

ofCD81(4).WhilstSR-B1istheonlyknownhepatocyteentryfactorforP.vivaxsporozoites,84

studying this parasite remains difficult, notably due to the limited access to infected85

mosquitoes.Inthiscontext,P.bergheiprovidesanattractivemodeltoinvestigatetheroleof86

SR-B1duringsporozoiteinfection.87

SR-B1 is a highly glycosylated transmembrane protein that belongs to the CD3688

family,whichalsoincludesCD36andthelysosomalintegralmembraneprotein2(LIMP-2).89

AtertiarystructureofSR-B1waspredictedusingLIMP-2crystalstructureasatemplate(6).90


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SR-B1 possesses two transmembrane regions, cytoplasmic N- and C-termini, and a large91

extracellulardomainconstitutedbyaß-strandtunneltoppedbyahelicalbundle(6,7).SR-92

B1apicalhelicesare involved inthebindingofhighdensity lipoproteins (HDLs) (8).The93

hydrophobic cavity traversing theentireprotein is implicated ina selective lipid transfer94

withcholesterylesterbidirectionalexchangesbetweenHDLsandthecellmembrane(8,9).95

In this study, we show thatmurine SR-B1 poorly supportsP. berghei infection as96

comparedtoitshumancounterpart.Wetookadvantageofthisfunctionaldifferencetostudy97

thestructuraldeterminantsoftheSR-B1receptorinPlasmodiuminvasion,usingastructure-98

guidedstrategybasedonchimericconstructscombiningmouseandhumanSR-B1domains.99

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RESULTS101

CRISPR-Cas9mediatedinactivationofCD81abrogatesP.bergheiinfectioninHepa1-6102

cells.103

ThemurinehepatomaHepa1-6cellsexpressCD81butnotSR-B1(4).Inthesecells,P.104

bergheisporozoiteinfectionthusoccursviaaCD81-dependentrouteexclusively,andcanbe105

blocked by CD81-specific antibodies or siRNA (10). To corroborate these results, we106

generatedaHepa1-6celllinedeficientformurineCD81(CD81knockout(KO)Hepa1-6or107

CD81KOH16) using the CRISPR-Cas9 system. Abrogation of cell surface and total CD81108

expressioninCD81KOH16cellswasconfirmedbyflowcytometry(Fig1A)andwesternblot109

(Fig1B), respectively.Wethenanalyzedthe infectionphenotypeof theCD81KOH16cells110

usingP.bergheisporozoites.Asexpected,adramaticreductionofthepercentageofP.berghei111

infected cells was observed in the CD81KOH16 cell line (Fig 1C). PV quantification by112

microscopyafterstainingofUIS4,aPVmembranemarker,revealedacompleteinhibitionof113

productive infection in CD81KOH16 cells (Fig 1D). Intranuclear UIS4-negative parasites114

wereobservedintheCD81-deficientcells,contrastingwiththewell-developedEEFswitha115

strongUIS4stainingfoundintheparentalHepa1-6cells(Fig1E).Wehaveshownbeforethat116

intranuclear parasites result from sporozoites arrested during cell traversal (11). The117

residualintracellularparasitepopulationobservedbyflowcytometryintheKOcells(Fig1C)118

thus likely corresponds to non-productive invasion associated with cell traversal. The119

CD81KOH16cellline,whichlacksCD81andhaslostsusceptibilitytoP.bergheiinfection,thus120

provides a suitable tool to investigate SR-B1 function through genetic complementation121

experiments.122

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Human and murine SR-B1 differ in their ability to support P. berghei sporozoite124

infection.125

WehavepreviouslyshownthattheectopicexpressionofhumanSR-B1canrestoreP.126

bergheiinfectioninHepa1-6cellswhereCD81expressionhasbeenpreviouslysilencedwith127

siRNA(4).Here,wecomparedthefunctionalityofSR-B1proteinsfromhumanandmouse128

origins(hereinafterreferredashSR-B1andmSR-B1,respectively)duringP.bergheiinfection129

after genetic complementationof CD81KOH16 cells. After transient cell transfectionwith130

plasmidsencodinghSR-B1ormSR-B1,weobservedasimilarexpressionofthetwoproteins131

by western blot (Fig 2A) and flow cytometry (Fig 2B).The transfected cells were then132

infected with GFP-expressing P. berghei sporozoites (PbGFP). In agreement with our133

previousobservationsinCD81-silencedcells(4),thetransfectionofhSR-B1inCD81KOH16134

cells restored their susceptibility to P. berghei infection (Fig 2C). Unexpectedly, despite135

similarproteinexpression,mSR-B1wasnotasefficientashSR-B1 in restoringP.berghei136

infection(Fig2C).WeperformedsimilartransfectionexperimentsintheparentalHepa1-6137

celllineafterCD81silencingwithsiRNA,whichconfirmedthelowerfunctionalityofmSR-B1138

proteinduringP.bergheisporozoiteinfectionascomparedtohSR-B1(Fig2D).139

ToanalyzewhetherthepoorfunctionalityofmSR-B1wasspecifictohepatomacells,140

we performed additional experiments in primary mouse hepatocytes. A previous study141

showedsimilarP.bergheiinfectionratesinSR-B1-/-andWTmice(2).However,thepresence142

ofafunctionalCD81pathwaywouldexplainwhyP.bergheicaninfecttheliverdespitethe143

absenceofSR-B1.Wethusperformedinfectionexperimentsinprimaryhepatocytesisolated144

fromWTor transgenicC57BL/6JmiceharboringaCre-mediatedSR-B1gene inactivation145

specifically in the liver (12),whileusing theneutralizinganti-CD81monoclonal antibody146


