Plasmodium P36 determines host cell receptor usage during sporozoite invasion
Hepatocytes are the main cellular component of the liver and the first replication niche for the malaria-causing parasite Plasmodium . Malaria begins with the inoculation of sporozoites into the host skin by infected Anopheles mosquitoes . Sporozoites rapidly migrate to the liver and actively invade hepatocytes by forming a specialized compartment , the parasitophorous vacuole ( PV ) , where they differentiate into thousands of merozoites ( Ménard et al . , 2013 ) . Once released in the blood , merozoites invade and multiply inside erythrocytes , causing the malaria disease . Under natural transmission conditions , infection of the liver is an essential , initial and clinically silent phase of malaria , and therefore constitutes an ideal target for prophylactic intervention strategies . However , the molecular mechanisms underlying Plasmodium sporozoite entry into hepatocytes remain poorly understood . Highly sulphated proteoglycans in the liver sinusoids are known to bind the circumsporozoite protein , which covers the parasite surface , and contribute to the homing and activation of sporozoites ( Frevert et al . , 1993; Coppi et al . , 2007 ) . Subsequent molecular interactions leading to sporozoite entry into hepatocytes have not been identified yet . Several parasite proteins have been implicated , such as the thrombospondin related anonymous protein ( TRAP ) ( Matuschewski et al . , 2002 ) , the apical membrane antigen 1 ( AMA-1 ) ( Silvie et al . , 2004 ) , or the 6-cysteine domain proteins P52 and P36 ( van Dijk et al . , 2005; Ishino et al . , 2005; van Schaijk et al . , 2008; Kaushansky et al . , 2015; Labaied et al . , 2007 ) , however their role during sporozoite invasion remains unclear ( Bargieri et al . , 2014 ) . Our previous work highlighted the central role of the host tetraspanin CD81 , one of the receptors for the hepatitis C virus ( HCV ) ( Pileri et al . , 1998 ) , during Plasmodium liver infection ( Silvie et al . , 2003 ) . CD81 is an essential host entry factor for human-infecting P . falciparum and rodent-infecting P . yoelii sporozoites ( Silvie et al . , 2003 , 2006a ) . CD81 acts at an early step of invasion , possibly by providing signals that trigger the secretion of rhoptries , a set of apical organelles involved in PV formation ( Risco-Castillo et al . , 2014 ) . Whereas CD81 binds the HCV E2 envelope protein ( Pileri et al . , 1998 ) , there is no evidence for such a direct interaction between CD81 and Plasmodium sporozoites ( Silvie et al . , 2003 ) . Rather , we proposed that CD81 acts indirectly , possibly by regulating an as yet unidentified receptor for sporozoites within cholesterol-dependent tetraspanin-enriched microdomains ( Silvie et al . , 2006b; Charrin et al . , 2009a ) . Intriguingly , the rodent malaria parasite P . berghei can infect cells lacking CD81 ( Silvie et al . , 2003 , 2007 ) , however the molecular basis of this alternative entry pathway was until now totally unknown . Another hepatocyte surface protein , the scavenger receptor BI ( SR-BI ) , was shown to play a dual role during malaria liver infection , first in promoting parasite entry and subsequently its development inside hepatocytes ( Yalaoui et al . , 2008a; Rodrigues et al . , 2008 ) . However , the contribution of SR-BI during parasite entry is still unclear . SR-BI , which is also a HCV entry factor ( Scarselli et al . , 2002; Bartosch et al . , 2003 ) , binds high-density lipoproteins with high affinity and mediates selective cellular uptake of cholesteryl esters ( Acton et al . , 1996 ) . Yalaoui et al . reported that SR-BI is involved indirectly during P . yoelii sporozoite invasion , by regulating the levels of membrane cholesterol and the expression of CD81 and its localization in tetraspanin-enriched microdomains ( Yalaoui et al . , 2008a ) . In another study , Rodrigues et al . observed a reduction of P . berghei invasion of Huh-7 cells upon SR-BI inhibition ( Rodrigues et al . , 2008 ) . Since CD81 is not required for P . berghei sporozoite entry into Huh-7 cells ( Silvie et al . , 2007 ) , these results suggested a CD81-independent role for SR-BI . More recently , Foquet et al . showed that anti-CD81 but not anti-SR-BI antibodies inhibit P . falciparum sporozoite infection in humanized mice engrafted with human hepatocytes ( Foquet et al . , 2015 ) , questioning the role of SR-BI during P . falciparum infection . These conflicting results prompted us to revisit the contribution of SR-BI during P . falciparum , P . yoelii and P . berghei sporozoite infections . For the first time , we also explored the role of CD81 and SR-BI during hepatocyte infection by P . vivax , a widely distributed yet highly neglected cause of malaria in humans , for which the contribution of hepatocyte surface receptors has not been investigated to date . Here , we show that SR-BI is an important entry factor for P . vivax but not for P . falciparum or P . yoelii sporozoites . Remarkably , SR-BI and CD81 fulfil redundant functions during host cell invasion by P . berghei sporozoites , which can use one or the other molecule . We further investigated parasite determinants associated with host cell receptor usage . We show that genetic depletion of P52 and P36 , two members of the Plasmodium 6-cysteine domain protein family , abrogates sporozoite productive invasion and mimics the inhibition of CD81 and/or SR-BI entry pathways , in both P . berghei and P . yoelii . Finally , we identify P36 as the molecular driver of P . berghei sporozoite entry via SR-BI . Our data , by revealing a functional link between parasite and host cell entry factors , pave the way towards the identification of ligand-receptor interactions mediating Plasmodium infection of hepatocytes , and open novel perspectives for preventive and therapeutic approaches .
To evaluate the contribution of CD81 and SR-BI during P . vivax infection , we tested the effects of neutralizing CD81- and/or SR-BI- specific antibodies on P . vivax sporozoite infection in primary human hepatocyte cultures ( Mazier et al . , 1984 ) . A monoclonal antibody ( mAb ) against the main extracellular domain of CD81 , previously shown to inhibit P . falciparum sporozoite infection ( Silvie et al . , 2003 ) , had no effect on the number of P . vivax-infected cells in vitro ( Figure 1A ) . In sharp contrast , a mouse mAb specific for SR-BI ( Zahid et al . , 2013 ) greatly reduced infection , with no additive effect of anti-CD81 antibodies ( Figure 1A ) . The same inhibitory effect was observed using polyclonal anti-SR-BI antibodies ( Figure 1B ) . We performed the same experiments with P . falciparum sporozoites and found that anti-CD81 but not anti-SR-BI antibodies inhibit P . falciparum infection in vitro ( Figure 1C ) , in agreement with the in vivo data from Foquet et al ( Foquet et al . , 2015 ) . These data strongly suggest that P . vivax and P . falciparum sporozoites use distinct entry pathways to infect hepatocytes , reminiscent of the differences between P . berghei and P . yoelii ( Silvie et al . , 2003 ) . 10 . 7554/eLife . 25903 . 003Figure 1 . Anti-SR-BI antibodies inhibit P . vivax but not P . falciparum sporozoite infection . ( A ) Primary human hepatocyte cultures were incubated with P . vivax sporozoites in the presence of anti-CD81 and/or anti-SR-BI mAbs at 20 μg/ml , and the number of EEF-infected cells was determined 5 days post-infection after labeling of the parasites with anti-HSP70 antibodies . ( B ) Primary human hepatocytes were incubated with P . vivax sporozoites in the presence or absence of anti-SR-BI polyclonal rabbit serum ( diluted 1/100 ) , and the number of EEFs was determined at day 5 by immunofluorescence . ( C ) Primary human hepatocyte cultures were incubated with P . falciparum sporozoites in the presence of anti-CD81 mAb ( 20 μg/ml ) and/or anti-SR-BI polyclonal rabbit serum ( diluted 1/100 ) , and the number of EEF-infected cells was determined 5 days post-infection after labeling of the parasites with anti-HSP70 antibodies . Results from three independent experiments are shown and expressed as the percentage of control ( mean ±SD ) . ( D ) Immunofluorescence analysis of P . vivax EEFs at day 5 post-infection of primary human hepatocytes . Parasites were labeled with anti-HSP70 antibodies ( green ) , and nuclei were stained with Hoechst 33342 ( blue ) . Large EEFs and small EEFs were observed in both control and anti-SR-BI antibody-treated cultures . Scale bars , 10 μm . ( E ) Inhibitory activity of anti-SR-BI antibodies on small EEFs ( white histograms ) and large EEFs ( black histograms ) . The results from four independent experiments are shown , and are expressed as the percentage of inhibition observed with anti-SR-BI antibodies as compared to the control . