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Human erythrocyte remodelling during Plasmodium falciparum malaria parasite growth and egress Alassane Mbengue,1 Xue Y. Yam1,2 and Catherine Braun-Breton1 1

CNRS UMR 5235, University Montpellier II, Dynamique des Interactions Membranaires Normales et Pathologiques, University Montpellier I, Montpellier, France and 2School of Biological Sciences, Nanyang Technological University, Singapore, Singapore

Summary The intra-erythrocyte growth and survival of the malarial parasite Plasmodium falciparum is responsible for both uncomplicated and severe malaria cases and depends on the parasite’s ability to remodel its host cell. Host cell remodelling has several functions for the parasite, such as acquiring nutrients from the extracellular milieu because of the loss of membrane transporters upon erythrocyte differentiation, avoiding splenic clearance by conferring cytoadhesive properties to the infected erythrocyte, escaping the host immune response by exporting antigenically variant proteins at the red blood cell surface. In addition, parasite-induced changes at the red blood cell membrane and sub-membrane skeleton are also necessary for the efficient release of the parasite progeny from the host cell. Here we review these cellular and molecular changes, which might not only sustain parasite growth but also prepare, at a very early stage, the last step of egress from the host cell. Keywords: malaria, Plasmodium falciparum, red blood cell, parasitophorous vacuole, Maurer’s clefts. Plasmodium falciparum, the most life-threatening species of parasites causing human malaria, is responsible for over 1 million deaths per year, predominantly in children under 5 years in Africa. Most clinical symptoms of malaria occur during the 48-h intra-erythrocytic and asexual development of the parasite, with one merozoite-stage parasite giving a progeny of about 16–32 merozoites (Fig 1). Most of that time is spent at the trophozoite stage, during which the parasite will grow to fill up to 50% of the volume of the red blood cell (RBC) while digesting the host cell cytoplasm (Krugliak et al, 2002). The last 12–15 h correspond to the multinucleate schizont stage and the differentiation and release of new merozoites. The pathogenesis of severe malaria

Correspondence: Catherine Braun-Breton, CNRS UMR 5235, University Montpellier II, Dynamique des Interactions Membranaires Normales et Pathologiques, 34095 Montpellier Cedex 5, France. E-mail: [email protected] ª 2012 Blackwell Publishing Ltd British Journal of Haematology, 2012, 157, 171–179

is largely due to the ability of Plasmodium falciparum to remodel its host’s erythrocyte (RBC) by exporting P. falciparum erythrocyte membrane protein-1 (PfEMP1) to the RBC surface. PfEMP1 is an adhesin that mediates the adhesion of infected RBCs to host cells by interacting with a variety of host cell surface receptors, and is anchored at the RBC surface in parasite-induced protuberances called knobs. However, export of parasite proteins to the host RBC is not restricted to the knob components. Indeed, an increasing number of exported parasite proteins, as well as parasitehijacked host cell proteins, have been identified that might be essential for the parasite growth, survival and dissemination. Here we present the current state of knowledge concerning RBC remodelling by the P. falciparum malaria parasite.

Living inside a red cell Malaria parasites grow and replicate inside a parasitophorous vacuole generated upon RBC invasion (for review Zuccala & Baum, 2011): active penetration of the merozoite promotes invagination of the host cell plasma membrane with the junction between the two cells acting as a sieve, excluding host integral membrane proteins from the nascent parasitophorous vacuole membrane (PVM) (Fig 2). Initially, the PVM essentially contains host cell plasma membrane phospholipids (Ward et al, 1993). Discharge of the merozoite rhoptry bulb provides proteins and lipids that contribute to the growth and modification of the parasitophorous vacuole (Bannister et al, 1986). Noteworthy, some of the rhoptry bulb proteins, such as the RhopH complex proteins, are secreted to the RBC cytosolic face of the parasitophorous vacuole (Hiller et al, 2003) and move later to the RBC periphery (Vincensini et al, 2008). The biological roles of the RhopH complex are still unknown although they are at least considered to be critical for RBC invasion (Sam-Yellowe et al, 1988; Sam-Yellowe & Perkins, 1991; Hiller et al, 2003). Noteably, changes at the RBC surface are also induced by the parasite very early upon invasion (reviewed in Zuccala & Baum, 2011): parasite rhoptry proteins are deposited on the RBC surface (Sterkers et al, 2007) and the major RBC transmembrane protein Band 3 is phosphorylated on tyrosine First published online 7 February 2012 doi:10.1111/j.1365-2141.2012.09044.x

