Cell, Vol. 90, 511–522, August 8, 1997, Copyright 1997 by Cell Press

TRAP Is Necessary for Gliding Motility andInfectivity of Plasmodium Sporozoites

Ali A. Sultan,* Vandana Thathy,*† Ute Frevert,† micronemes. Invasive stages of Apicomplexa also ex-hibit gliding motility, a form of substrate-dependent cellKathryn J. H. Robson,‡ Andrea Crisanti,§locomotion characterized by the absence of locomotoryVictor Nussenzweig,* Ruth S. Nussenzweig,†organelles, such as cilia or flagella, and by the absenceand Robert Menard*†

of any obvious change in cell shape during translocation*Michael Heidelberger Division of Immunology(Russell, 1983; King, 1988).Department of Pathology

Malaria infection is initiated when the insect vectorKaplan Cancer Centerinjects Plasmodium sporozoites into a susceptible ver-New York University Medical Centertebrate host. Sporozoites rapidly leave the circulatoryNew York, New York 10016system to invade hepatocytes, where parasite develop-†Department of Medical and Molecularment eventually generates the parasite form that in-Parasitologyvades and multiplies within erythrocytes. Two proteins,New York University Medical Centerthe circumsporozoite protein (CS) (Yoshida et al., 1980)New York, New York 10016and the thrombospondin-related anonymous protein‡MRC Molecular Haematology Unit(TRAP) (Robson et al., 1988), also named sporozoiteInstitute of Molecular Medicinesurface protein 2 (SSP2) (Rogers et al., 1992b), are con-John Radcliffe Hospitalsidered to play an important role in sporozoite infectivityHeadington, Oxford, OX3 9DUto hepatocytes (Nussenzweig and Nussenzweig, 1985;United KingdomCerami et al., 1992a; Rogers et al., 1992a; Muller et al.,§Department of Biology1993). These proteins are conserved in all plasmodialImperial College of Science, Technologyspecies examined to date (McCutchan et al., 1996; Tem-and Medicinepleton and Kaslow, 1997) and are present on the parasitePrince Consort Roadsurface and in the micronemes (Fine et al., 1984; Naga-London, SW7 2BBsawa et al., 1988; Rogers et al., 1992a). CS uniformlyUnited Kingdomcovers the external surface of sporozoites and repre-sents up to 15% of the total protein synthesized bysporozoites obtained from mosquito salivary glands

Summary (Yoshida et al., 1981). It contains a highly conserved 18-residue motif homologous to a portion of the type 1

Many protozoans of the phylum Apicomplexa are inva- repeat of thrombospondin. This motif, named region IIplus, binds to heparan sulfate proteoglycans (HSPGs)sive parasites that exhibit a substrate-dependent glid-associated with the hepatocyte membrane (Cerami eting motility. Plasmodium (malaria) sporozoites, theal., 1992a, 1992b; Pancake et al., 1992; Frevert et al.,stage of the parasite that invades the salivary glands1993; Sinnis et al., 1994). TRAP has also been reportedof the mosquito vector and the liver of the vertebrateto be expressed on the surface of salivary gland sporo-host, express a surface protein called thrombospon-zoites (Cowan et al., 1992; Rogers et al., 1992a), wheredin-related anonymous protein (TRAP) that has homo-it displays a patchy distribution. TRAP contains a regionlogs in other Apicomplexa. By gene targeting in aII plus–like motif that binds to sulfated glycoconjugatesrodent Plasmodium, we demonstrate that TRAP is crit-(Muller et al., 1993; Robson et al., 1995) and a region ofical for sporozoite infection of the mosquito salivaryz200 residues homologous to the A domain presentglands and the rat liver, and is essential for sporozoitein many proteins involved in cell–cell, cell–matrix, andgliding motility in vitro. This suggests that in Plasmo-matrix–matrix interactions (Colombatti et al., 1993). Bothdium sporozoites, and likely in other Apicomplexa,CS and TRAP bind specifically to the region of the hepa-gliding locomotion and cell invasion have a commontocyte plasma membrane facing the circulating bloodmolecular basis.in the space of Disse (Cerami et al., 1992a; Robson etal., 1995), and antibodies raised against each of theseproteins inhibit sporozoite invasion of hepatocytes in

Introduction vitro (Hollingdale et al., 1982; Rogers et al., 1992a; Mulleret al., 1993).

Apicomplexa, one of the largest phyla of protozoa, con- Plasmodium sporozoites are formed within oocyststains more than 5,000 named species, many of which in the insect midgut. Thousands of sporozoites are re-are obligate intracellular parasites. Among the most leased by a mature oocyst into the body cavity of thepathogenic and prevalent of these parasites for humans mosquito and are carried by the hemolymph to theare Plasmodia, the etiological agents of malaria, as well mosquito salivary glands. Sporozoites then need toas Toxoplasma and Cryptosporidium, which cause op- invade the glands to be injected into the mammalianportunistic infections. All invasive stages of Apicom- host during subsequent mosquito blood meals. Immu-plexa exhibit a common structural organization at their nolabeling has shown that CS synthesis starts in younganterior pole called the apical complex (Jensen and Ed- oocysts before sporozoites are formed (Nagasawa etgar, 1978; Dubremetz and Torpier, 1978), which includes al., 1988), whereas TRAP distribution has been a matter

of controversy. The TRAP gene, originally described ina group of secretory organelles known as rhoptries and

Cell512

Plasmodium falciparum, was found to be expressed inerythrocytic stages (Robson et al., 1988). However, P.falciparum TRAP and Plasmodium yoelii TRAP/SSP2were later detected predominantly in the sporozoitestage of the parasite (Rogers et al., 1992a; Cowan etal., 1992). More recently, TRAP was reported to be pro-duced only by sporozoites collected from the mosquitosalivary glands and not by sporozoites contained withinoocysts (Robson et al., 1995).

The recent advent of a gene targeting technology inPlasmodium parasites (van Dijk et al., 1996; Wu et al.,1996; Crabb and Cowman, 1996) now allows for agenetic approach to study Plasmodium protein function.Disruption of the CS gene in the rodent parasite Plasmo-dium berghei hasshown that CS is essential for sporozo-ite formation within oocysts in the mosquito midgut(Menard et al., 1997). Here, we disrupted the TRAP genein P. berghei to analyze the role of TRAP in the develop-ment of sporozoites and their interactions with hostcells.

Results

Generation of Plasmodium berghei TRAPMutant Lines INT1 and INT2To investigate the role of TRAP in the life cycle of Plas-modium parasites, we disrupted by homologous recom-

Figure 1. Gene Targeting at the TRAP Locus of P. berghei and Gen-bination the single-copy TRAP gene in erythrocytic eration of Clones T1, INT1, and INT2stages of P. berghei. In erythrocytic stages of P. berghei

(a) Plasmid constructs and integration plasmid pINT (not drawn tostrain NK65, TRAP mRNA was not detected by reverse scale). Open boxes, TRAP coding region; stippled boxes, DHFR-transcriptase-polymerase chain reaction (RT-PCR) and TS cassette consisting of the coding region of the pyrimethamine-

resistant mutant gene and 2.5 and 1 kb of its 59 and 39 untranslatedthe TRAP protein was not detected by immunofluores-regions, respectively. Double line, pCRII vector sequences; wavycence and Western blot analysis (data not shown). Inlines, pUC18 vector sequences; thick lines, pBSKS vector se-contrast, TRAP was detected in P. berghei NK65 sporo-quences. Abbreviations: B, BamHI; E, EcoRI; H2, HincII; K, KpnI; N,zoites collected from midguts or salivary glands of in-NsiI; P, PvuII; S, SpeI.

fected mosquitoes (see below). For targeting the TRAP (b) Wild-type (WT) TRAP genomic locus and INT locus generatedgene of P. berghei NK65, we cloned this gene by PCR by integration of plasmid pINT via a single cross-over between the

