The journey of the malaria sporozoite through its hosts: two parasite proteins leadthe way

Review

The journey of the malaria sporozoitethrough its hosts: two parasite proteins lead

the wayRobert Ménard

Department of Medical and Molecular Parasitology, NYU School of Medicine, New York, NY, USA

ABSTRACT – Malaria is transmitted to a mammalian host when the sporozoite stage of thePlasmodium parasite is injected by a mosquito vector. Sporozoites are unique in being able to interactwith both hosts. Formed and released in the mosquito midgut, sporozoites bind to the salivary glandsand invade their secretory cells. Once injected into the mammalian host, they home to the liver andinvade hepatocytes. Recent work has shown that two sporozoite surface proteins, CS and TRAP, act inboth hosts, perform multiple functions, and are each essential for the parasite at more than one step ofits life cycle. © 2000 Éditions scientifiques et médicales Elsevier SAS

malaria / cell invasion / gliding motility / CS / TRAP

Malaria remains one of the most important infectiousdiseases in the world. Its incidence has increased in thepast few years, and resistance of the causative Plasmo-dium parasite to available drugs is now widespread.Despite this alarming situation, our knowledge of theinfectious process at a molecular level is still limited. Theparasite has a complex life cycle (figure 1), in which onlythe red blood cell (RBC) phase can, in some plasmodialspecies, be completed in vitro. Plasmodium was the lastprotozoan of major medical importance (after species ofLeishmania, Trypanosoma, Toxoplasma, and Entamoeba)to become, in 1996, genetically transformable [1, 2].Because transformation takes place in RBC stages of theparasite, it has not been possible to study essential eventsoccurring during parasite replication in RBCs, which leadto all the symptoms and complications of the disease.Conversely, the bases of the infectious process by otherparasite forms, including the infective mosquito stage, arenow open to investigation.

1. The sporozoite stageThe genus Plasmodium is a member of the Apicom-

plexa phylum of protozoa that contains more than 5 000named species, many of which are obligate intracellularparasites. The phylum includes other human pathogenslike Toxoplasma and Cryptosporidium, which cause oppor-tunistic infections. Invasive stages of Apicomplexa, whichare almost always motile, exhibit a remarkably conservedstructural organization. They are polarized cells, with a

typical crescent shape. Their anterior pole (apical com-plex) contains secretory organelles, called rhoptries andmicronemes, which store and secrete their contents duringcell invasion through a narrow duct at the anterior tip ofthe parasite.

The Plasmodium sporozoite is a uninucleated cell gen-erated by budding from an oocyst, a multinucleated stageof the parasite that develops in the insect midgut (figure 2).Sporozoites are released by a mature oocyst into thehemolymph, the fluid that bathes the body cavity of themosquito, and are carried to the salivary glands. They thenselectively bind to, and invade the salivary glands. Onceinjected into the blood circulation of the mammalian hostduring the mosquito blood meal, sporozoites are arrestedin the liver and rapidly invade hepatocytes. The intracel-lular sporozoite then generates over two to ten days,depending on the species, thousands of merozoites thatwill invade RBCs.

Three molecules have been identified that are expressedspecifically in the sporozoite stage of the parasite: CS(circumsporozoite protein [3, 4]), TRAP (thrombospondin-related anonymous protein [5]), and sporozoite threonine-and asparagine-rich protein [6]. Only CS and TRAP havebeen extensively studied, including using the moleculargenetic techniques developed in Plasmodium berghei, aplasmodial species that infects rodents and therefore allowsin vivo analysis of parasite-liver interactions. These twoproteins emerge as multifunctional proteins essential formany steps of the sporozoite’s life: formation inside

Microbes and Infection, 2, 2000, 633−642© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved

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oocysts, adhesion to target organs, substrate-dependentgliding motility, and invasion into host cells.

