plasmodium sporozoite molecular cell biology - Semantic Scholar

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Annu. Rev. Cell Dev. Biol. 2004. 20:29–59 doi: 10.1146/annurev.cellbio.20.011603.150935 c 2004 by Annual Reviews. All rights reserved Copyright First published online as a Review in Advance on April 9, 2004

Annu. Rev. Cell. Dev. Biol. 2004.20:29-59. Downloaded from arjournals.annualreviews.org by NEW YORK UNIVERSITY - BOBST LIBRARY on 04/28/06. For personal use only.

PLASMODIUM SPOROZOITE MOLECULAR CELL BIOLOGY Stefan H.I. Kappe,1 Carlos A. Buscaglia,2 and Victor Nussenzweig2 1

Seattle Biomedical Research Institute, Seattle, Washington 98109-1651; email: [email protected] 2 Michael Heidelberger Division of Immunology, Department of Pathology, New York University School of Medicine, New York 10016; email: [email protected]; [email protected]

Key Words malaria, Apicomplexa, hepatocyte, gliding motility, cell invasion ■ Abstract Plasmodium sporozoites display complex phenotypes including gliding motility and invasion of and transmigration through cells in the mosquito vector and the vertebrate host. Sporozoite studies have been difficult to perform because of technical concerns. Nevertheless, they have already provided insights into several aspects of sporozoite biology, shared in part with other apicomplexan invasive stages. Structure/function analysis of the thrombospondin-related anonymous protein paved the way to the understanding of the molecular mechanisms of apicomplexan gliding motility and host cell invasion. Functional studies of circumsporozoite protein revealed its role in Plasmodium sporozoite morphogenesis in addition to its well-known function in host cell invasion. Transcriptional surveys, which facilitate the investigation of gene expression programs that control sporozoite phenotypes, have revealed a high degree of previously unappreciated complexity and novel proteins that mediate sporozoite host cell infection.

CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GENE EXPRESSION IN SPOROZOITES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SPOROZOITE PROTEINS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Circumsporozoite Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Thrombospondin-Related Anonymous Protein . . . . . . . . . . . . . . . . . . . . . . . . . Apical Membrane Antigen/Erythrocyte Binding-Like Protein . . . . . . . . . . . . . . . . . Scavenger Receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SPOROZOITE DEVELOPMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . GLIDING MOTILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SPOROZOITE INVASION OF MOSQUITO SALIVARY GLANDS . . . . . . . . . . . . . DIFFERENCES IN OOCYST AND SALIVARY GLAND SPOROZOITES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1081-0706/04/1115-0029$14.00

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SPOROZOITE MIGRATION AND INVASION OF HEPATOCYTES . . . . . . . . . . . . 47 CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50


INTRODUCTION Malaria is a widespread infectious disease of humans, with an estimated toll of 300 million new cases that result in ∼1–3 millions deaths every year. Malaria parasites (Plasmodium spp.) are part of the phylum Apicomplexa that includes other important intracellular pathogens such as Toxoplasma, Cryptosporidium, Eimeria, Babesia, and Theileria. The phylum is characterized by the presence of a chloroplast-like juxta-nuclear apicoplast (Foth & McFadden 2003), the apical polar ring, which serves as a microtubule (MT)-organizing center, and a unique set of secretory vesicles, termed micronemes and rhoptries, that define the apical complex (Figure 1). Micronemes are small vesicles of varying electron density in Plasmodium sporozoites that frequently show a neck-like extension. Rhoptries are large, usually paired, pear-shaped organelles filled with proteins and phospholipids. Both organelles discharge at the anterior tip of the parasite, and their contents (and that of dense granules, not yet identified in Plasmodium sporozoites) are involved in apicomplexan motility, host cell invasion, and the generation of the nonphagosomal parasitophorous vacuole (PV), where the parasite resides and replicates inside the host cell. The cell cortex of apicomplexan invasive forms is surrounded by rows of MTs that support a composite triple-membrane pellicle, consisting of the outer plasma membrane and a closely juxtaposed inner membrane complex (IMC) (Morrissette & Sibley 2002). An actin-myosin motor essential for parasite motility and invasion is located in the narrow space between the plasma membrane and the outer membrane of the IMC (Kappe et al. 2004). Malaria infection starts when infected Anopheles mosquitoes take their blood meal and inject sporozoites into the host skin. Sporozoites enter blood vessels, are transported to the liver, invade hepatocytes, and develop into liver stages, also termed exo-erythrocytic forms (EEFs). EEFs grow and differentiate into thousands of merozoites. Upon rupture of the infected hepatocytes, merozoites are released and rapidly enter red cells, where they undergo schizogony and propagate the blood cycle of the infection that causes the malaria symptoms. Some intra-erythrocytic merozoites develop into gametocytes that are taken up by mosquitoes with the blood meal and fuse to form zygotes, which subsequently transform into the invasive ookinetes. Ookinetes reach the space between the midgut epithelium and the basal lamina of the mosquito where they transform into oocysts (Figure 2). Thousands of sporozoites develop from the multinucleated oocysts and are released into the hemolymph. They invade the mosquito salivary glands, where they remain viable but do not replicate (Beier & Vanderberg 1998). Investigations into the biology of sporozoites have long lagged behind those in blood stages, mainly because of the inherent inaccessibility of this parasite stage. Sporozoites have to be isolated from infected mosquitoes, are always contaminated

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Apical end

Polar ring

Apical complex

Rhoptries


Micronemes

Golgi

Apicoplast

Nucleus Ribosomes Endoplasmic reticulum Mitochondria Inner membrane complex (IMC)

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actin actin myosin motor

Figure 1 Schematic representation of a Plasmodium sporozoite showing some of its organelles and subcellular structures.

by mosquito tissue, and can be obtained only in relatively small numbers. However, recent advances in genomics and proteomics and the development of new molecular and cellular tools such as green fluorescent protein (GFP)-expressing parasites (Natarajan et al. 2001) have contributed to a wealth of new information about sporozoites. In addition, the possibility of in vitro culturing of sporozoites (AlOlayan et al. 2002) should accelerate the understanding of the parasite biology. In this review, we focus on the molecular mechanisms of Plasmodium sporozoite development, migration, and host cell infection.

