Transport processes in Plasmodium falciparum-infected erythrocytes: potential as new drug targets

International Journal for Parasitology 32 (2002) 1567–1573 www.parasitology-online.com

Invited review

Transport processes in Plasmodium falciparum-infected erythrocytes: potential as new drug targets Sanjeev Krishna a,*, Ursula Eckstein-Ludwig a, Thierry Joe¨t a, Anne-Catrin Uhlemann a, Christophe Morin b, Richard Webb a, Charles Woodrow a, Ju¨rgen F.J. Kun c, Peter G. Kremsner c a

Department of Infectious Diseases, St. George’s Hospital Medical School, Cranmer Terrace, London, SW17 ORE, UK b Laboratoire d’Etudes Dynamiques et Structurales de la Se´lectivite´, B.P. 53-38041 Grenoble, Cedex 9, France c Sektion Humanparasitologie, Institut fuer Tropenmedizin, Universitaet Tuebingen, Wilhelmstrasse 27, 72074 Tuebingen, Germany Received 18 February 2002; received in revised form 6 August 2002; accepted 13 August 2002

Abstract Plasmodium falciparum infection induces alterations in the transport properties of infected erythrocytes that have recently been defined using electrophysiological techniques. Mechanisms responsible for transport of substrates into intraerythrocytic parasites have also been clarified by studies of three substrate-specific (hexose, nucleoside and aquaglyceroporin) parasite plasma membrane transporters. These have been characterised functionally using the Xenopus laevis oocyte heterologous expression system. The same expression system is currently being used to define the function of parasite ‘P’ type ATPases responsible for intraparasitic [Ca 21] homeostasis. We review studies on these transport processes and examine their potential as novel drug targets. q 2002 Published by Elsevier Science Ltd. on behalf of Australian Society for Parasitology Inc. Keywords: Plasmodium; Transporter; ATPase; Channel; Drug target

1. Introduction Plasmodium falciparum has faced the highly complex problem of obtaining nutrients and disposing of waste products, whilst at the same time growing and multiplying within the human erythrocyte. The delivery of substrates and disposal of metabolites depends upon parasite-encoded transport proteins that facilitate exchange across the parasite plasma membrane. Identifying mechanisms responsible for transport processes that sustain this infection is of potential importance because they may become new drug targets. Multidisciplinary methods to study these transport processes in P. falciparum-infected erythrocytes have progressed rapidly in recent years, and are now providing novel molecular and cellular insights into intracellular survival of parasites. In contrast to substrate-specific transporters at the parasite plasma membrane and perhaps in the parasitophorous vacuolar membrane, changes in the transport properties of infected erythrocyte membranes are a result (perhaps not exclusively) of alterations in properties of host cell proteins. We review some mechanisms that are involved in transport processes and that may be critical for * Corresponding author. Tel.: 144-20-8725-5827; fax: 144-20-87253487. E-mail address: [email protected] (S. Krishna).

parasite survival, with particular emphasis on recent advances. The attractiveness of some transport processes for investigation as potential drug targets is also mentioned. 2. Transport processes and metabolism in the P. falciparum-infected erythrocyte There are dramatic parasite-associated developmental changes in the metabolism of infected erythrocytes which are illustrated in Fig. 1 (ter Kuile et al., 1993). This figure summarises data obtained from parasites that have been tightly synchronised (to give a maximum age differential of 6 h by sequential sorbitol lysis and Percoll centrifugation). Not only are there large increases in the uptake of synthetic precursors (such as amino and nucleic acids), but also the timing of these changes varies with the substrate that is utilised. The increase in glycolytic activity is also stagedependent. A further technically useful consequence of infection of erythrocytes by P. falciparum is that as parasites mature into trophozoites, the erythrocyte becomes susceptible to lysis by osmotic solutes (for example to sorbitol and mannitol), which do not lyse uninfected erythrocytes (Lambros and Vanderberg, 1979). These changes in permeability properties of infected erythrocytes are a consequence of alterations in the erythrocyte membrane, and assist in