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 003 Two distinct populations of P . vivax exo-erythrocytic forms ( EEFs ) could be distinguished in the infected cultures , large EEFs representing replicating schizonts , and small EEFs that may correspond to hypnozoites ( Dembele et al . , 2011 ) ( Figure 1D ) . Anti-SR-BI antibodies reduced the numbers of large and small EEFs to the same extent ( Figure 1E ) , suggesting an effect on sporozoite invasion rather than on parasite intracellular development . In order to investigate in more details the role of SR-BI during sporozoite entry , we used the more tractable rodent malaria parasite P . berghei . Indeed , P . berghei sporozoites readily infect HepG2 cells , which lack CD81 ( Charrin et al . , 2001; Berditchevski et al . , 1996 ) but express high levels of SR-BI ( Figure 2—figure supplement 1 ) , raising the possibility that this rodent parasite uses a SR-BI route to infect CD81-null cells . To test this hypothesis , P . berghei sporozoites constitutively expressing GFP ( PbGFP ) ( Manzoni et al . , 2014 ) were incubated with HepG2 cells in the presence of increasing concentrations of polyclonal anti-SR-BI rabbit antibodies . We observed a dramatic and dose-dependent reduction of the number of EEF-infected cells induced by anti-SR-BI antibodies ( Figure 2A ) . Quantification of host cell invasion by FACS demonstrated that the rabbit anti-SR-BI antibodies block infection at the invasion step ( Figure 2B ) . Similar results were obtained with polyclonal rat antibodies and a mouse mAb directed against human SR-BI ( Figure 2—figure supplement 2 ) . 10 . 7554/eLife . 25903 . 004Figure 2 . Infection of human HepG2 cells by P . berghei sporozoites depends on SR-BI . ( A ) HepG2 cells were incubated with P . berghei sporozoites for 3 hr in the absence ( Control ) or presence of increasing concentrations of rabbit polyclonal SR-BI antisera . Infected cultures were further incubated for 24 hr before quantification of EEFs-infected cells by fluorescence microscopy . ( B ) HepG2 cell cultures were infected with PbGFP sporozoites as in A , and the number of invaded cells ( GFP+ ) was quantified by FACS 3 hr post-infection . ( C ) HepG2 cells were incubated for 3 hr with PbGFP sporozoites and rhodamine-labeled dextran , in the presence or absence of anti-SR-BI antibodies . The percentage of traversed ( dextran-positive ) cells was then determined by FACS . ( D ) HepG2 cells transfected with siRNA oligonucleotides targeting SR-BI ( siSR-BI , red histogram ) or with a control siRNA ( siCont , blue histogram ) were stained with anti-SR-BI antibodies and analyzed by flow cytometry . The negative staining control is in grey . ( E ) P . berghei EEF number in HepG2 cells transfected with siRNA oligonucleotides targeting SR-BI ( siSR-BI ) or a control siRNA ( siCont ) . ( F ) HepG2 cells transfected with siRNA oligonucleotides targeting SR-BI ( siSR-BI ) or a control siRNA ( siCont ) were infected with PbGFP sporozoites and incubated for 24 hr , before measurement of the mean fluorescence intensity ( GFP MFI ) of infected ( GFP-positive ) cells by FACS . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 00410 . 7554/eLife . 25903 . 005Figure 2—figure supplement 1 . CD81 and SR-BI surface expression in HepG2 and HepG2/CD81 cells . HepG2 ( A ) and HepG2/CD81 cells ( B ) were stained with anti-CD81 ( red histograms ) or anti-SR-BI ( green histograms ) antibodies and analyzed by FACS . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 00510 . 7554/eLife . 25903 . 006Figure 2—figure supplement 2 . Anti-SR-BI antibodies neutralize P . berghei infection of HepG2 cells . Effect of anti-SR-BI rat polyclonal antibodies ( pAb ) and mouse mAb on P . berghei EEF numbers in HepG2 cells . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 00610 . 7554/eLife . 25903 . 007Figure 2—figure supplement 3 . Effect of SR-BI silencing on sporozoite cell traversal and invasion . HepG2 cells transfected with siRNA oligonucleotides targeting SR-BI ( siSR-BI ) or a control siRNA ( siCont ) were incubated for 3 hr with PbGFP sporozoites and rhodamine-labeled dextran , and the percentage of invaded cells ( GFP-positive , green bars ) and traversed cells ( dextran-positive , red bars ) was determined by FACS . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 007 As a complementary approach , we used small interfering RNA ( siRNA ) to specifically knockdown SR-BI expression in HepG2 cells ( Figure 2D ) . SR-BI silencing caused a dramatic reduction of the number of EEF-infected cells ( Figure 2E ) , but had no significant effect on the intracellular development of the few invaded parasites ( Figure 2F ) . Plasmodium sporozoites migrate through several cells before invading a final one inside a PV ( Mota et al . , 2001 ) . Sporozoite cell traversal was increased in SR-BI-depleted HepG2 cells , as compared to the control ( Figure 2—figure supplement 3 ) . This is likely due to the robust cell traversal activity of P . berghei sporozoites , which continue to migrate through cells when productive invasion is impaired . Altogether , these data establish that SR-BI is a major entry factor for P . berghei sporozoites in CD81-null HepG2 cells . We next tested whether the presence of CD81 would affect SR-BI function during P . berghei sporozoite infection . We monitored invasion and replication of P . berghei sporozoites in HepG2 cells genetically engineered to express CD81 ( HepG2/CD81 ) ( Figure 2—figure supplement 1 ) ( Silvie et al . , 2006a ) , in the presence of anti-SR-BI and/or anti-CD81 antibodies . Strikingly , unlike in CD81-null HepG2 cells , anti-SR-BI antibodies had no inhibitory effect on P . berghei infection in HepG2/CD81 cells ( Figure 3A ) . Remarkably , whilst neither anti-SR-BI nor anti-CD81 antibodies alone had any significant impact on invasion ( Figure 3A ) or parasite intracellular development ( Figure 3—figure supplement 1 ) , the combination of CD81 and SR-BI antibodies markedly reduced the number of infected cells ( Figure 3A ) . Similarly , siRNA-mediated silencing of either CD81 or SR-BI alone had no effect on infection , whereas simultaneous silencing of both factors greatly reduced infection ( Figure 3B ) . 10 . 7554/eLife . 25903 . 008Figure 3 . CD81 and SR-BI define alternative entry routes for P . berghei sporozoites . ( A ) HepG2/CD81 cells were incubated with P . berghei sporozoites in the presence or absence of anti-human CD81 and/or SR-BI mAbs , and the number of EEFs-infected cells was determined by fluorescence microscopy 24 hr post-infection . ( B ) P . berghei EEF numbers in HepG2/CD81 cells transfected with siRNA oligonucleotides targeting CD81 ( siCD81 ) and/or SR-BI ( siSR-BI ) . ( C ) HepG2/CD81 cells were incubated with PbGFP sporozoites for 15–120 min , in the presence or absence of anti-CD81 and/or anti-SR-BI antibodies , then trypsinized and directly analyzed by FACS to quantify invaded ( GFP-positive ) cells . ( D ) Protein extracts from Hepa1-6 cells and Hepa1-6 cells transiently transfected with a human SR-BI expression plasmid were analyzed by Western blot using antibodies recognizing mouse and human SR-BI ( Abcam ) or mouse CD81 ( MT81 ) . ( E ) Hepa1-6 cells were transfected first with siRNA oligonucleotides targeting endogenous mouse CD81 ( simCD81 ) , then with plasmids encoding human CD81 ( hCD81 ) or SR-BI ( hSR-BI ) . Cells were then incubated with PbGFP sporozoites , and the number of infected ( GFP-positive ) cells was determined 24 hr post-infection by FACS . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 00810 . 7554/eLife . 25903 . 009Figure 3—figure supplement 1 . Effect of anti-CD81 and anti-SR-BI antibodies on P . berghei sporozoite cell traversal and intracellular development . ( A ) HepG2/CD81 cells were incubated for 3 hr with PbGFP sporozoites and rhodamine-labeled dextran , in the presence or absence of anti-CD81 and/or SR-BI antibodies . The percentage of traversed ( dextran-positive ) cells was then determined by FACS . ( B ) HepG2/CD81 cells were infected with PbGFP sporozoites in the presence or absence of anti-CD81 or anti-SR-BI antibodies , and incubated for 24 hr before measurement of the MFI of infected ( GFP-positive ) cells by FACS . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 009 Blocking both CD81 and SR-BI was associated with an increase in cell traversal activity ( Figure 3—figure supplement 1 ) , suggesting that the concomitant neutralization of the two host factors prevented commitment to productive invasion . To directly test this hypothesis , we analysed the invasion kinetics of PbGFP sporozoites in HepG2/CD81 cells , in the presence of anti-SR-BI and/or anti-CD81 neutralizing antibodies . We have shown that in vitro sporozoite invasion follows a two-step kinetics ( Risco-Castillo et al . , 2015 ) , with initially low invasion rates at early time points , reflecting cell traversal activity , followed by a second phase of productive invasion associated with PV formation . In HepG2/CD81 cells , the invasion kinetics of P . berghei sporozoites in the presence of anti-SR-BI or anti-CD81 specific antibodies were comparable to those of the control without antibody ( Figure 3C ) . In sharp contrast , blocking both CD81 and SR-BI simultaneously suppressed the second phase of productive invasion ( Figure 3C ) . Based on these results , we conclude that SR-BI and CD81 are involved in the commitment to productive entry . P . berghei sporozoites infect mouse hepatocytic Hepa1-6 cells via a CD81-dependent pathway , as shown by efficient inhibition of infection by CD81 specific antibodies or siRNA ( Silvie et al . , 2007 ) . Interestingly , we failed to detect SR-BI in Hepa1-6 cells ( Figure 3D ) , providing a plausible explanation as to why CD81 is required for P . berghei infection in this model . We tested whether ectopic expression of SR-BI would rescue P . berghei infection of Hepa1-6 cells upon silencing of endogenous murine CD81 by siRNA . CD81-silenced Hepa1-6 cells were transiently transfected with plasmids encoding human SR-BI or CD81 , before exposure to P . berghei sporozoites . The number of infected cells was greatly reduced in CD81-silenced cells as compared to control non-silenced cells ( Figure 3E ) , in agreement with our previous observations ( Silvie et al . , 2007 ) . Remarkably , transfection of either human CD81 or human SR-BI was sufficient to rescue infection in CD81-silenced cells ( Figure 3E ) , demonstrating that the two receptors can independently perform the same function to support P . berghei infection . Collectively , our data provide direct evidence that CD81 and SR-BI play redundant roles during productive invasion of hepatocytic cells by P . berghei sporozoites . We next investigated the contribution of SR-BI to P . yoelii sporozoite infection in HepG2/CD81 cells . P . yoelii infection was dramatically reduced by anti-CD81 antibodies , consistent with our previous work ( Silvie et al . , 2006a ) , but strikingly was not affected by anti-SR-BI antibodies ( Figure 4A ) . In addition , P . yoelii infection was not affected by siRNA-mediated silencing of SR-BI , but was almost abolished upon knockdown of CD81 ( Figure 4B ) . These results indicate that CD81 but not SR-BI plays a central role during P . yoelii sporozoite invasion in HepG2/CD81 cells , similarly to P . falciparum in human hepatocytes . 10 . 7554/eLife . 25903 . 010Figure 4 . Infection of HepG2/CD81 cells by P . yoelii sporozoites depends on CD81 but not SR-BI . ( A ) HepG2/CD81 cells were incubated with P . yoelii sporozoites in the presence of anti-CD81 mAb ( 20 μg/ml ) or anti-SR-BI polyclonal rabbit serum ( diluted 1/100 ) , and the number of EEF-infected cells was determined 24 hr post-infection by fluorescence microscopy . Results from three independent experiments are shown and expressed as the percentage of control ( mean ± SD ) . ( B ) P . yoelii EEF numbers in HepG2/CD81 cells transfected with siRNA oligonucleotides targeting CD81 ( siCD81 ) and/or SR-BI ( siSR-BI ) . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 010 The data above show that P . berghei sporozoites can use either CD81 or SR-BI to infect hepatocytic cells , whereas P . yoelii utilizes CD81 but not SR-BI , suggesting that one or several P . berghei factors may be specifically associated with SR-BI usage . Among potential candidate parasite factors involved in sporozoite entry , we focused on the 6-cysteine domain proteins P52 and P36 , two micronemal proteins of unknown function previously implicated during liver infection ( van Dijk et al . , 2005; Ishino et al . , 2005; van Schaijk et al . , 2008; Kaushansky et al . , 2015; Labaied et al . , 2007; VanBuskirk et al . , 2009; Mikolajczak et al . , 2014 ) . To facilitate monitoring of the role of P52 and P36 during host cell invasion , we used a Gene Out Marker Out ( GOMO ) strategy ( Manzoni et al . , 2014 ) to generate highly fluorescent p52/p36-knockout parasite lines in P . berghei ( Figure 5—figure supplement 1 ) and P . yoelii ( Figure 5—figure supplement 2 ) . Pure populations of GFP-expressing , drug selectable marker-free PbΔp52/p36 and PyΔp52/p36 blood stage parasites were obtained and transmitted to mosquitoes in order to produce sporozoites . Analysis of the kinetics of PbΔp52/p36 sporozoite invasion by FACS , in comparison to control PbGFP sporozoites , revealed that genetic ablation of p52/p36 abrogates sporozoite productive invasion of HepG2 ( SR-BI-dependent entry pathway ) and Hepa1-6 cells ( CD81-dependent entry pathway ) ( Figure 5A and B ) . PbΔp52/p36 sporozoite invasion followed similar kinetics to those observed for PbGFP sporozoites upon blockage of SR-BI or CD81 , respectively , and was not modified by addition of anti-SRBI or anti-CD81 antibodies . Using antibodies specific for UIS4 , a marker of the PV membrane ( PVM ) that specifically labels productive vacuoles ( Risco-Castillo et al . , 2015; Mueller et al . , 2005 ) , we confirmed that PbGFP but not PbΔp52/p36 parasites could form replicative PVs , in both HepG2 and HepG2/CD81 cells ( Figure 5C ) . In both cell types , only very low numbers of EEFs were observed with PbΔp52/p36 parasites ( Figure 5D ) , all of which were seemingly intranuclear and lacked a UIS4-labeled PVM ( Figure 5E ) . We have shown before that intranuclear EEFs result from cell traversal events ( Silvie et al . , 2006a ) . Altogether these data demonstrate that PbΔp52/p36 sporozoites fail to productively invade host cells , irrespective of the entry route . Similar results were obtained with a P . berghei Δp36 single knockout line using the GOMO strategy ( Figure 5—figure supplement 3 ) . PbΔp36 sporozoites did not invade HepG2 cells or HepG2/CD81 cells , reproducing a similar phenotype as PbGFP parasites in the presence of anti-CD81 and anti-SR-BI neutralizing antibodies ( Figure 5—figure supplement 4 ) . 10 . 7554/eLife . 25903 . 011Figure 5 . The 6-cys proteins P52 and P36 are required for productive host cell invasion . ( A–B ) HepG2 ( A ) or Hepa1-6 cells ( B ) were incubated with PbGFP ( blue lines ) or PbΔp52/p36 sporozoites ( red lines ) for 15–120 min , in the presence ( dotted lines ) or absence ( solid lines ) of anti-SR-BI ( A ) or anti-CD81 ( B ) antibodies . Cells were then trypsinized and directly analyzed by FACS to quantify invaded ( GFP-positive ) cells . ( C ) HepG2 and HepG2/CD81 cells were infected with PbGFP or PbΔp52/p36 sporozoites and the number of EEFs was determined 28 hr post-infection by fluorescence microscopy . ( D ) HepG2 and HepG2/CD81 cells infected with PbGFP or PbΔp52/p36 sporozoites were fixed at 28 hr post-infection , stained with anti-UIS4 antibodies ( red ) and the nuclear stain Hoechst 33342 ( blue ) , and examined by fluorescence microscopy . Parasites were detected based on GFP expression ( green ) . Scale bars , 10 μm . ( E ) Quantification of UIS4 expression in HepG2 and HepG2/CD81 cells infected with PbGFP ( red ) or PbΔp52/p36 ( black ) . ( F ) HepG2 ( dotted lines ) and HepG2/CD81 cells ( solid lines ) were incubated with PyGFP ( blue lines ) or PyΔp52/p36 sporozoites ( red lines ) for 15–180 min , trypsinized , and directly analyzed by FACS to quantify invaded ( GFP-positive ) cells . ( G ) HepG2/CD81 cells were infected with PyGFP or PyΔp52/p36 sporozoites and the number of EEFs was determined 24 hr post-infection by fluorescence microscopy . ( H ) HepG2 and HepG2/CD81 cells infected with PyGFP or PyΔp52/p36 sporozoites were fixed at 24 hr post-infection , stained with anti-UIS4 antibodies ( red ) and the nuclear stain Hoechst 33342 ( blue ) , and examined by fluorescence microscopy . Parasites were detected based on GFP expression ( green ) . Scale bars , 10 μm . ( I ) Quantification of UIS4 expression in HepG2 and HepG2/CD81 cells infected with PyGFP ( red ) or PyΔp52/p36 ( black ) . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 01110 . 7554/eLife . 25903 . 012Figure 5—figure supplement 1 . Targeted gene deletion of p52 and p36 in P . berghei . ( A ) Replacement strategy to generate PbΔp52p36 parasites . The wild-type ( WT ) genomic locus of P . berghei p52/p36 was targeted with a GOMO-GFP replacement plasmid containing a 5’ and a 3’ homologous sequence inserted on each side of the plasmid GFP/hDHFR-yFCU/mCherry triple cassette . Upon double crossover recombination , the adjacent p52 and p36 genes are replaced by the plasmid cassettes . Subsequent recombination between the two identical PbDHFR/TS 3’ UTR sequences ( pink lollipops ) results in excision of hDHFR-yFCU and mCherry . Genotyping primers and expected PCR fragments are indicated by arrows and lines , respectively . ( B–C ) PCR analysis of genomic DNA isolated from control PbGFP and PbΔp52p36 parasites recovered after positive selection with pyrimethamine ( B ) and after negative selection with 5-fluorocytosine ( C ) . Confirmation of the predicted recombination events was achieved with primer combinations specific for 5’ integration ( 5’ integr . ) , 3’ integration ( 3’ integr . ) or 3’ integration followed by marker excision ( 3’ excised ) . Primers used for genotyping are indicated in the Materials and methods . The absence of amplification with primer combinations specific for the WT locus ( WT ) and the non-excised integrated construct ( 3’ integration ) confirms that the final populations contain only PbΔp52p36 drug-selectable marker-free P . berghei parasites . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 01210 . 7554/eLife . 25903 . 