TRAP homologous sequences. The site of linearization in pINT isusing erythrocytic stage genomic DNA and oligonucleo-shown by a break in the TRAP target sequence. The TRAP probe (1.6tides designed on the basis of the TRAP sequence ofkb-NsiI internal fragment) used in genomic Southern hybridization isP. berghei strain ANKA (Robson et al., 1997) and con-symbolized by a bold line within the TRAP coding region.structed the integration plasmid pINT (Figure 1a). This(c) Genomic Southern hybridization of WT P. berghei strain NK65,

plasmid contained an internal fragment of TRAP up- and of clones T1, INT1, and INT2. Digestions with EcoRI, whichstream from a pyrimethamine-resistant dihydrofolate re- does not cut in pINT, and with BamHI, which cuts once in pINT,ductase thymidylate synthase (DHFR-TS) mutant gene show that the increase in the size of the TRAP locus in INT clones

corresponds to the size of one pINT (9.7 kb). Digestions with HincIIconstitutively expressed by its flanking regions (van Dijkor with SpeI, which both cut in the duplicatedTRAP target sequence,et al., 1995). P. berghei NK65 merozoites were thenshow the presence of a band of the exact size of pINT in INT clones,transformed by electroporation with plasmid pINT. Toin addition to the two bands also detected in the wild type.

favor integration of pINT in the TRAP genomic locus,the plasmid was linearized with the restriction enzymeSpeI that cuts once in the TRAP target sequence (Figure1b). Rats were injected with electroporated merozoites,

from a spontaneous mutation in the DHFR-TS endog-and pyrimethamine-resistant redblood cell stages of theenous gene or from a gene replacement of this gene byparasite were selected. At day 10 postelectroporation,the mutated copy borne by pINT. The disruptant clonesresistant parasites were cloned by limiting dilution intohad integrated a single copy of the entire pINT into theten rats, and at day 20 postelectroporation the genomicTRAP genomic locus, via a recombination process thatDNA of the parasite clones was analyzed by Southernhad repaired the SpeI site used to promote integration.hybridization using a TRAP probe (Figure 1c). One cloneThese clones thus contained two truncated copies ofhaving a wild-type TRAP, named T1, and two TRAPthe TRAP gene, the first lacking the last 66 codons ofdisruptant clones obtained from independent electro-the gene and downstream untranslated region and theporation experiments, named INT1 and INT2, were se-second lacking the 59 promoter sequences and the firstlected for further analysis. The T1 clone, which con-

tained a single copy of the DHFR-TS gene, arose either 22 codons of the gene (Figure 1b).

TRAP-Dependent Infectivity and Motility of Malaria Parasites513

Table 1. Developmental Cycle of Wild-Type (WT) P. berghei, and of Clones T1, INT, and REP in Anopheles stephensi Mosquitoesa

Midgut Hemolymph Salivary Glands (SG)

Number of Percentage Number of Midgut Number of Hemolymph Percentage Number of SG-AssociatedOocysts per of Infected Sporozoites per Sporozoites per of SG Bearing Sporozoites perInfected Mosquito Midguts Infected Mosquito Infected Mosquito Sporozoites Infected Mosquito

ParasitePopulation Day 14 Day 14 Day 18 Day 18 Day 18 Day 14 Day 18 Day 18

WT 31 80 70 21,000 4,000 85 77 19,000T1 25 90 67 24,000 2,000 62 72 20,000INT1 35 71 75 18,000 8,000 22 29 360INT2 46 84 70 27,000 ND 45 25 310REP1 38 74 62 22,000 6,000 24 36 700REP2 42 92 90 25,000 ND 36 40 500

Each value is the mean of at least three independent feeding experiments. The following numbers of mosquitoes were examined for eachparasite population: at day 14 postfeeding, 150 mosquitoes; at day 18 postfeeding, 700 mosquitoes; and 200 mosquitoes to determine thenumber of hemolymph sporozoites. ND, not determined.a T1, wild-type TRAP-containing parasite clone; INT and REP, TRAP mutant parasites obtained with plasmid pINT and pREP, respectively.

TRAP Is Not Necessary for Sporozoite Formation micronemes were seen in the anterior pole of TRAP(2)sporozoites.The successful selection of TRAP mutant parasites im-

plied that TRAP is not necessary for development oferythrocytic stages of the parasite. To test whether theINT mutation had any effect on parasite replication in TRAP Is Critical for Sporozoite Invasion

of the Mosquito Salivary Glandserythrocytes, we compared the duration of the schizo-gonic blood cycle of wild-type and INT parasites. For Throughout this paper, midgut sporozoites refer to the

sporozoites inside midgut oocysts or free but still mid-this, we analyzed the outcome of rat infections initiatedby a mixture of identical numbers of T1 and INT1 eryth- gut-associated; hemolymph sporozoites are those pres-

ent in the hemocele; and salivary gland–associated spo-rocytic stages. Fifteen days after injection (z15 schizo-gonic cycles), we subjected the mixed parasite popu- rozoites consist of those adherent to or inside of the

glands (see Experimental Procedures). At day 18 post-lations to Southern blot hybridization. The signalscorresponding to a wild-type and a disrupted TRAP were feeding, the number of midgut sporozoites was compa-

rable in mosquitoes infected with T1, INT1, or INT2of similar intensities (data not shown), indicating thatlack of TRAP does not affect merozoite invasion of, and populations (Table 1). At thesame day postfeeding,simi-

lar numbers of hemolymph sporozoites were countedparasite development in, erythrocytes.The levels of mature gametocytes and the ability of in mosquitoes infected with either parasite population,

indicating that INT sporozoites were normally releasedmale gametocytes to exflagellate in vitro were also simi-lar in the wild type and in clones T1, INT1, and INT2 (data from oocysts into the hemocele (Table 1). In contrast,

at day 18 postfeeding, the number of salivary gland–not shown). To study their sporogonic cycle, Anophelesstephensi mosquitoes were fed on rats bearing high associated sporozoites per infected mosquito was on

average z60 times smaller in INT than TRAP(1) popula-levels of gametocytes that exflagellated in vitro. P. ber-ghei oocysts develop asynchronously in the mosquito tions (Table 1). Likewise, the percentage of infected sali-

vary glands in mosquitoes harboring INT populationsmidgut, and starting at day 10–12 postfeeding, sporozo-ites are released from oocysts into the hemolymph that was 2.2 and 2.7 times lower at days 14 and 18 postfeed-

ing, respectively. We next measured the proportion ofcirculates in the body cavity (hemocele) of the mosquito.To examine oocyst development, midguts of mosqui- salivary gland–associated sporozoites that had actually

invaded the glands. In two separate experiments fortoes were observed by light microscopy at days 14 and18 postfeeding (Table 1). The number of oocysts per each of the wild-type and INT populations, salivary

glands of mosquitoes at day 18 postfeeding were dis-infected mosquito and the percentage of infected mid-guts were within the range of variability observed in sected, treated with trypsin to remove surface-asso-

ciated sporozoites, washed, and then ruptured in aindividual populations. Oocysts in mosquito midgutscollected at day 14 postfeeding were also examined ground-fitting homogenizer to free internalized sporozo-

ites. In mosquitoes infected with the wild-type popula-by transmission electron microscopy. The sporozoitebudding process from the sporoblastoid body of the tion, the proportion of total salivary gland–associated

sporozoites released from within the glands was 77%,oocyst (Figures 2A and 2C) was typical of Plasmodiumparasites (Figure 2B; Vanderberg et al., 1967). The ultra- confirming the intracellular location of most of the wild-

type sporozoites. In contrast, only 13% of the salivarystructure of sporozoites inside oocysts (Figures 2C–2E)and of free sporozoites released in the hemocele (Fig- gland–associated sporozoites werereleased from within

the glands in mosquitoes infected with the INT popula-ures 2F and 2G) was undistinguishable from that of thewild type. In particular, ultrastructurally normal subpelli- tions. This indicates that the TRAP disruptants poorly

invade, if at all, the salivary glands of mosquitoes.cular microtubules and electron-dense rhoptries and

Cell514

Figure 2. Transmission Electron Microscopyof TRAP(2) Sporozoites

(A and B) Toluidin blue-stained semithin sec-tions of wild-type and INT1 P. berghei oo-cysts, respectively, in the midgut of A. ste-phensi mosquitoes 14 days after the infectiveblood meal. TRAP(2) parasites form matureoocysts that contain numerous sporozoites(S). RB, residual body. Bars, 10 mm.(C) Budding TRAP(2) sporozoites in a matur-ing oocyst in the process of importing a nu-cleus (N) from the sporoblastoid body (SB).The anterior pole of the cell is formed firstand contains electron-dense rhoptries (ar-rows). Bar, 1 mm.(D) The apical complex of mature TRAP(2)sporozoites appears unaltered. Parasitescontain a normal set of rhoptries and micro-nemes at the anterior pole of the cell (arrows).Bar, 1 mm.(E) A tangential section of the TRAP(2) sporo-zoite pellicle reveals the array of subpellicularmicrotubules (arrowheads). Bar, 1 mm.(F and G) After their release from a matureoocyst, TRAP(2) sporozoites possess welldeveloped rhoptries and micronemes at theirapical pole (arrows). Bars, 1 mm.