2. The CS protein

The CS protein is the most abundant protein in sporo-zoites present in the salivary glands of mosquitoes [7]. Itsstructure is schematized in figure 3. CS, which ends by ahydrophobic sequence, is most likely anchored to theparasite membrane via a glycosylphosphatidylinositolgroup. It contains, as do most plasmodial surface proteins,a large immunodominant domain that consists of repeatsof a short, species-specific, peptide motif. On either side ofthis repeat domain are two sequences highly conserved inCS proteins of the different plasmodial species. Region Iencompasses an invariable KLKQP sequence and region IIis homologous to the type I repeat of thrombospondin(TSR). The TSR is a ∼ 60 residue-long module found in asuperfamily of adhesive proteins that includes thrombo-spondin, properdin, complement factors C6 to C9, andproteins involved in axon growth in Caenorhabdtis elegansand vertebrates. A basic function of the TSR is to mediatebinding to sulfatide and sulfated glycoconjugates [8].

3. CS and sporozoite homing to the liverof the mammalian host

Sporozoite arrest in the liver is an efficient process.Only a small number of sporozoites are injected into thehost during the mosquito’s blood meal (estimated at 5 to50 in laboratory-infected mosquitoes), and sporozoitescan be found in hepatocytes only two minutes after injec-tion in rodents. The route that sporozoites take to invadehepatocytes, however, is unknown. They may traverse thefenestrated endothelium, despite the much smaller diam-eter of the fenestrae (0.1 µm) than of the sporozoites (1µm), or directly invade endothelial cells. The phagocyticKuppfer cells that line the sinusoids are unlikely to consti-tute a port of entry, since they interact poorly with sporo-zoites in vitro and their depletion has little effect onsporozoite infectivity in vivo [9]. After leaving the circula-tion, sporozoites must also cross the space of Disse, aloose extracellular matrix, before making contact with thebasolateral pole of hepatocytes.

The participation of CS in sporozoite infection of hepa-tocytes was first suggested by the neutralizing activity ofantibodies to the CS repeats on sporozoite infectivity inrodents [10]. Staining of liver sections revealed that CS

Figure 1. Life cycle of the Plasmodium parasite. 1. The sporozoite stage is transmited by the mosquito to the mammalian host during ablood meal. 2. Sporozoites rapidly invade hepatocytes and transform into liver schizonts [or exo-erythrocytic forms of the parasite]. 3.When mature, the liver schizont ruptures and releases up to 30 000 merozoites. 4. Each merozoite can invade a RBC and develop into aRBC-stage schizont. 5. When mature, the RBC-stage schizont ruptures and releases ∼ 10 to 20 merozoites, each of which can initiate a newerythrocytic cycle or develop within the erythrocyte into sexual stages, the gametocytes. 6. Shortly after a mosquito ingests gametocyte-infected erythrocytes, gametocytes are liberated from RBC and fertilize into a diploid zygote. 7. The zygote rapidly transforms into a motileookinete. 8. The ookinete traverses the midgut epithelial cells and transforms, between epithelial cells and the basal lamina of the midgut,into an oocyst. The oocyst generates, in 5–15 days, 5–10 000 haploid sporozoites. Sporozoites are liberated into the hemocele (body cavity),are carried to the salivary glands by the hemolymph and eventually traverse the secretory cells of the salivary glands. They then awaittransmission via the next mosquito blood meal.

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634 Microbes and Infection2000, 633-642

