Vacuolar type H+ pumping pyrophosphatases of parasitic protozoa

International Journal for Parasitology 32 (2002) 1–14 www.parasitology-online.com

Invited Review

Vacuolar type H 1 pumping pyrophosphatases of parasitic protozoa Michael T. McIntosh*, Akhil B. Vaidya Department of Microbiology and Immunology, MCP Hahnemann School of Medicine, Philadelphia, PA 19129, USA Received 18 June 2001; received in revised form 24 August 2001; accepted 28 August 2001

Abstract Trans-membrane proton pumping is responsible for a myriad of physiological processes including the generation of proton motive force that drives bioenergetics. Among the various proton pumping enzymes, vacuolar pyrophosphatases (V-PPases) form a distinct class of proton pumps, which are characterised by their ability to translocate protons across a membrane by using the potential energy released by hydrolysis of the phosphoanhydride bond of inorganic pyrophosphate. Until recently, V-PPases were known to be the purview of only plant vacuoles and plasma membranes of phototrophic bacteria. Recent discoveries of V-PPases in kinetoplastid and apicomplexan parasites, however, have expanded our view of the evolutionary reach of these enzymes. The lack of V-PPases in the vertebrate hosts of these parasites makes them potentially excellent targets for developing broad-spectrum antiparasitic agents. This review surveys the current understanding of V-PPases in parasitic protozoa with an emphasis on malaria parasites. Topological predictions suggest remarkable similarity of the parasite enzymes to their plant homologues with 15–16 membrane spanning domains and conserved sequences shown to constitute critical catalytic residues. Remarkably, malaria parasites have been shown to possess two V-PPase genes, one is an apparent orthologue of the canonical plant enzyme, whereas the other is a more distantly related paralogue with homology to a recently identified new class of K 1-insensitive plant V-PPases. VPPases appear to localise both to the plasma membrane and cytoplasmic organelles believed to be acidocalcisomes or polyphosphate bodies. Gene transfer experiments suggest that one of the malarial V-PPases is predominantly localised to the surface of intraerythrocytic parasites. We suggest a model in which V-PPase localised to the malaria parasite plasma membrane may serve as an electrogenic pump utilising pyrophosphate as an energy source, thus sparing the more precious ATP. Searching for V-PPase inhibitors could prove fruitful as a novel means of antiparasitic chemotherapy. q 2002 Published by Elsevier Science Ltd. on behalf of Australian Society for Parasitology. Keywords: Inorganic pyrophosphate; Vacuolar-type H 1-pyrophosphatase; Proton pump; Bisphosphonates; Apicomplexa; Plasmodium falciparum; Plasmodium vivax; Plasmodium yoelii; Toxoplasma gondii; Trypanosoma cruzi; Trypanosoma brucei; Leishmania donovani

1. A distinct class of proton pump Throughout nature high-energy phosphoanhydride bonds of ATP have been utilised as the principle currency for energy exchange among biochemical reactions. Discoveries of abundant polyphosphate reserves and specialised enzymes that hydrolyse pyrophosphate among diverse microbial organisms and plants have led to the conclusion that high-energy phosphoanhydride bonds of pyrophosphate or polyphosphate can serve as important alternative or adjunct energy sources (Kornberg et al., 1999). The vacuolar type H 1 pumping pyrophosphatase (V-PPase) represents one unique example as it can couple some of the free energy of phosphoanhydride bond cleavage to H 1 translocation across membranes, thereby creating electrochemical poten* Corresponding author. Infectious Diseases Section, Department of Internal Medicine, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520, USA. Tel.: 11-203-785-7573; fax: 11-203-7853864. E-mail address: [email protected] (M.T. McIntosh).

tial gradients across membranes in a role analogous to that of the H 1/ion pumping ATPases (Rea and Poole, 1993). Indeed, V-PPase enzymes constitute a novel class of H 1 pumps, distinct from the V-, P-, and F-type H 1 pumping ATPases (Rea et al., 1992a), and from the simplest of the known H 1 pumps, bacteriorhodopsin. Compared with ATP, pyrophosphate can be an inexpensive and abundant source of energy. It is a byproduct of several major biosynthetic pathways including DNA and RNA syntheses, the aminoacylation of tRNAs, and in mitochondria fatty acyl-CoA synthesis. In addition, syntheses of sucrose, cellulose, and starch make substantial contributions to the pyrophosphate pool in plants. The novel capacity of V-PPase to couple some of the free energy of pyrophosphate hydrolysis to H 1 translocation permits the efficient conversion of stored energy to a H 1 and/or electrical transmembrane gradient, which can be utilised for a multitude of diverse cellular transport processes (reviewed in Maeshima, 2001). This is in contrast to the soluble PPases (S-PPases), which are ubiquitous throughout nature and confer a favour-

0020-7519/02/$20.00 q 2002 Published by Elsevier Science Ltd. on behalf of Australian Society for Parasitology. PII: S 0020-751 9(01)00325-3

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able poise on otherwise thermodynamically unfavourable reactions by catalysing the dissipative release of the free energy of pyrophosphate hydrolysis as heat (Cooperman, 1992). To date, there is no evidence for the existence of V-PPase within human cells or among the rest of the animal kingdom, and V-PPase homologues have not been found in the completed genome sequences of Escherishia coli, the cyanobacterium Synechocystis PCC 6803, or the yeast Synechocystis cerevisiae. While membrane-associated PPase activities have been identified in yeast mitochondria (Mansurova, 1989; Lundin et al., 1991) and rat liver Golgi (Brightman et al., 1992), pea mitochondria (Zancani et al., 1995), and spinach chloroplast thalakoid (Jiang et al., 1997), their involvement in H 1 translocation is unclear. Furthermore, these enzymes more closely resemble the S-PPase in size, sequence, and hydrophilicity, and are therefore likely to belong to yet another class of enzymes termed, “membrane-associated PPases”, which may form complexes with additional integral membrane subunits responsible for their associations with membranes (reviewed in Maeshima, 2000, and Mansurova, 1989). Previously believed to exist only in the vacuoles of higher plants and plasma membrane folds of phototrophic bacteria, the recent discoveries of V-PPase enzymes in the Kinetoplastida (Scott et al., 1998; Rodrigues et al., 1999a,b) and apicomplexan parasites (Luo et al., 1999; Rodrigues et al., 2000; Marchesini et al., 2000; McIntosh et al., 2001) have presented the scientific community with a fresh perspective on ways in which these parasites might utilise energy. These discoveries have also led to the exciting possibility that insights into this class of H 1 pump may spawn new therapies for the treatment of such widespread and debilitating diseases as malaria, toxoplasmosis, visceral leishmaniasis, African sleeping sickness and Chagas’ disease.