GENE EXPRESSION IN SPOROZOITES The past few years have seen considerable progress in the identification of sporozoite-expressed genes. An expressed sequence tag project of Plasmodium yoelii (rodent parasite) salivary gland sporozoites identified many novel genes

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Kupffer cell 5

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Figure 2 The Plasmodium life cycle. (1) Ookinetes penetrate the mosquito midgut epithelium and transmigrate to the side facing the mosquito body cavity. They develop into an oocyst under the midgut basement membrane. (2) Within the oocyst sporozoites differentiate and emerge by budding. (3) Completely formed sporozoites are released into the body cavity and invade the salivary glands. Sporozoites accumulate in the salivary ducts of the glands and are transmitted to the next mammalian host. (4) They are transported to the liver by the blood stream and are arrested on the liver sinusoid endothelium. They move on the endothelium until they encounter a Kupffer cell. (5) Sporozoites traverse Kupffer cells and invade the underlying hepatocytes. During invasion a vacuolar compartment forms in which the sporozoite transforms into EEFs. (6) EEFs rupture, releasing merozoites into the blood where they invade erythrocytes and initiate the erythrocytic cycle. (7) Some parasites differentiate into gametocytes, which are transmitted to the mosquito vector during a bite. (8) Male gametocytes undergo exflagellation, generating male microgametes. (9) A female gamete and a male microgamete fuse to form a zygote, which develops into an ookinete (10). Tissues and parasite stages are not drawn to scale.

and others not previously reported to be transcriptionally active in this parasite stage (Kappe et al. 2001). In addition, differential gene expression analysis using suppression subtractive hybridization (SSH) revealed extensive differences in the transcriptomes of sporozoites emerging from the oocysts and those within the mosquito salivary glands (Matuschewski et al. 2002b). This methodology was also successfully used to profile gene expression during oocyst development and sporozoite differentiation (Srinivasan et al. 2004) and to identify genes whose expression is restricted to sporozoites such as those coding for a novel thrombospondin-related

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sporozoite protein (Kaiser et al. 2004b), which possibly localizes to the rhoptries, and a perforin-like protein that localizes to the micronemes (Kaiser et al. 2004a). Publication of the complete or nearly complete genome sequences of different Plasmodium spp. (Carlton et al. 2002, Gardner et al. 2002) has tremendously eased gene expression analysis and allowed the construction of oligonucleotide microarrays with genome-wide coverage (Bozdech et al. 2003a, Le Roch et al. 2003). The use of these microarrays led to the identification of ∼2000 genes that are transcriptionally active in salivary gland sporozoites, including ∼500 genes that are highly expressed. More than 100 of these genes are not significantly expressed in blood stages (Le Roch et al. 2003). A sizable number of sporozoite-specific genes had previously been identified by SSH, confirming both data sets. Microarray studies also confirmed sporozoite expression of the blood stage multigene families rifin, stevor, and var, which were previously identified in a proteomic analysis of sporozoites (Florens et al. 2002). Proteins encoded by var genes (PfEMP1) are expressed on the infected red cell surface and mediate binding to endothelial receptors (Craig & Scherf 2001). The functional significance of their expression in sporozoites is unclear but warrants further investigations. Sporozoiteexpressed genes are distributed over all chromosomes and do not necessarily cluster in transcriptionally active regions except for the multigene families mentioned above.

SPOROZOITE PROTEINS The detailed discussion of each sporozoite protein is beyond the scope of this review, and we therefore focus on those that have been well characterized and for which functional data are available.

Circumsporozoite Protein Circumsporozoite (CS) protein is a distinctive molecule of the Plasmodium genus not found in other Apicomplexa. It is encoded by a single copy gene and covers the entire surface of sporozoites (Nussenzweig & Nussenzweig 1989). Following sporozoite invasion of hepatocytes, CS is also detected on the plasma membrane of early EEFs and in the cytoplasm of the infected cells (Hamilton et al. 1988). Due to its abundance, surface localization, immunogenicity, and key role in parasite invasion, CS constitutes the leading candidate molecule for the development of malaria pre-erythrocytic vaccines (Saul et al. 2004). CS proteins from different Plasmodium spp. display common structural features, including a signal peptide, a central domain composed mostly of amino acids repeats, and a C-terminal hydrophobic sequence (Figure 3) (McCutchan et al. 1996). The conserved motifs among CS proteins are called region I, II-plus, and III. Region I consists of the pentapeptide KLKQP, and it is involved in the attachment of sporozoites to different cells (see below). Region II-plus is an 18– amino acid sequence embedded at the proximal region of the thrombospondin

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Tandem Repeats

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TRAP Annu. Rev. Cell. Dev. Biol. 2004.20:29-59. Downloaded from arjournals.annualreviews.org by NEW YORK UNIVERSITY - BOBST LIBRARY on 04/28/06. For personal use only.

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PxSR Figure 3 Schematic representations of the CS, TRAP, MAEBL, and SR proteins. CD, cytoplasmic domain; GPI, glycosyl phosphatidylinositol; SP, signal peptide; RI, region I; RII-plus, region II-plus; RIII, region III; TD, transmembrane domain; TSR, thrombospondin type I repeat domain. Details regarding these domains are described in the text. The MAEBL MI and MII domains are cysteine-rich domains similar to that in AMA-1 and the c-cys domain, and the exon/intron structure of the C terminus is similar to that in the erythrocyte binding-like proteins (exons are depicted as boxes, introns as lines). The SR protein displays four Limulus factor C, Coch-5b2, Lgl1 (LCCL) domains, two copies of the scavenger receptor cysteine-rich (SRCR), one pentraxin domain (PTX), and one lipooxygenase domain (LH2).

repeat (TSR) type 1 domain. It includes a conserved tryptophan residue, a pair of cysteines, and a cluster of basic residues interspersed with hydrophobic amino acids. This region mediates the adhesion of sporozoites to the heparan sulfate proteoglycans (HSPGs) in liver sinusoids and might also be relevant for the invasion of mosquito salivary glands (see below). TSR is an ancient eukaryotic domain present in a large number of proteins involved in immunity, cell adhesion, angiogenesis, and the development of the nervous system (Adams & Tucker 2000). It has a novel three-stranded fold, and its front contains a positively charged groove that might accommodate sulfate groups of HSPGs (Tan et al. 2002). In addition to CS proteins, canonical or altered TSR motifs are present in several apicomplexan proteins (Chattopadhyay et al. 2003, Deng et al. 2002, Kaiser et al. 2004b, Kappe et al. 2001, Menard 2001). Region III is predicted to form an amphipathic α-helix that may provide the proper framework for the neighboring region II-plus adhesion motif.