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MT81toblocktheCD81entryroute(13).CD81inhibitiondidnotimpedeP.bergheiinfection147

ofSR-B1-deficienthepatocytes,but,attheopposite,substantiallyincreasedtheinfectionrate,148

similarly toWThepatocytes(Fig2E).Thisenhancingeffectofanti-CD81antibodiesonP.149

berghei-infectionhasbeenreportedbeforeinC57BL/6mousehepatocytecultures,butthe150

underlying mechanism remains unknown (10). Altogether, these results support the151

hypothesis thatmouseSR-B1doesnotplayaprominentroleduringP.berghei sporozoite152

invasioninthemouseliver,andsuggestthat,inadditiontoCD81,otheryetunidentifiedhost153

proteinsareimplicated.154

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Human and mouse SR-B1 protein sequence analysis and structure-homology156

modeling.157

Wenextinvestigatedthestructuralbasisthatcouldexplainthedifferentialfunctionality158

betweenhumanandmouseSR-B1duringP.bergheisporozoiteinvasion.hSR-B1(isoform1)159

contains509aminoacids(AA)andpresentsalargeextracellulardomain(404AA)flanked160

bytwotransmembranedomains(both23AA)andtwocytoplasmictails(N-terminal:12AA;161

C-terminal:47AA)(6).ThemodelingofhSR-B1usingCD36asatemplate(PDBID:5lgd)(7)162

shows that the extracellular part of the receptor can be divided into three regions: a N-163

terminal region (AA 36-136) harboring a thrombospondin-binding domain in the164

homologous CD36 protein (14), an apical region (AA 137-214) consisting of four alpha165

helices (α4, 5, 6 and 7), and a large C-terminal region (AA 215-439) contributing to the166

hydrophobicchannel(Fig3A,C).ThepairwisesequencealignmentofhSR-B1andmSR-B1167

showedthattheN-terminalandC-terminalextracellularregionswerethemostsimilar,with168

81.1%and85.7% identity, respectively,whilst theapicaldomain ismoredivergent,with169


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66.2%identity(Fig3CandD).ThehSR-B1proteinharbors9N-glycosylationsites,against170

11 sites for mSR-B1 (15) (Fig 3C). The superposition of hSR-B1 andmSR-B1 structural171

modelsrevealeddifferencesfortwoloopsattheverytopoftheapex,betweentheα4andα5172

helices and after the α7 helix (Fig3B).We also observed differences in the electrostatic173

surfacepotentialsinthisarea(Fig3E).Whenthestructureisorientatedinasideviewto174

present itshydrophobic tunnel entrance, the apex lateral surfaceofmSR-B1seems tobe175

mainlyelectropositivewhereaselectronegativityispredominantinthehumanmodel(Fig176

3E).Remarkably,whilstthetopoftheapicalsurfaceisstrictlyneutraltoelectropositivein177

hSR-B1,mSR-B1displaysadenseelectronegativeregion(Fig3E).178

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TheapicaldomainofSR-B1playsacrucialroleduringP.bergheiinfection180

TodeterminewhetherthepredictedstructuraldifferencesattheapicaldomainofSR-B1181

could explain the differential functionality of human andmouse SR-B1,we analyzed the182

functionalpropertiesoftwochimericconstructsmadeofhumanandmousesequencesofSR-183

B1.TheApicalHchimeracorrespondstoamSR-B1backboneproteinwithahumanapical184

region(AA137-214)(Fig4A,B).Reciprocally,theApicalMchimeracorrespondstoahSR-B1185

proteinbearingamurineApical region(Fig4A,B).Theelectrostaticsurfacepotentialsof186

ApicalHandApicalMapextoparesimilartohumanandmouseSR-B1,respectively,withonly187

ApicalM showing a dense negatively charged region (Fig 4C). CD81KOH16 cells were188

transiently transfectedwithplasmidsencodinghSR-B1,mSR-B1,ApicalHorApicalM.The189

two chimeras were expressed at the surface of transfected cells and detected by flow190

cytometry using anti-human and anti-mouse SR-B1 polyclonal antibodies (Fig 4D). They191

werealsodetectedbywesternblotanalysisofwholecellularextracts(FigS1).Aslightly192


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higherbandwasobservedinthelanescorrespondingtocellstransfectedwithmSR-B1and193

ApicalH constructs a compared to hSR-B1 and ApicalM,which is likely explained by the194

differentialglycosylationpatternofthemSR-B1backbone(Fig4A).Cellstransfectedwith195

ApicalHandApicalMconstructsboundCy5-labelledHDLs(SupplementalFigS2),similarly196

tohSR-B1andmSR-B1,suggestingthatbothchimerasarefunctional.197

TransfectedcellswerethenincubatedwithP.bergheisporozoites,andthenumberof198

infectedcellswasdeterminedat24hourspost-infection.Theseexperimentsrevealedthat199

replacementoftheapexofmSR-B1bythatofhSR-B1inApicalHyieldedachimerawitha2-200

3foldincreaseinP.bergheiinfectionratesascomparedtomSR-B1(Fig4E).Attheopposite,201

replacementoftheapexofhSR-B1bythatofmSR-B1intheApicalMchimeraresultedina202

lossoffunction,withinfectionlevelssimilartothoseobservedaftertransfectionofmSR-B1203

(Fig4E).Altogether,theseresultsdemonstratethatthehSR-B1apicalhelixbundle(AA137-204

214) is functionallydeterminantduringP.bergheisporozoite invasionofhepatocytic cell205

lines.206

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AshortportionoftheapicaldomainofSR-B1supportsP.bergheiinfection208

WethensoughttodefinemorepreciselythefunctionalregionsimplicatedinP.berghei209

infection within the apex domain. We designed three new chimeras made of a mSR-B1210

backbone harboring short hSR-B1 sequences, based on both the amino acid differences211

between themouseand thehumansequences, and theputative interacting sites inother212

CD36familyreceptors.TheD1chimera(AA150-164)includestheloopbetweentheα4and213

α5helices,wheretheEnterovirus71interactingsiteislocatedintheSR-B1homologLIMP-214

2,andencompassesalargepartoftheα5helixincludingthePfEMP1-interactingsiteinCD36215


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(Fig5A-B).TheD2chimera(AA193-203)comprisestheexternaltipoftheα7helixbutalso216

three phenylalanine residues in the downstream loop, exclusively present in the human217

sequence(Fig5A-B).TheD3chimera(AA201-211)includesonlyoneofthesephenylalanine218

residues(Fig5A-B).ThepredictedelectrostaticsurfacepotentialofD1andD3apextopis219

similartomSR-B1(Fig5C),whereasD2apexismostlyelectropositive,likehSR-B1,withno220

markofelectronegativity.221

AfterthetransienttransfectionofCD81KOH16cells,D1,D2,andD3chimeraswereall222

detectedbyflowcytometryonthecellsurfaceusingthe“αM”antibody.Interestingly,only223

