Plasmodium falciparum is a protozoan parasite that is responsible for the most virulent form of human malaria. Transmission from mosquitoes to humans involves a remarkable series of morphological trans-formations. Following injection of motile sporozoites into the host’s blood stream, the parasite enters the liver where it multiplies and then re-differentiates to gener-ate thousands of merozoites. Upon release, these mero-zoites invade the host’s red blood cells (RBCs) to initiate the blood stage of the infection. The intra erythrocytic parasite morphs through the so-called ring, tropho-zoite and schizont stages, eventually bursting to release 16–32 daughter merozoites1,2 (FIG. 1). Each asexual cycle takes ~48 hours and cell rupture induces periodic waves of fever in patients as the disease progresses3. Symptoms range in severity from headaches, hypo-glycaemia, anaemia and fevers to renal failure, cerebral malaria and death3.

To enter the RBC the malaria parasite induces a con-trolled and transient disruption of the normal organiza-tion of the RBC membrane. Following entry, the parasite modifies the permeability and adhesive characteristics of its host cell to promote its own survival4–6 (FIG. 2). Remodelling of the host RBC is initiated by the export of many parasite proteins, called the exportome. These proteins are transported beyond the plasma membrane of the parasite and the membrane of the parasitopho-rous vacuole (PV) in which the parasite resides, and

are selectively trafficked to sites in the host cell7–10. The exported proteins alter the architecture of the RBC membrane, compromise membrane deformability and facilitate the delivery of adhesins to the RBC surface. To traffic virulence proteins across the RBC cytoplasm, the parasite elaborates novel membrane structures in the RBC cytoplasm, known as the tubulovesicular network and the Maurer’s clefts10–13. This Review summarizes recent studies of the trafficking, organization and functions of exported proteins that re-sculpt the RBC cytoplasm, interact with the erythrocyte membrane skeleton or insert into the host cell plasma membrane.

Properties and functions of the RBC membraneTo understand the nature of the modifications to the human RBC membrane that are initiated by the intracel-lular parasite, it is useful to first discuss the properties of the uninfected RBC. The human RBC has been described as a simple ‘sack’ of haemoglobin, evolutionarily tailored to perform the specialized tasks of O2 and CO2 transport. During its terminal differentiation it loses its nucleus and its ability to synthesize new proteins. However the mem-brane surrounding the cell enables the RBC to undertake a journey of hundreds of kilometres, transiting through the circulation approximately a million times without repair during the four month lifespan of the cell. The RBC can undergo remarkable deformation without fragmentation, most dramatically illustrated by the

*The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Melbourne 3050, Victoria, Australia. ‡Department of Biochemistry and ||ARC Centre of Excellence for Coherent X‑ray Science, La Trobe University, Melbourne 3086, Victoria, Australia. §Department of Microbiology, Monash University, 3800, Victoria, Australia. Correspondence to L. T. e‑mail: l.tilley@latrobe.edu.audoi:10.1038/nrmicro2110

Malaria parasite proteins that remodel the host erythrocyteAlexander G. Maier*‡, Brian M. Cooke§, Alan F. Cowman* and Leann Tilley‡||

Abstract | Exported proteins of the malaria parasite Plasmodium falciparum interact with proteins of the erythrocyte membrane and induce substantial changes in the morphology, physiology and function of the host cell. These changes underlie the pathology that is responsible for the deaths of 1–2 million children every year due to malaria infections. The advent of molecular transfection technology, including the ability to generate deletion mutants and to introduce fluorescent reporter proteins that track the locations and dynamics of parasite proteins, has increased our understanding of the processes and machinery for export of proteins in P. falciparum-infected erythrocytes and has provided us with insights into the functions of the parasite protein exportome. We review these developments, focusing on parasite proteins that interact with the erythrocyte membrane skeleton or that promote delivery of the major virulence protein, PfEMP1, to the erythrocyte membrane.

Cerebral malariaComplication that is observed in a small subset of P. falciparum infections and is associated with changes in mental status and coma. The mortality ratio is between 25–50%. The histopathological feature of this encephalopathy is the sequestration of parasitized red blood cells (RBCs) and uninfected RBCs to cerebral capillaries and venules.

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Cleft and loopstructures

Knobs with parasiteadhesin PfEMP1Merozoite

MicronemesRhoptries

Dense granule

Maurer’s clefts

Maurer’s clefts

24 hours 40 hours 48 hours12 hours0–5 min 36 hours

ExportomeUp to 8% of the parasite’s gene products contain a putative PEXEL/HT motif and are predicted to be exported to sites in the red blood cell (RBC) cytoplasm and at the RBC membrane.

Tubulovesicular networkMembrane-bound extensions and whorls that emanate from the parasitophorous vacuole membrane. These structures are thought to be involved in the trafficking of lipids and other molecules.

Maurer’s cleftsMembrane-bound organelles in the host cell cytoplasm of infected red blood cells (RBCs). These flat and roughly disc-shaped structures are connected to the RBC membrane by tubular tethers. The Maurer’s clefts are thought to be involved in the transport of cargo between the parasite and the RBC membrane.

Erythrocyte membrane skeletonA regular hexagonal array of proteins forming a two-dimensional meshwork at the cytoplasmic surface of the red blood cell comprising spectrin tetramers, actin oligomers, protein 4.1R and accessory proteins.

transit of RBCs (diameter ~8 μm) through the 1–2 μm interendothelial slits that separate the splenic cords and venous sinuses14.

The RBC membrane owes its remarkable deform-ability and durability to the membrane skeleton15. The skeleton is composed of a regular hexagonal array of proteins that form a two-dimensional meshwork at the cytoplasmic surface of the cell16,17 (FIG. 3). Spectrin heterodimers (comprising αi- and βiΣ1-spectrin) con-stitute the cross-beams of the molecular architecture. Spectrin is an elongated molecule built from tandem, homologous, 106-residue, triple-helical domains that are linked by flexible hinge regions18–20. The spectrin het-erodimers self-associate, head-to-head, to form tetra-mers — a process that is mediated by lateral interactions between a single helical repeat at the end of α-spectrin in one heterodimer and a two-helical repeat at the end of β-spectrin in the other heterodimer21,22. The tails of the spectrin heterodimers are linked into a junctional com-plex that contains actin oligomers (each with 14–16 pro-tomers), protein 4.1R, tropomyosin, adducin, dematin (protein 4.9), p55 and tropomodulin23,24 (FIG. 3a).

The RBC membrane is deformable in part because of the structural flexibility of some of the helical linkers between the spectrin repeats18,25 and in part because of a ‘breath-ing’ action whereby tetramer association and dissociation accommodates the distortions imposed by shear forces in the circulation26,27. Genetic defects in proteins of the mem-brane skeleton often lead to haemolytic diseases14,28,29.