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The central domain of the CS protein is formed by a variable number of intandem repeats that contain B-cell immunodominant epitopes (Godson et al. 1983), a common theme among proteins from protozoan parasites (Buscaglia et al. 1999, Kemp et al. 1987). The sequence of the CS repeats is species specific, thus constituting a useful antigenic signature to label sporozoites in extracts of mosquitoes captured in endemic areas (Zavala et al. 1982). In Plasmodium falciparum (human parasite), the CS repetitive unit consists of the tetrapeptide NANP and three NVDP variants. In spite of this amino acid conservation, a large number of synonymous nucleotide polymorphisms are detected among the repeat units from different P. falciparum isolates and even among the repeat units of the same parasite stock (Rich et al. 2000), suggesting that they are under selective pressure. Other Plasmodium spp. show diversity in the repeat sequence of isolates from different endemic areas, although the repeat units are invariant within each strain (Arnot et al. 1985, de la Cruz et al. 1987, Galinski et al. 1987). The functional role of the CS repeats is unknown. They are unlikely to represent the sporozoite liver ligand because the repeat sequence differs between species of Plasmodium that infect the same host. Furthermore, the phenotype of the rodent parasite Plasmodium berghei bearing a hybrid CS protein that displays the repeats of P. falciparum CS is indistinguishable from the wild type (Persson et al. 2002), indicating that the identity of the repeats is not important for the parasite viability. Perhaps CS repeats fulfill a structural role, i.e., repeats from neighboring CS molecules may interlock to form a rigid sheath surrounding the parasite (Godson et al. 1983). Sequence variation within the repeats might preclude the assembly of this quaternary structure. The C terminus of CS proteins consists of a hydrophobic stretch of residues that typically encodes a GPI attachment signal, but this has not been formally demonstrated. Recent biosynthetic studies documented the incorporation of labeled palmytic acid in CS and the release of surface-associated CS by GPI-specific phospholipase D (P. Sinnis, unpublished data). CS protein in sporozoite extracts migrates as a triple band on SDS-PAGE. A precursor-product relationship between these forms has been demonstrated (Yoshida et al. 1981). The shorter CS form appears to begin at the boundary of region I. CS N-terminal trimming is mediated by a parasite-derived cystein protease(s) and is likely to be critical for the sporozoite infectivity toward mammalian cells both in vitro and in vivo (P. Sinnis, unpublished data). In Toxoplasma tachyzoites and Plasmodium merozoites, different attachment/invasion-associated proteins also undergo complex intra- or extracellular proteolytic processing critical for cell invasion (Blackman 2000, Carruthers et al. 2000, Cerede et al. 2002). Salivary gland sporozoites undergo a characteristic morphological change when incubated with antibodies to CS. A thick precipitate is formed on their surface, and it is gradually shed from the posterior end of the parasites [circumsporozoite protein (CSP) reaction] (Cochrane et al. 1976, Vanderberg 1974). The CSP reaction is the morphological representation of the continuous secretion of CS from the anterior end of the parasite, its incorporation on the plasma membrane, and

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its release at the posterior end. When the parasites move on glass surfaces, the released CS appears in circular trails and is embedded in vesicle-like particles of unknown composition (Stewart & Vanderberg 1991). The apical sporozoite secretory organelles appear to contain CS (Aikawa et al. 1990, Nagasawa et al. 1987, Posthuma et al. 1989). However, definitive colocalization studies of CS with known markers have not been done. In Toxoplasma the sorting of GPI-anchored molecules to the surface is in fact independent of micronemes and/or rhoptries (Joiner & Roos 2002).

The Thrombospondin-Related Anonymous Protein The thrombospondin-related anonymous protein (TRAP) is the founding member of a family of apicomplexan type I trans-membrane proteins (Menard 2001). This family is characterized by the presence of an acidic cytoplasmic domain containing a conserved tryptophan residue and single or multiple copies of two extracellular adhesive domains, the A domain (or I domain of integrins), and the TSR (Figure 3). The TRAP family arose early in apicomplexan evolution because TRAP-like proteins are found throughout the phylum. The family includes MIC2 of Toxoplasma (Wan et al. 1997), Etp100/EtMIC1 of Eimeria (Tomley et al. 2001), NcMIC2 of Neospora (Lovett et al. 2000), and a recently described BbTRAP from Babesia (E. de Vries, unpublished data). Cryptosporidium TRAPC1 is an unusual TRAP-like protein that lacks an A domain (Spano et al. 1998). Several apicomplexan transmembrane molecules displaying a cytoplasmic tail resembling that of TRAP and different adhesive motifs on the extracellular portion have been described (Tomley et al. 2001, Meissner et al. 2002a). Some of these proteins might have a role analogous to TRAP in parasite motility (see below), although engaging different counter-receptors on the surface of the respective target host cells. In Plasmodium, two identified members of the TRAP family are expressed: TRAP in sporozoites and CS- and TRAP-related protein (CTRP) in ookinetes (Trottein et al. 1995). Whereas TRAP has single adhesive modules, CTRP has multiple A domains and TSRs. The TRAP family members connect the host cell receptors with the molecular motor that drives Apicomplexa motility and cell invasion. Indeed, P. berghei parasites carrying a targeted disruption of TRAP (TRAP-) develop normally within oocysts, but the free sporozoites are immobile and do not infect cells (Sultan et al. 1997b, 2001). Similarly, ookinetes from CTRP-parasites have impaired motility and do not invade the mosquito midgut (Dessens et al. 1999, Templeton et al. 2000, Yuda et al. 1999). In addition, TRAP may modulate the development of malaria-associated pathology by triggering an antiinflammatory TGF-β response in the infected mammalian host (Omer et al. 2003). TRAP is found in micronemes and on the plasma membrane of sporozoites with a characteristic patchy distribution. A tyrosine-containing motif present in the TRAP cytoplasmic domain leads to its subcellular localization (Bhanot et al. 2003).

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This motif (Yxx, where stands for a bulky hydrophobic residue) is found in numerous eukaryotic proteins and participates in endosome and lysosome sorting (Bonifacino & Dell’Angelica 1999). This same motif directs rhoptry localization of Toxoplasma ROP2 (Hoppe et al. 2000) and micronemal localization of MIC2 (Di Cristina et al. 2000). In the case of MIC2, its microneme/surface localization requires an escorter protein termed M2AP (Huynh et al. 2003), and the formation of a multimeric MIC2/M2AP complex (Jewett & Sibley 2004). No M2AP orthologue is found in the Plasmodium genome. The A (or I) domains are formed by ∼200 amino acids, and are found in the α-chains of nine integrins; in von Willebrand factor; in several matrix proteins such as collagens, undulin, and cartilage matrix protein; and in complement components (Michishita et al. 1993). The crystal structure of the A domain reveals the presence of seven α-helices and a β-sheet (Emsley et al. 1998, Li et al. 1998). At the top of the sheet there is a crevice containing the conserved 5–amino acid motif (metal ion-dependent adhesion site or MIDAS) that coordinates Mg2+ or Mn2+ (Michishita et al. 1993). Structural/functional studies of integrins implicate the MIDAS motif in ligand binding (Lee et al. 1995). The A domain of TRAP has been modeled on that obtained from integrin CD11B/CD18, and it includes the MIDAS motif (Gantt et al. 2000). The adhesive functions of A domains are allosterically controlled by conformational changes triggered by interactions involving the integrin cytoplasmic domains and vice versa (bidirectional signaling) (Hynes 2002). It is conceivable that TRAP function is controlled in an analogous fashion. As in CS, the ligands of the TSR of TRAP-like proteins are most likely HSPGs (Robson et al. 1995). When expressed as a fusion protein in bacteria, the A domain also binds to heparin and other HSPGs in a multimeric state, but the binding is independent of the MIDAS (Harper et al. 2004, McCormick et al. 1999). In vivo studies using parasites expressing TRAP mutant proteins suggest instead that the A domain and TSR recognize distinct cellular receptors (Matuschewski et al. 2002a). Integrins containing A domains recognize several collagens, laminin, ICAM-1 and ICAM-2, complement fragments of C3 and C4, Factor X, and fibrinogen. Some of these molecules can be excluded as essential TRAP ligands: Knockout mice for ICAM-1, ICAM-2, C3, and C4 were as susceptible to sporozoite infection as control mice (Sultan et al. 1997a). The amino acid sequences of the cytoplasmic domains of TRAP family members have little in common: the Yxx motif (see above), a conserved tryptophan close to the C terminus, and an abundance of acidic residues. Notwithstanding the sequence differences, these domains are functionally identical (Kappe et al. 1999), suggesting that only a few shared features are required for a productive interaction with the actin-myosin motor. Indeed, a small C-terminal deletion of the TRAP tail or the substitution of the conserved tryptophan or of neighboring acidic residues abolishes sporozoite gliding and invasion but does not affect protein localization (Kappe et al. 1999).