D2wasdetectedbythe“αH”antibody,similarlytohSR-B1andApicalHproteins(Fig5D).224

InfectionofthetransfectedcellswithP.bergheisporozoitesrevealedthatreplacementofthe225

AA193-203sequenceofmSR-B1bythatofhSR-B1intheD2chimeraresultedina2-fold226

increaseinP.bergheiinfectioninCD81KOH16cells(Fig5E).Incontrast,replacementofthe227

AA 150-164 or AA 201-211 sequences in the D1 and D3 chimera, respectively, did not228

increase infection as compared to mSR-B1 (Fig 5E). These results thus highlight the229

functionalimportanceofashort11aminoacidsequencewithinthehSR-B1apicaldomain,230

whichissufficienttopromoteefficientP.bergheiinfection.231

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ThelipidtransferactivityofhumanSR-B1isnotrequiredforP.bergheiinfection233

SR-B1mediates selective efflux and uptake of cholesteryl esters between the plasma234

membraneandHDLs(16),whichismediatedbySR-B1hydrophobicchannelthatspansthe235

entirelengthofthemolecule(6).Rodriguesetal.reportedthatblocklipidtransport(BLT)236

inhibitors,whichblock the lipid transferactivityof SR-B1, inhibitboth theentryand the237

developmentofP.bergheiinsideHuh7cells(2).Wethereforesoughttodeterminewhether238


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inadditiontotheapicaldomain,theSR-B1lipidtransferactivityisalsoinvolvedduringSR-239

B1-dependentP.berghei infectionofHepG2cells.Weobservedan inhibitionofP.berghei240

invasioninHepG2cellswhensporozoiteswereco-incubatedwitha20μMconcentrationof241

BLT-1(Fig6A).However,thesameinhibitionwasalsoobservedinHepa1-6cellslackingSR-242

B1 receptor (Fig 6C). Pre-incubation of cells with BLT inhibitors before sporozoite243

inoculationcausednoinhibitionofinfectioninanyofthecelllinestested(Fig6Band6D).244

BLT-1 at high concentration also blocked sporozoite cell traversal activity,monitored by245

dextran-rhodaminecellularuptake(FigS3).Thesedatastronglysuggestthattheinhibition246

causedbyBLT1isduetothetoxicityofthecompoundonsporozoites,ratherthanblockage247

ofSR-B1function.Infavorofthishypothesis,anotherBLTinhibitor,BLT-4,hadnoeffecton248

eithercelltraversalorinvasionbyP.bergheisporozoites(Fig6A-DandFigS2).249

Altogether,thesedataindicatethattheapicaldomainbutnotthelipidtransferactivityof250

SR-B1isimportantduringP.bergheisporozoiteentry.251

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DISCUSSION253

Previous studieshighlighted the dual role of SR-B1duringPlasmodium sporozoite254

invasionandintracellularliverstagedevelopment(2,5).Morerecently,wehaveshownthat255

SR-B1isanimportanthostfactorforP.vivaxbutnotforP.falciparuminfection,andthatP.256

berghei sporozoites canusehSR-B1asanalternativeentry route to theCD81-dependent257

pathway(4).P.bergheiisarodent-infectingparasite,yetP.bergheisporozoitescanreadily258

infecthumanhepatocyticcells,usingeitheraCD81oraSR-B1entryroute(4).Here,weshow259

thatmSR-B1, in contrast to its human counterpart, does not support efficientP. berghei260

sporozoiteinvasionofhepatocyticcells.P.bergheiwasoriginallyisolatedfromtheAfrican261

tree rat Grammomys surdaster, and artificially introduced for scientific purposes in the262

domestic mouse Mus musculus (17). We cannot exclude that SR-B1 function during263

Plasmodium infection may vary depending on the rodent host. In previous studies, we264

reportedthatP.bergheisporozoitesreadilyinfectCD81-deficientmousehepatocytesinvivo265

and in vitro (3,10), supporting theexistenceof alternativeentrypathways.Whilst SR-B1266

providesaCD81-independentrouteforP.bergheiinhumanhepatocyticcells(4),wereport267

herethatconcomitantblockageofmurineCD81andSR-B1receptorsdoesnotpreventP.268

bergheiinfectioninprimarymousehepatocytecultures.Theseresultssupporttheexistence269

of alternative entry routes for the parasite, which still remain to be identified. Possible270

candidate host receptors include the SR-B1-related proteins CD36 and LIMP-2. Although271

LIMP-2ispredominantlyexpressedinlysosomes,afractionoftheproteinpoollocalizesat272

thecellplasmamembrane,whereLIMP-2actsasareceptorthatmediatestheEnterovirus71273

hostcellentry(18,19).LIMP-2roleduringPlasmodiuminfectionhasnotbeeninvestigated274

so far.At theopposite,CD36 isknowntoplaymajorrolesduringmalaria infection.CD36275


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bindsPfEMP1variantsexpressedatthesurfaceofP.falciparum-infectederythrocytes,and276

contributestothecytoadherenceofP.falciparumtovascularendothelialcells(20–22).Itis277

alsoamajorreceptorfortissuesequestrationofP.berghei-infectederythrocytesinmice(23).278