Review

Fig 1. The 48 h intra-erythrocytic development of Plasmodium falciparum. The different stages of the parasite intra-erythrocytic development are presented as Giemsa-stained infected RBC (from early trophozoite to merozoite release), a snap-shot of live-imaging (for RBC invasion by a merozoite) and as schemes (right panel). Following entry into the RBC, the merozoite differentiates into a trophozoite, which grows during the first 30–35 h of development. Haemoglobin digestion by the parasite results in the accumulation of haemozoin, also known as malaria pigment. Mature trophozoites differentiate into multinucleate schizonts. Following nuclei divisions, merozoites are individualized and further released in the external milieu. hpi, hours post-invasion.

residues, a process that might be important for parasite entry by de-connecting Band 3 from the sub-membrane skeleton (Pantaleo et al, 2010; Ferru et al, 2011).

Nutrient uptake and induction of new permeability pathways

Fig 2. schematic representation of the RBC major changes induced by P. falciparum. The parasite grows inside a self-made parasitophorous vacuole, the membrane of which constitutes the interface between the parasite and its external environment. Extensions of the parasitophorous vacuole membrane (PVM) form the tubuvesicular network (TVN) extending into the host cell cytosol. Various parasite structures are transposed into the RBC cytosol: the Maurer’s clefts are flat and elongated membrane vesicles at the host cell periphery and linked to the host cell membrane and sub-membrane skeleton; J dots are probably membrane structures that might traffic some parasite proteins through the RBC cytosol. Complexes of exported parasite proteins interacting with the RBC membrane and sub-membrane skeleton forms protrusions of the RBC membrane, referred to as knobs, that mediate adhesion of the infected RBCs to host cells.

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Trophozoite growth is accompanied by extensive digestion of the RBC cytoplasm. However, this does not provide the parasite with all the nutrients it needs to sustain its growth because haemoglobin does not contain isoleucine and several other amino acids, such as glutamate, methionine, cysteine and proline, are under represented (Ginsburg et al, 1985; Kirk et al, 1994; Martin & Kirk, 2007). In addition, the RBC loses many membrane transporter activities upon differentiation from the reticulocyte, thus limiting the parasite’s access to extracellular nutrients. Various RBC changes appear to ensure the supply of nutrients to the parasite: (i) the parasite alters the permeability of the RBC membrane by creating new permeability pathways (Kirk & Saliba, 2007), (ii) the parasite exports a network of tubovesicular membranes (TVN) that extends from the parasitophorous vacuole to the RBC periphery and might specialize in providing the parasite with efficient access to nutrients (Lauer et al, 1997). At the trophozoite stage, the RBC membrane undergoes a marked increase in its permeability to a variety of solutes, ª 2012 Blackwell Publishing Ltd British Journal of Haematology, 2012, 157, 171–179

Review including anions and cations, present in the extracellular milieu (Kirk, 2001; Baumeister et al, 2006). The origin of these new permeability pathways is still the subject of an animated debate; some propose that they are generated by parasite-encoded transporters (Alkhalil et al, 2004), while others favour the modulation of endogenous transporters of the RBC (Huber et al, 2005). It has been reported that both RBC Band 3 and the new permeability pathways of intact infected RBC can be inactivated by chymotrypsin treatment (Baumeister et al, 2006): re-suspension of the cells in a chymotrypsin-free medium resulted in the progressive re-appearance of the new permeability pathways, which was dependent on the parasite viability and ability for protein secretion. These results clearly showed that the new permeability pathways depend on parasite proteins either as components of the new permeability pathways (Alkhalil et al, 2004; Nguitragool et al, 2011) or as modulators of endogenous erythrocytic transporters. Accordingly, several parasite protein-kinases are exported to the RBC cytosol (Nunes et al, 2007) that might modulate the activity and specificity of preexisting inactive membrane transporters. Another illustration of the importance of both parasite and host proteins in the induction of new permeability pathways is the specific and high affinity interaction of serum albumin with the surface of infected RBCs that stimulates anion conductance in the host RBC membrane (Duranton et al, 2008).