013Figure 5—figure supplement 2 . Targeted gene deletion of p52 and p36 in P . yoelii . ( A ) Replacement strategy to generate PyΔp52p36 parasites . The wild-type ( WT ) genomic locus of P . yoelii p52/p36 in the PyGFP parental line was targeted with a GOMO replacement plasmid containing a 5’ and a 3’ homologous sequence from pyp52 inserted on each side of a hDHFR-yFCU/mCherry double cassette . An additional 3’ homologous sequence from pyp36 ( 36–3’ ) was inserted immediately downstream of the 5’ homologous sequence from pyp52 ( 52–5’ ) . Upon double crossover recombination , pyp52 is replaced by the plasmid cassettes . Subsequent recombination between the two identical 36–3' sequences results in excision of hDHFR-yFCU , mCherry and pyp36 . Genotyping primers and expected PCR fragments are indicated by arrows and lines , respectively . ( B–C ) PCR analysis of genomic DNA isolated from parental PyGFP and PyΔp52p36 parasites recovered after positive selection with pyrimethamine ( B ) and after negative selection with 5-fluorocytosine ( C ) . Confirmation of the predicted recombination events was achieved with primer combinations specific for 5’ integration ( 5’ integr . ) , 3’ integration ( 3’ integr . ) or 3’ integration followed by marker excision ( 3’ excised ) . Primers used for genotyping are indicated in Materials and methods . The absence of amplification with primer combinations specific for the WT locus ( WT ) and the non-excised integrated construct ( 3’ integration ) confirms that the final populations contain only PyΔp52p36 drug-selectable marker-free P . yoelii parasites . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 01310 . 7554/eLife . 25903 . 014Figure 5—figure supplement 3 . Targeted gene deletion of p36 in P . berghei . ( A ) Replacement strategy to generate PbΔp36 parasites . The wild-type ( WT ) genomic locus of P . berghei p36 was targeted with a GOMO-GFP replacement plasmid containing a 5’ and a 3’ homologous sequence inserted on each side of the plasmid GFP/hDHFR-yFCU/mCherry triple cassette . Upon double crossover recombination , the p36 gene is replaced by the plasmid cassettes . Subsequent recombination between the two identical PbDHFR/TS 3’ UTR sequences ( pink lollipops ) results in excision of hDHFR-yFCU and mCherry . Genotyping primers and expected PCR fragments are indicated by arrows and lines , respectively . ( B–C ) PCR analysis of genomic DNA isolated from control PbGFP and PbΔp36 parasites recovered after positive selection with pyrimethamine ( B ) and after negative selection with 5-fluorocytosine ( C ) . Confirmation of the predicted recombination events was achieved with primer combinations specific for 5’ integration ( 5’ integr . ) , 3’ integration ( 3’ integr . ) or 3’ integration followed by marker excision ( 3’ excised ) . Primers used for genotyping are indicated in the Materials and methods . The absence of amplification with primer combinations specific for the WT locus ( WT ) and the non-excised integrated construct ( 3’ integration ) confirms that the final populations contain only PbΔp36 drug-selectable marker-free P . berghei parasites . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 01410 . 7554/eLife . 25903 . 015Figure 5—figure supplement 4 . P36 is required for P . berghei sporozoite entry via both SR-BI- and CD81-dependent routes . HepG2 and HepG2/CD81 cells were incubated with PbGFP or PbΔp36 sporozoites in the presence or absence of anti-CD81 and anti-SR-BI antibodies , and the percentage of infected ( GFP-positive ) cells was determined 24 hr post-infection by FACS . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 015 We then examined the kinetics of P . yoelii Δp52/p36 sporozoite invasion , in comparison to those of PyGFP sporozoites , in HepG2/CD81 versus HepG2 cells . PyΔp52/p36 sporozoites showed a lack of productive invasion in HepG2/CD81 cells , reproducing the invasion kinetics of PyGFP in the CD81-null HepG2 cells ( Figure 5F ) . The PyΔp52/p36 mutant failed to form PV in HepG2/CD81 cells ( Figure 5G ) , where only intranuclear EEFs lacking a UIS4-labeled PVM were observed , similarly to the control PyGFP parasites in HepG2 cells ( Figure 5H and I ) . Altogether , these results reveal that P52 and P36 are required for sporozoite productive invasion , in both P . berghei and P . yoelii , irrespective of the entry route . We further sought to investigate whether P52 and/or P36 proteins contribute to the selective usage of host cell receptors by different sporozoite species . We designed a trans-species genetic complementation strategy in which copies of P . berghei ( Pb ) , P . yoelii ( Py ) , P . falciparum ( Pf ) or P . vivax ( Pv ) P52 and P36 were introduced in the Δp52/p36 parasites . For this purpose , we used centromeric plasmid constructs for stable expression of the transgenes from episomes ( Iwanaga et al . , 2010 ) . Complementing PbΔp52/p36 sporozoites with PbP52 and PbP36 restored sporozoite infectivity to both HepG2 and HepG2/CD81 cells ( Figure 6A ) , where the parasite formed UIS4-positive vacuoles ( Figure 6B , C and D ) , confirming that genetic complementation was efficient . Remarkably , complementation of PbΔp52/p36 with PyP52 and PyP36 restored infection in HepG2/CD81 but not in HepG2 cells ( Figure 6A ) , where only low numbers of UIS4-negative intranuclear EEFs were observed ( Figure 6B and D ) . Thus , the concomitant replacement of PbP52 and PbP36 by their P . yoelii counterparts reproduced a P . yoelii-like invasion phenotype in chimeric P . berghei sporozoites , indicating that P52 and/or P36 contribute to the selective usage of a CD81-independent entry pathway in P . berghei sporozoites . Complementation of PbΔp52/p36 parasites with either P . falciparum or P . vivax P52 and P36 coding sequences did not restore infectivity of transgenic sporozoites , not only in HepG2 and HepG2/CD81 cells ( Figure 6—figure supplement 1A ) , but also in primary human hepatocytes , the most permissive cellular system for human malaria sporozoites in vitro ( Figure 6—figure supplement 1B ) . Hence it was not possible using this approach to assess the function of P . falciparum or P . vivax P52 and P36 in transgenic P . berghei sporozoites . 10 . 7554/eLife . 25903 . 016Figure 6 . P36 mediates CD81-independent entry in P . berghei sporozoites . ( A ) HepG2 ( blue histograms ) or HepG2/CD81 cells ( red histograms ) were incubated with sporozoites from PbΔp52/p36 parasites genetically complemented with P . berghei and/or P . yoelii P52 and P36 , and analysed by FACS or fluorescence microscopy to determine the number of GFP-positive cells 24 hr post-infection . Results from three independent experiments are shown and are expressed as the percentage of infection in comparison to control PbGFP-infected cultures ( mean ±SD ) . ( B ) Immunofluorescence analysis of UIS4 expression in HepG2 or HepG2/CD81 cells infected with genetically complemented PbΔp52/p36 sporozoites . Cells were fixed with PFA 28 hr post-infection , permeabilized , and stained with anti-UIS4 antibodies ( red ) and the nuclear stain Hoechst 33342 ( blue ) . Parasites were detected based on GFP expression ( green ) . Scale bars , 10 μm . ( C–D ) HepG2/CD81 ( C ) and HepG2 ( D ) cells were infected with PbGFP , PbΔp52/p36 and complemented PbΔp52/p36 sporozoites . The numbers of UIS4-positive ( red histograms ) and UIS4-negative ( black histograms ) EEFs were determined by fluorescence microscopy 24 hr post-infection . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 01610 . 7554/eLife . 25903 . 017Figure 6—figure supplement 1 . Genetic complementation with p52 and p36 from P . falciparum or P . vivax does not restore sporozoite infectivity in PbΔp52p36 parasites . ( A ) HepG2 and HepG2/CD81 cells were incubated with PbGFP , PbΔp52p36 and PbΔp52p36 complemented with p52 and p36 from P . falciparum or P . vivax . The percentage of infected ( GFP-positive ) cells 24 hr post-infection was determined by FACS . ( B ) Primary human hepatocytes were incubated with PbGFP , PbΔp52p36 and PbΔp52p36 complemented with p52 and p36 from P . falciparum or P . vivax . The number of EEFs was determined 24 hr post-infection by fluorescence microscopy . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 017 We next dissected the individual contribution of P52 and P36 by complementing PbΔp52/p36 parasites with mixed combinations of either PyP52 and PbP36 or PbP52 and PyP36 . This approach revealed that P36 determines the ability of P . berghei sporozoites to enter cells via a CD81-independent route . Indeed , PbΔp52/p36 complemented with PyP52 and PbP36 infected both HepG2/CD81 and HepG2 cells ( Figure 6A ) , forming UIS4-labeled PVs in both cell types ( Figure 6B , C and D ) . P52 therefore is not responsible for the phenotypic difference between P . berghei and P . yoelii . In sharp contrast , complementation of PbΔp52/p36 with PbP52 and PyP36 restored sporozoite infectivity to HepG2/CD81 but not HepG2 cells , thus reproducing a P . yoelii-like invasion phenotype ( Figure 6A , B and D ) . In reciprocal experiments , we analysed whether expression of PbP36 would be sufficient to allow P . yoelii sporozoites to invade CD81-null cells . For this purpose , we performed genetic complementation experiments in PyΔp52/p36 parasites , using the same constructs employed with the P . berghei mutant . Strikingly , whilst HepG2 cells are normally refractory to P . yoelii productive invasion , complementation with P . berghei P52 and P36 protein was sufficient to confer chimeric P . yoelii mutants the capacity to infect HepG2 cells ( Figure 7A ) . Most importantly , PyΔp52/p36 parasites complemented with PyP52 and PbP36 infected both HepG2 and HepG2/CD81 cells ( Figure 7A ) . In particular , PyΔp52/p36 parasites expressing PbP36 became capable of forming UIS4-positive PVs in HepG2 cells ( Figure 7B ) . Thus , the transgenic expression of PbP36 appears to be sufficient to recapitulate a P . berghei-like invasion phenotype in P . yoelii sporozoites . In contrast , complementation of PyΔp52/p36 parasites with PbP52 and PyP36 restored sporozoite infectivity in HepG2/CD81 cells only , but not in HepG2 cells ( Figure 7A and B ) . This confirms that P52 , although essential for sporozoite entry , is not directly associated with host receptor usage . Finally , invasion of PbP36-expressing PyΔp52/p36 sporozoites was abrogated by anti-SR-BI antibodies in HepG2 cells ( Figure 7C ) , demonstrating that P . berghei P36 is a key determinant of CD81-independent entry via SR-BI . 