Figure 2. Formation of the Plasmodium sporozoite inside oocysts. A-C. Sporozoite budding inside an oocyst. A. Oocysts can be seen in theguts of mosquitoes 4 to 5 days after the infective blood meal. The oocyst (oo) is a large, multinucleated cell limited from mosquito tissue(mo) by a thick, electron-dense capsule (ca), which frequently protrudes in the body cavity. B. The oocyst outer membrane then retractsfrom the capsule and invaginates into the oocyst cytoplasm, subdividing the oocyst into several sporoblasts (spr). At this developmentalstage, budding sites (arrows) are revealed by the densification of the oocyst outer membrane. C. A budding process from the periphery ofsporoblasts generates sporozoites, which are typically 10 µm-long, 1 µm-wide, crescent-shaped, uninucleated cells. D-E. Longitudinalsections of sporozoites. Like most invasive stages of Apicomplexa, sporozoites are limited by a tri-membranous pellicle composed of anouter membrane (om) and a double inner membrane (dim). The pellicle is apposed onto a microtubule (mt) structure, which maintains theform and the rigidity of the cell. The anterior pole of the sporozoite contains electron-dense secretory vesicles, micronemes and rhoptries.E. The club-shaped rhoptries (Rh) secrete their content through the anterior, typically truncated tip of the sporozoite.

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binds specifically to the basolateral pole of the hepatocytemembrane exposed to the circulating blood [11]. Recom-binant CS protein injected intravenously into mice wereshown to rapidly enter the space of Disse and bind to thebasolateral surface of hepatocytes, but not to endothelialor Kuppfer cells [12].

The cell-binding properties of CS were shown to bedependent on its TSR. Only TSR-containing recombinantCS bound to frozen liver sections or to fixed HepG2 cells,and synthetic peptides representing the TSR, but not otherregions of CS, inhibited sporozoite invasion into HepG2cells [11, 13]. The residues of the TSR crucial for thecell-binding activity of CS have been mapped using TSR-derived peptides and various cell lines (see figure 3). Theconserved CSVTCG sequence, which has been reported tomediate cell adhesion [14] and act as a CD36-bindingdomain [15], does not appear to be important [13]. Incontrast, the downstream basic residues play a key role inthe cell-binding activity of CS [13, 16]. Moreover, thebinding activity of the TSR-containing peptides increaseswith the degree of aggregation, suggesting that assembly ofmultimers of the downstream basic residues may increaseaffinity to their receptors. In agreement with this, CS mul-timers, but not monomers, bind to hepatocytes in vivo[12].

The nature of the CS receptor(s) has been the subject ofmuch investigation, and there is now ample evidence thatCS binds to glycosaminoglycan (GAG) chains of heparansulfate proteoglycans (HSPGs) on the surface of culturedcells. The relevance of the CS-HSPG interaction for sporo-zoite infectivity is supported by the inhibition of sporozo-ite infectivity to mice by a subset of sulfated glycoconju-gates [11, 17]. CHO cells treated with heparitinase [18], or

mutant CHO cells deficient in HS or GAG biosynthesis[13, 17], do not bind CS. Also, CS and the physiologicalsubstrates of hepatic HSPGs, such as lipoprotein remnantsand lactoferrin, target the same set of HSPGs on thesurface of HepG2 cells and compete for clearance fromthe circulation in mice [19].

The rapid and specific sequestration of sporozoites inthe liver is thus thought to result from CS-HSPG interac-tions, which are likely to involve ionic bonds between thepositive charges of the basic residues of the TSR and thenegative charges of the sulfate moieties of the GAG chains.The unique architecture of the liver sinusoids, which arelined by endothelial cells that have open fenestrae, mayallow the long GAG chains of the HSPG to protrude intothe circulation. Although CS can recognize HSPG of otherorgans in tissue sections [11, 18], the fenestrae of theirvascular endothelium closed by diaphragms precludescontact with circulating CS [12], and presumably sporo-zoites. Sporozoite homing to hepatocytes may also befacilitated by the combination of the preferential bindingof CS to highly sulfated, heparin-like glycoconjugates [18,20, 21] and the sulfation levels at least 50% higher inhepatocyte HSPGs than in most other HSPGs [22].