Networks of membrane compartments in the infected RBC cytoplasm At the trophozoite stage, the parasitophorous vacuole extends in the RBC cytosol as a network of tubovesicular membranes that might form loops and whorls, penetrate deep in the host cell but do not appear to fuse with the RBC membrane (Atkinson & Aikawa, 1990; Elmendorf & Haldar, 1994; Bannister et al, 2000) (Fig 2). Inhibition of the parasite sphingomyelin synthase by dl-threo-1-phenyl-2-palmitoylamino-3morpholino-1-propanol (PPMP) blocked the assembly of this network and the delivery of some specific extracellular nutrients to the parasite such as adenosine, glutamate and orotic acid (Lauer et al, 1997), suggesting an important role of the TVN in nutrient import. Moreover, comparative transcriptomic analyses of PPMP-treated P. falciparum-infected RBCs led to the identification of a new TVN-associated parasite protein and to the description of a novel vesicular membrane compartment in the host cell cytoplasm proposed to be implicated in lipid import from the RBC surface (Tamez et al, 2008). However, the TVN is established in the infected RBC far later than the new permeability pathways (Elford et al, 1995), suggesting complementary rather than connected uptake processes. In addition to the TVN, a variety of vesicular-like profiles have been observed in the infected RBC that might be more implicated in export of parasite proteins and waste products to the host cell and external milieu (see below). ª 2012 Blackwell Publishing Ltd British Journal of Haematology, 2012, 157, 171–179

Knobs focally display the major P. falciparum virulence factor PfEMP1 Changes at the RBC surface, together with a loss of deformability of the infected RBC (Nash et al, 1989; Deplaine et al, 2011), would result in a very efficient splenic removal of infected RBCs from the blood circulation, had the parasite not been able to confer adhesive properties to its host cell, resulting in the sequestration of mature trophozoite and schizont stages by cytoadherence to the microvasculature endothelium. Cytoadherence is mediated by the parasite adhesin, PfEMP1, exposed at electron-dense protrusions of the RBC surface, referred to as knobs (Baruch et al, 1995; Smith et al, 1995; Su et al, 1995; Fairhurst & Wellems, 2006) (Fig 2). Knob formation is a dynamic process that occurs as the parasite matures from trophozoite to schizont. Indeed, the knobs increase in density (from 10–35 to 45–75 knobs/ lm2) as the parasite matures and eventually cover the entire RBC surface, while their size varies inversely from 160–110 to 70–100 nm in diameter (Gruenberg et al, 1983). This also implies dynamic changes to the RBC membrane and submembrane skeleton, which involve redistribution and organization of constituents from both parasite and host cell origin. The knob-associated histidine-rich protein (KAHRP) selfaggregates (Kilejian et al, 1991) and anchors the carboxy-terminal domain of PfEMP1 to the RBC sub-membrane skeleton by interacting with the actin-protein 4.1-spectrin junction (Waller et al, 1999, 2002). In addition, extractability data strongly suggest that the insertion of PfEMP1 in the RBC membrane relies more on protein-protein interactions than protein-lipid interactions, thus suggesting that other RBC membrane-associated proteins are implicated (Papakrivos et al, 2005). Like KAHRP, other parasite and RBC proteins affect the amount and distribution of PfEMP1 at the RBC surface (Allred et al, 1986; Fairhurst & Wellems, 2006). Many studies have contributed to provide an integrated model of the knob structure (Maier et al, 2009), implicating RBC cytoskeletal components, such as spectrin, ankyrin and actin (Pei et al, 2005). The interactions of knob components with RBC skeletal proteins probably alter the architecture of the sub-membrane skeleton and its interactions with membrane proteins (reviewed in Mohandas & An, 2006), and result in increased rigidity and adhesiveness of the RBC membrane (Rug et al, 2006). However, while the 5′ repeat region of KAHRP is required for the knob protrusion (Rug et al, 2006), the precise interactions at the RBC membrane and sub-membrane skeleton that cause the protrusion of the RBC plasma membrane still need further investigation.

The Maurer’s clefts, a novel type of secretory organelle Tens of seconds after entry, while the merozoite is still close to the entry site, the RBC membrane deforms from its biconcave discoidal shape to an echinocyte shape, returning to its normal state after several minutes (Gilson & Crabb, 2009). 173