The membrane skeleton is connected to the overly-ing plasma membrane via a series of vertical interac-tions with different integral membrane proteins (FIG. 3a). a well-studied tethering interaction involves the binding of ankyrin to the head region of β-spectrin30,31 (14th–15th repeat units). Erythroid ankyrin is a large (210 kD) protein with a central spectrin-binding domain, a car-boxy-terminal regulatory domain and an amino-termi-nal domain that interacts with the cytoplasmic domain of band 3 (also known as anion exchange protein 1)32. Band 3 is a multiple transmembrane segment protein that forms dimers and higher oligomers33,34. The binding

of ankyrin to the cytoplasmic domain of band 3 pro-motes tetramerization of this protein and decreases its diffusional mobility35,36. Protein 4.2 (also known as pal-lidin) and Rhesus proteins also help to stabilize the ver-tical interaction between spectrin, ankyrin and band 3 (REFs 37,38). in addition, protein 4.1R and p55 form a ternary complex with the cytoplasmic domain of glyco-phorin C, which provides a second point of attachment to the plasma membrane39,40. Recently it was proposed that dematin and adducin are also linked directly to the membrane bilayer by an interaction with the RBC glucose transporter GlUT1 (REF. 41).

Modifications to the RBC membraneInvasion of the RBC: breaking and entering. The invad-ing merozoite faces the logistical problem of gaining entry into a cell that does not normally undergo phago-cytosis. merozoites initiate invasion by binding to RBC surface ligands. For example the merozoite surface pro-teins EBa175 and EBa140 bind to the RBC membrane sialoglycoproteins as well as to glycophorins a and C42–44. alternatively, the parasite can use sialic acid-independent invasion pathways45. For example P. falciparum reticulocyte binding-like (PfRh) proteins appear to play key roles in merozoite invasion with PfRh2b, acting through a neurami-nidase-resistant, chymotrypsin-sensitive host receptor46.

Following the establishment of a tight junction between the parasite and the RBC, entry is mediated by the activa-tion of an actin–myosin motor in the pellicle of the invad-ing merozoite47. This coincides with the secretion of lipids and proteins (including proteases) from organelles, known as the rhoptries, micronemes and mononemes, onto the RBC surface48–53. it has been proposed that proteolytic cleavage of integral membrane proteins, such as band 3, disrupts the normal nexus of integral membrane proteins and skeletal proteins54–56. The parasite is thought to induce the invagination of a protein-free patch of membrane57, thereby initiating the formation of the PV58, and to then use gliding motility to enter the host cell47. During and following invasion, proteins from the rhoptries and dense granules are secreted into the PV and are trafficked, along

Figure 1 | Development of Plasmodium falciparum in human red blood cells. The different stages of Plasmodium falciparum development. P. falciparum merozoites attach to and invade mature human red blood cells (RBCs) and the parasite develops in a parasitophorous vacuole (PV) through the ring (0–24 hours), trophozoite (24–36 hours) and schizont stages (40–48 hours). In mature-stage parasites (>24 h), membrane-bound structures appear in the RBC cytoplasm and knobby deformations are formed at the RBC membrane. After approximately 48 hours the infected RBC ruptures, releasing 16–32 daughter merozoites. Degradation of haemoglobin results in the deposition of crystals of haemozoin in a digestive vacuole. PfEMP1, Plasmodium falciparum erythrocyte membrane protein 1.

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a b

EN

P

KnobsMC

Maurer’sclefts

KnobDistortions on the surface of P. falciparum-infected red blood cells (RBCs) caused by the deposition and self-assembly of the knob-associated His-rich protein (KAHRP) at the cytoplasmic face of the RBC membrane.

CytoadherenceAn important pathological characteristic of P. falciparum infections is the adherence of mature-stage-infected RBCs to host endothelial cells and placental syncytiotrophoblasts.

Placental malariaCommon complication of malaria in pregnancy in areas of stable transmission, and particularly frequent and severe in women who are pregnant for the first time. Associated with a subpopulation of P. falciparum that sequesters massively in the placenta.

Parasitophorous vacuole membraneMembrane formed initially by invagination of the red blood cell (RBC) membrane during merozoite invasion. The parasitophorous vacuole membrane expands with the developing parasite and separates the parasite from the RBC cytoplasm.

with early ring-stage proteins, to the RBC cytoplasm to initiate the cascade of events that are required for remodelling of the host cell59,60.

Parasite-induced modifications to the host cell compart-ment: renovating and decorating. Ring-stage-infected RBCs are observed in the peripheral circulation of patients with P. falciparum infections. mature-stage-infected RBCs are sequestered in different organs and do not circulate. The adhesive phenotype is associated with changes in the RBC membrane, whereby it becomes distorted with knobby protrusions, comprised mainly of the knob-associated His-rich protein (KaHRP) (FIGs 2b,

3b). These knobs act as platforms for the presentation of a membrane-embedded cytoadherence protein, P. fal-ciparum erythrocyte membrane protein 1 (PfEmP1; FIG. 3b, BOX 1). PfEmP1 is responsible for adhesion to endothelial cells (FIG. 2) and other cells in the host vascu-lature. a movie showing KaHRP+ and KaHRP– infected RBCs adhering to CD36-expressing platelets is available in Supplementary information S1 (movie). The adhe-sion process prevents phagocytic clearance in the spleen and can be associated with lethal complications, such as cerebral and placental malaria61,62. Recently it was shown that a subpopulation of ring-stage-infected RBCs are also removed by the spleen63, indicating that some changes in membrane properties are already apparent at this stage.

membrane-bound organelles appear in the host cell cytoplasm as the parasite develops in its RBC. Electron microscopy reveals extensions and whorls that emanate from the PV membrane to form the tubulovesicular net-work (TVn)61,64. a second set of structures, with a distinct protein composition, known as the maurer’s clefts, are observed as slender features (~30 nm wide) with electron-dense coats and electron-lucent lumens65,66 (reviewed in REFs 10,11,67) (FIG. 2b). Electron tomography and serial sec-tioning have revealed the complexity of the maurer’s cleft

structures13,67,68. The bodies of these organelles are flat and disc-shaped with irregular edges, and are connected to each other by regions with more slender profiles. Tubular tethers, with electron-dense contents and a diameter of about 30 nm, attach the maurer’s clefts to the RBC mem-brane13,67. Vesicle-like structures with diameters of about 25 nm13 or 80 nm69 may be involved in the transport of cargo between membrane-bound compartments. The maurer’s clefts are thought to arise from the PV mem-brane or the TVn, and in some cases can remain physi-cally attached to TVn extensions11,68, although functional connectivity between these compartments appears to be lost13. The maurer’s clefts appear to act as secretory organelles that concentrate virulence proteins for delivery to the host RBC membrane70–72.

none of the maurer’s cleft-associated proteins that have been characterized so far are obvious close homo-logues of the coat proteins and fission and fusion media-tors that are used in the protein trafficking pathways of higher eukaryotes. moreover the vesicles observed in the infected RBC cytoplasm do not possess the characteristic morphology of the transport vesicles of other eukaryotes (that is, 50-nm coated vesicles). and, the parasite can-not commandeer host cell trafficking machinery, as the mature RBC is devoid of trafficking components. Early antibody-based studies suggested that components of the parasite’s endogenous trafficking machinery might be exported to the host73–75, although a recent study does not support this proposal76. This suggests that the para-site has developed a novel system for trafficking proteins through the RBC cytoplasm.