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Fig. 1. Stage-dependent metabolic activities in synchronous cultures of P. falciparum. Metabolic activities observed following invasion of erythrocytes are given for incorporation of [ 3H]hypoxanthine (open squares), [ 3H]isoleucine (filled circles) and lactate production (open circles). Results represent a change (D) in the given value compared with the preceding time point. For example, for [ 3H]hypoxanthine incorporation at 21 h, the counts represent an increase of approximately 10,000 cpm in the previous 6 h. Modified from ter Kuile et al. (1993) with permission.

synchronising parasite cultures. Kirk and colleagues defined novel permeation pathways that allow entry of solutes in some detail by using a variety of substrates and inhibitors (Kirk et al., 1992a,b, 1994, 1999). Membrane transport in P. falciparum-infected erythrocytes has been reviewed from different perspectives in recent publications (Bock and Cardew, 1999; Kirk, 2001). The novel permeation pathways have channel-like properties which do not saturate, with no stereospecificity, and with a broad overall specificity and preference for anions. Studies with inhibitors suggest that a large range of compounds can block entry through novel permeation pathways. The biophysical nature of novel permeation pathways has also recently been established, and relevant data are summarised in Table 1 (modified from Thomas et al., 2001 with data from Desai et al., 2000; Huber et al., 2002). Included are some data from uninfected erythrocytes, for comparison (Schwarz et al., 1989; Freedman et al., 1994; Freedman and Novak, 1997; Huber et al., 2001). Single anion channel activities in uninfected erythrocytes are difficult to record because conductances are low. The appearance of a conductance detected by whole cell patch-clamp studies on infected erythrocytes, its conductance characteristics (a reversal potential Erev ¼ 247 mV, approximating to the Nernst potential for Cl 2) and its inhibitor susceptibility profile (abolition by 125 mM furosemide) were postulated as sufficient to explain the transport properties of a novel permeation pathway (Desai et al., 2000). This single voltage-dependent predominantly anion selective conductance described in trophozoite-infected erythrocytes was approximately 150-fold larger than that detected in uninfected erythrocytes. On-cell patch-clamp characterisation showed an inwardly rectifying, fast gated small conductance (20 pS) ion channel whose activity was reduced by furosemide (200 mM) (Desai et al., 2000).

Further studies have revealed greater complexity in changes in conductance of infected erythrocyte membranes. Plasmodium gallinaceum-infected chicken erythrocytes have been studied by electrophysiological approaches. Results show that modifications in electrophysiological properties of erythrocyte proteins may arise from changes in the ionic status of infected erythrocytes, for example, increased intraerythrocyte cytosolic [Ca 21]free may activate non-selective cationic and Ca 21-activated channels (Thomas et al., 2001). In infected chicken cells, at least three conductances are detectable (24, 62 and 248 pS) with differing inhibitor susceptibility and ion selectivity profiles (Table 1). All three channels show linear voltage sensitivity. Two channels are relatively cation selective (and blocked by gadolinium or quinine), whereas the channel with the highest conductance is anion selective and blocked by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and tamoxifen. This anion channel is observed less commonly than the other channels (5–10% of membrane patches). Alternatively or additionally, in P. falciparum-infected cells conductances may arise because of alterations in oxidation status that modify endogenous host proteins. A dramatic demonstration of these modifications can be produced by subjecting uninfected erythrocytes to oxidative stress for a short time. Exposure to 1 mM t-butylhydroperoxide (t-BHP, for 15 min) generates conductances that are comparable with those measured electrophysiologically in infected erythrocytes (Fig. 2) (Huber et al., 2002). Furthermore, uninfected erythrocytes now lyse in the presence of sorbitol and mannitol – the same solutes that lyse infected erythrocytes. Results from these experiments illustrating some of these observations are shown in Fig. 2. Infection-induced conductances are also susceptible to inactivation within minutes by reducing agents such as dithioerythritol, which reduce sorbitol-depen-