A previous study investigated the contribution of CD36 during P. yoelii and P. berghei279

sporozoiteinfection,usingCD36-deficientmice.Thedatashowedthatbothparasitescould280

still infect hepatocytes in the absence of CD36 (24). However, in these experiments, the281

presenceofafunctionalCD81-entrypathwaycouldhavemaskedanyimportantroleofCD36.282

HencethecontributionofCD36andLIMP-2deservesfurtherinvestigation.283

WetookadvantageofthedifferentialfunctionalitybetweenhumanandmurineSR-B1284

toinvestigatetheSR-B1moleculardeterminantsinvolvedduringP.bergheiinfection.Using285

a series of complementary chimeras designed through a structure-guided strategy, we286

demonstrateherethecriticalroleofan11aminoaciddomainwithinhSR-B1apicalhelices287

(AA193to203)duringP.bergheisporozoiteinfection.ThisisconsistentwithCD36family288

proteinstypicallybindingtoavarietyofligandsviatheirhelicalbundle.Forinstance,theß-289

glucocerebrosidasebindstoLIMP-2apicaldomaintobedeliveredintothelysosome(25).290

BindingofEnterovirus71dependsona7aminoacidsequence(AA144-151)inLIMP-2(18,291

26).Furthermore,anapicalphenylalanineofCD36(F153)bindstoPlasmodiumPfEMP1(7).292

ThesesitescanbemappedontheSR-B1predictedstructureattheintersectionbetweenthe293

α4andα5helices,at theverytopof theapex.Thiscrucialphenylalanine isreplacedbya294

threonine in hSR-B1, and no other phenylalanine residue seems close to this area in the295

tertiarystructure(Fig3D).Interestingly,the11aminoacidhSR-B1functionaldomainwe296

haveidentifiedcontains3phenylalanineresiduesandcanbemappedinthesameregionat297

thetopoftheapexbutatadistinctsiteintheSR-B1model(Fig3B).298


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OurdataindicatethatthedifferentialactivityofhumanandmurineSR-B1isnotdue299

totheN-orC-terminalregionsofSR-B1ectodomain,whichparticipateinthehydrophobic300

channelmediatingthelipidtransfer.Furthermore,wedidnotobserveanyspecificinhibition301

ofSR-B1-dependentinfectionbyBLTinhibitors.Takentogether,ourdatasuggestthatthe302

lipidtransferactivityofSR-B1isnotinvolvedduringP.bergheisporozoiteinfection.Rather,303

we speculate that the apical helical domain of the proteinmay serve as a receptor for a304

hithertounidentifiedsporozoiteligand.ThedenseelectronegativespotattheapexofmSR-305

B1andofpoorlyfunctionalchimeras(ApicalM,D1andD3),whichisabsentinhSR-B1and306

functionalchimeras(ApicalHandD2),maybeunfavourableforthebindingtothisputative307

ligand.Onecandidateisthe6-cysteinedomainproteinP36,whichisrequiredforsporozoite308

productive invasion of hepatocytes, and is functionally linked to host receptor usage. In309

particular, we have shown that P. yoelii sporozoites genetically complemented with P36310

protein from P. berghei can infect host cells through a SR-B1-dependent pathway (4).311

WhetherP36protein fromP.berghei or fromthemedically-relevantP. vivax binds to the312

apicalhelixbundleofSR-B1remainstobedetermined.313

In conclusion, this study provides new insights into the function of SR-B1 during314

malaria infection, and paves theway towards a better characterization of themolecular315

interactions leading to parasite entry into hepatocytes. Our results may be particularly316

relevanttoP.vivaxmalaria,asSR-B1isthefirstanduptonowonlyknownhostentryfactor317

for P. vivax sporozoites (4). The characterization of SR-B1 molecular function and the318

identification of interacting parasite ligands may lead to the development of novel319

interventionstrategiestopreventP.vivaxsporozoiteentry,beforetheestablishmentofthe320

liverstageandthehypnozoitereservoir.321


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322

MATERIALSANDMETHODS323

Ethicsstatement324

AllanimalworkwasconductedinstrictaccordancewiththeDirective2010/63/EUofthe325

EuropeanParliamentandCouncil‘Ontheprotectionofanimalsusedforscientificpurposes’.326

ProtocolswereapprovedbytheEthicalCommitteeCharlesDarwinN°005(approval#7475-327

2016110315516522).328

329

Experimentalanimals,parasiteandcelllines330

We used GFP-expressing P. berghei (PbGFP, ANKA strain) parasites, obtained after331

integrationofaGFPexpressioncassetteatthedispensablep230plocus(27).PbGFPblood332

stageparasiteswerepropagatedinfemaleSwissmice(6–8weeksold,fromJanvierLabs).333

AnophelesstephensimosquitoeswerefedonPbGFP-infectedmiceusingstandardmethods334

(28),andkeptat21°C.PbGFPsporozoiteswerecollectedfromthesalivaryglandsofinfected335

mosquitoes21–28dayspost-feeding.Hepa1-6cells(ATCCCRL-1830)andHepG2(ATCCHB-336

8065)were cultured at 37°Cunder 5%CO2 inDMEM supplementedwith10% fetal calf337

serum(10500064,LifeTechnologies), L-glutamine20µM(25030024,LifeTechnologies),338

and1%penicillin-streptomycin,asdescribed(10).Primarymousehepatocyteswereisolated339

bycollagenaseperfusion(C5138,Sigma),asdescribedin(29),fromC57BL/6miceharboring340

aCre-mediatedSR-B1gene inactivationspecifically in the liver(12).Primaryhepatocytes341

wereseededatconfluencyin96wellplatesandculturedat37°Cin4%CO2inWilliam’sE342

medium (22551022, Life Technologies) with 10% fetal calf serum, 1% penicillin-343

streptomycin (15140122, Life Technologies), hydrocortisone 50µM (Upjohn laboratories344


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SERB) and 1% L-glutamine, Bovine insulin 5 µg/ml (I5500, Sigma) for 24 hours before345

sporozoiteinfection.346

347

SmallinterferingRNAsilencingofCD81348

ThesiRNAoligonucleotideagainstCD81(5’-CGUGUCACCUUCAACUGUA-3’)wasvalidatedin349

previous studies (10). Transfection of siRNA oligonucleotides was performed by350

electroporationinpresenceof10µLofsiRNA20µM,asdescribed(11).Cellswerecultured351

during48hoursbeforeinfectionoranalysisbyimmunofluorescence.Asnegativecontrols,352

weusedcellselectroporatedintheabsenceofsiRNAoligonucleotide.353

354

GenerationofaCD81KOH16celllineusingCRISPR-Cas9355

Thedaybeforetransfection,Hepa1-6cellswereplatedin24wellplatesatadensityof90356

000cellsperwell.Cellsweretransfectedwith500ngofLentiCrispRV2(Addgeneplasmid357

#52961)containingaguideRNAtargetingmouseCD81(GCAACCACAGAGCTACACCT)using358

Lipofectamine2000(11668027,LifeTechnologies).Puromycinselectionwascarriedout36359

hoursaftertransfectionusinga5µg/mlsolution.Cellswereexposedtopuromycinefor48360

hours, then washed and expanded for two weeks in complete medium before analysis.361

