Protoplasma (2010) 240:3–12 DOI 10.1007/s00709-009-0090-3
REVIEW ARTICLE
The malaria parasite Plasmodium falciparum: cell biological peculiarities and nutritional consequences Stefan Baumeister & Markus Winterberg & Jude M. Przyborski & Klaus Lingelbach
Received: 4 November 2009 / Accepted: 9 November 2009 / Published online: 25 November 2009 # Springer-Verlag 2009
Abstract Apicomplexan parasites obligatorily invade and multiply within eukaryotic cells. Phylogenetically, they are related to a group of algae which, during their evolution, have acquired a secondary endosymbiont. This organelle, which in the parasite is called the apicoplast, is highly reduced compared to the endosymbionts of algae, but still contains many plant-specific biosynthetic pathways. The malaria parasite Plasmodium falciparum infects mammalian erythrocytes which are devoid of intracellular compartments and which largely lack biosynthetic pathways. Despite the limited resources of nutrition, the parasite grows and generates up to 32 merozoites which are the infectious stages of the complex life cycle. A large part of the intra-erythrocytic development takes place in the socalled parasitophorous vacuole, a compartment which forms an interface between the parasite and the cytoplasm of the host cell. In the course of parasite growth, the host cell undergoes dramatic alterations which on one hand contribute directly to the symptoms of severe malaria and which, on the other hand, are also required for parasite survival. Some of these alterations facilitate the acquisition of nutrients from the extracellular environment which are not provided by the host cell. Here, we describe the cell biologically unique interactions between an intracellular S. Baumeister : M. Winterberg : J. M. Przyborski : K. Lingelbach (*) Department of Parasitology, Faculty of Biology, Philipps Universität, 35032 Marburg, Germany e-mail:
[email protected] Present Address: M. Winterberg School of Biochemistry and Molecular Biology, Australian National University, Canberra, Australia
eukaryotic pathogen and its metabolically highly reduced host cell. We further discuss current models to explain the appearance of pathogen-induced novel physiological properties in a host cell which has lost its genetic programme. Keywords Apicomplexa . Erythrocyte . Malaria . New permeability pathways . Plasmodium falciparum Abbreviations NPP new permeability pathways PV parasitophorous vacuole PVM parasitophorous vacuolar membrane RBC red blood cells iRBC infected red blood cells
Introduction Plasmodium falciparum is a protozoan parasite and the causative agent of the most virulent form of malaria in humans. In endemic areas, this disease is responsible for almost one million deaths (WHO 2009), primarily among children under the age of five. The parasite has a complex life cycle, which includes the formation of a zygote, meiosis and subsequent asexual replication in anopheline mosquitoes from which so-called sporozoites are transmitted to the human host. Here, the sporozoites invade liver cells (extraerythrocytic development) where the parasite replicates to form up to 30,000 merozoites which then go on to invade differentiated red blood cells (RBC). It is the infection of, and multiplication within, the RBC that cause the severe symptoms of malaria tropica. P. falciparum belongs to a group of parasites called Apicomplexa which are characterised by an apical complex, a morphologically
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conspicuous structure consisting of specialised organelles such as micronemes and rhoptries, which are located at one pole of the respective invasive stages. Most apicomplexans are obligatory intracellular pathogens with only short-lived extracellular stages within their respective hosts. Another cell biological peculiarity shared by many apicomplexans is a rudimentary plastid, the apicoplast, which was acquired by secondary endosymbiosis between a free-living ancestor of these parasites and a red alga. Although the apicoplast no longer has photosynthetic properties, it is nevertheless essential for parasite survival. Several biosynthetic pathways are located within the apicoplast which, in general terms, are more akin to the equivalent pathways in plants and bacteria than to the pathways found in animals. The existence of a relict plastid in a heterotrophic organism is not only a fascinating evolutionary phenomenon, but also opens new avenues for the identification of novel drug targets (Fichera and Roos 1997; Soldati 1999; Goodman and McFadden 2007; Lizundia et al. 2009). Although apicomplexan parasites share many cell biological properties that are unique to this group of organisms, there are also distinct differences, relating to the choice of the host cell, the principles of nutrient acquisition, and the morphological development inside the infected cell. The process of initial host cell invasion appears to follow a common general theme; first, the parasite attaches with any part of its surface to the plasma membrane of the host cell, and then re-orients such that the apical end comes into juxtaposition with the target membrane. Re-orientation involves a gliding motility, mediated by the glideosome, an actinomyosin-based protein complex (for a review: Soldati and Meissner 2004). This molecular motor spans the multiple membrane layers which form the inner membrane complex. Following an indentation of the host cell plasma membrane, the parasite enters the host cell and resides within a parasitophorous vacuole (Bannister et al. 1975). The molecular mechanisms leading to the formation of the parasitophorous