Export of parasite proteins to the RBC compartment: gaining access to the extension. Proteins of parasite ori-gin are exported to sites in the RBC cytoplasm and at the RBC membrane by what appears to be a novel secre-tory system67,77. The unusual nature of the export process is indicated by the identification of a novel pentameric amino acid sequence motif that directs the export of parasite proteins78,79. interestingly, recent studies dem-onstrate that the potato blight pathogen Phytophthora infestans uses a similar host-targeting signal to deliver avirulence gene products into plant cells80. The protein export element/host targeting (PEXEL/HT) motif (RXlXE) is located about 35 amino acids downstream from the hydrophobic signal sequence and is usually encoded near the start of exon 2 in a two-exon gene. Both soluble and membrane-embedded proteins can contain a PEXEl/HT motif, indicating that the transport machinery can export both classes of proteins. Recent work indi-cates that the 5-residue motif is recognized by a novel endoplasmic reticulum (ER) peptidase that cleaves on the C-terminal side of the leu residue in the PEXEl/HT motif, before acetylation of the new n terminus81,82. Exposure of the new n terminus may present a motif that is recognized by a specific transporter in the PV membrane. This transporter has not yet been structurally characterized but may be aTP-dependent83.

Up to 8% of the parasite gene products contain a predicted PEXEl/HT motif 84, although recent stud-ies indicate that the algorithms that are currently used

Figure 2 | Adhesion of Plasmodium falciparum-infected red blood cells to endothelial cells. a | Transmission electron micrograph of a knobby parasite (P)-infected red blood cell (RBC) adhering to the surface of a microvascular endothelial cell (EN). Scale bar is 1 μm. b | Detail of the interface between an infected RBC and an endothelial cell showing strands of electron dense connecting material located at knobs (arrows). Note the presence of a Maurer’s cleft (MC). Scale bar is 100 nm. Images modified with permission from REF. 125 (2005) The Company of Biologists Ltd.

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Pf332

MESA

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4.2

Ankyrin

4.1

ActinRESA

REX1

MAHRP1

a b

Glycophorin CBand 3

RhAG

Dematin

p55

Adducin

Tropomyosin

PfEMP3

PfEMP1

KAHRP

Maurer’s cleft

Spectrin repeat

4.1

PfSBP1

Electron tomographyAn electron-microscope-based method for obtaining detailed three-dimensional images of biological samples. semi-thin sections (~300 nm) of cells are used to collect a series of images at different tilt angles. The images are aligned and tomographic 3D reconstructions of the sample are generated computationally using segmentation tools.

Protein export element/ host targeting (PEXEL/HT) motifsequence element (RXLXE) located about 35 amino acids downstream from the hydrophobic signal sequence and usually encoded near the start of exon 2 in a two-exon gene. This motif appears to be cleaved and acetylated in the endoplasmic reticulum and then recognized by a specific transporter in the PV membrane.

may over-predict the exportome85. Some parasite pro-teins that do not contain an obvious PEXEl/HT motif are also exported86,87, indicating that additional signals can be recognized by the machinery or that additional pathways are possible. indeed, some integral membrane proteins appear to be inserted into the maurer’s clefts as they form86,88. The exported proteins can be located in the cytoplasm, at the maurer’s clefts and in or under-neath the RBC plasma membrane.

Functions of exported parasite proteinsThe contributions of individual exported parasite pro-teins to changes in RBC membrane properties are now being elucidated (TABLEs 1,2). Early studies were assisted by the fact that a number of genes encoding exported proteins are located in subtelomeric chromosome regions. During prolonged in vitro culture these regions frequently undergo double-strand breaks followed by repair and the addition of telomere repeats89–91. For exam-ple losses of the subtelomeric regions of chromosomes

1, 2 and 9 lead to loss of genes that encode ring-infected erythrocyte surface antigen92 (RESa), KaHRP and PfEmP3 (REF. 93) and a series of ring-stage proteins that play parts in gametocytogenesis and expression of viru-lence proteins94,95. more recently, targeted disruption of individual genes has become possible, allowing for a more defined analysis of the functions of individual proteins96,97.

a large-scale reverse genetic screen was undertaken recently for the functional analysis of exported pro-teins by constructing loss-of-function mutants of the genes that encode these proteins96. This study gener-ated deletion mutants of 46 proteins with predicted PEXEl/HT motifs, 5 genes encoding known exported proteins that lack the PEXEl/HT motif and 32 genes with no PEXEl/HT motifs96. Below we describe some of the exported proteins with known or postulated functions, concentrating on those proteins for which export has been verified and functions have been investigated.

Figure 3 | The membrane skeleton in uninfected and Plasmodium falciparum-infected red blood cells. a | Uninfected red blood cells (RBCs). Spectrin heterodimers, comprising repeat units linked by flexible hinges, can expand and unfold in response to deformation stress. Spectrin dimers are linked head-to-head to form tetramers and at their tails by junction complexes comprising actin oligomers that are stabilized by protein 4.1R and other molecules. Vertical interactions connecting the underlying skeleton to the membrane proper include the band 3–ankyrin–spectrin link and the ternary complex between protein 4.1R, p55 and glycophorin C. b | Infected RBCs. In ring-stage-infected RBCs, ring-infected erythrocyte surface antigen (RESA) associates with spectrin and stabilizes the membrane skeleton. In mature-stage parasitized RBCs, knob-associated His-rich protein (KAHRP) molecules self-associate to form conical structures that interact with spectrin. Pf332 and mature-parasite-infected erythrocyte surface antigen (MESA) bind to the junction complex while Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) binds to spectrin, further compromising RBC membrane deformability. The cytoadherence-mediating protein PfEMP1 is concentrated in the region of the knobs by an interaction of its cytoplasmic domain with KAHRP. The amino-terminal domain of PfEMP1 is presented at the extracellular surface where it can interact with host cell receptors to mediate cytoadherence. Parasite-derived, membrane-bound Maurer’s clefts are involved in PfEMP1 trafficking. The formation and architecture of the organelles is controlled by resident proteins such as the ring exported protein 1 (REX1), membrane-associated His-rich protein 1 (MAHRP1) and P. falciparum skeleton binding protein 1 (PfSBP1), and they are attached to the RBC membrane by tether-like structures (not shown).