Immunostainingwasperformedusing the ratmonoclonal antibodyMT81 to labelmouse362

CD81(Silvieetal.,2006a).Allincubationswereperformedat4°Cduringonehour.Weused363

AlexaFluor-488Goatanti-ratantibody(A1106,Lifetechnologies)asasecondaryantibody.364

Cellswerethenfixedwith1%formaldehydesolutionandanalyzedusingaGuavaEasyCyte365

6/2Lbenchcytometerequippedwith488nmand532nmlasers(Millipore).366

367


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HomologymodelingofSR-B1chimeras368

TheSR-B1aminoacidsequenceofH.sapiens(Uniprot:Q8WTV0)wassubmittedtothe369

HHpredinteractiveserverforremoteproteinhomologydetection(30).Theserveridentified370

theX-raystructureofthescavengerreceptorCD36(PDBID:5lgd)at2.07Åresolution(7)as371

the best template to model the SR-B1 protein (probability: 100%, e-value: 2.3e-91).372

Sequences of SR-B1 chimeraswere aligned andmodeled using Swiss-Model through the373

ExPAsy molecular biology suite (31). Each SR-B1 model was then subjected to loop374

refinementandenergyminimizationusingGalaxyRefine(32)andYASARA(33),respectively.375

SR-B1modelswerevalidatedforqualityusingMolProbityforlocalstereochemistry(34),and376

Prosa II for global 3D quality metrics (35). Additionally, we validated the structure by377

checkingthatalltheN-glycosylationsitesweresolvent-exposed.378

The protein electrostatic surface potential was calculated using Adaptive Poisson-379

Boltzmann Solver (APBS) (36), after determining the per-atom charge and radius of the380

structurewithPDB2PQRv.2.1.1(37).ThePoisson-Boltzmannequationwassolvedat298K381

usingagrid-basedmethod,withsoluteandsolventdielectricconstantsfixedat2and78.5,382

respectively.Weusedascaleof-2kT/eto+2kT/etomaptheelectrostaticsurfacepotential383

inaradiusof1.4Å.AllmoleculardrawingswereproducedusingUCSFChimera(38).384

385

SR-B1chimericconstructdesignandplasmidtransfection386

PlasmidsencodinghumanandmouseSR-B1havebeendescribedpreviously(39,40).The387

ApicalH and ApicalM chimera were obtained by cloning a single insert amplified from388

chimeric synthetic genes (Eurofins Genomics) into the mSR-B1 and hSR-B1 plasmids,389

respectively. The D1, D2 and D3 chimera were generated by inserting into the mSR-B1390


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plasmidtwofragmentsamplifiedwithprimerscontaininghSR-B1sequences.Thesequence391

ofalloligonucleotidesusedtoamplifyDNAinsertsandthesequenceofsyntheticgenesused392

as templates are indicated in Supplemental Table 1. Information on plasmid sequence is393

availableondemand.AllcloningstepswereperformedusingIn-fusioncloningkit(639649,394

Ozyme) and controlled by Sanger sequencing (Eurofins genomics). High concentration395

plasmid solutions were produced using XL1-Blue Competent Cells (200249, Agilent396

technology)andplasmidextractionwasperformedusingQiagenPlasmidMaxikit(12163,397

QIAGEN) according to the manufacturer’s recommendations. Transfection of SR-B1 or398

chimerasencodingplasmidswasperformed24hoursaftersiRNAelectroporation,ordirectly399

onCD81KOH16cells,usingtheLipofectamine2000reagent(11668027,LifeTechnologies)400

according to themanufacturer’sspecifications.Followingplasmid transfection, cellswere401

cultured for an additional 24 hours before sporozoite infection or protein expression402

analysis.403

404

Westernblot405

After cell lysis in1%NP-40, soluble fractionswereanalyzed bywesternblotundernon-406

reducingconditions,usingaBioradMini-Protean®electrophoresischamberforSDS-PAGE407

andtransferonpolyvinylidenefluoride(PVDF)membranes.Membraneswereprobedwith408

anti-mouseCD81MT81(13)at2µg/ml,anti-mSR-B1polyclonalantibody(Ab24603)diluted409

at 0.9 µg/ml, and anti-mouse GADPH (TAB1001) as a loading control (0.5 µg/ml).410

Chemiluminescence detection was performed using ECL Prime reagents (RPN2232,GE411

healthcareLifesciences)andanImageQuantLAS4000system(GEHealthcare).412

413


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Immunofluorescenceassays414

For the immunolabeling of SR-B1 and chimeric proteins, cells were harvested using an415

enzyme-free Cell Dissociation buffer (13151014, Thermofisher). All incubations were416

performedat4°C inPBS/BSA3%duringonehourwitheither“αH”anti-SR-B1polyclonal417

rabbit serum (40) or “αM” anti-SR-B1 polyclonal rabbit antibodies NB400-113 (Novus418

Biological). We used AlexaFluor-488 Donkey anti-rabbit antibody (Ab150073, Life419

technologies) as secondary antibody with a 45 minutes incubation. After fixation in 1%420

formaldehyde,cellswereanalyzedusingaGuavaEasyCyte6/2Lbenchcytometerequipped421

with488nmand532nmlasers(Millipore).Flowcytometryplotsarerepresentativeofat422

leastthreeindependentexperiments.423

424

Invitroinfectionassays425

Hepa1-6 cells were seeded in 96 well plates (2×104 per well seeded the day before426

transfection)andincubatedwith1×104PbGFPsporozoitesfor3hours,washed,andfurther427

cultureduntil24hourspost-infection.HepG2andHepG2/CD81,platedin96wellplateswith428

3×104 cells per well seeded the day before infection, were infected using 5×103 PbGFP429

sporozoites. In some experiments, anti-mouse CD81 MT81 at 20 µg/ml (13), BLT-1430