4. CS and sporozoite homing tothe mosquito salivary glands

Once released by the oocyst, sporozoites are thought toreach the mosquito salivary glands passively via thehemolymph that bathes the mosquito body cavity. The CSprotein appears to be involved in sporozoite binding to themosquito salivary glands. After injection into the bodycavity of mosquitoes, antibodies to CS inhibit infection ofthe salivary glands [23], and recombinant CS binds exclu-sively to the salivary glands and preferentially to the sameareas that are invaded by sporozoites [24]. A peptideencompassing region I of CS also appears to inhibit bind-ing of recombinant CS to the salivary glands of Anophelesstephensi mosquitoes [24]. The hypothesis of a role ofregion I of CS in sporozoite attachment to the salivaryglands is also supported by the notion that Plasmodiumgallinaceum, which infects birds and is transmitted byCulex and not Anopheles mosquitoes, is the only plasmo-dial species not to possess the consensus KLKQP sequencewithin the region I of CS [25]. CS may therefore mediatesporozoite binding to both the insect salivary glands andthe mammalian liver, possibly via distinct regions of theprotein.

5. CS and sporozoite formation withinoocysts

An unexpected function of CS was revealed by disrupt-ing the single-copy CS gene in P. berghei [26]. CS knock-out parasites, which infect mosquitoes and produce num-bers of oocysts similar to those of the wild-type (WT), donot generate sporozoites. In the WT, sporozoites are formedby budding from the periphery of a large, multinucleated

Figure 3. Schematic representation of the CS and TRAP pro-teins. The CS protein starts with a leader sequence and ends by ahydrophobic sequence (hatched boxes). CS contains a centralrepeat domain flanked by two conserved regions, region I and athrombospondin type I repeat (TSR). The TRAP protein is a typeI transmembrane protein containing a ∼ 45 residue-long cyto-plasmic tail; its ectodomain contains an A domain, a TSR and arepeat region. Shown below are the consensus TSR, which consistsof 55–60 residues, and the metal ion-dependent adhesion sitemotif in A domains, which is composed of five conserved residues(underlined). In the αL and αM chains of LFA-1 and Mac1leucocyte integrin receptors, respectively, the five residues coor-dinate a divalent cation (Mg2+ or Mn2+). Numbers in parenthesissymbolize the number of intervening residues.

Review Ménard


oocyst (figure 2A–C). The first event in bud formationrecognizable by transmission electron microscopy, andschematized in figure 4, is the focal emergence of twoclosely apposed membranes (called the double inner mem-brane or DIM) that lie immediately underneath the oocystouter membrane (OM). As the area evaginates, the OMand the DIM become the tri-membrane pellicle of thenascent sporozoites, typical of invasive stages of mostApicomplexa. In CS– parasites, however, the DIM is nolonger limited to the budding sites but extends to the entireOM, impeding organized budding (unpublished results).The DIM in Apicomplexa is known to be made of adjacentflattened vesicles, and CS in oocysts is mainly present atthe OM [27]. Therefore, CS may prevent/regulate dockingto the OM of the cytoplasmic vesicles that eventuallyconstitute the DIM network. The basis for this function ofCS is unknown, but may be mediated via the glyco-sylphosphatidylinositol anchors of OM-associated CS.

6. The TRAP proteinTRAP (thrombospondin-related anonymous protein) [5]

is found in the micronemes and on the surface of sporo-zoites [28]. TRAP is a type I transmembrane protein (figure3) whose ectodomain consists of (i) an A domain, (ii) aTSR, and (iii) a repeat region of variable length and

sequence, depending on the plasmodial species. The Adomain is a ∼ 200 residue-long adhesive module that wasfirst recognized in the plasma protein von Willebrandfactor. It now defines a superfamily of soluble proteins,including complement proteins factor B and C2, extracel-lular matrix proteins, including numerous types of non-fibrillar and FACIT (fibril associated collagen with inter-rupted triple helix) collagens, and integral membraneproteins, including seven integrin α chains.