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The first proteins to be discussed are proteins that modify host cell architecture and RBC deformability or that enhance PfEmP1 presentation. as described above, maturation of the intraerythrocytic parasite is accompa-nied by striking changes in the surface topology of the infected RBC64,98 and by a marked loss of cellular deform-ability99,100 (Supplementary information S2 (movie)). The membrane undergoes dramatic changes in fluidity, permeability and adhesiveness (reviewed in REFs 101,102) and the RBC integral membrane proteins show a marked decrease in diffusional mobility103,104. These changes are initiated in the ring stage of infection and are increased during the trophozoite stage of growth99,103,105. what are the proteins that bring about these changes?

RESA. RESa106 is produced in the final stages of schizont development and is stored in dense granules in indi-vidual merozoites107. During or immediately following invasion of the RBC, RESa is secreted into the newly formed PV and is probably one of the first proteins that is transported across the PV membrane. indeed timing of expression is crucial for correct delivery of this pro-tein108. it is transferred to the RBC membrane skeleton59, where it interacts with spectrin109–111. The spectrin-binding domain has been mapped to a 48 amino acid region located adjacent to a DnaJ domain and between

two blocks of repetitive sequence109,112, whereas a RESa fragment binds to repeat 16 of β-spectrin, close to the dimer–dimer self-association site112.

Recombinant RESa stabilizes purified spectrin against thermally-induced denaturation and dissocia-tion112,113 and RBCs infected with RESa– parasites are more susceptible to heat-induced vesiculation113,114. This suggests that RESa may stabilize the RBC mem-brane against the febrile shock that occurs in patients following rupture and re-invasion of infected RBCs. Targeted disruption of the RESA gene abolishes an increase in rigidity that is observed at febrile temper-atures in ring-stage-infected RBCs115. Furthermore, RESa disruption is associated with a modest increase in cytoadhesion of mature-stage-infected RBCs to CD36 under flow conditions114,116. RESa is found in all of the field strains that have been examined, sug-gesting that it is necessary for survival in vivo. in the squirrel monkey, however, its presence or absence does not modulate parasite virulence115.

KAHRP. From about 16 hours post-invasion, knob-like protrusions (~100 nm in diameter) appear at the sur-face of infected RBCs12,117 (FIG. 2). These distortions are caused by the deposition and self-assembly of KaHRP at the cytoplasmic face of the RBC membrane118. KaHRP interacts with spectrin and actin119–121 and is essential for knob formation122. KaHRP is not essential for delivery of PfEmP1 to the RBC surface, although RBCs infected with KaHRP– parasite exhibits a dramatically reduced level of adhesion under flow conditions91,122–124 (Supplementary information S1 (movie)), suggest-ing that knobs promote the presentation of PfEmP1 (REF. 125). Thus, KaHRP is likely to be an important virulence factor in vivo.

KaHRP binds to repeat 4 of the spectrin α-chain126 and also to ankyrin127. The deposition of KaHRP sig-nificantly compromises the deformability of infected RBCs99,122. Binding to the membrane skeleton involves the n-terminal region of KaHRP, whereas the C-terminal repeats are necessary for the formation of mature knobs and for efficient adhesion of infected RBCs under flow conditions71,128. Both the n- and C-terminal regions of KaHRP contain binding sites for the cytoplasmic domain of PfEmP1 (REFs 120,129). Phosphorylation of the cytoplasmic domain of PfEmP1 by RBC casein kinase ii is proposed to enhance binding to KaHRP130.

Mature-parasite-infected erythrocyte surface antigen. The mature-parasite-infected erythrocyte surface anti-gen (mESa) is a high-molecular-mass protein that is expressed in trophozoites and schizonts131–135. mESa interacts with the n-terminal 30 kDa domain of pro-tein 4.1R, specifically to a 51-residue sequence in a region that is involved in the formation of the ternary complex with p55 and glycophorin C136. The interaction involves 19 residues of mESa adjacent to the PEXEl/HT motif136,137. mESa fragments compete with p55 for bind-ing to 4.1R and may modulate the 4.1R–p55 interaction in vivo136,137.

Nature Reviews | Microbiology

N-terminalsegment DBL1α DBL2

Bindingspecificity

• CD36• CD31• IgM

• Heparan sulfate• CR1

• ICAM1

DBL3

Cys-richinterdomainregion 1

Cys-richinterdomainregion 1

Transmembranedomain

Acidic terminalsegmentC2

Box 1 | PfEMP1

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is encoded by the var multigene family (there are 59 var genes in the 3D7 genome) with each parasite producing a single PfEMP1 variant per infected red blood cell139 (RBC). The 200- to 250-kDa multidomain protein is exported to the RBC surface where it mediates adhesion to host molecules, and different variants of PfEMP1 mediate adhesion to different receptors, which include ICAM1, CD31, CD36 and glucosaminoglycans193–195

(see the figure). Splenic macrophages recognize and remove RBCs with compromised deformability or altered antigenicity. Adhesion of mature-stage-infected RBCs to the vascular endothelium allows the parasite to escape scrutiny by the spleen. Adhesion to the vascular endothelium underlies much of the pathology associated with malaria.

PfEMP1 appears to be a target of protective immunity in humans196 and immunization of Aotus monkeys with a domain of PfEMP1 induces protection against a lethal Plasmodium parasite line197. However switching expression between different var genes allows the parasite to undergo clonal antigenic variation, thus evading the host’s protective antibody response4.

Depending on the particular variant of the surface-expressed PfEMP1, P. falciparum-infected RBCs may adhere in the capillaries or in the larger microvasculature of a range of organs, and the precise location of the sequestered parasites is thought to influence the outcome of the disease process. Cytoadherence to receptors in organs such as the liver or lungs appears be associated with less severe symptoms. Binding of infected RBCs to brain venule epithelial cells or (in pregnant women) to placental syncytiotrophoblasts seems to be central to the pathogenic events that underlie the life-threatening complications of cerebral and placental malaria. DBL, Duffy-binding-like.

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Novel proteins that affect knob formation and deform-ability of infected RBCs. The gene deletion screen of maier et al.96 identified two novel genes encoding proteins that are required for correct knob assembly (TABLEs 1,2). Disruption of PFD1170c and PF10_0381 results in infected RBCs with no knobs or with a greatly decreased number of knobs and substantially reduced adhesion. PFD1170c has been identified previously in a proteomics study138 as a protein whose level of expression varies between parasite strains with different adhesion properties. This protein may play a part in promoting self-assembly of KaHRP or in the interaction of KaHRP with the RBC membrane skeleton. This would in turn affect the level of virulence of different strains of P. falciparum.