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Apical Membrane Antigen/Erythrocyte Binding-Like Protein Apical membrane antigen/erythrocyte binding-like (MAEBL) protein is a type I trans-membrane protein with a chimeric structure (Figure 3), highly conserved among Plasmodium spp. (Kappe et al. 1998). MAEBL shares similarity with apical membrane antigen-1 (AMA-1) in the N-terminal portion and, accordingly, it shows in vitro binding activity to red blood cells (Kappe et al. 1998). The C terminus of MAEBL is constituted by tandem amino acid repeats followed by a cysteinerich region highly similar to that present in the erythrocyte-binding protein family (Adams et al. 1992). MAEBL localizes to micronemes and/or the parasite surface and is expressed in blood stage merozoites, late oocysts, and sporozoites (Kappe et al. 2001, Kariu et al. 2002, Noe & Adams 1998, Srinivasan et al. 2004). Targeted disruption of P. berghei MAEBL has no apparent effect in the parasite life cycle in the blood, but MAEBL-sporozoites are incapable of infecting the mosquito salivary glands (Kariu et al. 2002).

Scavenger Receptor The scavenger receptor (SR) protein displays a multi-domain structure that includes four copies of the LCCL module found in Limulus clotting factor C (Dessens et al. 2004), two copies of the SR cysteine-rich domains present in proteins of the immune system, one pentraxin domain highly similar to that of agglutinating serum proteins, and one domain likely involved in protein-lipid interactions (Figure 3) (Claudianos et al. 2002). SR protein was originally described as a gametocyterestricted molecule (Delrieu et al. 2002), but its role in this stage of the parasite is not essential, because SR- parasites formed normal numbers of gametocytes in the blood of mice and normal ookinetes and oocysts in infected mosquitoes. However, SR-parasites are incapable of forming sporozoites (Claudianos et al. 2002).

SPOROZOITE DEVELOPMENT The diploid zygote formed in the mosquito blood meal transforms into the motile and invasive ookinete that undergoes meiosis, which leads to the formation of four haploid genomes within its single nucleus (Sinden & Hartley 1985). The ookinete traverses several midgut epithelial cells and, after migration through the intercellular space, reaches the basal lamina where its transformation into the spherical oocyst takes place. The molecular aspects leading to ookinete invasion of the mosquito peritrophic matrix and midgut have been described elsewhere (Dessens et al. 2003, Dimopoulos et al. 2002, Han et al. 2000, Langer & Vinetz 2001). Early in its development, the subpellicullar MTs are disassembled; the oocyst loses the apical organelles and the IMC and secretes a cyst wall rich in transglutaminase (Adini et al. 2001) that separates the parasite from the basal lamina. Nutrients for oocyst development most likely originate from the hemolymph, which bathes the basal lamina (Beier & Vanderberg 1998). The large nucleus is initially polyploid

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and may contain two parental and two recombinant genotypes. The genome rapidly divides and after ∼10 days about 6–8000 haploid nuclei are formed. At the same time the cytoplasm is subdivided by multiple clefts that appear to originate from expansion of the endoplasmic reticulum cisternal space. The plasmalemma and the clefts form large vesicular structures called sporoblasts that are densely covered with the CS protein (Aikawa et al. 1990, Nagasawa et al. 1987, Posthuma et al. 1988). Sporozoites bud from the sporoblast surface into the cisternal space. The mature oocyst is about 50 µm in diameter and contains thousands of sporozoites packed in a multi-lobed structure that may be compared to a pomegranate, the seeds representing the budding parasites. The first morphological evidence for sporozoite bud formation is the appearance of the IMC and the associated MTs under the cytoplasmic face of the sporoblast plasma membrane. The sporozoite bud grows by simultaneous extension of the plasma membrane, the IMC, and underlying MTs, thus forming the triple-membrane pellicle structure of the nascent sporozoite. Whereas the sporozoite plasma membrane is derived from the sporoblast plasma membrane, the IMC is presumably made de novo from Golgi-derived cytoplasmic vesicles that fuse and flatten concurrent with sporozoite outgrowth (Beier & Vanderberg 1998, Sinden & Strong 1978). The emergent sporozoites contain an elongate nucleus, mitochondria, endoplasmic reticulum, and a Golgi apparatus. They are delimited by the plasma membrane, the underlying IMC and, facing the cytoplasmic side of the IMC, 16 subpellicular MTs (variable among species) connected to the polar ring at the apical end and extending about halfway to the posterior end. In cross-sections of sporozoites, the arrangement of MTs appears asymetrical with 15 evenly spaced MTs occupying about two thirds of the circumference and a single MT in the remaining third of the circumference (Morrissette & Sibley 2002). Microneme and rhoptry formation are initiated concurrently with sporozoite budding. In merozoites, rhoptries are formed by the fusion of Golgiderived vesicles (Bannister et al. 2000), and micronemes are translocated along MTs from a single Golgi-like cysterna near the nucleus to the anterior pole of the parasite (Bannister et al. 2003). There is little information about the molecular events governing sporogenesis. Both CS- and SR-parasites show normal formation of oocysts; however, sporozoite budding is severely impaired (Claudianos et al. 2002, Menard et al. 1997). Ultrastructural analysis of CS-oocysts reveals that the IMC is no longer restricted to discrete budding sites but extends underneath the entire oocyst plasma membrane. Transgenic parasites with a partial deletion of the CS locus 3 untranslated region produce ∼fivefold less CS than wild-type parasites (Thathy et al. 2002). In these CS underproducers an intermediate phenotype is observed with regard to sporozoite development. The IMC appears restricted to discrete sites under the plasma membrane; however, it extends much more widely than observed in wild-type parasites. Sporozoite formation is abnormal with more than 50% of the sporozoites being shortened and deformed. How CS exerts its crucial function in sporogenesis remains to be elucidated. Perhaps its GPI anchor might function in shaping the coordinated extension of the pellicle. Ultrastructural analysis of the “empty”

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oocysts in the SR-parasites has not been performed, and it is not known whether the morphological changes are similar to those described for the CS-oocysts.