Review The consequences of these peculiar RBC membrane deformations are not known, but might be related to some maturation of the parasitophorous vacuole and/or to the very first generation of Maurer’s clefts, a membrane compartment transposed by the parasite to the host cell cytoplasm (Maurer, 1902; Atkinson & Aikawa, 1990; Lanzer et al, 2006 for review) (Fig 2). Immunofluorescence microscopy and proteomic studies revealed that the parasitophorous vacuole, the tubovesicular network and the Maurer’s clefts have different sets of resident proteins (Gunther et al, 1991; Spielmann et al, 2003; Haeggstrom et al, 2004; Vincensini et al, 2005; Nyalwidhe & Lingelbach, 2006). Moreover, photobleaching studies using a fluorescent lipid probe that labelled the PVM, Maurer’s clefts and RBC membranes (Hanssen et al, 2008), determined the absence of fluid continuum between individual clefts or between the Maurer’s clefts and the PVM or the host cell membrane. In conclusion, once formed, the Maurer’s clefts lose connection with their site of budding. In early trophozoites, the Maurer’s clefts appeared to diffuse within the host cell cytoplasm before mostly residing at the RBC periphery (Gru¨ring et al, 2011). However, Maurer’s clefts are attached to the RBC sub-membrane skeleton from the very early trophozoite stage to merozoite egress (Blisnick et al, 2000, 2005). Binding of the Maurer’s clefts to the RBC membrane probably depends on multiple protein-protein interactions implicating parasite as well as host proteins: stalk-like extensions of the Maurer’s clefts, identified by MAHRP2 (membrane-associated histidine-rich protein 2), might tether the clefts to the host cell sub-membrane skeleton (Hanssen et al, 2008; Pachlatko et al, 2010); moreover, the host plasma membrane-associated protein LANCL1 binds to the Maurer’s clefts resident protein PfSBP1 (Blisnick et al, 2000, 2005); in addition, actin filaments, polymerizing from the Maurer’s clefts to domains of the RBC sub-membrane skeleton underneath the knobs, link the clefts to the RBC periphery (Cyrklaff et al, 2011). Given that most, if not all, parasite proteins exported to the RBC are at least transiently associated with the Maurer’s clefts, this novel organelle appears to act as a marshal platform for proteins before reaching their final destination to the host RBC. To our knowledge, such sorting organelles have not been reported in other organisms and might thus be specific to the malaria parasites. Maurer’s clefts do not represent a surrogate, extracellular Golgi for the parasite because they lack important protein markers and metabolic functions that usually characterize a Golgi organelle. Interestingly, they might host metabolic pathways that are unexpected for a secretory compartment (Vincensini et al, 2005). There is growing evidence that Maurer’s clefts play a crucial role for the parasite intra-erythrocytic development and represent a new type of organelle specific to malaria parasites. Maurer’s clefts have no direct connection to the RBC membrane, and several studies strongly support a vesicular trafficking of proteins between these two compartments. 174

Uncoated vesicles of c. 25 nm in diameter were observed in the erythrocyte cytoplasm and appeared to fuse with the RBC membrane (Hanssen et al, 2010). Moreover, the actin filaments polymerizing from the Maurer’s clefts to the knobs seem to provide support and guidance for the transport of vesicles of similar size loaded with the PfEMP1 virulence factor, to the host cell plasma membrane (Cyrklaff et al, 2011). Differently, the export of parasite proteins to the Maurer’s clefts could take various routes, involving either vesicular traffic or soluble protein complexes (Fig 3). Highly mobile membranous structures, referred to as ‘J-dots’, have been recently proposed to traffic proteins across the RBC cytosol (Ku¨lzer et al, 2010). It has also been proposed that Maurer’s clefts membrane-associated proteins traffic from the parasitophorous vacuole to pre-formed clefts as soluble protein complexes (Papakrivos et al, 2005). This hypothesis is consistent with a similar host cell-targeting motif present in both soluble and membrane exported parasite proteins (Hiller et al, 2004; van Ooij et al, 2008).

Crossing the parasitophorous vacuole Indeed, malaria parasites export numerous proteins into their host cell that are defined by two amino-terminal export motifs: a signal peptide addressing the proteins to the parasite endoplasmic reticulum (ER) and a short amino acid sequence with the RxI/LxE/Q/D consensus addressing the proteins to the host cell and known as PEXEL (Plasmodium export element) or VTS (vacuole transport signal) (Hiller et al, 2004; Marti et al, 2004). The PEXEL motif is cleaved in the ER and the new N-terminus acylated (Boddey et al, 2010; Chang et al, 2008; Russo et al, 2010). These features appear to be necessary for efficient export to the host cell (Boddey et al, 2009). Once released in the parasitophorous vacuole, PEXEL-positive proteins interact with a PEXEL-protein translocation machine (PTEX) at the parasitophorous vacuole membrane (de Koning-Ward et al, 2009), facilitating their export to the host cell (Fig 3). Based on the PEXEL/VTS motif, up to 8% of the P. falciparum-encoded proteins are predicted to be exported to the host cell (Hiller et al, 2004; van Ooij et al, 2008). However, this predicted exportome is not exclusive because an increasing number of proteins lacking a PEXEL motif (PEXEL-negative proteins) are exported to the RBC, such as the Maurer’s cleft proteins PfSBP1 and MAHRP1 (Saridaki et al, 2009; Spycher et al, 2006; Spielmann & Gilberger, 2010 for review). Although short amino acid sequences have been identified in individual PEXEL-negative exported proteins that are sufficient to address the protein to the host cell, no obvious export motif has been characterized (Dixon et al, 2008; Haase et al, 2009; Saridaki et al, 2009) and how PEXEL-negative proteins do cross the PVM remains to be determined. The PVM forms a barrier between the parasite and the host cell that can be crossed by parasite proteins exported to the host cell but also by host proteins recruited by the parasite at ª 2012 Blackwell Publishing Ltd British Journal of Haematology, 2012, 157, 171–179