TRAP Is Critical for Sporozoite Infection parasites induced by INT1 or INT2 sporozoites con-tained only wild-type TRAP, with no trace of disruptedof the Mammalian Liver

Plasmodium sporozoites do not need to enter the mos- TRAP (Figure 3a). This raised the possibility that a num-berof INT parasiteshad excised plasmid pINT by intralo-quito salivary glands tobecome infective to the mamma-

lian host (Ball and Chao, 1961). Nonetheless, salivary cus recombination via the TRAP duplicated sequence,and that the revertants to a wild-type TRAP had infectedgland–associated sporozoites are at least 1000-fold

more infective than midgut sporozoites (Vanderberg, the rats.To test this hypothesis and to search for revertants1975; shown also in Table 2). To assess the role of TRAP

in sporozoite infectivity to the mammalian host, midgut, among INT parasites, we designed three oligonucleo-tides, named O1, O2, and O3 (see Figure 1b), to discrimi-and salivary gland–associated sporozoites were iso-

lated from mosquitoes at day 18 postfeeding and in- nate between a wild-type and a disruptant TRAP locusin PCR reactions. As shown in Figure 3b, both wild-typejected into young rats, which are highly susceptible to

infection by P. berghei sporozoites (Table 2). Although and disruptant TRAP were amplified from erythrocyticstages of INT1 and INT2 clones prior to mosquito feed-both INT1 and INT2 sporozoites induced rat blood infec-

tion, the prepatent periods were prolonged compared ing. The wild-type TRAP-containing parasites were notcontaminants of the cloning procedure but originatedwith T1 and wild-type sporozoite-induced infections.

Based on the numbers of wild-type sporozoites that from disruptant parasites for the following reasons. Onthe basis of the Southern analysis of INT1 and INT2infect rats with similar prepatent periods, we estimate

that midgut and salivary gland–associated INT sporozo- populations performed immediately prior to mosquitofeeding (Figure 1c), the proportion of wild-type parasitesites were at least 100-fold less infective than the corre-

sponding wild-type sporozoites. Surprisingly, Southern in INT populations must be very small (,10%). As statedabove, wild-type and disruptant parasites replicate withhybridization experiments showed that the blood stage

TRAP-Dependent Infectivity and Motility of Malaria Parasites515

Table 2. Infectivity to Rats of Wild-Type (WT) P. berghei, and of Clones T1, INT, and REPa

Midgut Sporozoites SG-Associated Sporozoites

Number of Number of Pre Number of Number of PreParasite Injected Infected Patent Injected Infected PatentPopulation Sporozoites Animalsb Periodc Sporozoites Animalsb Periodc

WT 50,000 2/2 5.5 30 2/2 5.5100,000 2/2 5 150 3/3 4.7

1,000,000 2/2 4 1,500 2/2 415,000 4/4 3

T1 100,000 2/2 5 30 3/3 5.31,000,000 2/2 4.5 150 3/3 5

1,500 3/3 415,000 4/4 3

INT1 1,000,000 0/3 — 15,000 6/6 55,000,000 1/1 6

INT2 1,000,000 0/2 — 15,000 5/5 55,000,000 3/3 5.3

REP1 5,000,000 0/2 — 20,000 0/3 —100,000 1/2 6

REP2 5,000,000 0/2 — 20,000 0/3 —100,000 1/1 6

a As defined in Table 1.b Number of infected animals/number of animals injected with the sporozoite suspension.c Number of days between sporozoite injection and detection of at least one erythrocytic stage upon a 10-min examination of a Giemsa-stained blood smear. The value is the mean prepatent period of successful infections.—, parasitemia undetectable up to day 10 after sporozoite injection.

equal efficiency in rat red blood cells. Therefore, if these copy, an event that does not generate sequence dupli-cation at the recombinant locus and therefore precludeswild-type parasites are contaminants, then numerous

parasites (at least 10) were injected simultaneously into reversion through homologous recombination (Figure4b). To favor the allelic exchange, plasmid pREP wasanimals during “cloning.” However, we only character-

ized clones derived from experiments in which not more digested with BamHI, which liberates the TRAPmutationas a linear molecule. Two clones originating from inde-than four out of ten injected animals became infected,

ruling out the possibility of injection of multiple parasites pendent electroporation experiments and having under-gone theexpected gene replacement (Figure 4c), namedin a single animal. In addition, wild-type TRAP parasites

were detected by PCR in all four independently gener- REP1 and REP2, were selected for further analysis.The developmental cycle of REP populations in A.ated INT clones tested, but were never found in TRAP

mutant lines that could not revert to the wild-type TRAP stephensi mosquitoes was in perfect agreement withthat of INT populations (Table 1). The ultrastructure ofthrough homologous recombination (see below).

After mosquito feeding, wild-type TRAP was also am- REP sporozoites inside oocysts and of released sporo-zoites, examined by transmission electron microscopyplified from INT midgut sporozoites (Figure 3b), although

not from INT salivary gland-associated sporozoites of mosquito midguts collected at day 14 postfeeding,was undistinguishable from that of the wild type (data(data not shown), probably because of their low number

and the small proportion of revertants. In sharp contrast not shown). The number of salivary gland–associatedsporozoites per infected mosquito was 34 times smallerwith these results, only the wild-type TRAP was ampli-

fied from erythrocytic stages in rats infected by INT in REP than TRAP(1) populations. Furthermore, ratblood infection was never obtained after injection ofmidgut or salivary gland–associated sporozoites. Since

lack of TRAP does not affect parasite replication in midgut REP sporozoites or 20,000 salivary gland–associated sporozoites (Table 2). However, injection oferythrocytes, the absence of TRAP-disruptant erythro-

cytic stages in animals infected with INT sporozoites 100,000 REP salivary gland–associated sporozoites,collected from z170 infected mosquitoes, induced aindicates that TRAP is required for sporozoite infection

of the liver of the host. blood infection in two out of three rats (Table 2). Asexpected by the replacement strategy, Southern hybrid-To provide further evidence for the role of TRAP in

sporozoite infectivity to the host, we generated TRAP ization indicated that the blood stage parasites inducedby REP sporozoites still contained the REP mutationmutants that cannot revert to a wild-type TRAP by ho-

mologous recombination. For this, P. berghei blood (data not shown). Based on the prepatent periods andthe minimal infective dose, these REP sporozoites werestages were transformed with the replacement plasmid

pREP obtained by inserting a DHFR-TS cassette into approximately 10,000 times less infective than the corre-sponding wild-type sporozoites. Finally, an additionalthe TRAP coding region (Figure 4a). A double cross-

over between the homologous TRAP sequences in the life cycle of the REP parasites that had infected ratswas analyzed. Their infectivity for both salivary glandsconstruct and in thegenome was expected to lead to the

exchange of the endogenous gene by the transformed of mosquitoes and liver of rodents was similar to that