vacuole (PV) are still rather elusive. Since the contents of rhoptries and micronemes are discharged during the process of invasion, it is a general view that the molecules contained in these compartments contribute to the formation of the vacuolar membrane (PVM); however, exactly how this takes place, and the molecules involved, remain unknown. The parasite grows to the metabolically active stage called trophozoite which, after approximately 36 h, begins to divide into up to 32 infectious merozoites (Fig. 1). In contrast to many other apicomplexan parasites which maintain the structures of the apical complex during intracellular replication, micronemes and rhoptries appear to disappear in the ring and trophozoite stages of P. falciparum. It remains a cell biological enigma how and from which precursors these compartments are recruited during merozoite formation. The
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detailed sequences of events whereby the merozoites egress from the infected cell are still a matter of controversy. While some authors suggest a direct release into the medium, other models propose that release from the red blood cell occurs within an intact PV (reviewed in Blackman 2008). Although this appears to be a purely academic argument, the release of parasites within an intact vacuole would delay the contact of liberated parasites with possibly protective antibodies present in the serum. The need to understand the molecular and cell biology of this unique pathogen and its interaction with the human red blood cell as a prerequisite to devise chemotherapeutic and prophylactic strategies to combat malaria has resulted in numerous recent reviews which deal in detail with various specific aspects. Here, we focus on the cell biological events allowing parasite survival in a nutritionally deprived cell and discuss current models trying to provide a molecular explanation for this phenomenon.
The compartmentation of the infected human erythrocyte The differentiated non-infected mammalian erythrocyte is a metabolically highly reduced host cell, which lacks compartments, a genetic programme and which has lost the ability to synthesise proteins or lipids de novo. Consequently, its requirements for nutrient supply are limited, and this highly specialised cell lacks trafficking pathways for proteins as well as transport pathways for many solutes or metabolites. In the infected erythrocyte, apart from the parasite itself, novel membrane systems and compartments appear (Fig. 1). Most conspicuous is the PV which is topologically distinct from the cytoplasms of the host and the parasite, respectively, and which contains a unique set of proteins (Nyalwidhe and Lingelbach 2006). In addition to the PVM, membrane structures are formed within the host cell cytoplasm. Depending on their morphology and their proximity to the erythrocyte plasma membrane, they are termed tubulovesicular network or Maurer's clefts, respectively (Hanssen et al. 2008). It is still a matter of debate whether these compartments, including the PV, are interconnected or whether they are independent entities (Wickert et al. 2004, Wickert and Krohne 2007; Hanssen et al. 2008). Electron-dense protrusions, called 'knobs' appear underneath the erythrocyte plasma membrane. These protein complexes consist of several parasite proteins which are exported to the erythrocyte membrane and which also contain proteins, collectively called P. falciparum erythrocyte membrane proteins (PfEMP-1). This family of proteins is exposed on the erythrocyte surface, undergoes antigenic variation, and it is responsible for the sequestration of infected erythrocytes in blood capillaries, thus directly
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Fig. 1 The infected erythrocyte and the increased permeability of its plasma membrane for solutes. a Prior to invasion, the extracellular infective stages (merozoites) attach to the surface of the erythrocyte and then re-orient such that the apical complex containing the clubshaped rhoptries (Rh) and slender micronemes (Mic) come into juxtaposition with the erythrocyte plasma membrane. Invasion and formation of the parasitophorous vacuole occur within seconds. Within the following 24-36 h, the parasite grows to become a trophozoite, concomitant with an increase in the vacuolar volume and a decrease in haemoglobin content. After 36–48 h, the parasite multiplies by schizogony, thereby forming up to 36 infectious
merozoites which egress from the infected cell. b Ultrastructural section of the trophozoite stage-infected erythrocyte. The parasite cell resides within the parasitophorous vacuole (PV) the lumen of which appears electron dense. Within the red blood cell cytosol (RBC) novel structures such as the Maurer's clefts (MC) appear. In addition, electron dense structures called knobs (K) are inserted into the erythrocyte cytoskeleton which underlies the red blood cell membrane. Conceptually, nutrient uptake from the extracellular milieu needs to occur across three membranes; the erythrocyte membrane, the parasitophorous vacuolar membrane and the parasite plasma membrane (PPM)
contributing to the clinical symptoms (Miller et al. 2002) Biochemical and morphological evidence suggests that these complexes are assembled from secreted soluble proteins at the Maurer's clefts which are instrumental in depositing these structures underneath the erythrocyte membrane (Kriek et al. 2003). It is appealing to hypothesise that this mechanism is a host-parasite adaptation which allows the delivery of parasite proteins in a cell which has lost the canonical protein trafficking pathways.