Table 1 Channel properties of uninfected and infected chicken and human erythrocytes Human uninfected

Human uninfected

Human uninfected (oxidised)

Human infected

Human infected

Human infected

Chicken

Chicken

Chicken

Anion channel

Anion channel

Anion channel

Anion channel

Anion channel

Anion channel

Anion channel

, 6 pS

Not determined

7 nS (whole cell)

18 nS (whole cell)

, 10 pS

248 pS

Non-selective cationic channel 62 pS

Ion selectivity

NCO32, Cl 2

Cl 2

Range as for in infected cells (7–18 nS, whole cell) Cl 2

Non-selective cationic channel 24 pS

Na 1, K 1, Rb 1, Cs 1, Li 1 . choline 1

Inward rectification

Inward/outward components not investigated separately

SCN 2 . I 2 . Br 2 . Cl 2 . acetate 2 . lactate 2 . glutamate 2 Inward rectification

Na 1, K 1, Rb 1, Cs 1, Li 1 . Ba 21 . Ca 21

Linear low open probability at positive potentials 4,4’-diisothiocyano-2,2’disulfonic acid stilbene (DIDS) None reported

I 2 . SCN 2, Br 2, Cl 2 . lactate 2 . gluconate 2 Outward rectification

Cl 2

Voltage sensitivity

I 2 . SCN 2, Br 2, Cl 2 . lactate 2 . gluconate 2 Inward rectification

Linear

Linear

Linear

DIDS-insensitive

DIDS

NPPB . furosemide . DIDS . glybenclamide

NPPB . DIDS, furosemide, glybenclamide

NPPB . furosemide, glybenclamide, niflumate q DIDS

NPPB, tamoxifen

Gadolinium q quinine . flufenamic acid

Quinine, flufenamic acid q gadolinium

None reported

Oxidative stress

Oxidative stress

Oxidative stress

None reported

2 120 mV prepulse Thomas et al., 2001

Stretch-activated

Ca 21-sensitive

Type Conductance

Inhibitor(s)

Activator(s) Reference(s)

a

a

Schwarz et al., 1989

Freedman et al., 1994; Freedman and Novak, 1997

Huber et al., 2002

Huber et al., 2002

Conductances were calculated from single channel recordings unless indicated otherwise.

Huber et al., 2002

Desai et al., 2000

Thomas et al., 2001

Thomas et al., 2001

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dent haemolysis of infected erythrocytes (Huber et al., 2002). There have been two major conductances identified, one with inwardly and the other with outwardly rectifying characteristics. The inwardly rectifying conductance (7 nS) was less susceptible to blockers of novel permeation pathways (such as NPPB where the IC50 was .1 mM at a holding potential of 2100 mV) compared with the outwardly rectifying conductance (NPPB IC50 ,100 nM at 1100 mV). These findings on the inwardly rectifying conductance are consistent with previous work by Desai (Desai et al., 2000). The inhibitor susceptibility for glybenclamide for this inward conductance (IC50 .1 mM), however, is somewhat less (higher IC50) than that reported previously (Table 1). Outwardly rectifying conductances are readily detectable in some studies. These types of electrophysiological discrepancies reported from different groups will need to be resolved. Whilst three apparent conductances arising from infection have been shown in two independent experimental systems, there may be more, as yet unidentified, alterations in channel-like behaviour of erythrocyte membranes. The mechanisms that cause conductances to appear after infection by Plasmodium may also vary with each species examined (for example, contrast the findings after infection with P. gallinaceum and P. falciparum, Table 1). How do these changes in properties relate to alterations in the bulk flow of solutes across the erythrocyte membrane? Linking these two disparate phenomena (electrophysiological properties of infected erythrocytes and their selective osmotic lysis) at present depends upon indirect studies such as those that examine inhibitor profiles, as the molecules that are responsible for such alterations have not been directly isolated. The recently published robust and reproducible experimental approaches will assist in identifying the molecular bases for changes in transport properties of erythrocyte membranes due to infection by parasites (Huber et al., 2002). Also, observable changes in the bulk flow of osmolytes (such as sorbitol) need to be related to stage-