(SML0059,Sigma),BLT-4(SML0512,Sigma)(bothpreparedinpureDMSO)ordilutedDMSO,431

wereaddedtosporozoitesduring infection.Forthedextranassay,0.5mg/mlrhodamine-432

conjugateddextran(Lifetechnologies)wasaddedtosporozoitesduringinfection.Infected433

cultureswere then either trypsinized for detection of GFP-positive cells and/or dextran-434

positivecellsbyflowcytometryonaGuavaEasyCyte6/2Lbenchcytometer(Millipore),or435


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fixedwith4%paraformaldehyde and analyzed by fluorescencemicroscopy after labeling436

withantibodiesspecificforUIS4(Sicgen)andthenuclearstainHoechst33342.437

438

HDLbindingassay439

HumanHDLlipoproteins(LP3,Calbiochem)werelabeledusingtheCy5monoreactiveDye440

pack (PA25001, GE Healthcare) and filtered using Illustra microspin G25 columns441

(27532501,GEHealthcare).CD81KOH16cellsweredissociatedat24hourspost-transfection442

usinganenzyme-freeCelldissociationbuffer(13151014,Thermofisher)andincubatedwith443

Cy5labeledHDLs(5µg/ml)for20minutesat37°C.Afterwashing,theywereincubatedwith444

Suramin(574625,MerckMillipore)at10mg/mlforonehourat4°C.HDLbindingtoSR-B1445

andchimeraswasthenevaluatedbyflowcytometryusingtheBDLSRFortessa™.446

447

Statisticalanalyses448

Statistical analyseswere performedwith GraphPad Prism on at least three independent449

experiments, each performed in triplicates, as indicated in the legend to the figures. All450

graphs show the mean ± SEM (unless otherwise indicated) expressed as percentage of451

control(WTcellsorCD81KOH16cellstransfectedwithhSR-B1,asindicated).452

453

ACKNOWLEDGMENTS454

We thank Jean-FrançoisFranetich,Maurel Tefit,MariemChoura andThierryHoupert for455

rearingofmosquitoesandtechnicalassistance,andDrsMaryseLebrunandJérômeClainfor456

fruitfuldiscussions.ThisworkwasfundedbytheEuropeanUnion(FP7PathCoCollaborative457

ProjectHEALTH-F3-2012-305578),theLaboratoired'ExcellenceParaFrap(ANR-11-LABX-458


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0024),andtheAgenceNationaledelaRecherche(ANR-16-CE15-0004).GMwassupported459

bya“DIMMalinf”doctoralfellowshipawardedbytheConseilRégionald'Ile-de-France.460

461


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FIGURELEGENDS586

Figure 1. CRISPR-mediated inactivation of CD81 abrogates P. berghei infection in587

Hepa1-6cells.588

(A) Hepa1-6 and CD81KOH16 cellswere stained for surface CD81with anti-CD81MT81589

monoclonalantibodyandfluorescentsecondaryantibodies,beforeflowcytometryanalysis.590

Histograms represent the fluorescence intensity of extracellular CD81 proteins for WT591

Hepa1-6(blue)andCD81KOH16cells(orange).Thegreyhistogramrepresentscellsstained592

withsecondaryantibodiesonly(Control).(B)Westernblotanalysisof totalCD81protein593

expressioninWTHepa1-6andCD81KOH16cells.GAPDHwasusedasloadingcontrol.(C-E)594

WTHepa1-6andCD81KOH16cellswereinfectedwithPbGFPsporozoitesandanalyzed24595

hoursafterinvasionbyflowcytometry(C)ormicroscopy(D,E)afterstainingwithanti-UIS4596

antibodies(red)andHoechst33342nuclearstain(blue).ThenumberofEEFsperwellranged597

from105to362(median175)incontrolcells.****,p<0.0001(ratiopairedttest).Theimages598

showPbGFPEEFs(green)surroundedbyaUIS4-positivePVmembrane(red)orintranuclear599

parasitesinCD81KOH16cells.Scalebar,10μm.600

601

Figure2.MouseSR-B1ispoorlyfunctionalduringP.bergheisporozoiteinvasion.602

(A-B)CD81KOH16cellsweretransfectedwitheithermouseorhumanSR-B1plasmids,orno603

plasmidasacontrol(Mock).Totalproteinexpressionwasanalyzedusingpolyclonalanti-SR-604

B1 antibodies (Ab24603) bywestern blot (A) with GAPDH as a loading control. Surface605

protein expression was analyzed by flow cytometry (B) using anti-human“α-H” SR-B1606

polyclonal rabbit serum (blue) and anti-mouse “α-M” polyclonal antibodies NB400-113607

(orange).Thegreyhistogramrepresentsstaineduntransfectedcellswiththecorresponding608


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antibody.(C-D)CD81KOH16(C)andWTHepa1-6cellstreatedwithsiRNAagainstCD8124609

hoursbefore(D),weretransfectedwithmouseorhumanSR-B1plasmids,ornoplasmidasa610

negative control (Mock), and then infected with PbGFP sporozoites. EEFs numbers were611

countedbymicroscopyafterUIS4stainingat24hoursaftersporozoiteaddition.Thenumber612

ofEEFsperwellrangedfrom43to334(median169)inhSR-B1-transfectedcells(C),and613

from19to300(median94)incontrolWTcells(D).*,p<0.05;**,p<0.01(repeatedmeasures614

one-wayANOVAfollowedbyTukey’smultiplecomparisonstest).(E)Primaryhepatocytes615

isolatedfromWTorSR-B1deficientC57BL/6micewereinfectedwithPbGFPsporozoitesin616

theabsenceorpresenceofneutralizinganti-mCD81mAbMT81,andculturedfor24hours617

beforeEEFsquantification.*,p<0.05(ratiopairedttest).618

619

Figure3.SR-B1modelingidentifiespotentialfunctionalregions.620

(A)PredictedtertiarystructureofhSR-B1extracellulardomainbyhomologymodelingusing621

CD36(PDBID:5lgd)asatemplate,withthethreeregionsreferredtoas“N-terminal”(green),622