harmful hydrolases, and MHC complexes able to signal the infection to the immune system, a distinct advantage of a 'life inside a phagosome' is the nutrient supply that results from the degradation of various macromolecules (Burchmore and Barrett 2001). In contrast, the PVM formed by apicomplexan parasites is unable to fuse with the host cell's endomembrane system (Joiner et al. 1990; Sinai and Joiner 1997). This has been studied for Toxoplasma, Eimeria spp. and Cryptosporidium, which invade nucleated cells. Unlike phagosomal compartments, the pH of the PV remains close to neutral. It is conceivable that, in nucleated cells, important functions of the PVM are to inhibit fusion with compartments containing lysogenic proteins and/or to prevent access of parasiticidal cytosolic proteins. On the other hand, it is noteworthy that Theileria and Babesia, which infect nucleated cells and differentiated erythrocytes, respectively, survive without maintaining a PVM. Although the PV may nevertheless act as an ideal shelter for the parasite it harbours, it is, in contrast to phagosomes, a nutrient-poor environment. All these considerations partic-
The parasitophorous vacuole as an interface between parasite and host cell Many prokaryotic and eukaryotic pathogens enter their host cells by taking advantage of the phagocytic properties of these cells. As a consequence, these organisms reside within a compartment that is able to fuse with the host cell's endosomal network. Although these pathogens are then confronted with a low pH environment, potentially
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ularly apply to the erythrocytic stages of P. falciparum which invades a nutritionally deprived and metabolically sluggish host. Owing to its role as interface between the parasite and the host cell, biogenesis and cell biological functions of the PVM have been of considerable interest. Since the PVM is devoid of the major erythrocyte membrane proteins such as the glycophorins or the anion transporter band 3, it has long been thought that host cell proteins are actively excluded when this membrane is formed during invasion (reviewed in Lingelbach and Joiner 1998). However, recent data suggests the presence of several erythrocyte membrane proteins in the PVM (Hiller et al. 2003; Bietz et al. 2009). It appears that these proteins are lipid raft associated (Murphy et al. 2004, 2007) and that they are recruited to the PVM during or soon after parasite invasion (Bietz et al. 2009) rather than by a process involving invasion followed by subsequent retrograde trafficking from the erythrocyte membrane to the parasite. It is unknown whether the erythrocyte proteins found in the PVM are required for parasite survival or whether they are recruited fortuitously. In the latter case, although still speculative, this might indicate a certain preference for detergent resistant microdomains as entry sites. From a nutritional standpoint, it is noteworthy that host cell proteins are excluded from the PV. Nevertheless, the PVM contains non-selective pores of 0.65–1.1 nm diameter (Desai et al. 1993) that allow passage of molecules up to ∼600 Da (Nyalwidhe et al. 2002). The proteins that constitute these pores have not yet been identified, but several proteins synthesised by the parasite are transported beyond the confines of the parasite plasma membrane and are integrated into the PVM (Günther et al. 1991, Marti et al. 2005). As host cell proteins are excluded from the PV, the protein content of this compartment represents proteins which are synthesised and secreted from the parasite cell. A recent proteome analysis of the PV has revealed a high proportion of chaperones and proteases (Nyalwidhe and Lingelbach 2006). Although access of host cell proteins to the PV is very restricted, if it occurs at all, there is an active unidirectional transport of parasite proteins, across the PVM into the RBC cytosol, and even to the RBC plasma