specific alterations in the uptake of synthetic precursors, and disposal of waste metabolites such as lactate (Fig. 1). In light of these data, a number of considerations must be addressed to validate the novel permeation pathways as potential drug targets. 1. As there is more than one kind of novel permeation pathway defined electrophysiologically, which of these is absolutely crucial for survival of intracellular parasites? 2. The extent to which delivery or disposal of crucial metabolites depends upon novel permeation pathways must be defined more precisely. For example, the delivery of hexoses to intraerythrocytic parasites is unlikely to be mediated in an important way through novel permeation pathways (Kirk et al., 1996). What of other essential substrates, such as nucleosides and amino acids? 3. If the novel permeation pathways arise through modifications of host (erythrocyte) proteins, will this make selective inhibition more difficult to achieve, compared with targeting parasite-encoded proteins? 4. IC50 values for inhibitors that have been used to characterise the novel permeation pathways have been obtained from cultured parasites (Table 1). Some of these inhibitors are also in clinical use in other contexts. However, there is a large (.50 mM) discrepancy between effective parasitocidal concentrations of these compounds, and levels that are safely achievable in vivo. Taken together, these points highlight some difficulties in selectively targeting novel permeation pathways to kill parasites.

3. Substrate-specific transporters By comparison with other lower non-parasitic eukaryotes, such as yeast, there may be relatively little redun-

Fig. 2. Substrate dependence of the infection-induced and oxidation-induced isosmotic haemolysis. (A,B) Enriched trophozoite-infected erythrocytes were suspended in isosmotic sorbitol, or in different isosmotic carbohydrate solutions. Incubation was stopped by centrifugation and haemolysis was indicated by haemoglobin in the supernatant. (A) Scanned images of supernatants from two individual experiments performed in duplicate. (B) The mean relative haemolysis of cells incubated in different isosmotic carbohydrate solutions is as shown (n ¼ 8–9, **P # 0:01, ***P # 0:001 compared with controls). Haemoglobin concentrations of supernatants were determined photometrically. (C) Substrate dependence of oxidation-induced isosmotic haemolysis. Imaged supernatants (C; individual experiments in duplicate) in different isosmotic carbohydrate solutions (n ¼ 7–10). Haemoglobin concentrations of supernatants were determined photometrically. Data modified from Huber et al. (2002) with permission.

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important role in the pathophysiology of cerebral malaria. Inhibiting this diversion may therefore be of immediate benefit to host tissues, whereas conventional antimalarials take many hours to inhibit parasite glycolysis significantly (ter Kuile et al., 1993). 4. There is no variation in the derived amino acid sequence of PfHT1 in eight laboratory isolates as well as in isolates obtained from patients with malaria, including hypoglycaemic individuals (Table 2).

dancy in the number of genes encoding substrate-specific transporters of P. falciparum. This may simply reflect adaptation to an intracellular microenvironment. The free-living baker’s yeast contains at least 18 hexose transporters with different physiological characteristics, expression profiles and functions (Boles and Hollenberg, 1997). By contrast, there is only a single copy gene with no closely similar paralogues that encodes a hexose transporter of P. falciparum (PfHT1) (Woodrow et al., 1999). PfHT1 was the first substrate-specific transporter to be characterised functionally in the Xenopus laevis oocyte heterologous expression system (Woodrow et al., 1999). The value of studying hexose transport mechanisms of P. falciparum is emphasised by the following facts (Krishna et al., 2000).