GLIDING MOTILITY Most apicomplexan invasive stages are motile and actively enter cells in a matter of seconds. They glide rapidly (1–10 µm/s) on solid substrates, although they have no cilia or flagella and do not extend pseudopods. In addition to circular gliding, they also exhibit flexing, turning, and twisting motions (Hakansson et al. 1999). Gliding motility, capping of parasite surface molecules, and host cell invasion are driven by the same actin-myosin motor (King 1988, Menard 2001, Opitz & Soldati 2002, Sibley 2004). The myosin involved is MyoA (Meissner et al. 2002b) and it belongs to a class of neckless myosins (XIV) that is unique to Apicomplexa (Heintzelman & Schwartzman 1997). The proteins linking the intracellular motor and the extracellular substrate belong to the TRAP family (Kappe et al. 1999, Sultan et al. 1997b) and act like a tank’s caterpillar tracks. The molecular motor pulls TRAP backward, and because the extracellular portion of the protein is anchored to a fixed substrate, this leads to a forward movement of the parasite. The nature of the substrate employed when sporozoites glide on glass is unknown, but it is likely provided by the parasite itself. If TRAP-like molecules engage cellular receptors, a moving junction is formed between the host cell membrane and the parasite and invasion follows (Miller et al. 1979). The moving junction is characterized by a thickened and highly selective membrane ring in the host cell. A constriction in the parasite body is formed as it squeezes through this structure, capping the moving junction along its body (Mordue et al. 1999). One requirement for the effective parasite penetration is the final separation from the cell plasma membrane at its posterior end. To achieve this, MIC2 and other Toxoplasma surface molecules are cleaved on their trans-membrane domains and released as soluble proteins by the action of a specific protease, MPP1 (Brossier et al. 2003, Carruthers et al. 2000, Opitz et al. 2002). MPP1 belongs to the family of rhomboid intramembrane proteases originally described in Drosophila (Urban & Freeman 2003). Although this has not been formally demonstrated in Plasmodium, a fragment of TRAP containing only the extracellular portion is left behind in the trail of moving sporozoites (Kappe et al. 1999). Large amounts of CS are also continuously released at the posterior end of the parasite during gliding (Stewart et al. 1986). This seems paradoxical because CS cannot establish a physical interaction with the motor. It could be argued that the extracellular domains of CS and TRAP interact, and that TRAP drags CS with it, but we failed to coimmunoprecipitate CS and TRAP (C.A. Buscaglia & V. Nussenzweig, unpublished data). The second possibility lies in the existence of specialized membrane structures or lipid rafts in the sporozoite surface. Although this notion is controversial, these microdomains enriched in cholesterol, sphingolipids, and GPI-anchored proteins have been clearly described in some cell types where they play a crucial role in transducing signals to the interior of the cells

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by recruiting specialized molecules in the inner membrane leaflet (Bagnat et al. 2000, Pizzo et al. 2002). The direct interaction of these structures with discrete actin filaments was proposed (Suzuki & Sheetz 2001). Thus the GPI anchor of CS might provide its connection to the parasite cytoskeleton. The presence of lipid rafts has been documented in Plasmodium blood stages (Wang et al. 2003). Our current understanding of the apicomplexan motor places MyoA tethered to the IMC, whereas the polymerized actin (F-actin) apposed to the plasma membrane interacts with the cytoplasmic tail of TRAP-like molecules (Figure 4a) (Kappe et al. 2004). Indeed, MTIP, a protein that binds to the tail of MyoA, has been identified and localized to the IMC (Bergman et al. 2003a, Herm-Gotz et al. 2002). Because MTIP has no membrane-spanning domain or potential lipid attachment signals, a linker is required to anchor the MTIP/MyoA complex to the IMC. A molecule called MyoA docking protein (MADP) has been copurified with the motor complex from extracts of Toxoplasma tachyzoites. MADP is myristoylated and palmitoylated in vivo and also localizes to the IMC (C.J. Beckers, unpublished data). The cytoplasmic tail of the TRAP family proteins is bridged to F-actin by the glycolytic enzyme aldolase (Buscaglia et al. 2003, Jewett & Sibley 2003). Indeed, the ternary complex formed by TRAP, aldolase, and F-actin can be obtained in vitro (Buscaglia et al. 2003). The binding of MIC2/TRAP to aldolase is strictly dependent on the presence of particular residues on their C termini (Buscaglia et al. 2003, Jewett & Sibley 2003), previously shown to be critical for normal parasite motility (Kappe et al. 1999). In resting Plasmodium sporozoites, aldolase is localized in the cytoplasm and on the surface of some micronemes (Buscaglia et al. 2003), most likely bound to the TRAP cytoplasmic tail that may project into the parasite cytoplasm. It has been speculated that upon parasite activation, TRAP-containing micronemes fuse with the sporozoite membrane at its extreme apical end (Carruthers et al. 1999, Gantt et al. 2000), bringing the aldolase along, which allows it access to the cortical space where the motor is engaged (Figure 4b). Aldolase is then translocated posteriorly along with TRAP/MIC2 during parasite gliding (Jewett & Sibley 2003). The substrate and the products of the reaction catalyzed by aldolase are excellent competitive inhibitors of its interaction with both TRAP and F-actin, indicating a possible metabolic regulation of the motor (Buscaglia et al. 2003). In addition to sporozoites, two other stages of Plasmodium depend on an actinmyosin motor to achieve their tasks: ookinetes and merozoites. CTRP, a TRAP paralogue, is essential for ookinete motility, which suggests an analogous architecture of the motor in this stage (Dessens et al. 1999, Yuda et al. 1999). On the other hand, the transcriptome analysis of the Plasmodium intra-erythrocytic stages indicates that, unlike other glycolytic enzymes that are downregulated on the late schizonts and invasive merozoites, the expression of aldolase is increased (Bozdech et al. 2003b). This should reduce the concentration of the intermediate glycolytic metabolites that interfere with the function of the motor (see above) and is consistent with an alternative (nonglycolytic) function for aldolase in invasive

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Figure 4 (a) Current model of the molecular motility machinery in Apicomplexa. The diagram shows a Plasmodium sporozoite and a magnification of its periphery, highlighting the possible arrangements of identified and hypothetical intracellular components of the motor. MTIP anchors to the outer membrane of the IMC by its putative interaction with MADP. MTIP binds the tail domain of MyoA, which in turn interacts with actin filaments (F-actin) that are connected to the cytoplasmic domain of TRAP via aldolase. MT, microtubules; IP, intramembranous particles; IMC, inner membrane complex; MTIP, MyoA tail domain–interacting protein; MyoA, myosin A; PM, plasma membrane; GPI, glycosyl phosphatidylinositol anchor. (b) Simplified model of TRAP/aldolase translocation from micronemes to the cortical space. (left) Contact with a specific cellular receptor causes Ca2+ signaling, which in turn triggers microneme movement to the apical end and subsequent discharge of its contents. (right) TRAP is sorted to the cortical space as a type I transmembrane molecule, bringing aldolase connected to its cytoplasmic tail, which binds to newly formed actin filaments (see a).