The reverse genetic screen also identified proteins for which deletion may be associated with changes in the deformability of the RBC membrane96. Several gene deletions are associated with moderate decreases in the deformability of the infected RBCs when measured by whole cell laser refractometry, confirming that several proteins contribute to the overall rigidity of parasitized RBCs. it is anticipated that these proteins may inter-act with the RBC membrane skeleton or may facilitate the transfer of skeleton-interacting proteins, but addi-tional studies are needed to confirm the roles of indi-vidual proteins. interestingly, disruption of some genes (PFB0920w, PF10_0159, PF13_0073 and PF14_0758) is associated with an increase in membrane rigidity96.

Table 1 | Characteristics of some soluble exported Plasmodium falciparum proteins

Protein (Mr in kDa)

location Predicted export signal

gene disruption

Putative function, interacting partners, remarkable features

refs

GBP130 (96) RBC cytoplasm, PVM

RXLXE Yes Unknown. Deletion increases RBC rigidity 96,198

KAHRP (71) RBC skeleton RXLXQ Yes Binds to RBC spectrin–actin and to the ATS of PfEMP1. Essential for knob formation. Deletion decreases RBC rigidity and adhesion under flow conditions

71,120, 122,129

MESA/PfEMP2 (168)

RBC skeleton RXLXE Natural deletion

Binds to protein 4.1R. May disrupt p55–4.1R interaction 136,199

RESA/Pf155 (127) RBC skeleton RXLXGE Yes Binds to spectrin. Deletion increases heat-induced membrane vesiculation. May stabilize RBC membrane. May prevent invasion of already parasitized RBCs

112,113, 115

PfEMP3 (274) MC, RBC skeleton

RXLXQ Yes Binds to spectrin. Disrupts spectrin–actin–4.1R interaction. Appears to be involved in PfEMP1 trafficking. Repeat region

159,162

HRP2 (32) RBC cytoplasm RXLXE No Binds haem. May promote haem detoxification 200,201

REX1 (83) MC (Alternative export element)

Yes Associates with the edges of Maurer’s cleft bodies. Affects Maurer’s cleft morphology. Gene located in locus associated with virulence

155,156, 202

REX3 (38) RBC cytoplasm RXLXE Yes Unknown. Highly expressed PEXEL-containing protein. Gene located in locus associated with virulence

94,156, 203,204

MAL7P1.172 (103) MC lumen RXLXE Yes Involved in PfEMP1 trafficking. PHIST domain. P. falciparum conserved region (TIGRFAM01639)‡

96

MAL7P1.171 (244) Unknown RXLXE Yes Involved in PfEMP1 trafficking. Deletion decreases RBC rigidity. Extensive repeat regions

96

Exoantigen PF70* (70)

Unknown RXLXE Yes Involved in PfEMP1 trafficking. Deletion decreases RBC rigidity. Repeat regions. Antibodies to Pf70 may have a protective role

96,205

PFD1170c (37) Unknown RXLXE Yes Deletion affects knobs. May promote KAHRP self-assembly. Differently expressed between strains. RESA-like molecule

96,206

PF10_0381 (47) Unknown RXLXE Yes Deletion affects knobs 96

PFB0920w (122) Unknown RXLXE Yes Deletion increases RBC rigidity. May promote host RBC survival

96

PF13_0073 (46) Unknown RXLXE Yes Deletion increases RBC rigidity 96

PFD1160w (286) Unknown None Yes Deletion decreases RBC rigidity 96

MAL8P1.154 (552) Unknown None Yes Deletion decreases RBC rigidity. FAD/NAD(P)-binding domain

96

PF14_0018 (58) Unknown RXLXE Yes Deletion decreases RBC rigidity. P. falciparum conserved region (TIGRFAM01639)‡

96

PFC0435w (154) TVN RXLXD (68 residues after signal sequence)

No Junctional TVN protein 85

*Accession number: PF10_0025. ‡Refers to The Institute for Genomic Research (TIGR) protein family. ATS, acidic terminal segment; KAHRP, knob-associated His-rich protein; MC, Maurer’s clefts; PfEMP1, P. falciparum erythrocyte membrane protein 1; PVM, parasitophorous vacuole membrane; RBC, red blood cell; RESA, ring-infected erythrocyte surface antigen; TVN, tubulovesicular network.

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One cell line (ΔPFB0920w) showed enhanced adhesion, whereas another (ΔPF14_0758) had no surface-exposed PfEmP1 (REF. 96), indicating differential effects. These proteins may control the interactions of other proteins with the membrane skeleton, thereby modulating their effects on RBC membrane deformability.

PfEMP1. The crucial role played by PfEmP1 in the virulence of P. falciparum is well known (BOX 1). This variant antigen comprises three regions. The cytoplas-mic tail, known as the acidic terminal segment (aTS), is anchored into the knobs129, whereas the transmem-brane region penetrates the RBC membrane (FIG. 3). Finally, the ectodomain comprises an n-terminal seg-ment followed by modular Duffy-binding-like (DBl) domains and Cys-rich interdomain regions (CiDR) of variable number. These domains interact with host endothelial cell receptors, thereby sequestering infected RBCs away from spleen-mediated destruction. adhesion to the vascular endothelium is essential for virulence of P. falciparum and underlies much of the associated pathology, including life-threatening com-plications such as cerebral and placental malaria139. By contrast another human parasite, Plasmodium vivax, does not cytoadhere and infections with this para-site are seldom fatal140. importantly, in the mouse

Plasmodium berghei model cytoadherence is not cru-cial for virulence or for cerebral malaria141, suggest-ing that it is an inappropriate immune response to the sequestered parasites that leads to severe complications rather than sequestration per se.

The structures of a condroitin sulphate a (CSa)-binding DBl domain (DBl3X) of the PfEmP1 encoded by var2csa (REF. 142) and of a CD36-binding CiDR1α domain, mC179 (REF. 143), were recently solved. Despite having less than 20% sequence identity, the DBl and CiDR domains have similar structures based on three-helix bundles. indeed, PfEmP1 appears to be constructed as a polymer of three-helix bundles143. The domain structure is similar to that of the DBl domains of proteins involved in invasion144,145. The helices are decorated with a number of polymorphic flexible loops that are likely to be involved in ligand binding and immune evasion142.