These reasons support further validation of PfHT1 as a potential drug target. To consolidate target validation, we are combining techniques of synthetic chemistry to generate potential inhibitors with screening of these compounds in heterologous expression systems by functional assay. An advantage of this approach is that it enables direct comparison of effects of potential inhibitors on GLUTs (the mammalian facilitative hexose transporter family) and PfHT1. The methodology for functional studies of parasiteencoded substrate-specific transport proteins has been reviewed in detail and is becoming more generally applied, as illustrated by work on two other transport proteins encoded by P. falciparum, a nucleoside transporter (PfENT1) (Carter et al., 2000; Parker et al., 2000) and most recently an aquaglyceroporin (PfAQ) (Hansen et al., 2001). These substrate-specific transporters (PfHT1, PfENT1 and PfAQ) all localise to the region of the parasite plasma membrane (see Fig. 3). Of these three, PfENT1 has been localised by higher resolution immunoelectronmicroscopy to the parasite plasma membrane (Rager et al., 2001). It is perhaps unsurprising that parasites that are capable of being axenically cultured (and therefore do not require erythrocyte membrane components to survive) contain a full complement of transport proteins to maintain a supply of essential substrates, dispose of waste products and regulate intracellular ion concentrations. In the short time that asexual stage

1. Asexual stage parasites need a continuous supply of hexose to survive, and requirements for energy may be up to 100-fold higher than for uninfected erythrocytes. Asexual stage parasites metabolise glucose relatively inefficiently via anaerobic glycolysis as a fully functioning Krebs’ cycle has not been demonstrated. 2. No energy stores (lipids or glycogen) have been reported in P. falciparum, although low amounts of energy stores in the form of amylopectin have been identified in other apicomplexan parasites, such as Toxoplasma gondii (Speer et al., 1998), Eimeria tenella (Heise et al., 1999), or Cryptosporidium parvum (Petry and Harris, 1999). The absolute dependence on glucose for energy supply for P. falciparum is demonstrated by experiments on freed trophozoites. Removal of extracellular glucose results in an immediate drop in intraparasite pH, an effect that is reversed by the addition of glucose (or fructose) to the extracellular medium. 3. Glucose may also be diverted from host tissue (for example cerebral capillaries in the syndrome of cerebral malaria) if glucose delivery becomes rate limiting for glycolysis (Krishna and Woodrow, 1999). This metabolic diversion plays an as yet untested but potentially very Table 2 Sequence variation in PfHT1 a 44

V GTG GTG

S AGT AGT

V GTG GTA

L TTA TTA

N AAT AAT

T ACA ACA

49

256

D GAT GAT

N AAT AAT

V GTA GTG

D GAT GAT

E GAA GAA

P CCA CCA

261

L TTA TTA

V GTT GTT

A GCT GCC

Y TAT TAT

L TTA TTA

P CCT CCT

374

Native sequence Mutant sequence

Native sequence Mutant sequence 369 Nucleotide sequence Nucleotide sequence a

Parasite strain/isolate 3D7; 13 non- and four hypoglycaemic patients HB3, K1, PAC 1 , T9, C10, D4, Dd2; three non- and two hypoglycaemic patients

3D7 HB3, K1, PAC 1 , T9, C10, D4, Dd2

3D7; 17 non- and four hypoglycaemic patients HB3, K1, PAC 1 , T9, C10, D4, Dd2; two non- and one hypoglycaemic patient

Amino acid sequences are shown with their positions numbered and accompanied by sense strand nucleotide sequence. In total, 19 parasite isolates from non-hypoglycaemic and six from hypoglycaemic (plasma glucose # 2.2 mM) patients were sequenced (some data are omitted because three patients had more than one parasite strain present).

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Fig. 3. Immunolocalisation of substrate transporters in asexual stages of P. falciparum. (I) Immunolocalisation of PfHT1. (a–c) Bright-field, immunofluorescence, and combined images of mature trophozoites of P. falciparum stained with anti-PfHT1 antibodies. Pb, pigment body; mem, parasite–host interface. Modified from Woodrow et al. (1999) with permission. (II) Immunolocalisation of PfNT1. Transmission electron micrograph of an ultrathin cryosection of schizont stage of P. falciparum stained with antiPfNT1 antibodies and detected with immunogold (magnification is £ 12,000). MC, Maurer’s clefts; N, nucleus; R, rhoptry; TVM, tubovesicular membrane; RBCM, red blood cell membrane; PVM, parasitophorous vacuole; PPM, parasite plasma membrane. Modified from Rager et al. (2001) with permission. (III) Immunolocalisation of PfAQP. (a,b) Brightfield and immunofluorescence of mature P. falciparum trophozoites stained with PfAQP antibodies and detected with a FITC-labelled secondary antibody. RC, red cell. Modified from Hansen et al. (2001) with permission.