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merozoites. It has been suggested that the trans-membrane protein EBA-175 is the TRAP paralogue in merozoites (Gilberger et al. 2003). However, its cytoplasmic tail has a phenylalanine instead of the conserved TRAP tryptophan. This kind of replacement abolishes the in vitro interaction between TRAP and aldolase (C.A. Buscaglia & V. Nussenzweig, unpublished data). Recently, merozoite proteins structurally related to TRAP have been identified, but it is not yet known whether they are part of the motor (Bergman et al. 2003b, Green et al. 2003, Morahan et al. 2003). One conflicting point in the motor model is that F-actin is hardly detectable in apicomplexan invasive stages. In Toxoplasma tachyzoites, F-actin is visualized only with use of powerful electron microscopy techniques (Schatten et al. 2003) or after treatment with jasplakinolide, an actin-polymerizing agent (Shaw & Tilney 1999, Wetzel et al. 2003). However, jasplakinolide-induced actin filaments are randomly oriented, which leads to increased but aberrant parasite motility (Wetzel et al. 2003). Overall, these findings indicate that actin polymerization is the rate-limiting step in apicomplexan gliding motility and that it is a highly dynamic and regulated process. Few actin-regulating proteins have been identified so far in Apicomplexa (Allen et al. 1997, Poupel et al. 2000, Tardieux et al. 1998), and they are involved in sequestering actin monomers such as toxofilin (homologous to mammalian cofilin) or in the shaping of preformed filaments. Recently, a functional pair of molecules (casein kinase II/phosphatase 2C) has been described that define the phosphorylation status of toxofilin (Delorme et al. 2003). The interplay of these molecules might be crucial in shaping the toxofilin affinity for monomeric actin and the whole actin dynamics in Toxoplasma and other apicomplexan parasites.

SPOROZOITE INVASION OF MOSQUITO SALIVARY GLANDS Sporozoites are released into the mosquito hemolymph from mature midgut oocysts between 10 and 14 days after the ingestion of an infective blood meal. About 20% of these sporozoites reach the mosquito salivary glands most likely by passive transport through the open circulatory system (Golenda et al. 1990, Rosenberg & Rungsiwongse 1991, Vaughan et al. 1992). The fact that sporozoites are rarely found attached to other mosquito organs and that they preferentially invade the median and distal lateral lobes of the salivary glands indicate the presence of specific receptors (Sterling et al. 1973). Sporozoites first interact with the basal lamina that covers the hemocoel-exposed side of the glands via filamentous connections (Pimenta et al. 1994). Plasmodium gallinaceum sporozoite invasion of Aedes aegypti salivary glands is inhibited by antibodies and lectins that react with the gland (Barreau et al. 1995). More recently, a female-specific Anopheles gambiae salivary gland protein was identified using monoclonal antibodies (mAbs) (Brennan et al. 2000). One mAb that recognized a still-uncharacterized 100 kDa protein present

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in the median and distal lobes reduced the number of sporozoites found within the salivary glands by ∼70%. Another study using a phage display peptide library identified one peptide that binds to the luminal side of the mosquito midgut and the distal lobes of salivary glands and strongly inhibits both oocyst formation and sporozoite invasion of salivary glands (Ghosh et al. 2001). To date there is clear evidence for the involvement of three sporozoite-expressed proteins in salivary gland invasion: CS, MAEBL, and TRAP. As the major surface protein of sporozoites, CS is a likely candidate ligand for their initial attachment to the salivary glands. In support of this hypothesis, recombinant CS protein binds specifically to the median and distal lateral lobes of salivary glands (Sidjanski et al. 1997). Furthermore, a peptide spanning the region I of CS blocks the binding of recombinant CS and sporozoites to the salivary glands (Myung et al. 2004). Unfortunately, CS-parasites do not develop sporozoites (Menard et al. 1997) and cannot therefore be used to study the role of other surface molecules in sporozoite interaction with the salivary glands. MAEBL seems to act along with CS in the initial adherence of the sporozoite to the salivary glands (Kariu et al. 2002). Because of its abundance on the sporozoite surface, CS may provide the initial recognition that triggers micronemal release and, subsequently, MAEBL may reinforce the attachment by interacting with additional gland receptors. In invading merozoites, micronemal proteins are necessary for formation of the moving junction with the red blood cell (Miller et al. 1979). MAEBL shares structural similarity with these proteins and might therefore mediate an analogous process that is essential for sporozoite salivary gland invasion. TRAP-sporozoites are deficient in gliding motility and unable to invade the salivary glands (Sultan et al. 1997b). However, TRAP-sporozoites adhere to the salivary glands in significant numbers, indicating that (a) gliding motility is not required for sporozoite homing and/or attachment to the salivary glands and (b) TRAP is critical for invasion rather than attachment to the salivary glands. Replacement of endogenous TRAP in P. berghei with P. falciparum TRAP leads to a reduced ability of sporozoites to infect the salivary glands of Anopheles stephensi mosquitoes (Wengelnik et al. 1999). Deletion of the TSR or an amino acid change in the A domain in these hybrid parasites leads to further decrease in salivary gland infection. A recent study addressed the role of the P. berghei TRAP adhesive domains in host cell invasion, but in contrast to a previous study, the mutations were introduced into the endogenous PbTRAP (Matuschewski et al. 2002a). Amino acid changes in the TSR lead to a ∼20% reduction in salivary gland infection, whereas amino acid changes in the MIDAS of the A domain lead to a ∼80% reduction. Combining both mutations leads to a ∼95% reduction in sporozoite salivary gland infection. Having traversed the basal lamina that covers the salivary gland, sporozoites come in contact with the plasma membrane of the secretory cells. For transmission to the vertebrate host, the sporozoites must go across the secretory cells and exit into the secretory cavity. It is not clear how the sporozoite accomplishes this.

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Electron microscopic data suggest that sporozoites invade the secretory cells with the formation of a junction, invagination of the plasma membrane, and formation of a vacuole containing the parasite. In a process of transmigration through the cell, the sporozoites exit the vacuole when its membrane fuses with the plasma membrane facing the secretory cavity (Pimenta et al. 1994). Recent videomicroscopic studies using GFP-expressing sporozoites document extensive sporozoite movement within the salivary ducts and extremely rapid ejection with the saliva during mosquito bite (Frischknecht et al. 2003).