The process for delivery of PfEmP1 to the RBC membrane is only poorly understood. it probably involves the loading of PfEmP1 into maurer’s clefts as they form at the PV membrane, followed by cisternal maturation of the maurer’s clefts and relocation closer to the RBC periphery (FIG. 4). Some studies suggest that PfEmP1 is transferred to the RBC membrane in transport vesicles69,146, whereas other studies indicate

Table 2 | Characteristics of some membrane-bound exported proteins of P. falciparum

Protein (Mr in kDa) location Predicted export signal

gene disruption

Putative function, interacting partners, remarkable features

refs

PfEMP1 (200–250)

MC, RBC membrane, RBC surface

PEXEL-like motif Yes Cytoadherence ligand. Involved in antigenic variation. Interacts with KAHRP

120,207, 208

MAHRP1 (29) MC None Yes Involved in Maurer’s cleft stability and PfEMP1 trafficking. Deletion decreases cytoadhesion

86,88, 209

PfSBP1 (36) MC and RBC skeleton

None Yes Involved in Maurer’s cleft morphology and PfEMP1 trafficking. Deletion decreases cytoadhesion

150,152, 153,210

RIFIN/STEVOR (~37) RBC cytoplasm, RBC surface

RXLXE/D, RXLXQ No§ May be surface-exposed in late stages. Possibly involved in antigenic variation

167, 168, 211

Pf332/Ag332 (689) MC, RBC skeleton None Yes Involved in Maurer’s cleft morphology and PfEMP1 trafficking. Deletion increases RBC rigidity and decreases cytoadhesion

165, 166

PFB0106c (34) MC, RBC cytoplasm RXLXE Yes Involved in PfEMP1 trafficking 96

PF14_0758 (144) RBC cytoplasm RXLXE Yes Involved in PfEMP1 trafficking. Deletion increases RBC rigidity. Repeat regions

96

PF13_0076 (37) Unknown RXLXE Yes Involved in PfEMP1 trafficking 96

PFD0225w (490) Unknown None Yes Deletion decreases RBC rigidity 96

PfMC-2TM* (~35) MC RXLXQ No§ Subfamily of STEVOR 96, 171

Cys repeat modular proteins (PCRMPs)

MC None Yes May mediate host–parasite interactions at different stages of the life cycle

212

ETRAMP/SEP‡ (~11) PVM/TVN and MC None No Unknown 96,213, 214

REX2 (13) MC None No Unknown 156, 215

EVP1 PFD0495c (101) TVN RXIXE Yes Promotes TVN-mediated lipid import. Repeat regions

85,96, 179

*PfMC-2TM family: PFB0985c, PFA0680c, PFC1080c, PF11_0014, MAL7P1.5, PF11_0025, MAL6P1.15, PF10_0390, PFA0065w, PFB0960c, MAL7P1.58. ‡ETRAMP/SEP family: PFB0120w, PFD1120c, PFE1590w, MAL8P1.6, PF10_0019, PF10_0323, PF10_0164, PF11_0039, PF11_0040, PFL1945c, PF13_0012, PF14_0016, PF14_0729. §Loss of multiple members of the STEVOR and MC-2TM families is tolerated216. KAHRP, knob-associated His-rich protein; MC, Maurer’s cleft; PfEMP1, P. falciparum erythrocyte membrane protein 1; PVM, parasitophorous vacuole membrane; RBC, red blood cell; STEVOR, subtelomeric variable open reading frame proteins; TVN, tubulovesicular network.

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a possible role for a chaperoned complex147,148 (FIG. 4). local disruption of the membrane skeleton may be necessary to allow the insertion of PfEmP1 into the RBC membrane, possibly at bilayer regions that are enriched in cholesterol149.

Below we describe proteins that are involved in sculpting the maurer’s clefts and controlling PfEmP1 trafficking. Recently the roles of several proteins that are exported to the maurer’s clefts were studied using transfectants expressing GFP-fusions or deletion mutants (TABLEs 1, 2). Some of these proteins seem to control maurer’s cleft architecture, whereas others affect PfEmP1 trafficking, either from the PV membrane or TVn to the maurer’s clefts, or from the maurer’s clefts to the RBC membrane.

P. falciparum skeleton binding protein 1. The P. falci-parum skeleton binding protein 1 (PfSBP1) is a maurer’s cleft-resident integral membrane protein. it is orientated with its C-terminal domain exposed to the RBC cyto-plasm150. it has been suggested that PfSBP1 plays a role in linking maurer’s clefts to the RBC membrane skel-eton150–153 and immunoelectron microscopy reveals that PfSBP1 is also associated with tether-like connecting structures13. maurer’s clefts still form in PfSBP1 dele-tion mutants, although they are narrower and farther from the RBC surface152. Deletion of PfSBP1 prevents export of PfEmP1 to the surface of the infected RBCs — a phenotype that is restored upon complementation of the PfSBP1 gene. This indicates that PfSBP1 plays an important role in parasite virulence152,153.

Membrane-associated His-rich protein 1. The mem-brane-associated His-rich protein 1 (maHRP1) is transcribed in ring-stage-infected RBCs86,154 but per-sists throughout the intraerythrocytic stages. like PfSBP1, maHRP1 is a transmembrane maurer’s cleft protein, with its His-rich C-terminal domain orien-tated towards the RBC cytoplasm154. maHRP1 is not essential for parasite viability or for maurer’s cleft for-mation, although in its absence these organelles are more fragile and susceptible to disruption88. Deletion of the MAHRP1 gene causes accumulation of PfEmP1 in the confines of the PV membrane, suggesting that maHRP1 plays a role in loading PfEmP1 into nascent maurer’s clefts88. This in turn leads to a decrease in surface-exposed PfEmP1 and to a decrease in adhe-sion of infected RBCs to endothelial cells, a phenotype that is restored upon complementation of the MAHRP1 gene88. Thus maHRP1 is also likely to be essential for parasite virulence in vivo.

Ring exported protein 1. The ring exported protein 1 (REX1) is a peripheral membrane protein that is asso-ciated with the cytoplasmic surface of the maurer’s clefts155,156. a non-PEXEl conforming region just after the n-terminal signal sequence directs protein export, whereas a predicted coiled-coil region, with some sequence similarity to a Golgi-tethering protein, is needed for maurer’s cleft association87. REX1 accumu-lates at the edges of the maurer’s cleft discs and plays a

part in sculpting these organelles157. Truncation of the C-terminal repeat region (or complete disruption of REX1) leads to maurer’s clefts with stacked and whorled cisternae157.

PfEMP3. PfEmP3 is a large, highly charged protein that is associated with the cytoplasmic surface of the RBC membrane in trophozoite stage parasites158,159. a PfEmP3 domain also shows some sequence similar-ity to a domain found in Golgi-tethering proteins159. Sequences in the n-terminal region interact with the membrane skeleton160 and are needed for delivery to the RBC membrane161. indeed a 60-residue fragment (residues 38–97) of PfEmP3 binds to spectrin at a site near the actin–protein 4.1R junction162. This disrupts the spectrin–actin–4.1R interaction162 and probably contributes to the loss of membrane deformability in mature-stage-infected RBCs99,116. PfEmP3 is not essen-tial for knob formation or for PfEmP1 surface expres-sion, although overexpression of a truncated form of PfEmP3 alters maurer’s cleft architecture and inhibits PfEmP1 trafficking159.