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Figure 3. INT Sporozoite-Induced Rat Blood Infections ContainWild-Type TRAP Revertant Parasites

(a) Southern hybridization of genomic DNA from erythrocytic stageparasites obtained after injection of WT, INT1, or INT2 sporozoitescollected at day 18 postfeeding from midguts or salivary glands ofinfected mosquitoes.Genomic DNA was cut with HincII (H2) or EcoRI

Figure 4. Gene Targeting at the TRAP Locus of P. berghei and Gen-(E) and hybridized with the 1.6 kb-NsiI TRAP probe.

eration of Clones REP1 and REP2(b) PCR analysis of the genomic DNA of T1, INT1, and INT2 parasite

(a) Construction of replacement plasmid pREP (not drawn to scale).clones at various stages of their life cycle. Genomic DNA was pre-Open boxes, TRAP coding region; stippled boxes, DHFR-TS cas-pared from erythrocytic stage parasites prior to mosquito feeding,sette consisting of the coding region of the pyrimethamine-resistantfrom midgut sporozoites in infected mosquitoes, and from erythro-mutant gene and 2.5 and 0.4 kb of its 59 and 39 untranslated regions,cytic stages induced by injection of INT sporozoites. PCR reactionsrespectively. Double line, pCRII vector sequences. Abbreviations:were performed using (A) either oligonucleotides O1 and O2, whichB, BamHI; EV, EcoRV; H2, HincII; N, NsiI; S, SpeI.amplify a 1.6 kb-PCR product specifically from a wild-type TRAP(b) Wild-type (WT) TRAP genomic locus and REP locus generatedlocus, (B) or with O1 and O3, which amplify a 1.6 kb-PCR productby a double cross-over between the homologous TRAP sequencesspecifically from a disruptant (Dis) TRAP locus.in the BamHI fragment of pREP and in the endogenous TRAP. TheTRAP probe (1.6 kb-NsiI internal fragment) used in genomic South-ern hybridization is symbolized by a bold line within the TRAP codingregion.of REP parasites of the previous cycle (data not shown),(c) Genomic Southern hybridization of WT P. berghei strain NK65,i.e., dramatically reduced. This indicates that infectionand of clones REP1 and REP2. Digestions with SpeI or HincII, both

of the liver by REP parasites is not associated with the cutting once in the TRAP coding region, show that the increase inselection of compensatory mutations, but rather sug- the size of one TRAP signal corresponds to the size of the DHFR-gests that infectivity of REP sporozoites results from TS cassette (4.5 kb). Digestions with BamHI or EcoRI, which do not

cut in the TRAP gene or in the DHFR-TS cassette, show that thestochastic events, such as host cell uptake of noninva-increase in the size of the entire TRAP locus in REP clones corre-sive sporozoites.sponds to the size of the cassette.

TRAP Is Expressed in Midgut SporozoitesThe essential role of TRAP in invasion of the mosquitosalivary glands implied that TRAP is expressed by mid- amino acid repeats of the protein (antiserum 2). In con-

trast, midgut and salivary gland INT sporozoites did notgut sporozoites. To verify this, wild-type sporozoitescollected from midguts of infected mosquitoes at day 18 react with any of the TRAP antisera (Figure 5b). The

finding that antibodies against the central region ofpostfeeding were air dried, permeabilized, and stainedusing antibodies directed against a 20-residue synthetic TRAP recognized wild-type but not INT sporozoites con-

firmed that the first truncated TRAP copy in the INTpeptide representing a portion of the C-terminal cyto-plasmic domain of TRAP (antiserum 1, see Figure 5a). locus did not give rise to a stable product.

On their way to the sporozoite plasma membrane,Virtually all wild-type midgut sporozoites displayed thecharacteristic pattern of polar and mottled fluorescence TRAP and CS transit through the micronemes (Fine et

al., 1984; Rogers et al., 1992a). To test whether the ab-(Figure 5b), described for TRAP/SSP2 in P. falciparumand P. yoelii salivary gland sporozoites (Rogers et al., sence of TRAP interfered with the trafficking of CS, live

wild-type and INT midgut and salivary gland–associated1992a; Cowan et al., 1992). The same staining patternwas obtained using a second antiserum directed against sporozoites collected from mosquitoes at day 18 post-

feeding were stained using monoclonal antibodiesthe central region of TRAP encompassing theconserved

TRAP-Dependent Infectivity and Motility of Malaria Parasites517

Figure 5. INT Sporozoites Do Not ProduceTRAP and Expose CS on Their Surface

(a) Schematic representation of P. bergheiTRAP and regions of the protein used to gen-erate anti-TRAP antisera. The 606-residue-long TRAP protein contains, from its N to Cterminus, a hydrophobic leader sequence(HLS), a z200-residue-long A domain, athrombospondin type 1–like domain (RegionII), an asparagine/proline-rich repeat regionfollowed by a region of degenerate aminoacid repeats, a transmembrane domain (TMD),and a short acidic cytoplasmic domain witha highly conserved C terminus. 1, region ofTRAP (amino acids 586–604) correspondingto the synthetic peptide used to raise antise-rum 1; 2, region of TRAP (amino acids263–428) corresponding to the recombinantpeptide used to raise antiserum 2; and 3, Nterminus of TRAP potentially expressed bythe first truncated TRAP copy of the INT locus(up to residue 540).(b) Immunofluorescence assays of midgutsporozoites of the WT and INT1 populationscollected at day 18 postfeeding. Upper pan-els, permeabilized sporozoites stained usingpolyclonal antiserum 1. Lower panels, livesporozoites stained using MAbs 3D11 di-rected against the repeats of the P. bergheiCS. Similar results were obtained with INT1and INT2 sporozoites.

(MAbs) directed against the amino acid repeats of CS. as flexing or stretching (Figure 6a). In contrast, midgutand salivary gland–associated sporozoites from INT orAs shown in Figure 5b, TRAP(2) sporozoites displayed

a circumsporozoite fluorescence similar to that of wild- REP populations did not glide; they were either nonmo-tile or exhibited the movements not associated withtype parasites, indicating that TRAP is not necessary

for exposure of CS on the surface of free sporozoites. locomotion (Figure 6b).To ascertain that lack of TRAP was responsible for

the inability of INT sporozoites to both express glidingTRAP Is Necessary for Sporozoite GlidingMotility In Vitro motility in vitro and invade the mosquito salivary glands,

we analyzed the sporogonic cycle of the revertant para-In view of the apparent correlation between infectivityof Plasmodium sporozoites to the mammalian host and sites found in the blood of rats injected with INT1 sporo-

zoites. Anopheles stephensi mosquitoes were fed ontheir capacity to glide in vitro (Vanderberg, 1974, 1975),we examined the role of TRAP in sporozoite motility. these rats, and at day 18 postfeeding, the number of

salivary gland–associated sporozoites, the proportionMidguts and salivary glands of infected mosquitoes atday 18 postfeeding were dissected, and sporozoites internalized in the glands, and the percentage of midgut

and salivary gland–associated sporozoites expressingreleased from these tissues in a motility enhancer me-dium were allowed to settle on microscope slides and gliding motility were determined. Each of these parame-

ters was restored to a wild-type level (data not shown),examined with phase contrast microscopy. A small pro-portion of midgut (z10%) and the majority of salivary demonstrating that the TRAP mutation causes all defec-

tive phenotypes of INT sporozoites.gland–associated (up to 85%) sporozoites of the wildtype exhibited gliding motility (Figure 6a), usually glidingin a circle without any obvious flexing or undulationof the sporozoite body. As shown by the time-lapse Discussionmicrographs presented in Figure 6b, a wild-type sporo-zoite circles twice as fast as the second hand of a clock. Plasmodium sporozoites need to invade the secretory

cells of the mosquito salivary glands and hepatocytesThe wild-type sporozoites that did not glide were eithernonmotile or displayed various patterns of motility that of the mammalian host to induce malaria infection. Car-

ried by the hemolymph in the mosquito and the blooddo not induce cell locomotion (Vanderberg, 1974), such