2. Characteristic properties of V-PPases V-PPases are electrogenic enzymes that couple H 1 translocation to the hydrolysis or synthesis of pyrophosphate (Baltscheffsky and Baltscheffsky, 1993; Rea and Poole, 1993). In plants, V-PPases favour the forward reaction of pyrophosphate hydrolysis and serve to acidify the tonoplast, a large acidic vacuole that stores sucrose and organic acids, regulates hydrostatic pressure through storage of inorganic ions, and detoxifies the cytoplasm; hence its designation as a “vacuolar-type” H 1 pump (Maeshima, 2001). V-PPases from several plant tonoplast membranes have been shown to be capable of establishing a H 1 gradient of equal or greater magnitude than that generated by V-H 1-ATPases, which reside in the tonoplast membranes along with the VPPases (reviewed in Zhen et al., 1997a). While it has been suggested that a proton motive force generated by pyrophosphate hydrolysis could be used by the V-ATPase acting in reverse to drive ATP synthesis, there is little consensus on

the potential reversibility of enzymatic activities for either V-ATPase or V-PPase (Maeshima, 2000). In isolated maize tonoplasts, a pre-existent trans-membrane H 1 gradient established either by ATP or pyrophosphate was shown to drive the incorporation of 32Pi into either pyrophosphate or ATP, respectively (Rocha Facanha and de Meis, 1998); and pyrophosphate-dependent ATP synthesis by yeast VATPase was demonstrated in pyrophosphate energised vacuolar membranes from yeast expressing the Arabidopsis thaliana V-PPase (Hirata et al., 2000). In the light-gathering plasma membrane folds of the phototrophic bacterium Rhodospirillum rubrum, V-PPase (pyrophosphate synthase) is freely reversible and utilises the H 1 gradient established by light induced electron transport to synthesise pyrophosphate, which can later be harvested to contribute to H 1 electrochemical gradients when ATP reserves are low (Baltscheffsky, 1996). The ability to utilise pyrophosphate hydrolysis to energise membranes is in contrast to the acidification of the lysosomal compartment of an animal or yeast cell, typically via a V-type H 1 ATPase, or to the charging mechanism of an animal cell’s plasma membrane, typically via action of the Na 1/K 1 ATPase and resting K 1 channels. Functions such as these are vital for all living cells and require enormous amounts of energy, often expending up to 50% of intracellular ATP (Lodish et al., 2000). Therefore, the ability to utilise pyrophosphate as an alternate energy source may ensure the maintenance of such vital functions when ATP demand for other cellular activities is high. V-PPases are highly homologous, polytopic membrane proteins ranging in sizes from 70–115 kDa (predicted) and 56–79 kDa (apparent). Variations in their Mr predicted from cDNA size and apparent Mr from PAGE are well documented (Zhen et al., 1997a), common to highly hydrophobic proteins and appear to be related to their extreme hydrophobicity and incomplete saturation by SDS (Maddy, 1976). In these circumstances, V-PPases do not migrate according to their true Mr and hence their motilities become somewhat dependent upon buffer conditions and the percentage of acrylamide used in the analyses (Drozdowicz and Rea, personal communication). Unlike the H 1/ion pumping ATPases, which are often large heteromultimeric complexes, all of the catalytic properties of V-PPases are imparted by a single polypeptide as demonstrated by the heterologous expression of several V-PPase genes in yeast (Kim et al., 1994; Drozdowicz et al., 1999,2000; Hill et al., 2000; Nakanishi et al., 2001; Perez-Castineira et al., 2001). This, however, does not preclude the likely possibility that V-PPase functions as a homodimer or homomultimer, as judged by native PAGE, cross-linking and gel filtration, and radiation inactivation studies (reviewed in Zhen et al., 1997a). While topological predictions vary with the choice of algorithm, TOPRED II (Claros and von Heijne, 1994) has been previously used to derive the putative topologies of the canonical plant V-PPase from A. thaliana (AVP1) (Zhen et al., 1997a,b), as well as an atypical V-PPase from A. thaliana (AVP2) (Drozdowicz et al., 2000) and a highly diver-

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Fig. 1. Topological predictions of aligned sequences from PfVP1 and AVP1. Putative V-PPase amino acid sequences from P. falciparum, PfVP1 (McIntosh et al., 2001) and A. thaliana, AVP1 (Sarafian et al, 1992) were aligned in PILEUP (GCGe). Sequence shown is that of PfVP1 and gaps in the alignment are indicated by dashes. Amino acid residues in PfVP1 with identity or similarity to AVP1 are highlighted (black) and (grey), respectively. Putative topologies of PfVP1 and AVP1 were modeled using the TopPred II algorithm (Claros and von Heijne, 1994). Transmembrane spans predicted with high confidence are indicated (pink shaded circles) and the position of the last predicted transmembrane span has been rendered by grouping of the amino acid residues in the Cterminal tail. Large well conserved loops on one aspect of the model and predicted to be cytosolic are numbered. The putative catalytic motif DX7KXE, and conserved C581:PfVP1 residue, corresponding to C634:AVP1 and shown to be required for the inhibition of AVP1 by N-ethylmaleimide (Zhen et al., 1994; Kim et al., 1995), are indicated. Also indicated (circled with yellow background) are acidic residues E254, E377, and D454 of PfVP1, which correspond to residues E305, E427, and D504 of AVP1 inferred from studies of AVP1 to be involved in PPi hydrolysis and/or H 1 translocation (Zhen et. al, 1997). Conserved epitopes for polyclonal antisera PABTK and PABHK (Kim et al, 1994) raised against peptides derived from the sequence of AVP1 have been circled.

gent V-PPase from the hyperthermophilic archaeon Pyrobaculum aerophilum (PVP) (Drozdowicz et al., 1999). Using this method, protozoan V-PPase homologues from Plasmodium falciparum (PfVP1 and PfVP2) (McIntosh et al., 2001) and Trypanosoma cruzi (TcVP) (Hill et al., 2000) display topologies which are remarkably similar to those of the orthologous enzymes found in A. thaliana, and P. aerophilum. While a 16 transmembrane span model was consistently favoured over a 15 span model by the algorithm, the

15 span model (shown for PfVP1 in Fig. 1) renders a cytosolically disposed C-terminus, which is supported by the evidence that fusion of a Ca 21 reactive protein apoaequorin with the C-terminus of AVP1 generates a vacuolar membrane-localised polypeptide capable of sensing cytosolic-free Ca 21 in transgenic A. thaliana (Knight et al., 1996). Though speculative, V-PPases from all three branches of life share this conserved 15–16 span topology despite variations in their primary structures (Fig. 1). Currently, the only

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exceptions include two highly divergent V-PPases recently identified, one from A. thaliana (AVP2) (Drozdowicz et al., 2000), and another from P. falciparum (PfVP2) (McIntosh et al., 2001), both of which are predicted to have an additional N-terminal transmembrane span which imposes a cytosolicly disposed N-terminus, while conserving the remaining predicted structures. The large hydrophilic loops present on one aspect of the topological models are predicted to be cytoplasmic as determined for heterologously expressed AVP1, by alkylation of a conserved AVP1:Cys634 residue using membrane permeant and impermeant maleimides (Zhen et al., 1994a,b). This topology is also predicted by the charge-difference rule (Hartmann et al., 1989) and positive-inside rule (von Heijne, 1986). In the current models of the available V-PPases, cytosolic loops III, V, VI, VII, and the C-terminal tail are presumed to interact with cytosolic ligands and represent the most conserved portions of the molecules, whereas the Nterminal sequences, cytosolic loop I, and extra-cytosolic loops display the greatest degree of sequence and size variations (Fig. 1). Plant V-PPases and the R. rubrum pyrophosphate synthase represent the most thoroughly characterised members of this unique class of H 1 pumps. These studies showed that V-PPases have an obligate requirement for Mg 21 and identified the substrate as a complex of Mg pyrophosphate, most likely Mg2 pyrophosphate (reviewed in Zhen et al., 1997a). In addition to Mg pyrophosphate or Mg2 pyrophosphate, free Mg 21 is also required to maintain proper conformation of the enzyme since the protein was protected from chemical inactivation or protease degradation in the presence of Mg 21 and Mg2 pyrophosphate but not when pyrophosphate alone was present (Rea and Poole, 1993; Gordon-Weeks et al., 1996; Maeshima, 1991). As described for the soluble PPase from yeast (Cooperman, 1982), V-PPases are likely to contain two distinct Mg 21 binding sites in addition to the substrate (Mg2 pyrophosphate) binding site, i.e. one of low affinity (Km ¼ 0.25– 0.46 mM) and the other of high affinity (Km ¼ 23–31 mM) (Baykov et al., 1993a; Zhen et al., 1997a,b; Maeshima, 2000). Most plant V-PPases, as well as the V-PPase activities characterised in parasitic protozoa, require 30–51 mM K 1 for maximum activation (Zhen et al., 1997a,b). Furthermore, binding sites for substrate, Mg 21, and/or K 1 are likely to be, in part, distinct based on differential protection to chemical inactivation (Baykov et al., 1993a). Other monovalent cations such as Rb 1 and NH41, but not Na 1 or Li 1, have also been shown to substitute for K 1 as activators of higher plant V-PPases (Obermeyer,et al., 1996; Zhen et al., 1997a,b). The possibility that some V-PPase enzymes have the additional capacity to transport K 1 (Davies et al., 1992; Obermeyer et al., 1996) remains uncertain based on subsequent studies, which found that neither purified reconstituted V-PPase (Sato et al., 1994) nor endogenous tonoplast V-PPase (Ros et al., 1995) were capable of K 1 transport. Yet another potential ligand includes Ca 21,