10 . 7554/eLife . 25903 . 018Figure 7 . Transgenic P . yoelii sporozoites expressing PbP36 can infect CD81-null cells via SR-BI . ( A ) HepG2 ( blue ) and HepG2/CD81 cells ( red ) were incubated with genetically complemented PyΔp52/p36 sporozoites , and fixed 24 hr post-infection . The number of UIS4-positive vacuoles was then determined by immunofluorescence . ( B ) Immunofluorescence analysis of UIS4 expression in HepG2 or HepG2/CD81 cells infected with sporozoites of PyΔp52/p36 parasites genetically complemented with P52 and P36 from P . berghei or P . yoelii . Cells were fixed with PFA , permeabilized , and stained with anti-UIS4 antibodies ( red ) and the nuclear stain Hoechst 33342 ( blue ) . Parasites were detected based on GFP expression ( green ) . Scale bars , 10 μm . ( C ) Quantification of UIS4 expression in HepG2 ( blue ) and HepG2/CD81 cells ( red ) infected with genetically complemented PyΔp52/p36 sporozoites and processed as in B for immunofluorescence . ( D ) HepG2 cells were incubated with PyΔp52/p36 sporozoites complemented with PbP36 and either PbP52 or PyP52 , in the presence or absence of anti-SR-BI antibodies . Infected cultures were fixed 24 hr post-infection , and the number of EEFs was then determined by fluorescence microscopy . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 018
Until now , the nature of the molecular interactions mediating Plasmodium sporozoite invasion of hepatocytes has remained elusive . Previous studies have identified CD81 and SR-BI as important host factors for infection of hepatocytes ( Silvie et al . , 2003; Yalaoui et al . , 2008a; Rodrigues et al . , 2008 ) . Still , the relation between CD81 and SR-BI and their contribution to parasite entry was unclear ( Silvie et al . , 2007; Foquet et al . , 2015 ) , and , importantly , parasite factors associated with CD81 or SR-BI usage had not been identified . Here we demonstrate that CD81 and SR-BI define independent entry pathways for sporozoites , and identify the parasite protein P36 as a critical parasite factor that determines host receptor usage during hepatocyte infection . Our data provide molecular insights into the host entry pathways used by different sporozoite species ( Figure 8A ) . We show that P . falciparum , like P . yoelii , relies on CD81 but not SR-BI , in agreement with the recent observation that antibodies against CD81 but not anti-SR-BI induce protection in humanized mice infected with P . falciparum ( Foquet et al . , 2015 ) . Our results are also consistent with the study from Yalaoui et al . showing that in primary mouse hepatocytes antibodies against SR-BI do not inhibit P . yoelii infection when co-incubated together with sporozoites ( Yalaoui et al . , 2008a ) . In the same study , the authors proposed a model where SR-BI indirectly contributes to P . yoelii infection through regulation of membrane cholesterol and CD81 expression , however our data in the HepG2/CD81 cell model with both P . yoelii and P . berghei clearly rule out a role of SR-BI during CD81-dependent sporozoite entry . For the first time , we also analysed the role of host factors during P . vivax sporozoite infection . We found that , in contrary to P . falciparum , antibodies against SR-BI but not against CD81 inhibit infection of primary human hepatocyte cultures by P . vivax sporozoites , illustrating that the two main species causing malaria in humans use distinct routes to infect hepatocytes . 10 . 7554/eLife . 25903 . 019Figure 8 . Model of host cell entry pathways for Plasmodium sporozoites . ( A ) Host cell membrane proteins CD81 and SR-BI define two independent entry routes for Plasmodium sporozoites . P . falciparum and P . yoelii sporozoites require CD81 for infection , whereas P . vivax sporozoites infect hepatocytes using SR-BI . P . berghei sporozoites can enter cells alternatively via CD81 or SR-BI . ( B ) The 6-cysteine domain protein P36 determines host cell receptor usage during P . yoelii and P . berghei sporozoite invasion . Whilst PyP36 supports only CD81-dependent sporozoite entry , PbP36 mediates sporozoite invasion through both CD81- and SR-BI-dependent pathways . ( C ) Model of the 3D structure of P . berghei P36 , established based on the crystal structure of PfP12 ( 2YMO ) . In the ribbon diagram , the tandem 6-cysteine domains are shown in blue ( D1 ) and green ( D2 ) , respectively , and the cysteine residues and disulphide bonds in yellow . The loop located between the third and fourth cysteine residues of the D2 domain ( inter-cys loop ) is indicated in red . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 01910 . 7554/eLife . 25903 . 020Figure 8—figure supplement 1 . P36 protein sequence analysis . ( A ) Alignment of P . berghei and P . yoelii P36 protein sequences . Identical , similar and different amino acids are shaded in black , grey and red , respectively . The tandem 6-cys domains D1 and D2 are indicated with blue and green lines , respectively , above the protein sequences . The six cysteine residues of each domain are indicated below the protein sequences . ( B ) Schematic representation of the tandem D1 and D2 6-cys domains of P36 , showing the disulfide bond arrangement . The position of the ‘inter-cys loop’ , located between the third and fourth cysteine residues of D2 , is indicated as a red line . ( C ) Alignment of P . falciparum , P . vivax , P . berghei and P . yoelii inter-cys loop sequences . Identical and similar amino acids are shaded in black and grey , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 25903 . 020 SR-BI and CD81 both have been shown to bind the HCV envelope glycoprotein E2 ( Pileri et al . , 1998; Scarselli et al . , 2002; Bartosch et al . , 2003 ) , and act in a sequential and cooperative manner to mediate virus entry ( Kapadia et al . , 2007 ) , together with several additional entry factors ( Colpitts and Baumert , 2016; Evans et al . , 2007; Ploss et al . , 2009 ) . By contrast , as shown here , SR-BI and CD81 operate independently during Plasmodium sporozoite infection . Remarkably , P . berghei can use alternatively CD81 or SR-BI to infect cells , which is surprising given that CD81 and SR-BI are structurally unrelated . CD81 belongs to the family of tetraspanins , which are notably characterized by their propensity to dynamically interact with other membrane proteins and organize tetraspanin-enriched membrane microdomains ( Charrin et al . , 2014 ) . CD81 might play an indirect role during Plasmodium sporozoite entry , possibly by interacting with other host receptors within these microdomains ( Silvie et al . , 2006b; Charrin et al . , 2009a , 2009b ) . Interestingly , SR-BI is structurally related to CD36 ( Neculai et al . , 2013 ) , which is known to bind several Plasmodium proteins , including P . falciparum erythrocyte membrane protein 1 ( PfEMP1 ) in the context of cytoadherence of infected erythrocytes to endothelial cells ( Baruch et al . , 1996; Oquendo et al . , 1989; Barnwell et al . , 1989 ) . CD36 was also reported to interact with P . falciparum sequestrin ( also called LISP2 ) ( Ockenhouse et al . , 1991 ) , a member of the 6-cys protein family involved in parasite liver stage development ( Orito et al . , 2013; Annoura et al . , 2014 ) , and is a major determinant of P . berghei asexual blood stage sequestration ( Franke-Fayard et al . , 2005 ) . CD36 was shown to be dispensable for mouse hepatocyte infection by P . yoelii and P . berghei sporozoites ( Sinnis and Febbraio , 2002 ) . However , it is conceivable that SR-BI , which shares a similar 3D structure as CD36 ( Neculai et al . , 2013 ) , may interact with parasite proteins expressed by sporozoites , such as the 6-cys protein P36 . The 6-cys protein family is characterized by the presence of a cysteine-rich domain , the 6-cysteine ( 6-cys ) or s48/45 domain . Plasmodium spp . possess a dozen 6-cys proteins , which perform important functions in different life cycle stages , and are often located on the parasite surface , consistent with a role in cellular interactions ( Annoura et al . , 2014 ) . Previous studies have shown that Plasmodium P52 and P36 are crucial for infection of the liver by sporozoites ( van Dijk et al . , 2005; Ishino et al . , 2005; van Schaijk et al . , 2008 ) , although it remained unclear