Cell518

Figure 6. TRAP(2) Sporozoites Do Not Ex-press Gliding Motility In Vitro

(a) Motility of midgut and salivary gland-asso-ciated WT, INT, and REP sporozoites. Midgutand salivary gland–associated sporozoiteswere isolated at day 18 postfeeding and ex-amined by phase contrast microscopy.Movement patterns were categorized as: (i)no motility; (ii) motility not associated withlocomotion, consisting mostly of flexing orstretching (Vanderberg, 1974); and (iii) thecharacteristic gliding motility. The frequen-cies are the mean values obtained upon ex-amination in two independent experiments ofat least 200 sporozoites for up to 15 s each.(b) Time-lapse micrographs of a wild-type ora TRAP(2) sporozoite at 5-s intervals. Thewild-type P. berghei sporozoite, whose glid-ing speed is z1–3 mm/s (Vanderberg, 1974;King, 1988), completes a circle in z20–30 s.TRAP(2) sporozoites did not glide, althoughbending and stretching of one or both sporo-zoite poles were frequently seen (see the firsttwo micrographs). Magnification, 3800.

stream in the mammalian host, sporozoites bind to tar- provided an internal complementation, demonstratingthat the defective phenotypes of these TRAP(2) sporo-get organs by a receptor-mediated process (Sinnis,

1996). Interaction betweenthe dense sporozoite CS coat zoites was due to the TRAP mutation. The specificstep(s) of the sporozoite infectious process that are im-and the abundant HSPGs of the hepatocyte basolateral

membrane probably accounts for the rapid sequestra- paired in the absence of TRAP, however, remain un-known. In mosquitoes, although relatively large numberstion of the parasites in the liver (Cerami et al., 1992a,

1992b; Pancake et al., 1992; Frevert et al., 1993; Sinnis of TRAP(2) sporozoites were present in the hemolymph,only a few of these sporozoites associated with theet al., 1994, 1996; Shakibaei and Frevert, 1996). In the

mosquito, the high levels of CS expression in hemocele salivary glands. In addition, the majority of the TRAP(2)salivary gland–associated sporozoites remained extra-sporozoites (Beier, 1993) and the inhibitory effect of

antibodies to CS on sporozoite infection of the salivary cellular. Therefore, TRAP may be important for sporozo-ite binding to, or traversing the basal lamina of, salivaryglands (Warburg et al., 1992) suggest that CS may also

be involved in attachment to these glands. After binding glands or for invading the underlying secretory cell. Inrodents, TRAP(2) midgut sporozoites were unable toto the target organ, the sporozoite must be motile to

both reach and enter the target cell. In the mosquito, it induce blood infection, and TRAP(2) salivary gland–associated sporozoites were 10,000-fold less infectivepenetrates the basal lamina of the salivary gland and

then enters the secretory cell. In the mammalian host, than the corresponding wild-type sporozoites. TRAP(2)erythrocytic stages, however, were not affected in theirthe sporozoite must traverse the cells lining the liver

sinusoids and cross the space of Disse, a loose extracel- ability to invade or replicate in red blood cells, pointingto the role of TRAP in the preceding step, i.e., infectionlular matrix separating endothelial cells from the under-

lying hepatocytes. Sporozoite entry into the target cell, of the liver. An in vivo role of TRAP in invasion of hepato-cytes is supported by earlier data on the inhibitory effecta process completed within seconds, is also associated

with active motility of the parasite. Sporozoite-host cell of antibodies toTRAP on sporozoite invasion of culturedhepatocytes (Rogers et al., 1992a; Muller et al., 1993).interactions in vitro include sporozoite gliding on the

surface of and penetration into the host cell without In view of the necessity for the sporozoite to move ac-tively to both reach and invade its target cells, and thebreaking stride (Vanderberg et al., 1990).

Previous reports have documented the relationship essential role of TRAP in gliding motility, it is also possi-ble that TRAP acts at multiple steps of the sporozoitebetween Plasmodium sporozoite gliding motility and in-

fectivity to the mammalian host (Vanderberg, 1974, infectious pathway in both of its hosts.Although little is known about the molecular basis of1975; Stewart et al., 1986; Vanderberg et al., 1990). The

results presented here suggest that gliding motility and gliding motility and cell invasion in Plasmodium or otherApicomplexa, these two processes are thought to beinfectivity for both the mosquito salivary glands and

the liver of the mammalian host, which are all TRAP- mechanistically related. Gliding motility and depositionof trails on the substrate during locomotion are com-dependent, have a common molecular basis. Concor-

dant phenotypes were obtained with independent mon features of Apicomplexa (Entzeroth et al., 1989;Arrowood et al., 1991; Dobrowolski and Sibley, 1996).TRAP(2) clones generated by two different gene-tar-

geting strategies. In addition, the reversion process The ability of these parasites to cap anionic site surfacemarkers and to translocate beads to their posterior pole(plasmid excision) in the TRAP locus of INT parasites

TRAP-Dependent Infectivity and Motility of Malaria Parasites519

by a submembranous microfilament system has led to its own extracellular domains, or may connect the con-tractile system to other substrate-binding molecules,the suggestion that gliding motility of Apicomplexa maysuch as CS.be mediated by a capping-like process (Russell and

The hypothesis that TRAP is involved in a capping-Sinden, 1981; Speer et al., 1985; King, 1988). Accordinglike process during cell invasion is supported by immu-to this hypothesis, parasite surface receptors would benolocalization of TRAP during sporozoite internalizationcross-linked upon binding to their ligand(s) and wouldin cultured cells. Upon contact with the host cell, TRAPbe translocated to the posterior pole of the cell via anis localized at the apical pole of the parasite and at theactin-dependent process (Bourguignon and Bourguig-junction between the anterior end of the parasite andnon, 1984). Binding of the parasite to a solid substratethe host cell (A. Crisanti and K. Robson, manuscript inwould then lead to the forward locomotion of the para-preparation). This TRAP-containing junction then slidessite and deposition of a trail containing the substrate–over the parasite during entry to concentrate graduallyreceptor complexes. In Plasmodium, CS may provideat its posterior end during cell penetration. This confirmsthe substrate-binding sites necessary for gliding loco-the involvement of TRAP in cell invasion and is inmotion. CS is released at the apical, leading end of theagreement with a capping-like process driving sporozo-sporozoite by the rhoptry–microneme complex (Fine etite entry into the host cell. As during gliding motility,al., 1984; Nagasawa et al., 1988), adheres to the sporo-TRAP may bind directly to the cell surface receptor,zoite surface at its anterior end, and is progressivelypossibly via its A domain, or indirectly via other sporozo-translocated to the posterior pole by a cytochalasin-ite surface molecules. Furthermore, proteins homolo-sensitive process (Stewart and Vanderberg, 1991; Stew-gous to TRAP have been described in a number ofart et al., 1986), before being shed at the trailing end ofApicomplexa, such as Eimeria (Tomley et al., 1991),the moving sporozoite (Stewart and Vanderberg, 1988,Toxoplasma (Wan et al., 1997), and Cryptosporidium1992).(Robson et al., 1997) species. The TRAP homolog inIt has also long been suggested that host cell invasionT. gondii, MIC2, displays by immunofluorescence anby Apicomplexa is dependent upon the actin-basedanterior-to-posterior moving pattern during parasite in-

contractile system of the parasite (Ryning and Reming-ternalization similar to that of TRAP (V. Carruthers and

ton, 1978; Russell and Sinden, 1981; Russell, 1983). TheD. Sibley, personal communication), suggesting that

seminal work of Dobrowolski and Sibley (1996) has nowTRAP-related molecules in other Apicomplexa may have

demonstrated that internalization of Toxoplasma gondii, similar functions.and likely other Apicomplexa, into host cells relies only TRAP-like molecules, along with the parasite actinon the actin cytoskeleton of the parasite. The last step of network (Dobrowolski and Sibley, 1996), may thus bethe host cell invasion process by Apicomplexa, namely central componentsof motility and invasion machineriesparasite translocation into an induced vacuole, is char- of Apicomplexa. Further investigations of parasite actin-acterized by the presence of a moving junction formed binding proteins and of parasite surface ligands bindingby the close apposition of the outer membrane of the to the substrate during gliding motility or to the cellparasite and of the host cell (Aikawa et al., 1978; Russell, surface during invasion should broaden our understand-1983; Pimenta et al., 1994). This junction, which initially ing of these events. These studies should also help ininvolves the anterior tip of the parasite, is progressively the development of malaria vaccines that aim at elicitingtranslocated to the posterior pole as the parasite antibodies against surface-exposed sporozoite ligands.squeezes into the host cell. Thus, parasite translocation Antibodies directed against crucial extracellular domainsis best explained by a capping-like process of a host- of TRAP should inhibit steps of the infectious processparasite tight junction (Dubremetz et al., 1985; King, 1988). subsequent to the region II plus–mediated attachment