which has been shown to directly inhibit V-PPases (Rea et al., 1992b), in addition to being able to prevent formation of Mg2 pyrophosphate by chelating pyrophosphate (Maeshima, 1991). Therefore, in addition to a substrate-binding site and H 1 conducting channel, V-PPases are likely to have domains required for dimerisation/multimerisation, and additional binding sites for Mg 21, Ca 21, and in some instances particular monovalent cations such as K 1.

3. Functionally conserved residues Important conserved features include sequence motifs with striking conservation among all V-PPase homologues (Fig. 2), and conserved residues that have been implicated or excluded from biochemical function by site-directed mutagenesis studies. Among these are two antibody binding epitopes for polyclonal antisera (PABTK and PABHK) raised against peptides of AVP1 (Kim et al., 1994), and used as reliable reagents for the detection of V-PPases from extremely diverse organisms, including plants (Kim et al., 1994; Drozdowicz et al., 2000), archaebacteria (Drozdowicz et al., 1999), Trypanosoma brucei and Trypanosoma cruzi (Scott et al., 1998; Rodrigues et al., 1999b), Leishmania (Rodrigues et al., 1999a), Plasmodium (Marchesini et al., 2000; McIntosh et al., 2001; Luo et al., 1999), and Toxoplasma (Luo et al., 2001; Rodrigues et al., 2000) (positions indicated in Fig. 1 and sequences aligned in Fig. 2). Encompassed by the PABTK epitope and located in cytosolic loop III (Fig. 1), is the putative substrate-binding motif DX7KXE, which is similar to the EX7KXE motif found in all S-PPases and membrane associated H 1-PPases (Cooperman, 1992) (Fig. 2). For the mung bean V-PPase (VVP), each conserved charged residue of the DX7KXE motif (D253, K261, and E263) (Fig. 2) has been shown to be essential for catalysis as judged by the loss of pyrophosphate hydrolytic activity and H 1 translocation upon non-conservative substitution by site-directed mutagenesis (Nakanishi et al., 2001). An additional indication that the enzyme can form a substratedependent conformation is resistance to trypsinisation conferred by the presence of substrate. Each non-conservative substitution of D253, K261, or E263 of VVP has been shown to confer susceptibility to trypsinisation in the presence of Mg pyrophosphate or Mg 21, implicating their involvement in substrate and/or Mg 21 binding (Nakanishi et al., 2001). Enzymes harbouring conservative substitutions of K261R or E263D were found to be incapable of H 1 translocation, but retained 25 and 50% of pyrophosphate hydrolytic activity, respectively, indicating that these residues are not only involved in substrate binding but also in the coupling of pyrophosphate hydrolysis to H 1 translocation (Nakanishi et al., 2001). An additional substitution, V259A, within the DX7KXE motif of VVP (Fig. 2) was also implicated in the coupling of pyrophosphate hydrolysis to H 1 translocation since the mutant enzyme retained 60% of the wild type pyrophosphate hydrolytic activity but none

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Fig. 2. The most conserved blocks of sequence from V-PPases. (Top panels) Putative V-PPase amino acid sequences derived from organisms spanning the three branches of life aligned by ClustalX (Thompson et al., 1994). Shown are only the blocks of sequence that display the greatest degree of conservation among all aligned V-PPases. Residues with 100% identity or similarity are shown with black or grey backgrounds, respectively. Epitopes for polyclonal antisera PABTK and PABHK (Kim et al, 1994), raised against peptides derived from the sequence of AVP1, are indicated above the alignments with dashed lines. Also indicated is the putative catalytic motif DX7KXE, and two acidic domains (DX3DX3) implicated in catalysis and/or substrate binding by site-directed mutagenesis of VVP (Nakanishi et. al, 2001). Acidic residues E305, E427 and D504 of AVP1 inferred from studies of AVP1 to be involved in PPi hydrolysis and/or H 1 tranlocation (Zhen et al., 1997) have been indicated above the alignment and highlighted (in red). Corresponding natural substitutions to non-acidic residues have also been highlighted (in red), and speculated compensatory substitutions in nearby residues are highlighted (in yellow). Sequences displayed are those from Vigna radiata, VVP, T07801 (Nakanishi and Maeshima, 1998); A. thaliana, AVP1, BAA32210 (Sarafian et al., 1992); AVP2, AAF31163 (Drozdowicz et al., 2000); Oryza sativa isoenzyme 1, OVP1, BAA08232 (Sakakibara et. al, 1996); T. cruzi, TcVP, AAF80381 (Hill et. al, 2000); Toxoplasma gondii, TVP1, (Drozdowicz, Y.M., Shaw, M.K., Striepen, B., Liwinski, H., Roos, D.S., and Rea, P.A., unpublished); P. falciparum, PfVP1, AAD17215 (McIntosh et al., 2001); PfVP2 (McIntosh et al., 2001); Acetabularia mediterranea, AmVP, BAA83103 (Ikeda et al., 1999); R. rubrum, RVP, AAC38615 (Baltscheffsky et al., 1998); Thermotoga maritima, TmVP, AAD35267 (Nelson et al., 1999); and P. aerophilum, PVP, AAF01029 (Drozdowicz et al., 1999). (Bottom panels) Alignments were performed as in top panels, using putative translations of DNA derived from PCR of P. vivax, P. cynomolgy, P. chaboudi, or P. yoelii DNA using degenerate primers [ggita(t/c)at(a/c/ t)ggiatgaa(a/g)at(a/c/t)gcigtitatgc] and [(c/t)tc(t/g/a)at(g/a)ta(t/c)ttigc(a/g)ttgtcccaigcicc]. Aligned type II V-PPase sequences include V-PPases from P. falciparum, PfVP2, and a preliminary P. yoelli sequence, PyVP2 from gnl|py|TIGR_233 P. yoelii contigs (www.tigr.org). Positions, motifs, and residues indicated as above, except that sequences are shown with respect to PfVP1 and identical residues are indicated by dashes.

of the H 1 translocation activity (Nakanishi et al., 2001). A non-conservative substitution of V262A also within the DX7KXE motif of VVP (Fig. 2) did not affect enzyme function and hence has been excluded from participation in catalysis (Nakanishi et al., 2001). Yet another residue implicated in coupling pyrophosphate hydrolysis to H 1 translocation is E427:AVP1, which was identified by a non-conservative E427Q substitution that yielded a mutant AVP1 enzyme still capable of

partial pyrophosphate hydrolytic activity but incapable of H 1 translocation and a E427D mutation which enhanced H 1 translocation (Kim et al., 1995). This residue, E427:AVP1, is located outside the DX7KXE motif, in another less conserved motif T[D/E]YYTS (Fig. 2) located at the interface between transmembrane span 11 and cytosolic loop VI (Fig. 1). Surprisingly, the residue corresponding to E427:AVP1 is naturally substituted by a basic residue in two sequence divergent V-PPases recently identified from