The crystal structures of the A domains in αL [of LFA1]and αM [of Mac1] have been determined [29–32]. Bothcomprise alternating amphiphatic α helices and hydro-phobic � strands conforming to the classic α� ’Rossmann’fold. Located at the top of the � sheet, 5 residues coordi-nate a divalent cation (Mg2+ or Mn2+) in both αL and αM Adomains and define the metal ion-dependent adhesionsite (MIDAS) motif (see figure 3), which is conserved in anumber of A domains including that of TRAP. Mutationalanalysis of the metal ion-dependent adhesion site motif inintegrin a chains has demonstrated the critical role of thesefive residues in ligand-binding [33–37].

7. TRAP is the transmembrane link of acapping machinery that drives bothsporozoite gliding motility and host cellinvasion

Gene knockout experiments in P. berghei have shownthat TRAP is essential for sporozoite invasion into mos-quito salivary glands, the rat liver, and HepG2 cells [38].TRAP is also crucial for sporozoite gliding motility [38], asubstrate-dependent form of locomotion during which,unlike crawling motility, the cell maintains a fixed shape(see figure 5). This type of locomotion is a unifying featureof many Apicomplexan parasites, most of which canactively invade host cells.

Cell invasion and gliding locomotion by Apicomplexa,which both depend on the microfilaments in the parasite[39], result from the backward translation of parasite sur-face ligands. During cell invasion, a tight junction thatforms between the anterior tip of the parasite and the cellsurface is redistributed to the rear of the parasite as itmoves into the host cell [40–42] (figure 5A). Cell penetra-tion is a rapid process, usually completed in a few sec-onds, which does not depend on remodeling of the hostcell cytoskeleton [39]. During gliding motility, parasitesshed from their posterior pole a trail of surface ligands thatare secreted at their anterior, leading pole [43–45] (figure5B). The redistribution activity is also evident from theability of most Apicomplexa to shed from their posteriorpole a variety of surface-bound ligands such as latex beadsor antibodies directed against surface proteins [46, 47](figure 5C). The backward translocation of parasite surfaceligands bound to substrate/cell surface receptors wouldthus induce forward locomotion on the substrate or pen-etration into the cell [48, 49].

There is now strong evidence that TRAP acts as thetransmembrane link of a capping machinery that drivesboth parasite gliding and cell invasion. TRAP is stored in

Figure 4. Schematics of CS role during sporozoite formation. Inthe WT, ∼ 5–10 000 sporozoites are formed per oocyst. CS ispresent at the oocyst outer membrane (OM) and associated with asubset of cytoplasmic vesicles. Sporozoite formation starts withthe emergence of flattened vesicles (that become the double innermembrane or DIM) at discrete areas of the oocyst OM (buddingsites). These areas then evaginate and become sporozoites asmicrotubules (MT) are polymerized underneath the DIM. In CS–

parasites, ∼ 10 sporozoites are formed per oocyst. The DIMappears prematurely, rapidly underlines the entire OM, and micro-tubules extend underneath the continuous DIM. CS thereforeappears to retard/circumscribe DIM formation, a phenomenonkey to organized sporozoite budding. In the WT, the DIM mayform/dock initially at zones of the oocyst OM free of CS, whichmay arise by a process similar to the confinement ofglycosylphosphatidylinositol-anchored proteins in lipid rafts inmammalian cells.



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micronemes, is exposed on the sporozoite surface, and isleft on the substrate during gliding motility [28, 50]. In P.berghei, deletion of the TRAP tail does not impede expres-sion of the TRAP truncate on the sporozoite surface but

precludes sporozoite gliding and cell invasion [50]. Inaddition, point mutations in the distal part of the TRAP taildo not abolish but modify the gliding phenotype (theytransform the WT circular gliding into a ’pendulum’ move-

