1
Molecular determinants of SR-B1-dependent Plasmodium sporozoite entry into1
hepatocyticcells.2
3
Anne-Claire Langloisa, Giulia Manzonia, Laetitia Vincensinia, Romain Coppéeb, Carine4
Marinacha, Maryse Guérinc, Thierry Hubyc, Véronique Carrièred, François-Loïc Cossete,5
MarlèneDreuxe,EricRubinsteina,OlivierSilviea6
7
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
16
17
Correspondingauthor:OlivierSilvie;[email protected] 18
19
Runningtitle:SR-B1structureandPlasmodiuminfection20
21
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2
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
40
41
42
43
44
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3
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
58
59
60
61
62
63
64
65
66
67
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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
100
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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
123
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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
155
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
179
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
207
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
232
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
252
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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
(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
16
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
(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
17
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
(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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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
(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
19
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
(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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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
(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
21
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
(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
22
0024),andtheAgenceNationaledelaRecherche(ANR-16-CE15-0004).GMwassupported459
bya“DIMMalinf”doctoralfellowshipawardedbytheConseilRégionald'Ile-de-France.460
461
(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
23
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29
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
(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
30
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
(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
31
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
(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
32
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
(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
33
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
Supplementalfigure2698
(A-B)CD81KOH16cellsweretransfectedwithhSR-B1,mSR-B1,ApicalHorApicalMchimeric699
constructs, or with a plasmid encoding mCD81 (negative control). (A) Protein surface700
(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
34
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
Supplementalfigure3708
HepG2cellswereinfectedwithPbGFPsporozoitesinthepresenceofrhodamine-conjugated709
dextranandBLTinhibitors(BLT1andBLT4)attwodifferentconcentrations(2μMand20710
μM),orDMSOasacontrol.Dextran-positivecellswereanalyzedbyflowcytometry3hours711
aftersporozoiteaddition.712
713
Supplementaltable1714
Sequencesofoligonucleotidesandsyntheticgenesusedinthisstudy.715
716
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35
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
mSRBIfor ACCGATCCAGCCTCCGCGGGCCCTGC
D2rev GGGGAACATGCCTGGAAAGTACTTGTTGAGAAAATGCACGAAGGGATCGTC
D2for CCAGGCATGTTCCCCTTCAAGGACAAATTTGGCCTGTTTGTTGGGATGAAC
mSRBIrev GAGGCGCACCAAACCTGCAGGTGCTG
D3 construct
mSRBIfor ACCGATCCAGCCTCCGCGGGCCCTGC
D3rev AAATAATCCGAACTTGTCCTTGAAGGGAAGCATGTCTGGGAGGTACGTG
D3for AAGTTCGGATTATTTGCTGAGCTCAACAACTCGAATTCTGGGGTCTTCACTGTC
mSRBIrev GAGGCGCACCAAACCTGCAGGTGCTG
ApicalH synthetic gene
accgatccagcctccgcgggccctgccaccatgggcggcagctccagggcgcgctgggtggccttggggttgggcgccctggggctgctgtttgctgcgctcggcgttgtcatgatcctcatggtgccctccctcatcaagcagcaggtgctcaagaatgtccgcatagacccgagcagcctgtccttcgggatgtggaaggagatccccgtccctttctacttgtctgtctacttcttcgaagtggtcaacccaaacgaggtcctcaacggccagaagccagtagtccgggagcgtggaccctatgtctacagggagttcagacaaaaggtcaacatcaccttcaatgacaacgacaccgtgtccttcgtggagaaccgcagcctccatttccagcctgacaagtcgcatggctcagagagtgactacattgtactgCCCAACATCCTGGTCTTGGGTGCGGCGGTGATGATGGAGAATAAGCCCATGACCCTGAAGCTCATCATGACCTTGGCATTCACCACCCTCGGCGAACGTGCCTTCATGAACCGCACTGTGGGTGAGATCATGTGGGGCTACAAGGACCCCCTTGTGAATCTCATCAACAAGTACTTTCCAGGCATGTTCCCCTTCAAGGACAAGTTCGGATTATTTGCTGAGCTCAACAACTCGaattctggggtcttcactgtcttcacgggcgtccagaatttcagcaggatccatctggtggacaaatggaacggactcagcaagatcgattattggcattcagagcagtgtaacatgatcaatgggacttccgggcagatgtgggcacccttcatgacacccgaatcctcgctggaattcttcagcccggaggcatgcaggtccatgaagctgacctacaacgaatcaagggtgtttgaaggcattcccacgtatcgcttcacggcccccgatactctgtttgccaacgggtccgtctacccacccaacgaaggcttctgcccatgccgagagtctggcattcagaatgtcagcacctgcaggtttggtgcgcctc
ApicalM synthetic gene
cgtgtccttcctcgagtaccgcaccttccagttccagccctccaagtcccacggctcggagagcgactacatcgtcatgCCCAACATCCTGGTCCTGGGGGGCTCGATATTGATGGAGAGCAAGCCTGTGAGCCTGAAGCTGATGATGACCTTGGCGCTGGTCACCATGGGCCAGCGTGCTTTTATGAACCGCACAGTTGGTGAGATCCTGTGGGGCTATGACGATCCCTTCGTGCATTTTCTCAACACGTACCTCCCAGACATGCTTCCCATAAAGGGCAAATTTGGCCTGTTTGCTGAGCTCAACAACTCCgactctgggctcttcacggtgttcacgggggtccagaacatctcgaggatccacctc
718
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Langlois et al Figure 1
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Langlois et al Figure 2
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