membrane, exemplified by the knob proteins. Whilst the P. falciparum erythrocyte membrane protein-1 family, actively contributes to the pathological effects of malaria, other proteins appear to be required for the correct trafficking to the erythrocyte surface (Crabb et al. 1997; reviewed in Maier et al. 2009). Briefly, these proteins are secreted into the PV before being translocated across the PVM (Ansorge et al. 1996). A pentapeptide towards the Nterminus of the protein, referred to as the Plasmodium Export Element (PEXEL; Marti et al. 2005) or vacuolar translocation signal (VTS) (Hiller et al. 2004) directs traffic
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of proteins to locations beyond the PVM (reviewed in Boddey et al. 2009). Since the pentapeptide is cleaved within the parasite from at least some proteins (Chang et al. 2008; Boddey et al. 2009), it is not clear at which point of the pathway PEXEL/VTS acts to determine the fate of the protein. Long hypothesised, a multimeric protein complex within the PVM which may act as a translocon has been recently described, and partially functionally characterised (Charpian and Przyborski 2008; de Koning et al. 2009; Gehde et al. 2009). The molecular mechanisms underlying protein transport within the RBC cytosol are less clear, in particular to what extent they involve conserved auxiliary proteins known to be required for such processes in nucleated cells. A detailed discussion of this fascinating cell biologically phenomenon is beyond the scope of this current article, and has been reviewed elsewhere (Maier et al. 2009; Charpian and Przyborski 2008; Lingelbach and Przyborski 2006).
Nutritional requirements and deposition of waste products The seclusion of apicomplexan parasites within a PV raises the need for novel mechanisms to acquire nutrients. Toxoplasma gondii tethers host cell mitochondria and endoplasmic reticulum to the PVM and even intersects the endosomal/lysosomal pathway, thereby acquiring nutrients which have been taken up by endocytic processes (Coppens et al. 2006). Obviously, as the mature erythrocyte lacks an endocytotic pathway, this option does not exist for the intraerythrocytic stages of P. falciparum but may be applicable in liver stages. Intraerythrocytic stages possess a structure called a cytostome, which becomes ultrastructurally visible in the trophozoite stage (Elliott et al. 2008). The cytosome can be considered as a large phagosome, with which the parasite engulfs the host cell cytosol. The early phagocytic compartments contain two membranes, the plasma membrane of the parasite and the PVM, respectively (Aikawa et al. 1966; Slomianny 1990). During transport to the food vacuole, the PVM is dissolved, and haemoglobin begins to be digested. The proteases involved in haemoglobin degradation are well-characterised and are obvious targets for chemotherapeutic strategies (Klemba and Goldberg 2002; Goldberg 2005). The potentially cytotoxic haem that is released during this process is inactivated by polymerisation to insoluble, crystalline haemozoin (Slater 1992). Although haemoglobin is the main source of amino acids for the parasite, the protein does not contain the amino acid isoleucine and several others such as glutamate, methionine, cysteine, and proline (Kirk 2001; Martin and Kirk 2007) are underrepresented. As well as requiring access to these “missing” amino acids, the parasite is furthermore
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reliant on the uptake of pantothenic acid (the precursor of coenzyme A), from the medium surrounding the host cell (Saliba et al. 1998; Kirk and Saliba 2007). Apart from the ingredients needed for the synthesis of biopolymers, the parasite's energy requirements are met primarily through glycolysis, and therefore the parasite has a large demand for glucose. In addition, there is a need to export waste products, in particular lactate resulting from the reduction of pyruvate.