whether their role was at the sporozoite entry step ( Ishino et al . , 2005; Labaied et al . , 2007 ) or for maintenance of the PV post-entry ( van Dijk et al . , 2005; VanBuskirk et al . , 2009; Mikolajczak et al . , 2014 ) . It should be noted that standard invasion assays , as performed in these studies , do not distinguish between sporozoite productive entry and non-productive invasion events associated with cell traversal , complicating the interpretation of phenotypic analysis of the mutants . Here , using GFP-expressing p52 and p36 mutants and a robust FACS-based invasion assay ( Risco-Castillo et al . , 2015 ) , we unequivocally establish that P . yoelii and P . berghei sporozoites lacking P52 and P36 efficiently migrate through cells but do not commit to productive invasion , reproducing the phenotype observed upon blockage of CD81 or SR-BI . Using a trans-species genetic complementation strategy , we identified P36 as a crucial parasite determinant of host receptor usage . Our data , combined with previous studies ( van Dijk et al . , 2005; Ishino et al . , 2005; van Schaijk et al . , 2008; Kaushansky et al . , 2015 ) , demonstrate that both P36 and P52 are necessary for sporozoite infection of hepatocytes , irrespective of the invasion route used by the parasite . Our study now reveals that P36 but not P52 is responsible for the phenotypic differences between P . berghei and P . yoelii sporozoites regarding host cell entry pathways . Importantly , P . berghei P36 mediates sporozoite entry via either CD81 or SR-BI , whereas P . yoelii P36 only supports CD81-dependent invasion ( Figure 8B ) . These results strongly suggest that PbP36 contains specific structural determinants that confer the ability of the protein to interact with SR-BI or SR-BI-dependent molecules . P . berghei and P . yoelii P36 proteins share 88% identity and 97% similarity in their amino acid sequence ( Figure 8—figure supplement 1 ) . Structural modelling of PbP36 , using the crystal structure of the 6-cys protein PfP12 ( Tonkin et al . , 2013 ) as a template , shows a typical beta sandwich fold for each of the tandem 6-cysteine domains ( Figure 8C ) . Most of the divergent residues between PbP36 and PyP36 are located in the second 6-cys domain ( Figure 8—figure supplement 1 ) , including in a loop located between the third and fourth cysteine residues . In this particular loop , PfP36 and PvP36 contain an inserted sequence of 21 and 3 amino acids , respectively , which may affect their binding properties and functions . Single gene deletions of p52 or p36 result in similar phenotypes as p52/p36 double knockouts , suggesting that the two proteins act in concert ( van Dijk et al . , 2005; Ishino et al . , 2005; van Schaijk et al . , 2008; Labaied et al . , 2007 ) . In P . falciparum blood stages , the two 6-cys proteins P41 and P12 interact to form stable heterodimers on the surface of merozoites ( Tonkin et al . , 2013; Taechalertpaisarn et al . , 2012; Parker et al . , 2015 ) . Whilst P12 is predicted to be GPI-anchored , P41 lacks a membrane-binding domain , similarly to P36 . By analogy , we hypothesize that P36 may form heterodimers with other GPI-anchored 6-cys proteins , including P52 . Our data show that complementation of PbΔp52/p36 parasites with P52 and P36 from P . falciparum or P . vivax does not restore sporozoite infectivity , supporting the idea that other yet unidentified parasite factors cooperate with P52 and P36 during invasion . In addition to P52 and P36 , sporozoites express at least three other 6-cys proteins , B9 , P12p and P38 ( Lindner et al . , 2013; Swearingen et al . , 2016 ) . Whereas gene deletion of p38 causes no detectable phenotypic defect in P . berghei ( van Dijk et al . , 2010 ) , B9 has been shown to play a critical role during liver stage infection , not only in P . berghei but also in P . yoelii and P . falciparum ( Annoura et al . , 2014 ) . Whether B9 , P38 and P12p associate with P52 and/or P36 and contribute to sporozoite invasion still deserves further investigations . Several 6-cys proteins have been implicated in molecular interactions with host factors . As mentioned above , sequestrin was reported to interact with CD36 ( Ockenhouse et al . , 1991 ) , although the functional relevance of this interaction remains to be determined , as sequestrin is only expressed towards the end of liver stage development ( Orito et al . , 2013 ) . Recent studies have shown that P . falciparum merozoites evade destruction by the human complement through binding of host factor H to the 6-cys protein Pf92 ( Rosa et al . , 2016; Kennedy et al . , 2016 ) . Pfs47 expressed by P . falciparum ookinetes plays a critical role in immune evasion in the mosquito midgut , by suppressing nitration responses that activate the complement-like system ( Molina-Cruz et al . , 2013; Ramphul et al . , 2015 ) . Pfs47 was proposed to act as a ‘key’ that allows the parasite to switch off the mosquito immune system by interacting with yet unidentified mosquito receptors ( ‘lock’ ) ( Molina-Cruz et al . , 2015 ) . By analogy , based on our results , one could envisage P36 as a crucial determinant of a sporozoite ‘key’ that opens SR-BI and/or CD81-dependent ‘locks’ for entry into hepatocytes . The function of P36 interaction with host cell receptors remains to be defined . P36 binding to SR-BI and/or CD81 , either directly or indirectly , may participate in a signalling cascade that triggers rhoptry secretion and assembly of the moving junction , key events committing the parasite to host cell entry ( Besteiro et al . , 2011 ) . Alternatively , P36 may induce signalling in the host cell by acting on SR-BI or other hepatocyte receptors . In this regard , Kaushansky et al . recently reported that sporozoites preferentially invade host cells expressing higher levels of the EphA2 receptor ( Kaushansky et al . , 2015 ) . Interestingly , this preference was still observed with p52/p36-deficient parasites , strongly suggesting that there is no direct link between EphA2 and P52/P36-dependent productive invasion . However , the same study showed that P36 interferes with Ephrin A1-mediated EphA2 phosphorylation ( Kaushansky et al . , 2015 ) , raising the possibility that P36 may affect EphA2 signalling indirectly , for example via SR-BI and the SRC pathway ( Naudin et al . , 2014; Mineo and Shaul , 2003 ) . In conclusion , our study reveals that host CD81 and SR-BI define two alternative pathways in human cells for sporozoite entry . Most importantly , we identified the parasite 6-cysteine domain protein P36 as a key determinant of host receptor usage during infection . These results pave the way toward the elucidation of the mechanisms of sporozoite invasion . The identification of the parasite ligands that mediate host cell entry may provide potential targets for the development of next-generation malaria vaccines . P36 is required for both CD81- and SR-BI-dependent sporozoite entry , suggesting that it may represent a relevant target in both P . falciparum and P . vivax . The understanding of host-parasite interactions may also contribute to novel therapeutic approaches . SR-BI-targeting agents have entered clinical development for prevention of HCV graft infection ( Felmlee et al . , 2016 ) . Our data suggest that SR-BI-targeting strategies may be effective to prevent establishment of the liver stages of P . vivax , including the dormant hypnozoite forms .
We used wild type P . berghei ( ANKA strain , clone 15cy1 ) and P . yoelii ( 17XNL strain , clone 1 . 1 ) , and GFP-expressing PyGFP and PbGFP parasite lines , obtained after integration of a GFP expression cassette at the dispensable p230p locus ( Manzoni et al . , 2014 ) . P . berghei and P . yoelii blood stage parasites were propagated in female Swiss mice ( 6–8 weeks old , from Janvier Labs ) . Anopheles stephensi mosquitoes were fed on P . berghei or P . yoelii-infected mice using standard methods ( Ramakrishnan et al . , 2013 ) , and kept at 21°C and 24°C , respectively . P . berghei and P . yoelii sporozoites were collected from the salivary glands of infected mosquitoes 21–28 or 14–18 days post-feeding , respectively . A . stephensi mosquitoes infected with P . falciparum sporozoites ( NF54 strain ) were obtained from the Department of Medical Microbiology , University Medical Centre , St Radboud , Nijmegen , the Netherlands . P . vivax sporozoites were isolated from A . cracens mosquitoes , 15–21 days after feeding on blood from infected patients on the Thailand-Myanmar border , as described ( Andolina et al . , 2015 ) . HepG2 ( ATCC HB-8065 ) , HepG2/CD81 ( Silvie et al . , 2006a ) and Hepa1-6 cells ( ATCC CRL-1830 ) were checked for the absence of mycoplasma contamination and cultured at 37°C under 5% CO2 in DMEM supplemented with 10% fetal calf serum and antibiotics ( Life Technologies ) , as described ( Silvie et al . , 2007 ) . HepG2 and HepG2/CD81 were cultured in culture dishes coated with rat tail collagen I ( Becton-Dickinson , Le Pont de Claix , France ) . Primary human hepatocytes were isolated and cultured as described previously ( Silvie et al . , 2004 ) . Primary human hepatocytes ( 5 × 104 per well in collagen-coated 96-well plates ) were infected with P . vivax or P . falciparum sporozoites ( 3 × 104 per well ) , as described ( Silvie et al . , 2004 ) , and cultured for 5 days before fixation with cold methanol and immunolabeling of EEFs with antibodies specific for Plasmodium HSP70 ( Tsuji et al . , 1994 ) . Nuclei were stained with Hoechst 33342 ( Life Technologies ) . Host cell invasion by GFP-expressing P . berghei and P . yoelii sporozoites was monitored by flow cytometry ( Prudêncio et al . , 2008 ) . Briefly , hepatoma cells ( 3 × 104 per well in collagen-coated 96-well plates ) were incubated with sporozoites ( 5 × 103 to 1 × 104 per well ) . At different time points , cell cultures were washed , trypsinized and analyzed on a Guava EasyCyte 6/2L bench cytometer equipped with a 488 nm laser ( Millipore ) , for detection of GFP-positive cells . To assess liver stage development , HepG2 or HepG2/CD81 cells were infected with GFP-expressing sporozoites and cultured for 24–36 hr before analysis either by FACS or by fluorescence microscopy , after fixation with 4% PFA and staining with antibodies specific for UIS4 ( Sicgen ) and Hoechst 33342 . For antibody-mediated inhibition assays , we used polyclonal antisera against human SR-BI raised after genetic immunization of rabbits and rats ( Maillard et al . , 2006; Zeisel et al . , 2007 ) , and monoclonal antibodies against human SR-BI ( NK-8H5-E3 ) ( Zahid et al . , 2013 ) , human CD81 ( 1D6 , Abcam ) or mouse CD81 ( MT81 ) ( Silvie et al . , 2006b ) . We used small double stranded RNA oligonucleotides targeting human CD81 ( 5’-GCACCAAGTGCATCAAGTA-3’ ) , human SR-BI ( 5’-GGACAAGTTCGGATTATTT-3’ ) or mouse CD81 ( 5’-CGTGTCACCTTCAACTGTA-3’ ) . An irrelevant siRNA oligonucleotide targeting human CD53 ( 5’-CAACTTCGGAGTGCTCTTC-3’ ) was used as a control . Transfection of siRNA oligonucleotides was performed by electroporation , as described ( Silvie et al . , 2006a ) . Following siRNA transfection , cells were cultured for 48 hr before flow cytometry analysis or sporozoite infection . Transfection of pcDNA3 plasmids encoding human CD81 ( Yalaoui et al . , 2008b ) or SR-BI ( Maillard et al . , 2006 ) was performed 24 hr after siRNA using the Lipofectamine 2000 reagent ( Invitrogen ) according to the manufacturer’s specifications . Following plasmid transfection , cells were cultured for an additional 24 hr before sporozoite infection . PbΔp52p36 and PbΔp36 mutant parasites were generated using a 'Gene Out Marker Out’ strategy ( Manzoni et al . , 2014 ) . For generation of PbΔp52p36 parasites , a 5’ fragment of PbP52 gene ( PBANKA_1002200 ) and a 3’ fragment of PbP36 gene ( PBANKA_1002100 ) were amplified by PCR from P . berghei ANKA WT genomic DNA , using primers PbP52rep1for ( 5’-TCCCCGCGGAATCGTGATGCTATGGATAACGTAACAC-3’ ) , PbP52rep2rev ( 5’-ATAAGAATGCGGCCGCAAAAAGAGACAAACACACTTTGTGAACACC-3’ ) , PbP36rep3for ( 5’-CCGCTCGAGTTAATATGTGATGTGTGTAGAAGAGTGAGG-3’ ) and PbP36rep4rev ( 5’-GGGGTACCTTGATATACATGCAACTTTTCACATAGG-3’ ) , and inserted into SacII/NotI and XhoI/KpnI restriction sites , respectively , of the GOMO-GFP vector . For generation of PbΔp36 parasites , a 5’ and a 3’ fragment of PbP36 gene were amplified by PCR from P . berghei ANKA WT genomic DNA , using primers PbP36repAfor ( 5’-AGCTGGAGCTCCACCGCGGGAAAAAAGGTTAACACATATATTGAAAAGC-3’ ) , PbP36rep-Arev ( 5’-CGGCTGAGGGCGGCCGCAATCAAAAAAAATAATAAAAACAAATATATGTACACTCG-3’ ) , and PbP36repBfor ( 5’-ATTAATTTCACTCGAGTATGTGATGTGTGTAGAAGAGTGAGG-3’ ) and PbP36repBrev ( 5’-TATAGGGCGAATTGGGTACCGCACGCCGGAAAAATTACAATACAAATGG-3’ ) , and inserted into SacII/NotI and XhoI/KpnI restriction sites , respectively , of the GOMO-GFP vector using the In-Fusion HD Cloning Kit ( Clontech ) . For generation of PyΔp52p36 parasites , 5’ and 3’ fragments of PyP52 gene ( PY17X_1003600 ) and a 3’ fragment of PyP36 gene ( PY17X_1003500 ) were amplified by PCR from P . yoelii 17XNL WT genomic DNA , using primers PyP52rep1for ( 5’-TCCCCGCGGAATCGCCATGCTATGGATAGTGTAGC-3’ ) , PyP52rep2rev ( 5’-ATAAGAATGCGGCCGCCATTGAAGGGGGGAACAAATCGACG-3’ ) , PyP52rep3for ( 5’-CCGCTCGAGTCAATATATGCCCACTATTCGAATTTTTGG-3’ ) , PyP52rep4rev ( 5’-GGGGTACCTTATTGATATGCATGCAACTTTCACATAGG-3’ ) , PyP36repFor ( 5’-ATAAGAATGCGGCCGCAAAATGCAAGGCGCCCGTTTAGAACC-3’ ) and PyP36repRev ( 5’-CCGGAATTCACAAAAAGATGCTACTGTGAAAAGCTCACC-3’ ) . The fragments were inserted into SacII/NotI , XhoI/KpnI and NotI/EcoRI restriction sites , respectively , of a GOMO vector backbone containing mCherry and hDHFR-yFCU cassettes . The resulting targeting constructs were verified by DNA sequencing ( GATC Biotech ) , and were linearized with SacII and KpnI before transfection . Wild type P . berghei ANKA blood stage parasites were transfected with pbp52p36 and pbp36 targeting constructs using standard transfection methods ( Janse et al . , 2006 ) . GFP-expressing parasite mutants were isolated by flow cytometry after positive and negative selection rounds , as described ( Manzoni et al . , 2014 ) . PyGFP blood stage parasites were transfected with a pyp52pyp36 targeting construct and a GFP-expressing drug selectable marker-free PyΔp52p36 mutant line was obtained using a two steps ‘Gene Out Marker Out’ strategy . Correct construct integration was confirmed by analytical PCR using specific primer combinations . For genetic complementation experiments , we used centromeric plasmids to achieve stable transgene expression from episomes ( Iwanaga et al . , 2010 ) . Complementation plasmids were obtained by replacing the GFP cassette of pCEN-SPECT2 plasmid ( kindly provided by Dr S . Iwanaga ) with a P52/P36 double expression cassette . Four complementing plasmids were generated , allowing expression of PbP52/PbP36 , PyP52/PyP36 , PbP52/PyP36 or PyP52/PbP36 . The centromeric plasmid constructs were assembled by In-Fusion cloning of 4 fragments in two-steps , into KpnI/SalI restriction sites of the pCEN-SPECT2 plasmid . For this purpose , fragments corresponding to the promoter region of PbP52 ( insert A , 1 . 5 kb ) , the open reading frame ( ORF ) of PbP52 ( insert B , 1 . 7 kb ) , the 3’ untranslated region ( UTR ) of PbP52 and promoter region of PbP36 ( insert C , 1 . 5 kb ) , the ORF and 3’ UTR of PbP36 ( insert D , 2 kb ) , the promoter region of PyP52 ( insert E , 1 . 5 kb ) , the open reading frame ( ORF ) of PyP52 ( insert F , 1 . 7 kb ) , the 3’ untranslated region ( UTR ) of PyP52 and promoter region of PyP36 ( insert G , 1 . 6 kb ) , and the ORF and 3’ UTR of PyP36 ( insert H , 2 kb ) were first amplified by PCR from P . berghei or P . yoelii WT genomic DNA , using the following primers: Afor ( 5’-TATAGGGCGAATTGGGTACCTTCACATGCATAAACCCGAAGTGTGC-3’ ) , Arev ( 5’-GAAAAAAGCAGCTAGCTTGCTTTAATGTAGAAAAAATATTTATGGATTTGG-3’ ) , Bfor ( 5’-ATTAAAGCAAGCTAGCAATATTACATTTGTGGTAAGGTAAAAC-3’ ) , Brev ( 5’-GAAGAGGTACCAAAAAGGTTTTGCCAAAATG-3’ ) , Cfor ( 5’-TTTTGGTACCTCTTCTTCTTATTATGAGG-3’ ) , Crev ( 5’-GAAAAAAGCAGCTAGCAGAAAGAAACAACAGTTATCGTAATAAAG-3’ ) , Dfor ( 5’-GCTAGCTGCTTTTTTCTTGAATCGACAATTATAATACTGAGGC-3’ ) , Drev ( 5’-TACAAGCATCGTCGACATTGCCATTACAATATGCTATAATCTG-3’ ) , Efor ( 5’-TATAGGGCGAATTGGGTACCTGCACATGCATAAACTCGAAGTGTGC-3’ ) , Erev ( 5’-AAAAAAGCAGCTAGCTTGCTTTAATGTAGAAAAAATATTTATGTATTTGG-3’ ) , Ffor ( 5’-ATTAAAGCAAGCTAGAATATTGCATTTGTGGTAAGGCAAATC-3’ ) , Frev ( 5’-GAAGACGTACCAAACATATTTTGCCAAAATG-3’ ) , Gfor ( 5’-GTTTGGTACGTCTTCTTCTTATTATGAGG-3’ ) , Grev ( 5’-GAAAAAAGCAGCTAGGATAACTGTCGATTCAAAGAAACAACC-3’ ) , Hfor ( 5’-GCTAGCTGCTTTTTTATACTTGAAGCATTTTTGTTGACTCTACC-3’ ) , Hrev ( 5’-TACAAGCATCGTCGACATTACCATTACGATATGCTATAATCTG-3’ ) . Cloning of fragments A and D followed by B and C into the pCEN vector resulted in the PbP52/PbP36 complementation plasmid . Cloning of fragments E and H followed by F and G into the pCEN vector resulted in the PyP52/PyP36 complementation plasmid . Cloning of fragments A and D followed by F and G into the pCEN vector resulted in the PyP52/PbP36 complementation plasmid . Cloning of fragments E and H followed by B and C into the pCEN vector resulted in the PbP52/PyP36 complementation plasmid . The centromeric plasmids for expression of P . falciparum and P . vivax P52 and P36 were assembled by In-Fusion cloning of 5 fragments in two steps . For this purpose , fragments corresponding to the promoter region of PbP52 ( insert B1 , 1 . 9 kb ) , the 3’ UTR of PbP52 and promoter region of PbP36 ( insert B2 , 1 . 6 kb ) , the 3’ UTR of PbP36 ( insert B3 , 2 kb ) , the ORF of PfP52 ( insert F1 , 1 . 4 kb ) , the ORF of PfP36 ( insert F2 , 1 . 1 kb ) , the ORF of PvP52 ( insert V1 , 1 . 4 kb ) and the ORF of PvP36 ( insert V2 , 1 . 