P. falciparum antigen 332. P. falciparum antigen 332 (Pf332) is the largest (~1 mDa) known malaria protein that is exported to the RBC membrane. it comprises a DBl domain at the n terminus of the protein followed by a predicted transmembrane domain and a large number of highly degenerate Glu-rich repeats163,164. RBCs infected with transgenic parasites in which Pf332 is deleted or truncated show altered rigidity compared with con-trols165,166, suggesting that Pf332 modulates the changes in the RBC membrane skeleton that are induced by P. fal-ciparum. RBCs infected with Pf332-deletion-mutant 3D7 parasites have decreased levels of PfEmP1 on the surface and are significantly less adhesive to CD36 (REF. 165). However, a similar mutant in a different parasite line, CS2, does not have any alterations in PfEmP1 levels in the RBC membrane in the absence of Pf332 (REF. 166). This may reflect parasite strain differences and redun-dancy: other proteins in CS2 may functionally overlap with Pf332 (REF. 166). Pf332 deletion causes a dramatic stacking of the maurer’s clefts, suggesting that this highly negatively charged protein may play a role in separating maurer’s clefts into individual lamella165.

Other exported proteins. maier et al.96 identified six additional proteins that are also required for export of PfEmP1 (TABLEs 1,2). PFB0106c, mal7P1.171 and PF10_0025 appear to interfere with early steps in PfEmP1 transport, leading to decreased levels of PfEmP1 in the Triton-insoluble fraction. This is consistent with the trapping of PfEmP1 in the para-site’s ER88,148. in parasite lines that are deficient in mal7P1.172, PF14_0758 or PF13_0076, PfEmP1 is trafficked to the maurer’s clefts, but is not efficiently transferred to the infected RBC surface96. The loca-tions of some of these proteins have been established: PFB0106c is localized in the RBC cytoplasm and asso-ciates with the maurer’s clefts; mal7P1.172 is present in the maurer’s cleft lumen; and PF14_0758 is in the

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dc

e

ba

Chaperone

Transporter

Vesicle-likestructure

Maurer’scleft

Endoplasmicreticulum

Nucleus

Knob

Chaperone

Nature Reviews | Microbiology

DnaJ proteinsAccessory proteins that are involved in the regulation of the molecular chaperone, heat shock protein 70. DnaJ proteins are implicated in protein folding, the assembly and disassembly of higher-order protein structures and the translocation of proteins across membranes.

RBC cytoplasm. The precise functions of these six gene products remain to be determined, but it is interesting to note that they are found either only in P. falciparum or in the plasmodia of primates. Thus, they may have roles in PfEmP1 export or in export of other proteins that are conserved across primate species.

Several other less well characterized maurer’s cleft proteins are listed in TABLEs 1,2. Some of these are members of expanded gene families, including the subtelomeric variable open reading frame proteins (STEVORs)167,168, the repetitive interspersed family pro-tein (RiFins169,170) and the maurer’s cleft two transmem-brane proteins (mC-2Tms171). Recently a conditional protein export system was established in P. falciparum, based on a domain that self-aggregates in the ER in a reversible manner172. The aggregation domain was fused to the first 80 amino acids of STEVOR and of full-length

PfSBP1, and was used to examine the events involved in the export of proteins to the RBC cytoplasm and the maurer’s clefts. This will be a useful tool for monitoring the trafficking and function of exported proteins.

Several enzymes are also exported to the host cell compartment. These include FiKK kinases173, falci-parum exported Ser/Thr protein kinase (FEST)174, a calcium-dependent protein kinase175 and a pro-tein phosphatase176. These enzymes may control the phosphorylation state, and thus function, of exported phospho-proteins such as PfSBP1, mESa and RESa. Exported enzymes that are involved in lipid remodel-ling, such as fatty acyl Coa synthetase177 and sphingo-myelin synthase178, may be involved in lipid storage or in lipid mobilization from the TVn. There is a pau-city of markers for the TVn, although a protein that appears to be associated with this compartment85, and a marker of a new vesicular compartment179, were described recently85. Few proteins (some examples are PfJ13, mal7P1.172 and protein phosphatase 1)96,176,180 are located in the maurer’s cleft lumen, suggesting that there is a mechanism for preventing non-selective transfer of soluble proteins from the PV into the lumen of the nascent maurer’s clefts. RBC proteins that appear to be recruited to the maurer’s clefts include lanCl1 (REF. 151) and ankyrin181.

Recent studies have shown that when RBCs from patients with haemoglobin S (HbS) and HbC are infected with P. falciparum, PfEmP1 surface exposure and cytoadherence are reduced182,183 and knob forma-tion is compromised184. These haemoglobinopathies are associated with deposition of denatured haemoglobin (hemichromes) at the RBC membrane, which in turn leads to aggregation of band 3 and crosslinking of the proteins of the membrane skeleton183,185. These changes to the RBC membrane may interfere with the traffick-ing of PfEmP1 or of KaHRP to the RBC membrane and are postulated to underlie the protection against severe malaria that is offered by these haemoglobin mutations182,183.

Proteins with chaperone-like roles. as exported P. fal-ciparum proteins have to cross several membranes to reach their final destinations (FIG. 4) an infrastructure is needed to regulate protein transport. in other systems molecular chaperones maintain cell homeostasis, both under normal and stress-response conditions, and are prime candidates for roles in controlling protein export in P. falciparum.

Exported DnaJ proteins. DnaJ proteins are involved in the regulation of the molecular chaperone heat shock protein 70 (HSP70). The family is implicated in the assembly and disassembly of higher-order protein structures and in the translocation of proteins across membranes. There are several potential sites for the involvement of exported DnaJ proteins in the traffick-ing of parasite proteins (FIG. 4). They might play roles in protein translocation across the PV membrane, in the loading or remodelling of membrane compart-ments in the RBC cytoplasm, in the assembly and

Figure 4 | Putative protein export pathways in parasitized red blood cells. Many of the proteins that are destined for export into the red blood cell (RBC) compartment possess a PEXEL/HT motif that is cleaved in the endoplasmic reticulum. The modified protein is thought to be recognized by a specific transporter in the parasitophorous vacuole (PV) or the nascent Maurer’s clefts, and the proteins are secreted into the RBC cytoplasm. Soluble proteins (such as knob-associated His-rich protein (KAHRP) and Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3)) may diffuse across the RBC cytoplasm and interact with the cytoplasmic surface of the Maurer’s clefts before redistributing to the RBC membrane skeleton. Integral membrane proteins destined for the Maurer’s clefts (such as membrane-associated His-rich protein 1 (MAHRP1), P. falciparum skeleton binding protein 1 (PfSBP1) and PfEMP1) are probably incorporated into the Maurer’s clefts as they form. Vesicle-like structures (VLS) are observed in the infected RBC cytoplasm and may be involved in PfEMP1 trafficking. Chaperones may be involved in (a) transport through the endomembrane system of the parasite, (b) loading exported proteins into the putative transporter in the PV membrane, (c) loading integral membrane proteins into nascent Maurer’s clefts, and (d) into vesicle-like structures or (e) delivering soluble proteins to the cytoplasmic surface of Maurer’s clefts and PfEMP1 to the RBC membrane.