New permeability pathways The growing and replicating parasite increases the metabolic activity of the infected erythrocyte. It was recognised a long time ago that the infected cell must undergo major physiological alterations in order to accommodate the metabolic requirements of the parasite. Early concepts and models are reviewed in Gero and Kirk (1994). Increased permeability of the erythrocyte for several solutes could be experimentally measured following infection, and this phenomenon was subsequently termed 'new permeability pathways' (NPP). The conceptualisation of a unifying model that explained the uptake of a variety of substances (see Table 1) was a major challenge. Since the differentiated mammalian erythrocyte, owing to the lack of protein and lipid biosynthetic pathways, has a very limited capacity for the formation of novel transport pathways, it has generally been inferred that such pathways are actively induced by synthetic activities of the parasite. Initially, three working hypotheses for the uptake of various solutes were proposed (Fig. 2). For a detailed discussion of the various models, see Gero and Kirk 1994. Briefly, two models propose a temporary (metabolic window) or permanent (parasitophorous duct) fusion of the PVM with the erythrocyte membrane, thereby allowing a direct access
Table 1 Known solutes which can permeate through NPP
*Solutes which are believed to be essential for parasite survival (adapted from Ginsburg et al., http://sites.huji.ac.il/malaria/)
Fig. 2 Working hypotheses for the acquisition of nutrients from the extracellular milieu. a Sequential uptake model. Access occurs via transport proteins located within the erythrocyte membrane, followed by passage through non-selective pores present in the PVM (Kirk 2001). b Parasitophorous duct model. The parasitophorous vacuole is permanently fused with the erythrocyte membrane, thereby allowing access of large molecules including antibodies (Pouvelle et al. 1991). c Metabolic window model. The PVM temporarily contacts the erythrocyte membrane and forms a yet to be characterised 'molecular sieve' (Lauer et al. 1997). In all models, the solutes are proposed to find access into the parasite via transporters within the parasite plasma membrane
of solutes to the surface of the parasite, bypassing the PVM. Uptake across the parasite plasma membrane would have to occur via appropriate parasite encoded transporters. Although these models have been debated controversially, they would explain the low selectivity of the solutes and occasional observations that macromolecules such as antibodies are internalised into seemingly intact infected RBC. A third model, initially suggested by Ginsburg et al. (1983), proposes a transport of solutes directly across the erythrocyte membrane. As this model has recently received
Carbohydrates
Amino acids
Nucleosides
Anions/Cations
Others
Glycerol Erythrytol D/L-arabitol Arabinose Ribose Sorbitol Xylitol Mannitol Dulcitol D/L-glucose 2-deoxyglucose etc.
Alanine Asparagine Aspartate Cysteine Glutamate Glycine Isoleucine* Leucine Serine NBD-taurine Carnitin etc.
D/L-adenosine D/L-thymidine Uridine NBMPR
ClGluconate Lactate DNDS Dipicolinic acid Pantothenic acid*
Thiourea Phloridzin di- and tri- peptides Glutathione GSSG Polypeptides Oligonucleotides Iron chelators Sulfo-NHS-Biotin
Na+, K+, Rb+ Mg2+, Ca2+ Fe2+ / Fe3+ Choline polyamines
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considerable attention as a working hypothesis, we will discuss the experimental evidence in support of it and their implications in more detail below. The observation that NPP allow access of a wide variety of solutes prompted us and others to exploit membrane impermeant biotin derivatives to identify the transport pathways, namely the transport proteins involved. Certain sulfoesters of biotin are excluded from non-infected erythrocytes but are imported into iRBC (Baumeister et al. 2003; Cohn et al. 2003). Import of the derivatives could be inhibited by compounds such as furosemide, 4,4′diisothiocyanatostilbene-2,2'-disulfonic acid and others, which also inhibit import of physiologically relevant solutes. Since the derivatives covalently bind to lysine residues of proteins, the rationale of these experiments was to delineate the import pathways by the identification of biotinylated proteins. The biotinylation of internal host cell proteins such as haemoglobin and spectrin, upon treatment of intact iRBC were strong arguments in favour of a pathway which involves direct transport across the erythrocyte membrane and release into the erythrocyte cytosol. Although the PVM was long considered a closed barrier between the parasite and the host cell cytosol, the identification of non-selective pores within, initially, the PVM of T. gondii (Joiner et al. 1990) and later in P. falciparum (Desai et al. 1993, Nyalwidhe et al. 2002), provided an explanation as to how solutes gain access from the erythrocyte cytosol to the lumen of the PV, and thus come into contact with the parasite plasma membrane. As previously noted, these pores allow the passage of small