DIFFERENCES IN OOCYST AND SALIVARY GLAND SPOROZOITES Sporozoites released from mature oocysts must be prepared to recognize and invade the mosquito salivary glands. Salivary gland sporozoites, on the other hand, must be ready to infect the mammalian host (Figure 2). Success in the completion of the cycle depends on their ability to perform numerous new interactions with host tissues in the skin and in the liver. Despite their morphological likeness, oocyst and salivary gland sporozoites display dramatically different phenotypes (Figure 5) (Vanderberg 1974, 1975). Salivary gland sporozoites are highly infectious to the mammalian host, migrate in a typical circular gliding pattern, and can elicit strong protective immune responses. Sporozoites that are released from the ooocyst are virtually noninfectious to the mammalian host, do not exhibit circular gliding, and fail to produce protective immunity. Sporozoites that entered the salivary glands are no longer capable of re-entering them when injected back into the mosquito hemocoel, suggesting that they are irreversibly programmed to infect the vertebrate host (Touray et al. 1992). Indeed, these profound phenotypic differences were documented to be associated with significant gene expression differences by using differential expression profiling between both sporozoite populations (Matuschewski et al. 2002b). This study identified 30 genes that are specifically turned on in infective salivary gland sporozoites (called UIS). Furthermore, all UIS with one exception are not expressed in blood stages either, suggesting that they are specifically involved in interactions with mammalian tissues and possibly in EEFs transformation. Indeed, P. berghei UIS4 codes for a 23-kDa protein with a predicted transmembrane domain that localizes to salivary gland sporozoite secretory organelles (Kaiser et al. 2004b) and is also expressed throughout EEFs development where it localizes to the PV (S.H.I. Kappe & K. Matuschewski, unpublished observation). P. berghei UIS4-parasites undergo normal blood stage replication, sporozoite development, and salivary gland and hepatocyte invasion but show a severe defect in EEFs development (S.H.I. Kappe & K. Matuschewski, unpublished observation). What are the signals that trigger this differential expression between both sporozoite populations? It is conceivable that sporozoites undergo a time-dependent endogenous maturation program, i.e., the sporozoite population in a later compartment (salivary glands) is simply older than that in an earlier compartment

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Figure 5 Two distinct sporozoite types. Sporozoites develop in mosquito midgut oocysts and are released into the hemolymph (left panel). After reaching the salivary glands, sporozoites attach to the basal lamina and invade the glands (right panel). Sporozoites in both panels express GFP, which aids visualization. (a) Phenotypic differences of oocyst sporozoites (OoSp) (left) and salivary gland sporozoites (SgSp) (right). (b) OoSp and SgSp express genes that are not expressed in blood stages. In addition, the two types of sporozoites differentially express some of these sporozoite-specific genes. Shown are sections of P. yoelii oligonucleotide microarrays (identical array positions on the left and right) that were hybridized to mixed blood stage RNA (red) and to OoSp RNA (green, left panel) or SgSp RNA (green, right panel). Highly expressed sporozoite-specific genes are boxed white. Some are expressed in both sporozoite populations; others are specific for one population (S.H.I. Kappe & L.W. Bergman, unpublished data).

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(midgut oocyst) (Vanderberg 1975). Alternatively, the switch in gene expression and maturation could be activated by an external signal coming from contact with the salivary glands.


SPOROZOITE MIGRATION AND INVASION OF HEPATOCYTES In their search for capillaries, mosquitoes probe the skin of the host and deposit saliva containing vasodilators, anticoagulants, and Plasmodium sporozoites. Sporozoites remain in the skin for a short time (Sidjanski & Vanderberg 1997) and probably traverse skin cells before entering the blood circulation. Sporozoites can enter cells by two distinct routes, either through a tight moving junction with the target cell that leads to the formation of a PV where EEF development proceeds, or by disrupting their plasma membrane (Mota et al. 2001). In the latter case, the parasite glides in the cytoplasm, and exits the cell by again rupturing the plasma membrane. Sporozoite migration through hepatocytes triggers the secretion of hepatocyte growth factor (HGF), which activates the HGF receptor and leads to an increase in the susceptibility of liver cells to infection (Carrolo et al. 2003). It has been suggested that the passage or transmigration of sporozoites through successive cells activates the parasite for the productive infection of hepatocytes (Mota et al. 2002). However, recent findings suggest an alternative scenario. Targeted disruption in P. berghei of the gene encoding a protein called SPECT renders sporozoites incapable of transmigration through cells, although SPECTsporozoites infect hepatocytes normally in vitro (Ishino et al. 2004). In addition, a phospholipase (PbPL, previously called UIS10) (Matuschewski et al. 2002b) found in the sporozoite plasma membrane seems to be involved in the penetration of target cells during transmigration. When injected by mosquito bite (but not following intravenous injection) PbPL-parasites are much less infective than the wild type. More important, PbPL-sporozoites display an impaired ability to cross epithelial cells in vitro (P. Bhanot, unpublished data). These studies suggest that the molecular mechanisms of transmigration are complex and that this sporozoite property is most likely required for traversing cells in the skin to reach the circulation, and for crossing the sinusoidal cell layer to enter hepatocytes (Ishino et al. 2004). Mosquitoes inject as few as ∼20–200 sporozoites into the skin (Beier & Vanderberg 1998). When injected directly into the blood stream, sporozoites invade hepatocytes within minutes (Shin et al. 1982). Thus the overall efficiency of the parasite’s migration and targeting is high. The precise route that sporozoites take from the skin to the blood is not known. Recent videomicroscopic analysis of GFP-expressing sporozoites in the skin revealed their high motile activity and subsequent active penetration through the vascular endothelium (Frischknecht et al. 2003). Most circulating sporozoites are arrested in the liver after a single passage,