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P. falciparum and A. thaliana, R:PfVP2 (TRYYTD) and K:AVP2 (SKYYTD), respectively (McIntosh et al., 2001; Drozdowicz et al., 2000) (Fig. 2). While PfVP2 has yet to be evaluated for H 1 pumping, AVP2 has been shown to be competent for H 1 translocation despite this natural substitution (Drozdowicz et al., 2000). Therefore, it seems likely that additional substitutions may compensate for the D/E-R/ K substitutions found in PfVP2 and AVP2, respectively (Fig. 2). Another important residue is C634:AVP1 which has been determined to be on the cytoplasmic aspect of the enzyme by its susceptibility to alkylation by membrane impermeant maleimides that irreversibly blocks catalysis (position indicated Fig. 1) (Zhen et al., 1994a; Kim et al., 1995). While this residue is conserved among most V-PPases, mutagenesis of this residue, or any of the additional eight cysteines in AVP1, did not interfere with catalysis (Kim et al., 1995). In addition, this residue is not conserved in the archaeal enzyme PVP. Thus, it seems likely that C634:AVP1 may be proximal to the active site but perhaps not directly involved (Kim et al., 1995). Mutagenesis of AVP1 has identified two additional residues, E305:AVP1 and D504:AVP1 (Fig. 1 and Fig. 2), required for pyrophosphate hydrolysis and pyrophosphatedependent H 1 translocation (Zhen et al., 1997b). Each acidic residue has been shown to contribute to the binding of the lipophilic carbodiimide, N,N 0 -dicyclohexylcarbodiimide (DCCD) (Zhen et al., 1997b). While residue E305:AVP1 is conserved among most higher plant VPPases, Plasmodium enzyme PfVP1 and algal enzyme CcVP, it does not appear to be conserved in any of the other enzymes (Fig. 2). Natural substitutions to aspartate among flanking residues (as shown in Fig. 2) may provide compensation for this lack of conservation among some VPPases (Fig. 2). Furthermore, despite the functional significance of residue D504:AVP1, this residue is not conserved in the sequence variant enzyme from P. falciparum, PfVP2 (Fig. 2) (McIntosh et al., 2001). Two additional conserved acidic motifs, both with the structure DX3DX3D (Fig. 2), were identified from alignments of V-PPases, and all acidic residues contained therein were found to be involved in enzyme catalysis, and/or substrate binding by site-directed mutagenesis of VVP (Nakanishi et al., 2001). The first DX3DX3D motif is adjacent to the putative substrate binding motif DX7KXE and the second DX3DX3D is encompassed by the binding epitope for antibody PABHK (Fig. 2). Each DX3DX3D motif was found to be embedded in the sequence clusters which exhibit the highest degree of conservation among all sequenced V-PPase enzymes (Fig. 2).

4. Evidence for a subtype of V-PPase While the existence of V-PPase isotypes in higher plants has been known for some time, these enzymes all appear nearly identical in sequence to the typical V-PPases found in

plant vacuolar membranes (Baltscheffsky et al., 1999). The recent characterisation of a sequence variant V-PPase from A. thaliana (AVP2), mentioned earlier with regard to its unique topology, revealed that AVP2 was more closely related to the archaebacterial and bacterial V-PPases than to any of the other higher plant V-PPases including its paralogue, AVP1 (Drozdowicz et al., 2000). In addition, unlike AVP1 and all previously characterised higher plant VPPases, AVP2 activity was insensitive to stimulation by K 1, a trait shared only by the characterised bacterial VPPase homologues from R. rubrum (Baltscheffsky and Baltscheffsky, 1993) and P. aerophilum (Drozdowicz et al., 1999). Based on these traits, phylogenetic divergence and biochemical distinctiveness, the possibility of distinct V-PPase types was suggested (Drozdowicz et al., 2000). The proposed type I enzymes would include the K 1 activated V-PPases which appear more closely related to the typical higher plant V-PPases such as AVP1, and type II enzymes would include the K 1 insensitive enzymes such as AVP2 which appear more closely related to the archaebacterial enzyme. Concurrent with the characterisation of a predominately K 1 stimulated V-PPase activity from P. falciparum and a P. falciparum V-PPase gene PfVP1, database searches using the AVP2 sequence revealed a second V-PPase in P. falciparum (PfVP2) that also appears to be more closely related to its orthologous counterparts in plants and bacteria than to its paralogue, PfVP1 (Fig. 3 ) (Drozdowicz et al., 2000; McIntosh et al., 2001). Genes encoding additional type I or type II related VPPases are evident among the gene sequence databases. Progress in the Plasmodium yoelii genome sequencing project (TIGR) and completion of the A. thaliana genome sequencing project, have revealed two additional genes PyVP2 and AVP3, respectively, which appear to be more closely related to AVP2 and PfVP2 than to their corresponding paralogous counterparts or any other higher plant VPPases (Fig. 3, McIntosh et al., 2001). In addition, gene sequence tags encoding portions of type I and type II VPPases are present in the preliminary genomic sequence database for Plasmodium vivax. Using PCR and degenerate primers rendered from type I V-PPases, we have identified additional genes encoding type I V-PPases in several primate and rodent specific species of Plasmodium (McIntosh and Vaidya, unpublished). Alignments of the predicted amino acid sequences from portions of these type I enzymes and other type II V-PPases present in the databases confirm the distinctions between the type I and type II enzymes from Plasmodium (Figs. 2 and 3). V-PPases from primate malaria parasites Plasmodium vivax and Plasmodium cynomolgy, and rodent parasites Plasmodium berghei, Plasmodium chabaudi and P. yoelii were all found to be highly homologous to PfVP1, despite the wide variation in G 1 C nucleotide content (50% vs. 28%) inherent in the nuclear DNA among these two groups (Fig. 2). Within the most conserved blocks of sequences from V-PPases, sequences from type II orthologues from P. yoelii and P. falciparum, were found to

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Fig. 3. Phylogenetic comparisons of V-PPases. Shared, aligned sequences displayed in Fig. 2 and corresponding sequences from an additional type II-like VPPase sequence from A. thaliana, AVP3, AAG09080, were subjected to parsimony and distance methods (PAUP 4.02b8) (Swofford, D.L., 1999. (PAUP) Phylogenetic analysis uisng parsimony (and other methods). Version 4.02b8. Sunderland, MA: Sinauer Associates). Full heuristic searches of 500 random addition replicates were employed and both parsimony and distance analyses were subjected to 1000 bootstrap replicates. Qualitatively similar results were obtained by both procedures and the consensus tree from the distance analysis is shown. Bootstrap percentages greater than 50% are indicated above the branches.

be correspondingly similar to each other yet quite divergent when compared with any of the type I enzymes from Plasmodium (Fig. 2). All of the acidic residues and conserved sequence motifs identified in other type I enzymes and in PfVP1 were found to be conserved in sequences from P. vivax, P. cynomolgy, P. berghei, P. chabaudi, and P. yoelii

(Fig. 2). Likewise, substitutions in the type I motif T[D/ E]YYTS to the type II motif T[R/K]YYTD were also found in the type II variants from P. falciparum and P. yoelii. As mentioned above, this motif includes a basic residue in place of the acidic residue corresponding to E427:AVP1 which has been implicated in coupling Pyrophosphate hydro-

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lysis to H 1 translocation (Kim et al., 1995). An additional novel substitution of D-S in residues corresponding to D504:AVP1, implicated in enzyme catalysis and DCCD binding (Zhen et al., 1997b),was found only among the type II enzyme variants from Plasmodium (Fig. 2). Given all of the conserved sequence differences, it is quite evident that Plasmodium spp., like A. thaliana, contain two distinct V-PPases whose divergence likely predates the evolution of Plasmodium spp.. Supporting the possibility that these two classes of V-PPase have distinct origins is the recent finding that the V-PPase of the hyperthermophilic bacterium Thermotoga maritima, which appears less related to the clade containing PfVP2 and AVP2/3 (Fig. 3) and is K 1 inducible (Perez-Castineira et al., 2001). While the overall activity in P. falciparum trophozoite membranes has been reported to be largely K 1 dependent (Luo et al., 1999; McIntosh et al., 2001), this may be attributable to the predominance of PfVP1 expression over that of PfVP2 (McIntosh et al., 2001). Biochemical distinctiveness of these enzymes, while evident in A. thaliana and bacterial enzymes, remains to be demonstrated for Plasmodium in a heterologous expression system. In trypanosomatids, the overall V-PPase activity has also been reported to be subject to activation by K 1 (Scott et al., 1998; Rodrigues et al., 1999a,b), and through expression in yeast, the cloned T. cruzi V-PPase TcVP has also been shown to be functionally competent in the presence of K 1 (Hill et al., 2000). Therefore, given the relatedness of TcVP to other K 1 activated VPPases it is likely that it too is activated by K 1; however, the possible existence of additional V-PPases, which may be K 1 insensitive, should also be considered in these parasites.