Figure 5. Host cell invasion, gliding motility, and the classical circumsporozoite precipitation (CSP) reaction by Plasmodium sporozoites.The three processes depend on the parasite actin cytoskeleton and are thought to result from the capping of parasite surface ligands. A. Cellinvasion. Penetration of a Plasmodium sporozoite into a mosquito salivary gland. Upper panel, note the intimate contact between theanterior tip of the sporozoite and the plasma membrane of the host cell (thick arrow). BL, basal lamina; PM, plasma membrane; S,sporozoite. Lower panel, a sporozoite inside a parasitophorous vacuole in a secretory cell of the salivary gland. Note the tight junctionbetween the cell membrane and the sporozoite outer surface (rectangle). (Reprinted with permission from [42]. B. Gliding motility.Plasmodium sporozoites glide when deposited on glass slides. Upper panel, the sporozoite typically glides in a circle at a constant speed of~ 2 µm/sec. Lower panel, scanning electron micrograph of the trail deposited on the substrate by a gliding Plasmodium sporozoite (spz)(immunolabeled with anti-CS primary mAb). The CS protein, secreted at the anterior pole of the sporozoite, is left behind the glidingsporozoite. (Reprinted with permission from [62]). C. Circumsporozoite precipitation (CSP) reaction. Sporozoites in suspension incubatedwith antibodies directed against the CS repeats shed from their posterior pole a threadlike CS-containing precipitate. Arrows indicate thejunction between the sporozoite posterior tip and the precipitate. Numbers indicate seconds. (Reprinted with permission from [46]).

Review Ménard


ment), further suggesting a direct role of TRAP in glidingand cell invasion [50].

There is also evidence that TRAP-related proteins inother Apicomplexa have the same function. These pro-teins, which contain several copies of A domain and/orTSR in their ectodomain, have been described in theookinete stage of the Plasmodium parasite (CTRP, [51]); inToxoplasma gondii (MIC2, [52]); in Cryptosporidium par-vum (TRAPC1, [53]); and in Eimeria tenella (Etp100, [54]).In T. gondii, immunofluorescence studies have shown thatMIC2, which is secreted at the anterior pole of the parasiteupon attachment to the host cell, undergoes progressiveanterior to posterior redistribution on the surface of thetachyzoite during cell penetration [55]. We have shownthat replacement of the cytoplasmic tail of TRAP by that ofToxoplasma MIC2 did not alter sporozoite gliding and cellinvasion by the P. berghei sporozoite [50], indicating thatthese proteins have similar functions and that their cyto-plasmic tails interact with homologous partners in therespective parasite. The mechanism by which the bridgingprotein is capped remains unknown, but is likely to involvemotor protein(s). In T. gondii, myosin colocalizes withactin in a circumferential pattern underneath the tachyzoiteplasma membrane and powers parasite gliding and cellinvasion [56]. Therefore, one model of gliding and inva-sion in Apicomplexa (figure 6A) is that parasite-substrateinteractions are capped via translocation of TRAP (oranalog) cytoplasmic tail-myosin complexes along the cor-tical actin filaments.

8. TRAP specifies two independent hostcell invasion pathways

To assess the contribution of the ectodomain of TRAP inthese processes, we have introduced various modifica-tions in the A domain and the TSR known to affect theirbinding capacities in other proteins (unpublished results).Although all mutations reduced sporozoite invasion invitro as well as in vivo (particularly mutations in the Adomain), none affected sporozoite gliding locomotion.Independent mutations in either the TSR or the A domainof TRAP always impaired, but never abolished, sporozoiteinvasion in vitro and in vivo. These results suggest that theTSR and the A domain of TRAP specifically interact withhost cell receptors during cell invasion, and that theseinteractions are used by the parasite to exert force andactively penetrate the cell. Modifications in both the Adomain and the TSR, while still not affecting gliding motil-ity, totally abolished sporozoite invasion in vitro and invivo. This suggests that the TRAP adhesive modules are theonly parasite ligands involved in productive interactionswith the cell surface during cell invasion. Therefore, a dualligand system involving the TSR and the A domain of TRAPappears to be necessary and sufficient for cell invasion(figure 6B). The likely receptor of the TSR of TRAP, theGAG chains of HSPGs [57], are as expected involved butnot essential for sporozoite invasion into cultured cells[20]. The receptor of the A domain, which confers most ofthe sporozoite invasive capacities both in vivo (invasion ofmosquito salivary glands and of the mammalian liver) and