molecules, which encompasses most essential nutrients. The observation that biotin derivatives eventually block uptake of other nutrients adds further support to the notion that import of the derivatives (and thus nutrients) occurs via one or several membrane bound transport proteins. These biochemical studies have been complemented by electrophysiological measurements which detected novel conductances in the membrane of iRBC (Desai et al. 2000). These conductances showed a clear preference for anions over cations, and similar responses to inhibitors of anion transporters as observed in tracer influx studies. Although there is a general agreement that the conductances which can be measured in iRBC are the electrophysiological correlates of NPP, controversial issues include the number of different channel types, the electrophysiological characteristics of the channels, and the origin of the channels (Fig. 3). The latter issue addresses the question whether the transport proteins are host- or parasite-encoded. Resolving these issues with singular experimental approaches is difficult owing to the high variability associated with individual physiological experiments. These variations include, apart from differences in experimental and instrumental details, the use of red blood cells obtained from
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Fig. 3 The origin and activation of solute transport proteins within the erythrocyte membrane. a A single transporter in the erythrocyte membrane, possibly encoded by the parasite, is responsible for the uptake of all solutes for which the membrane of the non-infected erythrocyte is impermeable (Alkhalil et al. 2004). b The phenomenon of NPP is mediated by at least four host encoded and thus endogenous channel proteins (three anion channels and one cation channel). Activation of the quiescent channels occurs via infection induced oxidative stress (Huber et al. 2002a). c The NPP constitute of three anion channels, one of which is parasite encoded. Activation of the two host encoded transport proteins occurs via phosphorylation (Egée et al. 2002; Merckx et al. 2008). d An infection induced CFTR dependent anion transport is proposed which is not essential for parasite survival. All other solutes gain access to the host cell cytosol via a separate channel/pore (Verloo et al. 2004). e Responsible for NPP are two distinct pores both of which are parasite encoded (Ginsburg and Stein 2004). PSAC plasmodial surface anion channel, ClC-2 chloride channel family member 2, IRC inward rectifying channel, ORCC outward rectifying conductance channel, NSCC nonspecific cation channel, ICAC intermediate conductance anion channel, SCAC small conductance anion channel, ORLCAC outwardly rectifying large conductance anion channel, CFTR cystic fibrosis transmembrane regulator, IRCAC inwardly rectifying conductance anion channel
different donors and cells that have undergone subtle but non-controllable differences in their respective physiological conditions.
New permeability pathways—host or parasite derived? While there is general consensus with respect to the sequential transport model (transport across the erythrocyte membrane, the PVM, and finally the parasite plasma membrane), the nature of the proteins mediating the transport across the erythrocyte membrane is still being debated (Ginsburg and Stein 2004, Staines et al. 2007). The
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fact that many solutes are excluded from non-infected cells and the loss of protein biosynthetic pathways have, for a long time, underscored a somewhat biassed view that NPP must be mediated by parasite transporters which are inserted into the host cell plasma membrane. Therefore, the observation by Huber and colleagues who were able to induce NPP-like activities in oxidised non-infected cells (Huber et al. 2002a) was unexpected and have kindled a controversial debate (Ginsburg 2002; Huber et al. 2002b). The results implicated an activation of endogenous but usually silent erythrocyte transporters following infection. Experiments from our laboratory showed that NPP in infected erythrocytes can be inactivated by chymotrypsin treatment of intact infected cells. They re-appear, and this re-appearance is dependent on parasite viability and protein secretion from the parasite (Baumeister et al. 2006). However, does this observation exclude an involvement of erythrocyte transporters? Recent experiments in our laboratory on non-infected erythrocytes showed that experimentally induced transport pathways which were also chymotrypsin sensitive could be 'restored' when the protease was removed, the erythrocytes transferred to normal culture conditions, and when the activation was repeated (Winterberg et al. unpublished). Most likely, each