The structural features of TRAP suggest that TRAP of sporozoites to the liver cells and thus increase themay link the substrate to the parasite microfilament sys- efficacy of existing CS-based vaccines (Sinnis and Nus-tem. TRAP is a typical single-pass transmembrane pro- senzweig, 1996; Stoute et al., 1997; Nussenzweig andtein. The extracellular region contains two highly con- Zavala, 1997).served matrix- or cell-binding domains (Figure 5a). The

Experimental Proceduresregion II plus, which is homologous to the region II plusof CS and to a portion of the thrombospondin type 1

Cloning of the P. berghei Strain NK65 TRAP Genedomain, binds to sulfated glycoconjugates (Muller etand Plasmid Constructions

al., 1993). TRAP also contains a typical A module. The The entire TRAP coding region of P. berghei strain NK65 was clonedproteins that incorporate type A modules, including the by PCR using erythrocytic stage genomic DNA and oligonucleotidesa chains of b1 and b2 integrins, interact with a large PbTRAP-FOR (sense, 59-CCCGGATCCATGAAGCTCTTAGGAAA

TAG-39) and PbTRAP-REV (antisense, 59-CCCGGATCCGTTCCAGTarray of cell surface– or extracellular matrix–associatedCATTATCTTC-39) designed on the basis of the TRAP sequence ofligands, such as proteoglycans, intercellular adhesionP. berghei ANKA strain (Robson et al., 1997). The 1.8-kb PCR prod-molecules (ICAMs), or collagens to participate in numer-uct was cloned into plasmid pCRII (Invitrogen) to yield plasmid

ous biological events such as cell adhesion, homing, pTRAP-1. A TRAP internal fragment was amplified from pTRAP-1and migration (Colombatti et al., 1993; Lee et al., 1995). using oligonucleotides P1 (sense, 59-CGGAATTCAATGGTCAG

GAAATTCTTGACGA-39) and P4 (antisense, 59-CCCAAGCTTTCCGTThe highly conserved cytoplasmic domain of the TRAPTATTAGATTTAGACTG-39). This fragment lacks nucleotides 1–67proteins of plasmodial species (Templeton and Kaslow,and the last 66 codons of TRAP. This fragment was digested with1997) could conceivably be involved in signal transduc-EcoRI and HindIII, filled in, and cloned into plasmid pUC18 digested

tion between the substrate and the parasite microfila- with HincII, yielding plasmid pTRAP-2. Plasmid pINT was then con-ment system. During gliding motility, TRAP may link the structed by cloning the KpnI–PvuII fragment of pTRAP-2 into plas-

mid pMD204 (van Dijk et al., 1995) lacking its SpeI site and digestedparasite contractile system to the substrate directly, via

Cell520

with KpnI and HincII. Plasmid pREP was constructed by inserting isothiocyanate (FITC)-labeled secondary antibodies (goat anti-rab-bit or goat anti-mouse) for 1 hr at 378C in a humid chamber, washedthe HincII–EcoRV fragment of PMD204 bearing the DHFR-TS mutant

gene and 2.5 and 0.5 kb of its 59 and 39 untranslated regions, respec- in PBS, and examined under a fluorescence microscope.tively, into pTRAP-1 cut with HincII.

Electron MicroscopyMosquito midguts, collected at day 13 postfeeding, or free midgutTransformation Experiments and Clone Selectionsporozoites, collected at day 17 postfeeding, were fixed with 1%Merozoites (109) of P. berghei strain NK65 were transformed byglutaraldehyde (Sigma, St. Louis, MO; grade I) and 4% paraformal-electroporation with 20 mg of digested DNA and injected intrave-dehyde (Sigma) in PBS, osmicated, dehydrated, and embedded innously into young rats. Recipient rats received a daily intraperitonealAraldite (Ted Pella, Redding, CA). Thin sections were cut with a RMCinjection of pyrimethamine (25 mg/kg of body weight) for 5 consecu-MT-7 ultramicrotome and examined with a Zeiss EM910 electrontive days, and resistant parasites were cloned by limiting dilutionmicroscope. Negatives were scanned with a AGFA horizon Plusinto ten rats. Cloning experiments were considered valid only whenflatbed scanner. After processing of the images with Adobe Pho-four or fewer of the injected animals became infected. The genomictoshop and QuarkXpress, final prints were prepared with a Fuji Pic-DNA of erythrocytic stages collected from animals containing moretography 3000 digital image printer.than 1% parasitized red blood cells was then prepared (day 20–25

postelectroporation) as previously described (Menard et al., 1997).Acknowledgments

Southern Blot and PCR Analysis of Parasite DNA A. S., V. T., U. F., V. N., R. N., and R. M. have participated in theSouthern blot analysis of parasite DNA was performed as previously planning and execution of the experiments. K. R. and A. C. provideddescribed (Menard et al., 1997). To differentiate parasites having a the TRAP DNA sequence of the P. berghei strain ANKA prior towild-type or an INT disruptant TRAP locus, oligonucleotides O1 publication. We thank Rita Altszuler and Claudio Cortes for their(sense, 59-GTTGTGCTTTTATTATGCATAAGTGTG-39) and O2 (anti- invaluable contribution to experiments involving animals and mos-sense, 59-GCTAATCCTCCAATAATACCACCAGC-39), correspond- quitoes, and E. Douglas MacDonald for technical assistance withing to sequences immediately upstream (nucleotides 34–60) and electron microscopy. We thank Photini Sinnis for helpful discussionsdownstream (nucleotides 1634–1659) from the TRAP internal frag- and reviewing the manuscript. This work was supported by a “Newment, respectively, and oligonucleotide O3 (antisense, 59-GACGTTG Initiative in Malaria Research” grant from the Burroughs WellcomeTAAAACGACGG CCAGTC-39), encompassing the (220) primer of Fund, a grant from the Institute of Allergy and Infectious DiseasespUC18 (New England Biolabs Inc., Beverly, MA), were used. Geno- of the National Institutes of Health, and a grant by UNDP/Worldmic DNA from sporozoiteswas prepared from z106 midgut sporozo- Bank/WHO Special Programme for Research and Training in Tropi-ites collected at day 18 postfeeding using QIAamp Tissue kit (Qiagen cal Diseases (TDR). A. A. S. is a recipient of a Fogarty internationalInc., Chatsworth, CA). PCR reactions were performedwith the geno- research fellowship and R. M. of an EMBO fellowship. Correspon-mic DNA corresponding to z107 red blood cell stage parasites or dence should be addressed to R. M.of z104 sporozoites.