in vitro, remains unknown. A domains can interact with alarge array of cell surface or extracellular matrix-associatedligands, including intercellular adhesion molecules,E-cadherin, collagens, or laminin. Perhaps more than onereceptor can be recognized by the TRAP A domain. Alter-natively, the A domain may recognize a phylogeneticallyconserved cell surface receptor, such as HSPGs. Eitherhypothesis would be in agreement with the capacity ofPlasmodium sporozoites, and other Apicomplexa, to pen-etrate virtually any adherent cell type in vitro.

Figure 6. Model of gliding locomotion and cell invasion byPlasmodium sporozoites. A. Both host cell invasion and glidingmotility result from actin- and TRAP-dependent capping ofparasite ligands bound to substrate/cell surface receptors. Thecytoplasmic tail of TRAP, which is necessary for these processes,may interact with a motor protein. The posterior translocation ofthe motor protein along the submembranous actin filamentswould lead to sporozoite penetration into the cell or forwardmovement on the substrate. B. Cell invasion, but not glidingmotility, depend on the TSR and the A domain of TRAP. Presu-mably, these ligands interact with cell surface receptors and allowtraction to be developped for active penetration. Both the TSRand the A domain are important for, and can independently allowcell invasion, and the two TRAP adhesive modules appear to bethe only parasite ligands directly involved in cell penetration.The receptors of the TSR and the A domain of TRAP remainunknown. PM, plasma membrane; IMC, inner membrane com-plex; ADO, A domain; TSR, thrombospondin type 1 repeat; R,repeats.



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9. Concluding remarks

In conclusion, the CS and TRAP proteins are crucial tomany basic steps of the sporozoite’s life. CS appears tohave different functions at different steps of the cycle. Inoocysts, CS is crucial for sporozoite formation, possibly byacting as an antidocking factor. On the sporozoite surface,CS is important for binding to the target organ in each host,and is probably also important for gliding locomotion. Ithas also recently been proposed that CS carried in bysporozoites during cell invasion can inhibit protein syn-thesis in the host cell by binding to ribosomes [58]. TRAPis essential for both sporozoite locomotion and cell inva-sion, by acting as a link between the parasite cytoskeletonand distinct parasite-substrate interactions involved inthese events. These multiple essential functions of CS andTRAP reinforce the value of these proteins as vaccinecandidates, as well as the need to further investigate theirstructure-function relationships. Conceivably, CS andTRAP interactions may also offer good targets for thecreation of transgenic mosquitoes refractory to Plasmo-dium transmission.

From a functional point of view, the nascent genetargeting technology in Plasmodium should be decisive inelucidating the molecular bases of host-parasite interac-tions. Most stage-specific genes in Plasmodium are single-copy genes, and knockout parasites reported so far (atvarious parasite stages) have clearly defective phenotypesand do not display efficient redundant pathways [26, 38,59, 60]. One challenge is now to identify new candidategenes involved in the cross-talk between the parasite andits hosts. The genome-based approach, combining thecurrent efforts of sequencing the parasite (Plasmodiumfalciparum) genome and the development of efficient meth-ods for identifying stage-specific transcripts [61], shouldprovide a wealth of candidate genes in the coming years.Another important goal is to develop ways for performinga genetic analysis of parasite products that are essential forcompletion of the RBC phase of the life cycle, whichcauses the malaria symptoms. We should soon have themeans to tackle many aspects of the physiopathology ofthis important disease at a molecular level.

Acknowledgments

I thank Jayne Raper and Arturo Zychlinski for theircareful review and suggestions for improving this manu-script, and Victor and Ruth Nussenzweig for their constantsupport.

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The journey of the malaria sporozoite through its hosts: two parasite proteins leadthe way