5. Functional relevance in parasitic protozoa Localisation of these enzymes has provided clues as to their potential functions in parasite physiology. Indirect immunofluorescence studies using polyclonal antisera PABTK and/or PABHK (Figs. 1 and 2) have revealed a parasite surface staining and intracellular punctate staining pattern in the intra-erythrocytic stages of P. falciparum and P. berghei (Luo et al., 1999; Marchesini et al., 2000; McIntosh et al., 2001). In addition, expression of a PfVP1GFP gene fusion in transfected P. falciparum has revealed a parasite surface fluorescence without an associated intracellular punctate fluorescence (McIntosh et al., 2001). An immunofluorescence pattern similar to that of P. falciparum has also been observed in T. gondii (Rodrigues et al., 2000), and immunoelectron microscopy has been used to show colocalisation of a P-type Ca 21 ATPase and V-PPase in the T. gondii plasma membrane, and in intracellular vacuoles morphologically and compositionally similar to acidocalcisomes (Luo et al., 2001) previously described in trypanosomatids (reviewed in Docampo and Moreno, 2001). An intense intracellular punctate and surface immunofluorescence staining pattern has also been noted in kinetoplastids

(Scott et al., 1998; Rodrigues et al., 1999b), and by immunoelectron microscopy, V-PPase has been localised to T. cruzi plasma membrane and acidocalcisomes (Scott et al., 1998). By exclusion, using various assays for marker enzymes in subcellular organelles such as plasma membrane, mitochondria, glycosome, and lysosome, subcellular fractionation studies in trypanosomatids have revealed that most of the V-PPase associated H 1 pumping activity and/or V-PPase polypeptide is present in discrete high-density membrane fractions that contain acidocalcisomes (Scott et al., 1998; Rodrigues et al., 1999a,b; Scott and Docampo, 2000). In contrast to Leishmania, in epimastigotes of T. cruzi and in procyclic and blood stream trypomastigote forms of T. brucei, an additional peak of V-PPase activity and associated V-PPase protein, was found in fractions containing more moderate density membranes coincident with parasite plasma membrane as judged by surface fluoresceination or a-glucosidase activity, respectively (Scott et al., 1998; Rodrigues et al., 1999b). In contrast, most of the V-PPase in P. falciparum, as determined by Western blot analysis of subcellular fractions, was present in subcellular membrane fractions of moderate density only (McIntosh and Vaidya, unpublished results). While data indicating a dual localisation of V-PPase to the parasite plasma membrane and acidocalcisome appear strong in kinetoplastid parasites and in T. gondii, several issues have the potential to confound such conclusions, especially with regard to Plasmodium. First, Ca 21 storage in acidified compartments within P. falciparum (Garcia et al., 1998) or P. berghei (Marchesini et al., 2000) have led to inference that Plasmodium too contains acidocalcisomes (Marchesini et al., 2000, Docampo and Moreno, 2001). These organelles in Plasmodium however, remain somewhat less defined than the acidocalcisomes of trypanosomatids. Second, in P. berghei, release of intracellular Ca 21, as indicated by the fluorescent Ca 21 indicator fura-2, upon exposure of parasites to the V-PPase inhibitor aminomethylene diphosphonate (AMDP) at 40 mM has been interpreted as indicating an association between V-PPase function and acidocalcisomes in Plasmodium (Marchesini et al., 2000). This conclusion, however, could be premature given that AMDP specificity has only been defined in vitro using plant membrane extracts (Baykov et al., 1993b; Zhen et al., 1994b), and its target enzyme(s) specificity in a whole cell or organism remains entirely undefined (Zhen et al., 1997a). Furthermore, as a competitive inhibitor, effects of AMDP on V-PPases, sPPases, or other phosphohydrolases are likely to be dependent on the prevailing pyrophosphate concentrations within the parasite cytosol or other intracellular compartments and the respective Km of those enzymes for pyrophosphate (see Zhen et al., 1997a). Third, the same generic antisera have been employed in all of the above-mentioned studies, and as some intracellular punctate background staining has been noted in P. falciaparum (McIntosh et al., 2001) and in T. gondii (Drozdowicz and Rea, personal communication), previous data should be confirmed by other methods. Fourth,

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the above-mentioned antisera are not likely to distinguish between distinct V-PPase types, and hence the presence of multiple types of V-PPase, as indicated in Plasmodium (McIntosh et al., 2001) but perhaps in other parasites as well, may confound localisation and interpretation of functions. Regardless of these confounding issues, if indeed VPPases are consistently present in both parasite plasma membrane and acidocalcisomes, as would appear to be the case in kinetoplastids (Scott et al., 1998; Rodrigues et al., 1999a,b), and Toxoplasma (Rodrigues et al., 2000), then an understanding of the sorting mechanisms for these polytopic membrane proteins may be of additional interest. Alternatively, a dual localisation for V-PPases may otherwise be a consequence of separate genes, each encoding a distinct type of V-PPase. In this regard, Mitsuda et al. (2001) have localised the type II V-PPase, AVP2 to the Golgi body of A. thaliana, while its counterpart, AVP1 resides primarily in the vacuolar membrane (Zhen et al., 1997a). While the orientation of V-PPases have not been determined for any protozoan parasites, it seems likely that VPPases localised to the parasite plasma membrane could contribute to the establishment of transmembrane pH gradients and cytosolic pH stasis by pumping H 1 out of the parasite. In the case of Plasmodium, where intraerythrocytic stages are surrounded by a parasitophorous vacuolar membrane, V-PPase would likely pump protons from the parasite cytosol into the parasitophorous vacuolar space. Given that the parasitophorous vacuolar membrane contains a non-selective large (,1400 Da) channel (Desai et al., 1993, 1997), it seems likely that a H 1 pump would have functional relevance in the parasite plasma membrane rather than the parasitophorous vacuolar membrane. In this regard, PfVP1 localisation to the parasite surface has been confirmed by the expression of a PfVP1-GFP gene fusion in transfected P. falciparum (McIntosh et al., 2001). By analogy, a similar arrangement can be envisioned for intracellular forms of T. gondii, which are also known to reside within a parasitophorous vacuolar membrane (Robibaro et al., 2001; Soldati et al., 2001). With regard to the contribution of these enzymes to electrochemical potential at the parasite plasma membrane, a build-up of counter anions on the inner side of the plasma membrane would permit H 1 accumulation on the outer surface of the parasite plasma membrane regardless of the permeability of the parasitophorous vacuolar membrane. Indeed, this configuration of an impermeant inner membrane and relatively permeant outer membrane is analogous to that of mitochondrial inner and outer membranes in which the combined electrochemical potential across the inner membrane can reach 2250 mV. Given the thickness (3.5 nm) of a non-conducting phospholipid bilayer, the potential across the inner mitochondrial membrane can reach more than 600 kV/cm, which has a significant and obligate effect on molecular transport into the mitochondria and on energy production (Rassow and Pfanner, 2000). The ability to have an analogous system at the parasite plasma membrane, which can