Received April 25, 1997; revised June 26, 1997.Analysis of Parasite Development and InfectivityAnopheles stephensi mosquitoes were fed on infected young rats Referencesand dissected at days 14 or 18 postfeeding. Sporozoite populationswere separated as described (Vanderberg, 1974, 1975) to minimize Aikawa, M.,Miller, L.H., Johnson, J., andRabbege, J. (1978). Erythro-cross-contamination between the two halves of the mosquito body cyte entry by malarial parasites. A moving junction between erythro-via hemolymph transfer. For hemolymph collection, a small hole cyte and parasite. J. Cell Biol. 77, 72–82.was pierced between the last two abdominal segments of mosqui-

Arrowood,M.J., Sterling, C.R., and Healey, M.C. (1991). Immunofluo-toes, and hemoceles were perfused with Medium 199 using a finelyrescent microscopical visualization of trails left by gliding Cryp-drawn pasteur pipet inserted into the membranous postspiraculartosporidium parvum sporozoites. J. Parasitol. 77, 315–317.area of the mesothorax. The first three drops of perfusate wereBall, G.H., and Chao, J. (1961). Infectivity to canaries of sporozoitescollected from each mosquito, and the perfusate from z30 mosqui-of Plasmodium relictum developing in vitro. J. Parasitol.47, 787–790.toes was pooled. Suspensions of sporozoites were centrifuged at

20 3 g for 5 min, the sporozoite-containing supernatant was col- Beier, J.C. (1993). Malaria sporozoites: survival, transmission andlected, and a sample of this supernatant was examined in a hemocy- disease control. Parasitol. Today 9, 210–215.tometer to determine the sporozoite concentration. To test sporozo- Bourguignon, L.Y.W., and Bourguignon, G.J. (1984). Capping andite infectivity, 0.2-ml sporozoite suspensions in Medium 199 were the cytoskeleton. Int. Rev. Cytol. 87, 195–224.injected intravenously into 21-day-old Sprague-Dawley rats, and Cerami, C., Frevert, U., Sinnis, P., Takacs, B., Clavijo, P., Santos,their parasitemia was checked daily by Giemsa-stained blood M.J., and Nussenzweig, V. (1992a). The basolateral domain of thesmears for 10 days. To analyze sporozoite motility, sporozoites were hepatocyte plasma membrane bears receptors for the circumsporo-maintained in 3% BSA-Medium 199 for up to 4 hr at 48C, which zoite protein of Plasmodium falciparum sporozoites. Cell 70,stimulates all patterns of sporozoite motility (Vanderberg, 1974), 1021–1033.before microscopic examination. To determine the proportion of

Cerami, C., Kwakye-Berko, F., and Nussenzweig, V. (1992b). Bindinginternalized sporozoites among salivary gland–associated sporozo-of malarial CS protein to sulfatides and cholesterol sulfate: depen-ites, salivary glands were dissected out at day 18 postfeeding anddency on disulfide bond formation between cysteines in region II.incubated in trypsin (50 mg/ml) in Medium 199 for 15 min at 378C.Mol. Biochem. Parasitol. 54, 1–12.Samples were then centrifugedfor 5 min, the supernatant containingColombatti, A., Bonaldo, P., and Doliana, R. (1993). Type A modules:the attached sporozoites was collected, and the salivary glandsinteracting domains found in several non-fibrillar collagens and inwere then ground to free internalized sporozoites.other extracellular matrix proteins. Matrix 13, 297–306.

Cowan, G., Krishna, S., Crisanti, A., and Robson, K. (1992). Expres-Sporozoite Immunostaining Assayssion of thrombospondin-related anonymous protein in PlasmodiumSporozoites were collected at day 18 postfeeding and processedfalciparum sporozoites. Lancet 339, 1412–1413.live or air-dried after fixationwith acetone for 15 min at room temper-Crabb, B.S., and Cowman, A.F. (1996). Characterization of promot-ature. Sporozoites werepreincubated in 0.5% bovine serum albuminers and stable transfection by homologous and nonhomologous(BSA) in PBS for 30 min at room temperature, washed in PBS,recombination in Plasmodium falciparum. Proc. Natl Acad. Sci. USAincubated with primary antibodies (anti-TRAP rabbit polyclonal anti-93, 7289–7294.sera 1 or 2, or anti-CS 3D11 mouse MAbs) for 1 hr at 378C in a humid

chamber, washed three times in PBS, incubated with fluorescein Dobrowolski, J.M., and Sibley, L.D. (1996). Toxoplasma invasion of

TRAP-Dependent Infectivity and Motility of Malaria Parasites521

mammalian cells is powered by the actin cytoskeleton of the para- Robson, K.J.H., Naitza, S., Barker, G., Sinden, R.E., and Crisanti,site. Cell 84, 933–939. A. (1997). Cloning and expression of the thrombospondin related

adhesive protein gene of Plasmodium berghei. Mol. Biochem. Para-Dubremetz, J.-F., and Torpier, G. (1978). Freeze fracture study ofsitol. 84, 1–12.the pellicle of an eimerine sporozoite. J. Ultrastruct. Res. 62, 94–109.Rogers, W.O., Malik, A., Mellouk, S., Nakamura, K., Robers, M.D.,Dubremetz, J.-F., Rodriguez, C., and Ferreira, E. (1985). ToxoplasmaSzarfman, A., Gordon, D.M., Nussler, A., Aikawa, M., and Hoffman,gondii: redistribution of monoclonal antibodies on tachyzoites dur-S.L. (1992a). Characterization of Plasmodium falciparum sporozoiteing host cell invasion. Exp. Parasitol. 59, 24–32.surface protein 2. Proc. Natl Acad. Sci. USA 89, 9176–9180.Entzeroth, R., Zgrzebski, G., and Dubremetz, J.-F. (1989). SecretionRogers, W.O., Rogers, M.D., Hedstrom, R.C., and Hoffman, S.L.of trails during gliding motility of Eimeria nieshulzi (Apicomplexa,(1992b). Characterization of the gene encoding sporozoite surfaceCoccidia) sporozoites visualized by a monoclonal antibody and im-protein 2, a protective Plasmodium yoelii sporozoite antigen. Mol.muno-gold-silver enhancement. Parasitol. Res. 76, 174–175.Biochem. Parasitol. 53, 45–52.Fine, E., Aikawa, M., Cochrane, A.H., and Nussenzweig, R.S.Russell, D.G. (1983). Host cell invasion by Apicomplexa: an expres-(1984). Immuno-electron microscopic observations on Plasmodiumsion of the parasite’s contractile system? Parasitology 87, 199–209.knowlesi sporozoites: localization of protective antigen and its pre-

cursors. Am. J. Trop. Med. Hyg. 33, 220–226. Russell, D.G., and Sinden, R.E. (1981). The role of the cytoskeletonin the motility of coccidian sporozoites. J. Cell Sci. 50, 345–359.Frevert, U., Sinnis, P., Cerami, C., Shreffler, W., Takacs, B., and

Nussenzweig, V. (1993). Malaria circumsporozoite protein binds to Ryning, F.W., and Remington, J.S. (1978). Effect of cytochalasin Dheparan sulfate proteoglycans associated with the surface mem- at Toxoplasma gondii cell entry. Infect. Immun. 20, 739–743.brane of hepatocytes. J. Exp. Med. 177, 1287–1298. Shakibaei, M., and Frevert, U. (1996). Dual interaction of the malariaHollingdale, M.R., Zavala, F., Nussenzweig, R.S., and Nussenzweig, circumsporozoite protein with the low density lipoprotein receptor-V. (1982). Antibodies to the protective antigen of Plasmodium ber- related protein (LRP) and heparan sulfate proteoglycans. J. Exp.ghei sporozoites prevent entry into cultured cells. J. Immunol. 128, Med. 184, 1699–1711.1929–1930. Sinnis, P. (1996). The malaria sporozoite’s journey into the liver.Jensen, J.B., and Edgar, S.A. (1978). Fine structure of penetration Infect. Agents Dis. 5, 182–189.of cultured cells by Isospora canis sporozoites. J. Protozool. 25, Sinnis, P., and Nussenzweig, V. (1996). Preventing sporozoite inva-169–173. sion of hepatocytes. In Malaria Vaccine Development: A Multi-King, C.A. (1988). Cell motility of Sporozoan protozoa. Parasitol. Immune Response Approach, S.L. Hoffman, ed. (Washington, DC:Today 4, 315–319. ASM Press), pp. 15–33.Lee, J.-O., Rieu, P., Arnaout, M.A., and Liddington, R. (1995). Crystal Sinnis, P., Clavijo, P., Fenyo, D., Chait, B.T., Cerami, C., and Nussen-structure of the A domain from the a subunit of integrin CR3 (CD11b/ zweig, V. (1994). Structural and functional properties of region II-CD18). Cell 80, 631–638. plus of the malaria circumsporozoite protein. J. Exp. Med. 180,