“apex” (red) and “C-terminal” (black). (B)A close-up viewof structural alignment of the623

apicalhelixbundleofmouse(orange)andhuman(blue)SR-B1,withtheirfouralphahelices624

(a4toa7).Themainstructuraldifferencesarecircledinblack.(C)Schematicrepresentation625

of SR-B1 N-glycosylation sites on human (blue) and mouse (orange) proteins. Two626

determinant sites for SR-B1 structure and function are in red (Asn108 and 173),mouse627

specificsitesareinyellow(Asn116and288)andconservedsitesareinblue.SR-B1modelis628

aschematicrepresentationofthedelineatedregions(“N-terminal”(green),“apex”(red),“C-629

terminal”(black))inSR-B1proteindisplayingallpotentialN-glycosylationsites.(D)Pairwise630

sequence alignment of mSR-B1 and hSR-B1 proteins for the 132-223 apical region with631


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correspondingpredictedhumansecondarystructure(alphahelicesinredandbetastrandin632

blue). Identical, similar and different amino acids are represented in black, blue and red633

respectively.ThethreonineresiduepositioncorrespondingtoPfEMP1bindingphenylalanine634

inCD36homologishighlightedinpurple.TheresiduesinSR-B1equivalenttoEnterovirus-635

interacting site in LIMP2 are highlighted in green and purple. (E) Electrostatic surface636

potentialofmSR-B1andhSR-B1extracellulardomainfromsideandtopviews.Valuesarein637

unitsofkT/eat298K,onascaleof-2kT/e(red)to+2kT/e(blue).Whitecolorindicatesa638

neutral potential. The black circle highlights a differential electrostatic surface potential639

betweenmSR-B1andhSR-B1atthetopofthe“apex”region.640

641

Figure4.TheapicaldomainofSR-B1playsacrucialroleduringP.bergheiinfection.642

(A)SchematicrepresentationoftheApicalHandApicalMchimericconstructs.(B)Predicted643

tertiarystructureofApicalHandApicalMchimerasbyhomologymodeling,highlightingthe644

portionsofmouse(orange)orhuman(blue)origins.(C)Topviewsoftheelectrostaticsurface645

potentialofApicalHandApicalMchimeras’apex.ValuesareinunitsofkT/eat298K,ona646

scaleof-2kT/e(red)to+2kT/e(blue).Whitecolorindicatesaneutralpotential.Theblack647

circle highlights a differential electrostatic surface potential between the two chimeric648

constructsatthetopofthe“apex”region.(D)CD81KOH16cellsweretransfectedwithhSR-649

B1,mSR-B1,ApicalHorApicalMchimeraplasmids,ornoplasmidasacontrol(Mock).Protein650

surfaceexpressionwasanalyzedusinganti-hSR-B1(“αH”,bluehistograms)andanti-mSR-B1651

(“αM”, orange histograms), 24 hours after transfection. The grey histogram represents652

untransfectedcellsstainedwiththecognateantibody.(E)CD81KOH16cellsweretransfected653

withhSR-B1,mSR-B1,ApicalHorApicalMconstructs,ornoplasmidasacontrol(Mock),and654


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infectedwithPbGFPsporozoites24hoursafter transfection.Thenumberof infectedcells655

(EEFs) was determined by microscopy after UIS4 staining at 24 hours after sporozoite656

addition.The number of EEFs perwell ranged from 43 to 334 (median 169) in hSR-B1-657

transfected wells. ns, non-significant; ***, p<0.001 (one-way ANOVA followed by Tukey’s658

multiplecomparisonstest).659

660

Figure5.AkeydomainwithinSR-B1apexisessentialforP.bergheiinfection.661

(A)MouseandhumanproteinsequencealignmentoftheapicalregionAA132-223withthe662

correspondingpredictedhumansecondarystructure(alphahelicesinredandbetastrandin663

blue). Identical, similar and different amino acids are represented in black, blue and red664

respectively. ShortdomainsD1,D2andD3aredelimitedby boxes.(B) Predicted tertiary665

structureofD1,D2andD3chimerasbyhomologymodeling,highlightingthesegmentsof666

mouse(orange)orhuman(blue)origins.(C)Topviewsoftheelectrostaticsurfacepotential667

ofD1,D2andD3chimeras’apex.ValuesareinunitsofkT/eat298K,onascaleof-2kT/e668

(red)to+2kT/e(blue).Whitecolor indicatesaneutralpotential.Blackscirclehighlighta669

differentialelectrostaticsurfacepotentialbetweenthedifferentchimericconstructsat the670

topofthe“apex”region.(D)CD81KOH16cellsweretransfectedwithhSR-B1,mSR-B1,D1,671

D2,orD3chimericconstructs.Proteinsurfaceexpressionwasanalyzedusinganti-hSR-B1672

(“αH”, blue histograms) and anti-mSR-B1 (“αM”, orange histograms), 24 hours after673

transfection. The grey histogram represents untransfected cells stainedwith the cognate674

antibody. (E) CD81KOH16 cells were transfected with hSR-B1, mSR-B1, D1, D2, or D3675

chimeric constructs, or no plasmid as a control (Mock), and then infected with PbGFP676

sporozoites24hoursaftertransfection.Thenumberofinfectedcells(EEFs)wasdetermined677


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bymicroscopyafterUIS4stainingat24hoursaftersporozoiteaddition.ThenumberofEEFs678

perwellrangedfrom43to334(median169)inhSR-B1-transfectedwells.ns,non-significant;679