9

utilise pyrophosphate as an alternate or adjunct energy source to ATP, could therefore be the basis for a vital physiological role for V-PPase in parasitic protozoa, particularly with regard to molecular import or export. In the context of malaria parasites, which as homolactate fermentors rely almost exclusively on glycolysis for production of ATP (Sherman, 1998), such an ATP conserving mechanism may indeed prove to be a necessity. V-PPase may also contribute to parasite cytosolic pH stasis by pumping H 1 out of the cytosol and into the acidocalcisome (Docampo and Moreno, 2001). Acidocalcisomes, first described in trypanosomes (Vercesi et al., 1994; Docampo et al., 1995), have been subsequently found to be morphologically and compositionally similar to volutin granules or polyphosphate bodies identified historically among many bacteria and unicellular eukaryotes (Kornberg et al., 1999; Docampo and Moreno, 2001). X-ray microanalysis in trypanosomatids and T. gondii have shown acidocalcisomes to contain molar-level concentrations of phosphate, and likely mM concentrations of Mg 21, Ca 21, Cu 21, Zn 21, and in some instances Na 1, and Cl 2 (Luo et al., 2001; Docampo and Moreno, 2001). Recently, 31P NMR studies have revealed that the high phosphorous content in T. cruzi, T. brucei, and L. major (estimated as high as 3–8 M) is likely to be in the form of short chain polyphosphates, such as di-, tri-, tetra-, and penta-phosphates (Urbina et al., 1999; Moreno et al., 2000). Thus while identified as acidic Ca 21 storage organelles, they appear to function in the sequestration or storage of several inorganic elements, with the principal constituent being high-energy oligomeric forms of inorganic phosphate (Docampo and Moreno, 2001). In accord with the above-mentioned V-PPase localisation studies and the initial findings of Ca 21 storage in acidocalcisomes, a role for V-PPase in intracellular Ca 21 storage by contribution to the acidification of these organelles has been proposed (Docampo and Moreno, 2001). In addition, a suggestion that these organelles may be capable of providing a source of releasable Ca 21 has also been made (Docampo and Moreno, 2001). In addition, V-PPase may affect pyrophosphate and polyphosphate storage in acidocalcisomes, perhaps permitting the mobilisation or utilisation of these high-energy molecules in times of stress or when energy demands are unusually great (Docampo and Moreno, 2001). This is supported by the recent observation that there are significant and rapid alterations in amounts of intracellular/acidocalcisomal short and long chain polyphosphate molecules during T. cruzi differentiation from trypomastigotes to amastigotes, and in response to environmental stimuli such as lag phase growth of epimastigotes, alkaline medium, and hypo- or hyper-osmotic medium (Ruiz et al., 2001). Additional support for pyrophosphate and polyphosphate in parasite metabolism, is provided by the identification of several other pyrophosphate-requiring enzymes such as pyrophosphate-dependent phosphofructokinase (pyrophosphate-PFK) in several parasitic protozoa (Mertens, 1991), the glycosomal enzyme pyrophosphate-dependent

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pyruvate phosphate dikinase (PPDK) in trypanosomatids (Bringaud et al., 1998), and the recently observed enzymatic activities in T. cruzi of exopolyphosphatase and polyphosphate kinase (Ruiz et al., 2001), which can catalyse the synthesis of ATP from ADP and polyphosphate (Kornberg et al., 1999). Taken together, the data concerning acidocalcisomes, polyphosphate metabolism, and V-PPase present a compelling argument for a role for polyphosphate metabolism in parasite adaptation to environmental stress.

6. V-PPases as targets for broad spectrum antiparasitic drugs The likely involvement of V-PPases in energy conservation, membrane transport, and polyphosphate metabolism/ storage, and the apparent absence of these enzymes in vertebrates, clearly places V-PPases as potential targets for the development of new chemotherapeutic strategies for the control of parasitic diseases. Indeed, the combined burdens of malaria, toxoplasmosis, Chagas’ disease, African sleeping sickness, and visceral leishmaniasis represent an unparalleled worldwide public health concern. Celis et al. (1998) have identified the membrane active organotin compound, triphenyltin chloride, as a non-competitive inhibitor of the pyrophosphate synthase of R. rubrum in vitro. However, given the inhibitory effects of triphenyltin on the H 1 conducting channels of H 1-ATPases, and the toxicity associated with many organotins, its utility is likely to be restricted to probing the structure–function relationships of H 1 pumping mechanisms in V-PPases and H 1-ATPases (Celis et al., 1998; White et al., 1999). The most obvious class of potentially therapeutic compounds includes pyro-

phosphate analogues such as the potent and specific in vitro V-PPase competitive inhibitor AMDP (Baykov et al., 1993b). AMDP is a member of a class of compounds called bisphosphonates in which the central oxygen atom of pyrophosphate has been substituted with a carbon atom. The resulting structure, P-C-P, has two phosphate groups linked by non-hydrolisable phosphoether bonds. The central carbon atom can further accommodate two additional covalent bonds each to carbon, oxygen, nitrogen, sulfur or halogen, thus giving rise to an enormous variety of compounds, which can vary dramatically in reactivity and chemistry (see Fig. 4 for representative structures). Several bisphosphonates, including pamidronate, alendronate, tiludronate, risedronate, etidronate, clodronate, and ibandronate, are already established as effective therapies in the treatment of bone resorption disorders such as osteoporosis and Paget’s disease, as well as myeloma, and bone metastases (Russell and Rogers, 1999). The potential use of bisphosphonates as anti-parasitic drugs became evident following the discoveries of pyrophosphate-PFKs in T. gondii, Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, and Naegleria fowleri (Peng et al., 1995; Byington et al., 1997; Bruchhaus et al., 1996). In addition, bisphosphonates have been shown to be toxic to Dictyostelium amoebae (Rogers et al., 1994a,b; and reviewed in Rogers et al., 2000). AMDP itself, while quite effective (I50 ¼ 5 mM) on V-PPase activity in membrane extracts from P. falciparum, was also found to inhibit growth of P. falciparum in culture, albeit at much higher concentrations (I50 ¼ 400 mM) (McIntosh et al., 2001). In initial investigations, some of the above mentioned compounds as well as AMDP, and/or imidodiphosphonate (IDP) have been shown to be effective in mM concentrations

Fig. 4. Comparison of pyrophosphate and bisphosphonate. For the bisphosphonates, the structures of R1 and R2 groups are given in the table to the right. Some of the compounds listed have been used for treating bone resorption disorders.