Review membrane physical properties necessary for egress of the newly formed merozoites from their host cell.

Outside the RBC, a split second event that might be set-up very early

Fig 3. one proposed model for P. falciparum protein export to the RBC. Parasite proteins exported to the host cell traffic within vesicles through the parasite constitutive secretory pathway as soluble proteins (1) (membrane proteins probably interact with chaperones to maintain them as unfolded and soluble) and are released in the lumen of the parasitophorous vacuole (2). Interacting with chaperones in the parasitophorous vacuole, they are addressed to a translocon in the parasitophorous vacuole membrane (PVM) (2) and released in the host cell cytosol (3) where they interact with proteins addressing them to the Maurer’s clefts (MC). The proteins are further addressed to the Maurer’s clefts, as soluble complexes. Alternatively, some proteins might traffic within J-dots (4). Finally, soluble proteins are sorted from the Maurer’s clefts to the RBC cytosol and sub-membrane skeleton (5b) and membrane proteins are trafficked to the RBC plasma membrane (5a), probably by vesicles that fuse with the host cell membrane.

the trophozoite stage. Indeed, integral and peripheral proteins located in lipid raft domains of the RBC membrane are detected at the PVM (Lauer et al, 2000) and, more recently, RBC dematin, an actin-binding protein of the RBC sub-membrane skeleton, was found internalized by the parasite and associated with the intra-cellular parasite 14-3-3 protein (Lalle et al, 2011). Recruitment of RBC membrane and sub-membrane skeleton proteins by the parasite might weaken the RBC membrane and thus participate in the changes of the RBC ª 2012 Blackwell Publishing Ltd British Journal of Haematology, 2012, 157, 171–179

Parasite release from the host cell requires the opening of the parasitophorous and RBC membranes. Swelling of the RBC precedes the egress of Plasmodium falciparum merozoites from the host cell by a few minutes (Dvorak et al, 1975) and the use of amphiphiles, osmotic stress and protease inhibitors strongly suggested that merozoite release is pressure-driven (Glushakova et al, 2005, 2009). Shortly before merozoite egress, the intracellular parasites seem to move more freely while the RBC membrane is still intact (Abkarian et al, 2011), concordant with the rupture of the parasitophorous vacuole membrane. Indeed, several studies have provided evidence that, when the merozoites are close to release, the PVM enlarges and ruptures before the RBC membrane (Wickham et al, 2003; Glushakova et al, 2010). Such a sudden increase in the osmotic pressure could have various origins. Indeed, a premature release of immature merozoites has been recently described as resulting from the inhibition of RNA degradation and concomitant swelling of the infected RBC (Balu et al, 2011). In addition, parasite proteases have been described that could also participate in the increased osmolarity: these enzymes are specifically active in infected RBCs just prior to merozoite release and their activity is necessary for efficient merozoite release (Koussis et al, 2009). Although first considered to be an explosive event, merozoite egress from the RBC has been shown recently to occur through the opening and stabilization of an osmotic pore in the RBC membrane allowing the release of a limited number of merozoites (Abkarian et al, 2011) (Fig 4). The opening of a pore in the RBC membrane is followed by its curling and buckling and the wide-angular dispersion of the remaining merozoites (Abkarian et al, 2011). Curling and buckling eversion of the RBC membrane happens when a critical radius of the osmotic pore is reached. Abkarian et al (2011) hypothesized that this instability is biologically relevant as it disperses the merozoites and contributes to their efficient separation from the infected RBC membrane. Indeed, abortive egress events have been observed with a stop of curling and no buckling, resulting in the merozoites remaining stuck together inside the open RBC and thus unable to further invade new RBCs (Abkarian et al, 2011). Abkarian et al (2011) proposed that the curling is related to a spontaneous curvature of the infected RBC membrane, specifically acquired during parasite development. The molecular actors of this spontaneous curvature are key factors in merozoite release and potential targets for new control strategies. Curling and buckling can originate from an additional membrane elastic energy due to an asymmetry between the membrane leaflets (Mabrouk et al, 2009). In P. falciparum -infected RBCs, this asymmetry between the two membrane leaflets 175