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display of correctly folded macromolecular complexes at the RBC membrane (such as knobs and transport-ers) or in the display of virulence proteins, such as PfEmP1. in addition they might play roles in protect-ing parasite proteins or the RBC membrane during febrile episodes.

a sequence-based classification system for DnaJ proteins has been proposed186–188. Type i and ii DnaJ proteins contain all the necessary domains to stimu-late aTP hydrolysis in HSP70, whereas type iii and iV DnaJ proteins are characterized by the presence of only a signature J-domain. intriguingly, Banumathy et al.189 found that PfHSP70 is restricted to the parasite cytoplasm, suggesting that exported PfDnaJ molecules interact with RBC HSP70. The P. falciparum genome contains 18 potentially exported proteins with DnaJ domains (3 are type i or ii, 4 are type iii and 11 are type iV)84. By contrast, rodent and other primate plasmodia have only one or two DnaJ proteins with PEXEl/HT motifs. in plasmodia, types iii and iV DnaJ proteins are entirely restricted to P. falciparum, where they seem to have undergone a major radiation. This suggests that DnaJ proteins may be important for P. falciparum virulence strategies, such as the display of PfEmP1. Of the 11 exported DnaJ molecules that have been disrupted, 4 deletions had an influence on PfEmP1 display or on membrane rigidity of infected RBCs96. interestingly three proteins with DnaJ domains (PFa0660w, PF11_0034 and PF11_0509) are needed for growth in culture, indicting that they have crucial core functions.

RESA-like proteins. as described above, RESa, which is thought to protect the RBC membrane of ring-stage parasites from thermal stress, contains a DnaJ domain. in fact the P. falciparum 3D7 genome contains 17 RESa paralogues, many of which contain a DnaJ domain. nine of these RESa paralogues have been successfully deleted (including RESa itself). This suggests that there may be some redundancy of function among this group of proteins or that their roles are more crucial in vivo. interestingly, the three exported type i and ii DnaJ molecules do not contain a domain with homology to RESa, whereas the majority of exported type iii and iV molecules do. RESa2 (PF11_0512) is not expressed in 3D7 or in any other laboratory-adapted strains that have been analysed106,190. By contrast it is increased in expression in some parasites that are isolated from the field191,192. This could indicate that this protein has a role that becomes obsolete (or deleterious) in cul-ture. interestingly PFD1170c, which influences knob formation, is a RESa-like protein96 (TABLE 1).

PerspectiveThe greatest insights into RBC–parasite interactions have been realized in P. falciparum. There are, how-ever, advantages to using other Plasmodium models that offer easier transfection strategies and the possi-bility of dissection of other parasite stages. it is of some interest, therefore, that only a subset of genes encod-ing exported proteins is conserved across human- and rodent-infecting Plasmodium species85,96. indeed the P. falciparum exportome is 5–10 times larger than that of other Plasmodium species, in part due to the expan-sion of particular gene families, such as PfEmP1, the STEVOR/RiFin family and DnaJ and Plasmodium helical interspersed subtelomeric (PHiST) domain families84. The P. falciparum-specific members of the exportome might play roles in the complex and novel system for export and display of virulence proteins; these genes may not be necessary for growth under culture conditions. By contrast, the conserved exported proteins might be involved in core functions and dele-tion of these genes might be deleterious to growth both in vitro and in vivo.

indeed, some exported proteins that are conserved across plasmodial species are essential for survival in vitro88 and in vivo85. Two P. falciparum-specific gene products, however, also appear to be essential for para-site growth in culture96. These proteins may play roles in repair of the host cell membrane after the invasion process, in uptake of lipids and other nutrients, in waste disposal, in maintenance of ion gradients or in parasite egress. They may also function as chaperones for the trafficking of crucial exported proteins or in the recon-ditioning of the RBC membrane skeleton after damage that is caused by increased levels of pro-oxidants.

The roles of exported proteins of blood-stage malaria parasites are gradually being elucidated. These pro-teins bring about the coordinated remodelling of the host cell cytoplasm and membrane skeleton to convert highly differentiated and inert host cells into adhesive vessels that act as incubators for parasite replication. Understanding these processes may lead to strategies that prevent the remodelling of the RBC membrane, the assembly of knobs and the delivery or presentation of PfEmP1. Bioinformatics, genetics and proteomics approaches have identified a substantial number of proteins with potentially interesting functions, although the detailed characterization of the roles of individual proteins remains a challenge for workers in the field. The unusual and sophisticated nature of the secre-tion system that is used by the malaria parasite may make it be particularly amenable to targeting by novel antimalarial agents.

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AcknowledgementsWe thank D. Ferguson, Oxford University, UK, for providing micrographs. A.G.M. is an Australian Research Council (ARC) Australian Research Fellow, B.M.C. is a National Health and Medical Research Council (NHMRC) Senior Research Fellow and A.F.C. is a Howard Hughes Medical Institute International Research Scholar. This work was supported by the ARC and the NHMRC.

DATABASESEntrez Genome Project: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genomeprj Phytophthora infestans | Plasmodium berghei | Plasmodium falciparum | Plasmodium vivaxuniProtKb: http://ca.expasy.org/sprotBand 3 | dematin | FEST | KAHRP | MAHRP1 | MAL7P1.171 | MAL7P1.172 | MESA | pallidin | PF10_0025 | PF10_0159 | PF10_0381 | PF11_0034 | PF11_0509| PF13_0073 | PF13_0076 | PF14_0758 | Pf332 | PFA0660w | PFB0106c | PFB0920w | PFD1170c | PfEMP1 | PfEMP3 | PfSBP1 | protein 4.1R | RESA | RESA2 | REX1

FURTHER INFORMATIONalexander Maier’s homepage: http://www.latrobe.edu.au/biochemistry/lab/maier/index.htmbrian cooke’s homepage: http://www.miin.monash.org/members/cooke-brian.htmlalan cowman’s homepage: http://www.wehi.edu.au/facweb/indexresearch.php?id=16leann tilley’s homepage: http://www.latrobe.edu.au/biochemistry/lab/tilley/index.htm

SUPPLEMENTARY INFORMATIONSee online article: S1 (movie) | S2 (movie)

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