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as in vitro inhibitors of V-PPase activities, though most not as potent and specific as AMDP (Gordon-Weeks et al., 1999; Rodrigues et al., 2000). Some of these compounds also have growth inhibitory effects on T. gondii cultures in human fibroblasts (Rodrigues et al., 2000; Martin et al., 2001) and/or in intracellular cultures of T. cruzi (Urbina et al., 1999; Martin et al., 2001). Furthermore, administration of pamidronate was found to block T. cruzi proliferation in vivo using a murine model of Chagas’ disease (Urbina et al., 1999). These initial studies have prompted a recent survey of a variety of synthesised and commercially available bisphosphonates for their effects on in vitro growth of T. brucei trypomastigotes, T. cruzi amastigotes, Leishmania amastigotes, T. gondii tachyzoites, and intraerythrocytic stages of P. falciparum (Martin et al., 2001). Cultures of T. brucei trypomastigotes were found to be more sensitive to inhibitory effects of bisphosphonates in general while P. falciparum appeared to be the most resistant. However, several compounds were identified which inhibited parasite growth in nM or low mM range in culture for some of the organisms. The aromatic nitrogen containing compound Risedronate (see Fig. 4 for the structure) demonstrated a broad spectrum but variable activity as indicated by: I50 ¼ 490 nM in T. gondii, 2.3 mM in L. donovani, 8.6 mM in T. brucei and 123 mM in T. cruzi and P. falciparum (Martin et al., 2001). As previously eluded to, in vivo effects of bisphosphonates, as competitive inhibitors, are likely to be dependent upon the Km for pyrophosphate of a particular enzyme and the intracellular concentration of Mg2 pyrophosphate as well as the permeability of cells and/or subcellular compartments to these anionic compounds (reviewed in Zhen et al., 1997a). Indeed, the anionic nature of these compounds is likely to be a significant impediment to their access to intracellular compartments. Hence, their uptake is believed to be primarily by fluid phase endocytosis (Rogers et al., 1997; and reviewed in Russell and Rogers, 1999; and Rogers et al., 2000). Since erythrocytes are incapable of endocytosis or phagocytosis, intraerythrocytic organisms such as Plasmodium are likely to be especially resistant as evident from the high concentrations of AMDP required to inhibit growth of P. falciparum in culture (McIntosh et al., 2001). Hence, targets of many bisphosphonates are likely to include a number of pyrophosphatases or phosphohydrolases in addition, or in preference, to V-PPases. For example, several of the simple (more pyrophosphate-like) bisphosphonates such as clodronate, etidronate, and tiludronate (see Fig. 4 for structures) have been found in Dictyostelium amoebae to be metabolised, presumably by the type 2 family of aminoacyl–tRNA synthetases (Rogers et al., 1994, 1996), into nonhydrolisable analogues of ATP which can potentially interfere with a plethora of cellular metabolic processes (reviewed in Rogers et al., 2000). Many of the more potent cytotoxic nitrogen-containing bisphosphonates (such as pamidronate, risedronate, olpa-

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dronate, and ibandronate) in Dictyostelium, and in mammalian osteoclast or macrophage cultures, have been found to inhibit farnesylpyrophosphate synthase (FPPS), a major branch point in the mevalonate pathway (reviewed in Russell and Rogers, 1999; Rogers et al., 2000). This inhibition results in a deficiency of farnesylpyrophosphate and geranylgeranylpyrophosphate, which can subsequently block sterol biosynthesis and the posttranslational lipid modification (prenylation) of proteins. Indeed, blockage of the mevalonate pathway and/or loss of protein prenylation of small GTPases such as Ras , Rho, and Rac, have been hypothesised to account for the major morphological, cytotoxic, and/or apoptotic effects of bisphosphonates on osteoclasts (reviewed in Russell and Rogers, 1999; Rogers et al., 2000). By analogy, it has been observed by Martin et al. (2001) that risedronate inhibited sterol biosynthesis at a presqualene level, and therefore, a similar mode of action was speculated for bisphosphonates on parasitic protozoa. Unlike trypanosomatids, the mevalonate pathway and sterol biosynthesis are absent from Plasmodium (Ridley, 1999). Instead, Jomaa et al. (1999) have identified the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway (DOXP/MEP pathway) in Plasmodium, a mevalonate-independent pathway for the synthesis of isoprenoid precursors, which in its own right presents several potential drug targets. Since the DOXP and mevalonate pathways share a common end product, isopentenyl diphosphate, required for the subsequent syntheses of farnesylpyrophosphate and geranylgeranyl pyrophosphate, it is conceivable that, as in mammalian cell cultures and Dictyostelium, bisphosphonates might interfere with prenylation of proteins rather than or in addition to V-PPases in Plasmodium. Martin et al. (2001) have also suggested the possible accumulation of bisphosphonates in acidocalcisomes as a mechanism for their anti-parasitic effects. Therefore, the potential promise of bisphosphonates as anti-parasitic drugs is tempered by the knowledge that many bisphosphonates, while specific in vitro, may lack specificity in vivo. Nevertheless, a greater dependence on pyrophosphate and/ or polyphosphate metabolism by parasites, suggested by the presence of V-PPase and other pyrophosphate-dependent enzymes, lends merit to the investigations of pyrophosphate analogues as anti-parasitic compounds. A major interest for future endeavors will be the crystallography and high-resolution X-ray diffraction of V-PPases from plants and/or parasitic protozoa to unveil the structure and mechanisms of PPase coupled H 1 translocation that will permit the rational design of non-competitive inhibitors.

Acknowledgements Preliminary sequence data for P. yoelii was obtained from The Institute for Genomic Research website (www.tigr.org). Sequencing of P. yoelii was part of the International Malaria Genome Sequencing Project and was supported by awards

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from the Burroughs Wellcome Fund and the US Department of Defense. A.B.V. is the recipient of a Burroughs Wellcome Fund New Initiatives in Malaria Research Award.

References Baltscheffsky, M., Baltscheffsky, H., 1993. Inorganic pyrophosphate and inorganic pyrophosphatases. In: Ernster, L. (Ed.). Molecular Mechanisms in Bioenergetics, Elsevier, Amsterdam, pp. 331–48. Baltscheffsky, H., 1996. In: Baltscheffsky, H. (Ed.). Origin and Evolution of Biological Energy Conversion, VCH Publishers, New York, NY, pp. 1–35. Baltscheffsky, M., Nadanaciva, S., Schultz, A., 1998. A pyrophosphate synthase gene: molecular cloning and sequencing of the cDNA encoding the inorganic pyrophosphate synthase from Rhodospirillum rubrum. Biochim. Biophys. Acta 1364, 301–6. Baltscheffsky, M., Schultz, A., Baltscheffsky, H., 1999. H 1-proton-pumping inorganic pyrophosphatase: a tightly membrane-bound family. FEBS Lett. 452, 121–7. Baykov, A.A., Bakuleva, N.P., Rea, P.A., 1993a. Steady-state kinetics of substrate hydrolysis by vacuolar H(1)-pyrophosphatase. A simple three-state model. Eur. J. Biochem. 217, 755–62. Baykov, A.A., Dubnova, E.B., Bakuleva, N.P., Evtushenko, O.A., Zhen, R.G., Rea, P.A., 1993b. Differential sensitivity of membrane-associated pyrophosphatases to inhibition by diphosphonates and fluoride delineates two classes of enzyme. FEBS Lett. 327, 199–202. Brightman, A.O., Navas, P., Minnifield, N.M., Morre, D.J., 1992. Pyrophosphate-induced acidification of trans cisternal elements of rat liver Golgi apparatus. Biochim. Biophys. Acta 1104, 188–94. Bringaud, F., Baltz, D., Baltz, T., 1998. Functional and molecular characterization of a glycosomal PPi-dependent enzyme in trypanosomatids: pyruvate, phosphate dikinase. Proc. Natl Acad. Sci. USA 95, 7963–8. Bruchhaus, I., Jacobs, T., Denart, M., Tannich, E., 1996. Pyrophosphatedependent phosphofructokinase of Entamoeba histolytica: molecular cloning, recombinant expression and inhibition by pyrophosphate analogues. Biochem. J. 316, 57–63. Byington, C.L., Dunbrack Jr., R.L., Cohen, F.E., Agabian, N., 1997. Molecular modeling of phosphofructokinase from Entamoeba histolytica for the prediction of new antiparasitic agents. Arch. Med. Res. 28, 86–88. Celis, H., Escobedo, S., Romero, I., 1998. Triphenyltin as an inhibitor of membrane-bound pyrophosphatase of Rhodospirillum rubrum. Arch. Biochem. Biophys. 358, 157–63. Claros, M.G., von Heijne, G., 1994. TopPred II: an improved software for membrane protein structure predictions. Comput. Appl. Biosci. 10, 685–6. Cooperman, B.S., 1982. The mechanism of action of yeast inorganic pyrophosphatase. Methods Enzymol. 87, 526–48. Cooperman, B.S., Baykov, A.A., Lahti, R., 1992. Evolutionary conservation of the active site of soluble inorganic pyrophosphatase. Trends Biochem. Sci. 17, 262–6. Davies, J.M., Poole, R.J., Rea, P.A., Sanders, D., 1992. Potassium transport into plant vacuoles energized directly by a proton-pumping inorganic pyrophosphatase. Proc. Natl Acad. Sci. USA 89, 11701–5. Desai, S.A., Krogstad, D.J., McCleskey, E.W., 1993. A nutrient-permeable channel on the intraerythrocytic malaria parasite. Nature 362, 643–6. Desai, S.A., Rosenberg, R.L., 1997. Pore size of the malaria parasite’s nutrient channel. Proc. Natl Acad. Sci. USA 94, 2045–9. Docampo, R., Moreno, S.N.J., 2001. The acidocalcisome. Mol. Biochem. Parasitol. 114, 151–9. Docampo, R., Scott, D.A., Vercesi, A.E., Moreno, S.N., 1995. Intracellular Ca21 storage in acidocalcisomes ot Trypanosoma cruzi. Biochem. J. 310, 1005–12. Drozdowicz, Y.M., Lu, Y.P., Patel, V., Fitz-Gibbon, S., Miller, J.H., Rea, P.A., 1999. A thermostable vacuolar-type membrane pyrophosphatase