Review

Fig 4. P. falciparum merozoite egress. Snapshots of the whole release process are presented: pressure-driven ejection of the first parasite (up to 1126 ms); curling of the RBC membrane (273–680 ms) and final buckling of the membrane (680–816 ms) pushing the remaining merozoites forwards, far from their initial position. These data were obtained in collaboration with Manouk Abkarian and Gladys Massiera (LCC, University Montpellier 2, France). The RBC membrane is schematically presented at each step of the release process.

could originate from a lipid excess in the inner leaflet caused by a lipid release of parasite origin, a modification of the mechanical properties of the RBC membrane through changes of the cytoskeleton/membrane interactions (reviewed in An & Mohandas, 2010) and/or interaction of the RBC membrane with the Maurer’s clefts (Blisnick et al, 2006). Parasite-induced changes at the RBC membrane that affect its stability occur as early as parasite entry and very early intra-erythrocytic growth with, for example, the tyrosinephosphorylation and clustering of the RBC Band 3 (Pantaleo et al, 2010) and the export of the parasite RESA protein to the RBC sub-membrane skeleton (Foley et al, 1991). Moreover, phosphorylation of host peripheral proteins increases upon parasite growth and might modulate the bio-physical properties of the RBC membrane (Pantaleo et al, 2010). However, the ability to curl and buckle has also been proposed to be an intrinsic property of the RBC membrane when the RBC is exposed to certain osmotic stresses (Lew, 2011) although with marked kinetic differences as compared to the infected RBC. Whether the parasite exploits a property of its host cell and at what extent the changes of the RBC membrane and sub-membrane skeleton induced by the parasite are essential for efficient merozoite release need further investigation.

Concluding remarks Changes in the RBCs closed environment, i.e. the RBC cytoplasm and plasma membrane, induced by the life-threatening 176

human malaria parasite Plasmodium falciparum have been extensively studied. Indeed, these changes are crucial for the parasite development and for its escape from the host immune response. In the last decade, P. falciparum genetic engineering progresses and the spectacular advances of cell imaging, have considerably highlighted our knowledge of the RBC remodelling by the parasite, the processes involved and their importance for the parasite survival. While new permeation pathways in the RBC membrane and extensions of the parasitophorous vacuole membrane in the host cell cytosol, named the tubovesicular network, participate in the import of nutrients from the extracellular milieu, Maurer’s clefts are central to the transport of parasite proteins to the RBC. They tightly interact with the host cell membrane even upon merozoite release. This interaction, together with exported parasite proteins interacting with the host cell sub-membrane skeleton might prevent the premature rupture of the RBC membrane and consequent release of immature merozoites. Indeed, the parasite has weakened its host cell membrane by altering the cohesion between the plasma membrane and sub-membrane skeleton via the phosphorylation and the recruitment of host cell membrane and skeletal proteins. On the other hand, one can consider that the parasite has prepared its host cell membrane not only for entry but also for egress: reversing the parasite-induced modifications, by the activation of phosphatases for example, would highly facilitate the opening of the RBC plasma membrane for merozoite release. ª 2012 Blackwell Publishing Ltd British Journal of Haematology, 2012, 157, 171–179

Review Merozoite release relies on a unique opening site in the RBC membrane, allowing the egress of the first one or two merozoites; the release of the remaining merozoites results from the curling and eversion of the RBC membrane. Importantly, the same sequence of events has been observed when infected RBC had adhered to a substrate (the usual status of P. falciparum-infected RBC that are cytoadhering in vivo to the micro-vessel endothelium and to non-infected RBCs). The physical parameters of curling and eversion of the RBC membrane emphasized once more the importance of parasite-induced changes to the host cell membrane. RBC remodelling by the malaria parasite necessitates both efficient export of parasite proteins to the host cell and extensive membrane synthesis. These processes, together with the parasite proteins involved in RBC remodelling, require

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