parasites are free-living (merozoites), they may not have critical requirements for many synthetic substrates. However, it is very likely that merozoites will continue to depend upon glucose uptake for energy, which is required for invasion of erythrocytes, as well as to generate ATP for action of membrane pumps such as ‘V’ type or ‘P’ type ATPases. These active (energy-dependent ATPase) transport proteins are important for cation homeostasis and several examples are encoded by P. falciparum.

4. ‘P’ type ATPases of P. falciparum ‘P’ type ATPases are one of the best understood families of membrane transport protein. Detailed study of their physiology has been greatly assisted by identification of highly selective inhibitors. Indeed, some inhibitors are already long established in clinical practice. For example, digitalis is one of the oldest drugs in the pharmacopoeia and selectively inhibits Na 1/K 1 ATPases, and omeprazole inhibits the stomach isoform of K 1/H 1 ATPases. The impetus

to examine ‘P’ type ATPases further as drug targets has derived from a recent publication of the crystal structure of a SERCA pump type Ca 21 ATPase. We began some years ago to identify sequences encoding putative ‘P’ type ATPases in P. falciparum using classical molecular techniques of screening genomic libraries of P. falciparum with degenerate oligonucleotides (Krishna et al., 1993, 1994). These approaches identified three partial sequences, which have subsequently been completed by others and ourselves (Kimura et al., 1993, 1999; Trottein and Cowman, 1995; Trottein et al., 1995; Dyer et al., 1996; Krishna et al., 2001a). Genome databases have also contributed further examples of this family of sequences (eight so far in total; Krishna et al., 2001b), and two are now functionally assayed in Xenopus oocytes (PfATPase 4 and 6) and confirmed to be Ca 21-dependent ‘P’ type ATPases. [Ca 21]free concentrations are ,1 mM in plasma and 0.1–1 mM in infected erythrocyte cytosol. Dealing with these abrupt large transitions in microenvironmental [Ca 21] is likely to involve mechanisms that actively translocate this cation. As both PfATP4 and 6 are expressed during the asexual stages of development, and there are no other identifiable Ca 21 ATPase paralogues in the P. falciparum genome, both ATPases are likely to be important elements in the homeostatic machinery that responds to large changes in [Ca 21]free that P. falciparum encounters. Clotrimazole is an antimycotic drug most frequently used in topical formulations. Recently, clotrimazole has also been shown to inhibit the SERCA pump of rabbit heart muscle (with an IC50 value of ,35 mM), with functional impairment of cardiac contractility demonstrable at concentrations of 50 mM (Snajdrova et al., 1998). Clotrimazole also inhibits in vitro growth of P. falciparum (IC50 ,1 mM), raising the possibility that it may act by inhibiting PfATP6 in the parasite (Tiffert et al., 2000). Our functional assays will allow this hypothesis to be tested directly.

5. Directions for future work Progress in examining the function of many transport proteins encoded by P. falciparum has been rapid. With heterologous assays, it is now possible to screen for selective inhibitors of particular transporters, based on functional comparisons with host proteins. These intensively analytical approaches coupled with expertise in synthetic chemistry may yield lead compounds that selectively inhibit parasite transporters, even when high-throughput screening of targets using combinatorial approaches is not feasible. It would therefore be of interest for such approaches to be applied to more recently identified substrate-specific transporters that may be essential for parasite survival. Finally, transport processes of P. falciparum-infected erythrocytes such as novel permeation pathways probably reflect alterations in host cell membrane proteins due to infection. Their properties will need to be defined in greater detail to deter-

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