297–306.McCutchan, T.F., Kissinger, J.C., Touray, M.G., Rogers, M.G., Li,J., Sullivan, M., Braga, E.M., Krettli, A.U., and Miller, L.H. (1996). Sinnis, P., Willnow, T.E., Briones, M.R.S., Herz, J., and Nussenzweig,Comparison of circumsporozoite proteins from avian and mamma- V. (1996). Remnant lipoproteins inhibit malaria sporozoite invasionlian malarias: biological and phylogenetic implications. Proc. Natl of hepatocytes. J. Exp. Med. 184, 945–954.Acad. Sci. USA 93, 11889–11894. Speer, C.A., Wong, R.B., Blixt, J.A., and Schenkel, R.H. (1985). Cap-Menard, R., Sultan, A.A., Cortes, C., Altszuler, R., van Dijk, M.R., ping of immune complexes by sporozoites of Eimeria tenella. J.Janse, C.J., Waters, A.P., Nussenzweig, R.S., and Nussenzweig, Parasitol. 71, 33–42.V. (1997). Circumsporozoite protein is required for development of Stewart, M.J., and Vanderberg, J.P. (1988). Malaria sporozoitesmalaria sporozoites in mosquitoes. Nature 385, 336–340. leave behind trails of Circumsporozoite protein during gliding motil-Muller, H.M., Reckman, I., Hollingdale, M.R., Bujard, H., Robson, ity. J. Protozool 35, 389–393.K.J.H., and Crisanti, A. (1993). Thrombospondin-related anonymous Stewart, M.J., and Vanderberg, J.P. (1991). Malaria sporozoites re-protein (TRAP) of Plasmodium falciparum binds specifically to sul- lease Circumsporozoiteprotein from their apical end andtranslocatefated glycoconjugates and to HepG2 hepatoma cells suggesting a it along their surface. J. Protozool. 38, 411–421.role for this molecule in sporozoite invasion of hepatocytes. EMBO

Stewart, M.J., and Vanderberg, J.P. (1992). Electron microscopicJ. 12, 2881–2889.analysis of circumsporozoite protein trail formation by gliding ma-

Nagasawa, H., Aikawa, M., Procell, P.M., Campbell, G.H., Collins,laria sporozoites. J. Protozool. 39, 663–671.W.E., and Campbell, C.C. (1988). Plasmodium malariae: distributionStewart, M.J., Nawrot, R.J., Schulman, S., and Vanderberg, J.P.of circumsporozoite protein in midgut oocysts and salivary gland(1986). Plasmodium berghei sporozoite invasion is blocked in vitrosporozoites. Exp. Parasitol. 66, 27–34.by sporozoite-immobilizing antibodies. Infect. Immun. 51, 859–864.Nussenzweig, R.S., and Zavala, F. (1997). A malaria vaccine basedStoute, J.A., Slaoui, M., Heppner, D.G., Momin, P., Kester, K.E.,on a sporozoite antigen. N. Engl. J. Med. 336, 128–130.Desmons, P., Wellde, B.T., Garcon, N., Krzych, U., Marchand, M.,Nussenzweig, V., and Nussenzweig, R.S. (1985). CircumsporozoiteBallou, R., and Cohen, J.D. (1997). A preliminary evaluation of aproteins of malaria parasites. Cell 42, 401–403.recombinant circumsporozoite protein vaccine against Plasmodium

Pancake, S.J., Holt, G.D., Mellouk, S., and Hoffman, S.L. (1992). falciparum malaria. N. Engl. J. Med. 336, 86–91.Malaria sporozoites and circumsporozoites proteins bind specifi-

Templeton, T.J., and Kaslow, D.C. (1997). Cloning and cross-speciescally to sulfated glycoconjugates. J. Cell Biol. 117, 1351–1357.comparison of the thrombospondin-related anonymous protein

Pimenta, P.F., Touray,M., and Miller, L. (1994). The journey of malaria (TRAP) gene from Plasmodium knowlesi, Plasmodium vivax, andsporozoites in the mosquito salivary gland. J. Euk. Microbiol. 41, Plasmodium gallinaceum. Mol. Biochem. Parasitol. 84, 13–24.608–624.

Tomley, F.M., Clarke, L.E., Kawazoe, U., Dijkema, R., and Kok, J.J.Robson, K.J.H., Hall, J.R.S., Jennings, M.W., Harris, T.J.R., Marsh, (1991). Sequence of the gene encoding an immunodominant micro-K., Newbold, C.I., Tate, V.E., and Weatherall, D.J. (1988). A highly neme protein of Eimeria tenella. Mol. Biochem. Parasitol. 49,conserved amino-acidsequence inthrombospondin, properdin, and 277–288.in proteins from sporozoites and blood stages of a human malaria

van Dijk, M.R., Waters, A.P., and Janse, C.J. (1995). Stable transfec-parasite. Nature 335, 79–82.tion of malaria parasite blood stages. Science 268, 1358–1362.

Robson, K.J.H., Frevert, U., Reckmann, I., Cowan, G., Beier, J.,van Dijk, M.R., Janse, C.J., and Waters, A.P. (1996). Expression ofScragg, I.G., Takehara, K., Bishop, D.H.L., Pradel, G., Sinden, R.,a Plasmodium gene introduced into subtelomeric regions of Plas-Saccheo, S., Muller, H.M., and Crisanti, A. (1995). Thrombospondin-modium berghei chromosomes. Science 271, 662–665.related adhesive protein (TRAP) of Plasmodium falciparum: expres-Vanderberg, J.P. (1974). Studies on the motility of Plasmodium spo-sion during sporozoite ontogeny and binding to human hepatocytes.

EMBO J. 14, 3883–3894. rozoites. J. Protozool. 21, 527–537.

Cell522

Vanderberg, J.P. (1975). Development of infectivity by the Plasmo-dium berghei sporozoite. J. Parasitol. 61, 43–50.

Vanderberg, J., Rdodin, J., and Yoeli, M. (1967). Electron micro-scopic andhistochemical studies of sporozoite formation in Plasmo-dium berghei. J. Protozool. 14, 82–103.

Vanderberg, J., Chew, S., and Stewart, M.J. (1990). Plasmodiumsporozoite interactions with macrophages in vitro: a videomicro-scopic analysis. J. Protozool. 37, 528–536.

Wan, K.-L., Carruthers, V.B., Sibley, L.D., and Ajioka, J.W. (1997).Molecular characterization of an expressed sequence tag locus ofToxoplasma gondii encoding the micronemal protein MIC2. Mol.Biochem. Parasitol. 84, 203–214.

Warburg, A., Touray, M., Krettli, A.U., and Miller, L.H. (1992). Plasmo-dium gallinaceum: antibodies to circumsporozoite prevent sporozo-ites from invading the salivary glands of Aedes aegypti. Exp. Parasi-tol. 75, 303–307.

Wu, Y., Kirkman, L.A., and Wellems, T.E. (1996). Transformation ofPlasmodium falciparum malaria parasites by homologous integra-tion of plasmids that confer resistance to pyrimethamine. Proc. NatlAcad. Sci. USA 93, 1130–1134.

Yoshida, N., Nussenzweig, R.S., Potocnjak, P., Aikawa, M., and Nus-senzweig, V. (1980). Hybridoma produces protective antibodies di-rected against the sporozoite stage of malaria parasite. Science207, 71–75.

Yoshida, N., Potocnjak, P., Nussenzweig, V., and Nussenzweig, R.S.(1981). Biosynthesis of Pb44, the protective antigen of sporozoitesof Plasmodium berghei. J. Exp. Med. 154, 1225–1236.