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suggesting that specific receptors are present on the cells lining the sinusoids. The liver sinusoid lining consists mostly of a fenestrated endothelium and Kupffer cells (Figure 2). The reduced speed in the blood circulation while percolating through the liver facilitates the encounter of the parasites with the putative sinusoidal receptor. Because CS covers the entire sporozoite plasma membrane, it is very likely that it contains the postulated liver ligand(s). Numerous observations indicate that the ligand is contained in the stretch of positively charged residues of region II-plus of CS, and that the binding sites in the liver are HSPGs (Sinnis & Nardin 2002, Tewari et al. 2002). There are intriguing similarities between the clearance patterns of CS protein and of apolipoprotein E-enriched chylomicron remnants generated from the metabolism of intestinal chylomicrons. The clearance of the remnants is mediated by apolipoprotein E, which has a positively charged motif similar to that of CS region II-plus (Libeu et al. 2001), and subsequent capture of the complex in the liver. The clearance of remnants and CS protein is inhibited by lactoferrin, and both apolipoprotein E-enriched chylomicrons and lactoferrin inhibit sporozoite invasion of HepG2 cells. Importantly, even though low-density lipoprotein (LDL) receptors are not required for sporozoite entry into hepatocytes, malaria sporozoites are less infective to LDL-receptor knockout mice maintained on a high-fat diet but not on a low-fat diet (Sinnis 1996). Region II-plus is found in the TSRs of a few host proteins (see above). Thus it was argued that the host proteins compete with sporozoites for the liver receptors (Rathore et al. 2001). This is unlikely because the concentration of those proteins in normal plasma is exceedingly low, and the surface of sporozoites contains a large number of representations of TSRs both in CS and in TRAP. In addition to region II-plus, the positively charged region I of CS may also bind to HSPGs and contribute to sporozoite arrest in the liver (Rathore et al. 2002). The nature of the liver HSPG receptor for the CS protein is not known. In addition to the liver, HSPGs are ubiquitously distributed in extracellular matrices and on cell surfaces. Among other functions, HSPGs bind growth factors and cytokines, are involved in lipoprotein metabolism, and participate in viral entry into cells. The multiple roles of HSPGs are associated with extensive chemical variation, imparting specificity to the various interactions (Iozzo 2001). Liver HSPGs include two members of the syndecan family (syndecan 1 and syndecan 2), which are type I integral membrane proteins that can function as coreceptors (Couchman 2003). Syndecan 1 is not an important sporozoite receptor because syndecan 1 knockout mice are as susceptible to sporozoite infection as the wild-type controls (Bhanot & Nussenzweig 2002). Syndecan 2 is more likely to be the CS receptor. It is an unusual member of the family of HSPGs, with a large proportion of heparinlike, highly sulfated structures at the distal end of the glycosaminoglycans chains (Lyon & Gallagher 1991, Pierce et al. 1992). Notably, among glycosaminoglycans, heparin is the most efficient inhibitor of CS binding to HepG2 cells. A well-defined decasaccharide isolated from heparin, which has a structure commonly found in liver syndecan 2, blocks the interaction between CS and HepG2 cells with an

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ID50 < 60 nM. The same decasaccharide binds specifically to apolipoprotein E (Rathore et al. 2001), providing additional support for the view that sporozoites and chylomicron remnants compete for the same liver sites. On the basis of these observations it is conceivable that the HSPG chains of hepatocyte syndecan 2 protrude through the fenestrae of the sinusoidal endothelium and bind to the positively charged groove of the TSR domain of CS (Sinnis & Sim 1997, Tan et al. 2002). The surface membrane of Toxoplasma is also coated with a family of GPI-linked molecules that bind HSPGs and mediate attachment to the host cells. The structure of one of these molecules (SAG-1) has been solved. It is a homodimer in which the dimeric interface contains an outward facing deep groove lined with positively charged residues that can accommodate the sulfated HSPGs of the target cells (He et al. 2002). To continue the cycle, the HSPG-attached parasites must shed the bound CS, traverse the sinusoidal lining probably through Kupffer cells (Pradel & Frevert 2001), and enter the Disse space where they reach the basolateral membrane of hepatocytes. The details of the molecular events that take place during in vivo invasion are not known. It is presumed that after micronemes discharge their content, including large amounts of TRAP, at the apical end, the adhesive extracellular domains of these molecules attach to ligands on the hepatocyte surface, which leads to the formation of a moving junction followed by parasite invasion. Sporozoites enter inside a vacuole lined with the host plasma membrane, which contains additional molecules secreted from the apical organelles (Carruthers & Sibley 1997). Similarly to what was described in the section on salivary gland invasion, amino acid changes in the TRAP A domain have a much greater inhibitory effect on sporozoite infectivity for rodents than mutations in the TRAP TSR (Matuschewski et al. 2002a). Recent reports show that AMA-1, a microneme protein involved in merozoite invasion of red blood cells (Triglia et al. 2000), is expressed in sporozoites (Silvie et al. 2004, Srinivasan et al. 2004). Furthermore, invasion of cultured human hepatocytes by sporozoites was inhibited in the presence of antibodies to AMA-1 (Silvie et al. 2004). During the intracellular gliding, the sporozoite leaves behind cytoplasmic CS trails, and CS is also found on the surface of early EEFs. In vitro CS binds to ribosomes and inhibits protein synthesis (Frevert et al. 1998), but it is not known whether this phenomenon occurs in vivo. If the CS trail is left within an antigenpresenting cell, it may be processed and presented on the cell surface in conjunction with class I MHC to initiate cellular immune responses to the parasite that have been detected in individuals living in endemic areas. One region in the C terminus of CS is highly polymorphic, and it appears to be positively selected to evade T cell recognition (Hughes 1991). Sporozoites of P. falciparum and P. yoelii do not infect CD81-deficient hepatocytes, most likely because the formation of the PV is impaired. Surprisingly, P. berghei, a rodent parasite closely related to P. yoelii, does not require CD81 to infect target cells. The CD81 ligand on the sporozoite surface has not been identified, raising the possibility that CD81, although essential for infection in some

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Plasmodium species, is a downstream participant of a chain of events initiated by the parasite encounter with the hepatocyte (Silvie et al. 2003). Finally it was recently shown that sporozoites do not have to invade hepatocytes to trigger their initial transformation into early EEFs (Kaiser et al. 2003). Indeed, a temperature shift similar to that encountered by sporozoites during transmission from mosquito to mammalian host and the presence of serum factors are sufficient to bring about the profound morphological and biochemical changes that occur during sporozoite-liver stage transition. These findings indicate that transformation follows a program that acts independently of sporozoite–host cell interactions.

CONCLUDING REMARKS Knowledge of Plasmodium sporozoite biology has increased rapidly, and new aspects of its complex behavior such as sporozoite-skin interactions and transmigration have been revealed. Complete gene expression profiles provide a fertile basis for understanding the genetic control of every aspect of sporozoite biology and have already led to the identification of gene products that are involved in transmigration or are responsible for the increased infectivity of salivary gland sporozoites in the mammalian host and even EEFs development. Their functional analysis has been greatly facilitated by gene targeting, which allows the inactivation or modification of pre-erythrocytically expressed genes without affecting the blood stage cycle. Some of the genes essential for the pre-erythrocytic part of the Plasmodium life cycle may present new targets for drug and vaccine development. In addition, it is now possible to create genetically attenuated malaria parasites that may be used as whole organism vaccines. We have known for over 30 years that immunization with radiation-attenuated sporozoites confers sterile protection against subsequent infectious sporozoites challenge (Nussenzweig et al. 1967). This protection is consistently achieved in rodent malaria models and in humans and is the only one that completely protects against infection. With new tools at hand and a detailed understanding of sporozoite-host interactions, we may now be able to merge sporozoite molecular cell biology and pre-erythrocytic immunology into a multi-pronged approach to defeat malaria.

ACKNOWLEDGMENTS We thank Con J. Beckers (University of Alabama in Birmingham), Photini Sinnis (NYU), Purnima Bhanot (NYU), and Erik de Vries (Utrecht University) for sharing with us their unpublished data. We apologize to people whose work was not referenced owing to limited space. Our work was supported by grants from the National Institutes of Health to S.H.I.K. and V.N. and the Bill and Melinda Gates foundation to S.H.I.K. C.A.B. is a recipient of a Bernard Levine fellowship.

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