1 kb ) , were first amplified by PCR from P . berghei , P . falciparum or P . vivax genomic DNA , using the following primers: B1for ( 5’-tatagggcgaattgggtaccTTCACATGCATAAACCCGAAGTGTGC-3’ ) , B1rev ( 5’-gctagcTTACTATTATTCTCAAAATGTGTATCACATTG-3’ ) , B2for ( 5’-ATCACAATATGTGCATAGTGTCAATATGCC-3’ ) , B2rev ( 5’-AATCAAAAAAAATAATAAAAACAAATATATGTACACTCG-3’ ) , B3for ( 5’-TAATAGTAAgctagcTATGTGATGTGTGTAGAAGAGTGAGGGAG-3’ ) , B3rev ( 5’-tacaagcatcgtcgacATTGCCATTACAATATGCTATAATCTG-3’ ) , F1for ( 5’-ATAATAGTAAgctagcAAAATGTATGTATTGGTGCTTATTCATATGTG-3’ ) , F1rev ( 5’-TGCACATATTGTGATTTATGTTGAATATATATATATTAAAAATATGAATAATATTAAG-3’ ) , F2for ( 5’-TTATTTTTTTTGATTATGGCTTATAATATTTGGGAGGAATATATAATGG-3’ ) , F2rev ( 5’-ACATCACATAgctagcCTAACTTTCTACAGTTTTATTTATGTTAAATAAACC-3’ ) , V1for ( 5’-ATAATAGTAAgctagcAAAATGAGGCGGATTCTGCTGGGCTGCTTGG-3’ ) , V1rev ( 5’-TGCACATATTGTGATTTACAGGGACGAGAAACCCGCGTAG-3’ ) , V2for ( 5’-TTATTTTTTTTGATTATGAGCACATGCCTTCCAGTAGTGTGG-3’ ) , and V2rev ( 5’-ACATCACATAgctagcTCACACCGCTTCAACCGCTGCG-3’ ) . An intermediate vector was first assembled by cloning inserts B1 and B3 into KpnI/SalI restriction sites of the pCEN-SPECT2 plasmid . Subsequently , In-Fusion cloning of inserts F1 , B2 and F2 or inserts V1 , B2 and V2 into the NheI restriction site of the intermediate vector resulted into PfP52/PfP36 and PvP52/PvP36 expression centromeric plasmids , respectively . All constructs were verified by DNA sequencing ( GATC Biotech ) before transfection . For double crossover replacement of P52 and P36 genes and generation of the PbΔp52p36 , PbΔp36 and PyΔp52p36 parasite lines , purified schizonts of wild type P . berghei ANKA or PyGFP were transfected with 5–10 μg of linearized construct by electroporation using the AMAXA NucleofectorTM device ( program U33 ) , as described elsewhere ( Janse et al . , 2006 ) , and immediately injected intravenously in the tail of one mouse . The day after transfection , pyrimethamine ( 70 or 7 mg/L for P . berghei and P . yoelii , respectively ) was administrated in the mouse drinking water , for selection of transgenic parasites . Pure transgenic parasite populations were isolated by flow cytometry-assisted sorting of GFP and mCherry-expressing blood stage parasites on a FACSAria II ( Becton-Dickinson ) , transferred into naïve mice , treated with 1 mg/ml 5-fluorocytosine ( Meda Pharma ) in the drinking water , and sorted again for selection of GFP+ parasites only , as described ( Manzoni et al . , 2014 ) . In the case of the PyΔp52p36 mutant , GFP+ mCherry+ recombinant parasites were first cloned by injection of limiting dilutions into mice prior to the negative selection step . For genetic complementation of the mutants , purified schizonts of PbΔp52p36 and PyΔp52p36 parasites were transfected with 5 μg of centromeric plasmids , followed by positive selection of transgenic parasites with pyrimethamine , as described above . Parasite genomic DNA was extracted using the Purelink Genomic DNA Kit ( Invitrogen ) , and analysed by PCR using primer combinations specific for WT and recombined loci . For genotyping of PbΔp52p36 parasites , we used primer combinations specific for the WT Pbp52 locus ( 5’-AATGAGATGTCAAAAAATATAGTGCTTCC-3’ and 5’-AAATGAGCAGTTTCTTCTACGTTGTTTCC-3’ ) , for the 5’ region of the recombined locus ( 5’-TATGTTTGGAATATCAGGACAAGGCATGG-3’ and 5’-TAATAATTGAGTCTTTAGTAACGAATTGCC-3’ ) , and for the 3’ region of the recombined locus before ( 5’-ATCGTGGAACAGTACGAACGCGCCGAGG-3’ and 5’-ATTGGACGTTTATTATTATTGCAAAAGCG-3’ ) or after excision of the selectable marker ( 5’-GATGGAAGCGTTCAACTAGCAGACC-3’ and 5’-ATTGGACGTTTATTATTATTGCAAAAGCG-3’ ) . For genotyping of PbΔp36 parasites , we used primer combinations specific for the WT Pbp36 locus ( 5’-GAGTTCGCACGCCATATTAACACG-3’ and 5’-CCATGATGAGATGCTAAATCGGG-3’ ) , for the 5’ region of the recombined locus ( 5’-GGAAGCATCATACAAAAAAGAAAGC-3’ and 5’-TAATAATTGAGTCTTTAGTAACGAATTGCC-3’ ) , and for the 3’ region of the recombined locus before ( 5’-ATCGTGGAACAGTACGAACGCGCCGAGG-3’ and 5’-CGTTATCTCTTTTTTTACTCATTAAGTATTG-3’ ) or after excision of the selectable marker ( 5’-GATGGAAGCGTTCAACTAGCAGACC-3’ and 5’-CGTTATCTCTTTTTTTACTCATTAAGTATTG-3’ ) . For genotyping of PyΔp52p36 parasites , we used primer combinations specific for the WT PyP52 locus ( 5’-ACTATATTTCAATTGGAGACATGTGG-3’ and 5’-ATGCAAAAAAAAGTTATCATTGCTAGTTGG-3’ ) , for the 5’ region of the recombined locus ( 5’-GTATGTTTGGAATGCCAGGATATGACATGG-3’ and 5’-CCGGAATTCACAAAAAGATGCTACTGTGAAAAGCTCACC-3’ ) , and for the 3’ region of the recombined locus before ( 5’-AGTTACACGTATATTACGCATACAACGATG-3’ and 5’-TAAGCATATATTGTATATTTGCCTTGTCC-3’ ) or after excision of the selectable marker ( 5’-GTATGTTTGGAATGCCAGGATATGACATGG-3’ and 5’-AATCTGATATGATAAATTATGGTATTGGAC-3’ ) . Amino-acid sequences of the P36 proteins from P . falciparum ( 379 aa , gi:296004390 , PF3D7_0404400 ) , P . vivax ( 320 aa , gi:156094683 , PVX_001025 ) , P . berghei ( 352 aa , gi:991456178 , PBANKA_1002100 ) and P . yoelii ( 356 aa , gi:675237743 , PY17X_1003500 ) were obtained from Genbank . Sequence alignments were carried out using Clustal Omega ( http://www . ebi . ac . uk/Tools/msa/clustalo/ ) . A 3D model of P . berghei P36 was generated by the prediction program I-Tasser ( Yang et al . , 2015 ) , using the 3D structure of Pf12 ( Pf12short in ref [Tonkin et al . , 2013] ) , which contains two 6-cys domains D1 and D2 arranged in tandem , as a template ( PDB access code 2YMO ) . The 3D model for PbP36 was then superimposed to the template Pf12short and visually inspected using the program Coot ( Emsley and Cowtan , 2004 ) , and the rotamers for the Cys residues adjusted such that the three disulfides bonds for each domain were formed following the pattern C1-C2 , C3-C6 and C4-C5 . Statistical significance was assessed by non-parametric analysis using the Mann-Whitney U and Kruskal-Wallis tests . All statistical tests were computed with GraphPad Prism 5 ( GraphPad Software ) . Significance was defined as p<0 . 05 ( ns , statistically non-significant; *p<0 . 05; **p<0 . 01 ) . In vitro experiments were performed at least three times , with a minimum of three technical replicates per experiment . All animal work was conducted in strict accordance with the Directive 2010/63/EU of the European Parliament and Council ‘On the protection of animals used for scientific purposes’ . The protocol was approved by the Charles Darwin Ethics Committee of the University Pierre et Marie Curie , Paris , France ( permit number Ce5/2012/001 ) . Blood samples were obtained from P . vivax-infected individuals attending the Shoklo Malaria Research Unit ( SMRU ) clinics on the western Thailand-Myanmar border , after signature of a consent form ( Andolina et al . , 2015 ) . Primary human hepatocytes were isolated from healthy parts of human liver fragments , which were collected from adult patients undergoing partial hepatectomy ( Service de Chirurgie Digestive , Hépato-Bilio-Pancréatique et Transplantation Hépatique , Groupe Hospitalier Pitié-Salpêtrière , Paris , France ) . The collection and use of this material were undertaken in accordance with French national ethical guidelines under Article L . 1121–1 of the ‘Code de la Santé Publique’ , and approved by the Institutional Review Board ( Comité de Protection des Personnes ) of the Centre Hospitalo-Universitaire Pitié-Salpêtrière , Assistance Publique-Hôpitaux de Paris , France .
Plasmodium sporozoites , the mosquito-transmitted forms of the malaria parasite , first infect the liver for an initial round of replication before the emergence of pathogenic blood stages . Sporozoites represent attractive targets for antimalarial preventive strategies , yet the mechanisms of parasite entry into hepatocytes remain poorly understood . Here we show that the two main species causing malaria in humans , Plasmodium falciparum and Plasmodium vivax , rely on two distinct host cell surface proteins , CD81 and the Scavenger Receptor BI ( SR-BI ) , respectively , to infect hepatocytes . By contrast , CD81 and SR-BI fulfil redundant functions during infection by the rodent parasite P . berghei . Genetic analysis of sporozoite factors reveals the 6-cysteine domain protein P36 as a major parasite determinant of host cell receptor usage . Our data provide molecular insights into the invasion pathways used by different malaria parasites to infect hepatocytes , and establish a functional link between a sporozoite putative ligand and host cell receptors .
Malaria is an infectious disease that affects millions of people around the world and remains a major cause of death , especially in Africa . It is caused by Plasmodium parasites , which are transmitted by mosquitoes to mammals . Once in the mammal , the parasites infect liver cells , where they multiply . Previous studies have suggested that proteins on the surface of the liver cells and on the parasite affect how Plasmodium infects liver cells . Understanding how these proteins enable the parasites to enter the cells may help researchers to develop treatments that interrupt the parasite life cycle and prevent infection . Manzoni et al . have now investigated how different malaria parasite species interact with liver cells . The main parasite species that infect humans are Plasmodium falciparum in Africa and Plasmodium vivax outside Africa . Manzoni et al . found that P . falciparum and P . vivax infect human liver cells by two different routes: P . falciparum interacts with a liver cell protein called CD81 , and P . vivax interacts with a liver cell protein called SR-BI . Further experiments that used mutant forms of malaria parasites that infect mice showed that a parasite protein called P36 determines which liver cell protein the parasite will interact with . The next step is to understand how P36 interacts with the liver cell proteins and to identify other parasite proteins that help Plasmodium to invade cells . In the future , such knowledge may help to develop a highly effective malaria vaccine .