**,p<0.01(one-wayANOVAfollowedbyTukey’smultiplecomparisonstest).680

681

Figure6.ThelipidtransferactivityofSR-B1isnotrequiredduringP.bergheiinfection.682

(A-D)HepG2orandHepa1-6cellsweretreatedwithBLTinhibitors(BLT1andBLT4)attwo683

differentconcentrations(2μMand20μM),eitheratthesametimeassporozoiteincubation684

(Coincubation:A,C)orpriortosporozoiteaddition(Preincubation:B,D).Controlcellswere685

treatedwiththesolvant(DMSO)alone.Thenumberofinfectedcellswasanalyzedafter24686

hoursbymicroscopyafterUIS4staining.ThenumberofEEFsperwellrangedfrom223to687

545(median428)incontrolHepG2cells,andfrom43to378(median161)incontrolHepa1-688

6cells.Alldatacomefromtwoindependentexperimentsandarerepresentedasmean+/-689

range.690

691

Supplementalfigure1692

CD81KOHepa1-6cellsweretransfectedwitheithermSR-B1,hSR-B1,ApicalHorApicalM693

constructplasmids,ornoplasmidasacontrol(Mock).Totalproteinexpressionwasanalyzed694

by western blot using polyclonal anti-SR-B1 antibodies (Ab24603) and anti-GAPDH695

antibodiesasaloadingcontrol.696

697


(A-B)CD81KOH16cellsweretransfectedwithhSR-B1,mSR-B1,ApicalHorApicalMchimeric699

constructs, or with a plasmid encoding mCD81 (negative control). (A) Protein surface700


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expressionwasanalyzedusinganti-hSR-B1(“αH”,bluehistograms)andanti-mSR-B1(“αM”,701

orange histograms), 24 hours after transfection. The grey histogram represents702

untransfectedcellsstainedwiththecorrespondingantibody.(B)Cy5fluorescentHDLswere703

addedtotransfectedcells24hoursaftertransfectiontomeasureHDLbinding(purplepeak).704

HDL binding to non-transfected cells (negative control) is shown as a white peak. Cells705

transfectedwithamouseCD81constructdidnotbindHDLs,asexpected.706

707


HepG2cellswereinfectedwithPbGFPsporozoitesinthepresenceofrhodamine-conjugated709

dextranandBLTinhibitors(BLT1andBLT4)attwodifferentconcentrations(2μMand20710

μM),orDMSOasacontrol.Dextran-positivecellswereanalyzedbyflowcytometry3hours711

aftersporozoiteaddition.712

713

Supplementaltable1714

Sequencesofoligonucleotidesandsyntheticgenesusedinthisstudy.715

716


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Supplemental table 1 717 Oligonucleotide Sequence 5´ à 3´

ApicalH construct

ApicalHfor ACCGATCCAGCCTCCGCGGGCCCTGC

ApicalHrev GAGGCGCACCAAACCTGCAGGTGCTG

ApicalM construct

ApicalMfor CGTGTCCTTCCTCGAGTACCGCACCTTCCAGTTCC

ApicalMrev GAGGTGGATCCTCGAGATGTTCTGGACCCCCGTG

D1 construct

mSRBIfor ACCGATCCAGCCTCCGCGGGCCCTGC

D1rev CATGATGAGCTTCAGGGTCATGGGCTTATTCTCCATCAATATCGAGCCCCCCAG

D1for CTGAAGCTCATCATGACCTTGGCATTCACCACGATGGGCCAGCGTGCTTTTATG

mSRBIrev GAGGCGCACCAAACCTGCAGGTGCTG

D2 construct


D2rev GGGGAACATGCCTGGAAAGTACTTGTTGAGAAAATGCACGAAGGGATCGTC

D2for CCAGGCATGTTCCCCTTCAAGGACAAATTTGGCCTGTTTGTTGGGATGAAC


D3 construct


D3rev AAATAATCCGAACTTGTCCTTGAAGGGAAGCATGTCTGGGAGGTACGTG

D3for AAGTTCGGATTATTTGCTGAGCTCAACAACTCGAATTCTGGGGTCTTCACTGTC


ApicalH synthetic gene

accgatccagcctccgcgggccctgccaccatgggcggcagctccagggcgcgctgggtggccttggggttgggcgccctggggctgctgtttgctgcgctcggcgttgtcatgatcctcatggtgccctccctcatcaagcagcaggtgctcaagaatgtccgcatagacccgagcagcctgtccttcgggatgtggaaggagatccccgtccctttctacttgtctgtctacttcttcgaagtggtcaacccaaacgaggtcctcaacggccagaagccagtagtccgggagcgtggaccctatgtctacagggagttcagacaaaaggtcaacatcaccttcaatgacaacgacaccgtgtccttcgtggagaaccgcagcctccatttccagcctgacaagtcgcatggctcagagagtgactacattgtactgCCCAACATCCTGGTCTTGGGTGCGGCGGTGATGATGGAGAATAAGCCCATGACCCTGAAGCTCATCATGACCTTGGCATTCACCACCCTCGGCGAACGTGCCTTCATGAACCGCACTGTGGGTGAGATCATGTGGGGCTACAAGGACCCCCTTGTGAATCTCATCAACAAGTACTTTCCAGGCATGTTCCCCTTCAAGGACAAGTTCGGATTATTTGCTGAGCTCAACAACTCGaattctggggtcttcactgtcttcacgggcgtccagaatttcagcaggatccatctggtggacaaatggaacggactcagcaagatcgattattggcattcagagcagtgtaacatgatcaatgggacttccgggcagatgtgggcacccttcatgacacccgaatcctcgctggaattcttcagcccggaggcatgcaggtccatgaagctgacctacaacgaatcaagggtgtttgaaggcattcccacgtatcgcttcacggcccccgatactctgtttgccaacgggtccgtctacccacccaacgaaggcttctgcccatgccgagagtctggcattcagaatgtcagcacctgcaggtttggtgcgcctc

ApicalM synthetic gene

cgtgtccttcctcgagtaccgcaccttccagttccagccctccaagtcccacggctcggagagcgactacatcgtcatgCCCAACATCCTGGTCCTGGGGGGCTCGATATTGATGGAGAGCAAGCCTGTGAGCCTGAAGCTGATGATGACCTTGGCGCTGGTCACCATGGGCCAGCGTGCTTTTATGAACCGCACAGTTGGTGAGATCCTGTGGGGCTATGACGATCCCTTCGTGCATTTTCTCAACACGTACCTCCCAGACATGCTTCCCATAAAGGGCAAATTTGGCCTGTTTGCTGAGCTCAACAACTCCgactctgggctcttcacggtgttcacgggggtccagaacatctcgaggatccacctc

718


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Langlois et al Supplemental Figure 1


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Molecular determinants of SR-B1-dependent Plasmodium … · 88 SR-B1 is a highly glycosylated...

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