protease treatment resulted in the inactivation of a subset of the transport proteins and thus made a further round of activation possible. Although far from being a complete mechanistic model, it is possible that the activation of silent channels in non-infected cells is the result of a rather controlled process that prevents a potentially deleterious activation of all available molecules. The following scenario for the uptake of some but certainly not all solutes into infected erythrocytes can be envisaged and used as a working hypothesis (Fig. 4); transport of solutes across the erythrocyte membrane is mediated by host cell proteins which are activated upon infection. Activation may occur through kinases which are exported from the parasite cell and which modulate activity and/or substrate specificity of otherwise silent transport proteins. Indeed, there are several examples of protein kinases in eukaryotic cells which modulate the activity of membrane transporters (Levitan 1994; Lev et al. 1995). The contribution of secreted parasite proteins to the activation would explain the effects of inhibitors of protein secretion and of parasiticidal drugs on the re-appearance of NPP in infected erythrocytes. In fact, a recently discovered family of parasite kinases (Schneider and Mercereau-Puijalon 2005; Nunes et al. 2007), some members of which are transported into the erythrocyte
Fig. 4 Model for the reappearance of solute transport mediated by an endogenous host cell protein. A transport protein is present in an inactive state in non-infected erythrocytes (left). In this state, the protein is resistant to chymotrypsin and possibly other proteases. Cellular stress induces the activation of the transporter (possibly through kinases, see Fig. 3c) which then becomes protease sensitive (a). After removal of the protease other, so far silent transport
molecules, are activated resulting in a reappearance of transport activity. In the infected cell (b and c), activation of silent endogenous transporters may, in addition, involve auxilliary parasite proteins. If these proteins have localisation which renders them susceptible to proteases (c), transport activity ceases and reappearance becomes dependent on parasite viability and protein secretion from the parasite as observed by Baumeister et al. (2006)
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cytosol, are possible candidates for modulating enzymes. In addition, some authors propose a protein kinase Adependent activation (Merckx et al. 2008, 2009).
The apicoplast, a algal-derived destination of nutrients As well as gaining access to the parasite, many nutrients must further be transported into organelles such as the apicoplast, where they are needed for essential biochemical pathways. The discovery, in 1996, of a second plastid organelle (the first being the mitochondrion) in malaria parasites (McFadden et al. 1996) led to a quantum shift in our understanding of parasite biology. Further studies revealed that this organelle contained several plant or bacteria specific pathways, and thus represents a highly significant target for anti-malarial drug development (Reviewed extensively in: Ralph et al. 2004). Importantly for the topic of this article, the biochemical pathways contained within the apicoplast rely on a constant delivery of raw materials from the parasite cytosol. Although the molecules mediating these transport processes remain largely uncharacterised, in silico reconstruction of biosynthetic pathways allow prediction of the nutritional requirements of the apicoplast (Foth et al. 2003; Ralph et al. 2004). The general model suggests that the apicoplast can be viewed as a chloroplast in the dark, with similar metabolic requirements. Carbon sources Several of the pathways taking place in the apicoplast require a carbon source. Phosphoenolpyruvate appears to be imported via a phosphoenolpyruvate/phosphate translocator, which has shown to be targeted to apicoplast membranes (Mullin et al. 2006). Additionally, dihydroxyacetone phosphate is predicted to be imported into the apicoplast via a triose phosphate transporter (Ralph et al. 2004). Haem biosynthesis Although the parasite has access to a large amount of haem from the host erythrocyte, it appears that the cell nevertheless engages in haem biosynthesis (Surolia and Padmanaban 1992). δ-Aminolevulinic acid is produced in the mitochondrion, and then seems to be transferred to the apicoplast via an as yet unknown transporter (Ralph et al. 2004). Energy source It is so far unknown how the apicoplast gains access to a source of energy. In dark chloroplasts, energy in the form of ATP is imported via an ATP/ADP antiporter, but until now,
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no such transporter has been found which could service the apicoplast (Ralph et al. 2004). In conclusion, the rapidly growing and replicating parasite has a high nutritional demand which cannot be satisfied by the host cell the parasite has chosen to invade. The need to induce novel pathways for the acquisition of a variety of nutrients from the extracellular milieu apparently opens avenues for the access of molecules others than nutrients. It is certainly an attractive strategy to utilise this phenomenon as a 'Trojan Horse' for the delivery of drugs preferentially into infected erythrocytes (Kirk and Saliba 2007).
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