from the archaeon Pyrobaculum aerophilum: implications for the origins of pyrophosphate-energized pumps. FEBS Lett. 460, 505–12. Drozdowicz, Y.M., Kissinger, J.C., Rea, P.A., 2000. AVP2, a sequencedivergent, K(1)-insensitive H(1)-translocating inorganic pyrophosphatase from Arabidopsis. Plant. Physiol. 123, 353–62. Garcia, R.S., Ann, S.E., Tavares, E.S., Dluzewski, A.R., Mason, W.T., Paiva, F.B., 1998. Acidic calcium pools in intraerythrocytic malaria parasites. Eur. J. Cell Biol. 76, 133–8. Gordon-Weeks, R., Steele, S.H., Leigh, R.A., 1996. The role of magnesium, pyrophosphate and their complexes as substrates and activators of the vacuolar H 1 -pumping inorganic pyrophosphatase: studies using ligand protection from covalent inhibitors. Plant. Physiol. 111, 195– 202. Gordon-Weeks, R., Parmar, S., Davies, T.G., Leigh, R.A., 1999. Structural aspects of the effectiveness of bisphosphonates as competitive inhibitors of the plant vacuolar proton-pumping pyrophosphatase. Biochem. J. 337, 373–7. Hartmann, E., Rapoport, T.A., Lodish, H.F., 1989. Predicting the orientation of eukaryotic membrane-spanning proteins. Proc. Natl Acad. Sci. USA 86, 5786–90. Hill, J.E., Scott, D.A., Luo, S., Docampo, R., 2000. Cloning and functional expression of a gene encoding a vacuolar-type proton-translocating pyrophosphatase from Trypanosoma cruzi. Biochem. J. 351, 281–8. Hirata, T., Nakamura, N., Omote, H., Wada, Y., Futai, M., 2000. Regulation and reversibility of vacuolar H(1)-ATPase. J. Biol. Chem. 275, 386–9. Ikeda, M., Tanabe, E., Rahman, M., Kadowaki, H., Moritani, C., Akagi, R., Tanaka, Y., Maeshima, M., Watanabe, Y., 1999. A vacuolar H 1 pyrophosphatase in Acetabularia acetabulum: molecular cloning and comparison with higher plant and a bacterium. J. Exp. Bot. 50, 139–40. Jiang, S.S., Fan, L.L., Yang, S.J., Kuo, S.Y., Pan, R.L., 1997. Arch. Biochem. Biophys. 346, 105–12. Jomaa, H., Wiesner, J., Sanderbrand, S., Altincicek, B., Weidemeyer, C., Hintz, M., Turbachova, I., Eberl, M., Zeidler, J., Lichtenthaler, H.K., Soldati, D., Beck, E., 1999. Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. Science 285, 1573–6. Kim, E.J., Zhen, R.G., Rea, P.A., 1994. Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport. Proc. Natl Acad. Sci. USA 91, 6128–32. Kim, E.J., Zhen, R.G., Rea, P.A., 1995. Site-directed mutagenesis of vacuolar H 1-pyrophosphatase. J. Biol. Chem. 270, 2630–5. Knight, H., Trewavas, A.J., Knight, M.R., 1996. Cold calcium signaling in Arabidopsis involved two cellular pools and a change in calcium signature after acclimation. Plant Cell 8, 489–503. Kornberg, A., Rao, N.N., Ault-Riche, D., 1999. Inorganic polyphosphate: a molecule of many functions. Annu. Rev. Biochem. 68, 89–125. Lodish, H., Berk, A., Zipursky, S.L., Matsudaira, P., Baltimore, D., Darnell, J.E., 2000. In: Lodish, H., Berk, A., Zipursky, S.L., Matsudaira, P., Baltimore, D., Darnell, J.E. (Eds.). Molecular Cell Biology, W.H. Freeman and Co, New York, NY, pp. 578–615. Lundin, M., Baltscheffsky, H., Ronne, H., 1991. Yeast PPA2 gene encodes a mitochondrial inorganic pyrophosphatase that is essential for mitochondrial function. J. Biol. Chem. 266, 12168–72. Luo, S., Marchesini, N., Moreno, S.N.J., Docampo, R.A., 1999. Plant-like vacuolar H 1-pyrophosphatase in Plasmodium falciparum. FEBS Lett. 460, 217–20. Luo, S., Vieira, M., Graves, J., Zhong, L., Moreno, S.N.J., 2001. A plasma membrane-type Ca(2 1 )-ATPase co-localizes with a vacuolar H(1)pyrophosphatase to acidocalcisomes of Toxoplasma gondii. EMBO J. 20, 55–64. Maddy, A.H., 1976. A critical evaluation of the analysis of membrane proteins by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. J. Theor. Biol. 62, 315–26. Maeshima, M., 1991. H 1-translocating inorganic pyrophosphatase of plant vacuoles. Inhibition by Ca 21, stabilization by Mg 21 and immunological

M.T. McIntosh, A.B. Vaidya / International Journal for Parasitology 32 (2002) 1–14 comparison with other inorganic pyrophosphatases. Eur. J. Biochem. 196, 11–17. Maeshima, M., 2000. Acuolar H 1-pyrophosphatase. Biochim. Biophys. Acta 1465, 37–51. Maeshima, M., 2001. Tonoplast transporters: organization and function. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 469–97. Mansurova, S.E., 1989. Inorganic pyrophosphate in mitochondrial metabolism. Biochim. Biophys. Acta 977, 237–47. Marchesini, N., Luo, S., Rodrigues, C.O., Moreno, S.N.J., Docampo, R., 2000. Acidocalcisomes and vacuolar H 1-pyrophosphatase in malaria parasites. Biochem. J. 347, 243–53. Martin, M.B., Grimley, J.S., Lewis, J.C., Heath 3rd, H.T., Bailey, B.N., Kendrick, H., Yardley, V., Caldera, A., Lira, R., Urbina, J.A., Moreno, S.N.J., Docampo, R., Croft, S.L., Oldfield, E., 2001. Bisphosphonates inhibit the growth of Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Plasmodium falciparum: a potential route to chemotherapy. J. Med. Chem. 44, 909–16. McIntosh, M.T., Drozdowicz, Y.M., Laroiya, K., Rea, P.A., Vaidya, A.B., 2001. Two classes of plant-like vacuolar-type H 1-pyrophosphatases in malaria parasites. Mol. Biochem. Parasitol. 114, 183–95. Mertens, E., 1991. Pyrophosphate-dependent phosphofructokinase, an anaerobic glycolytic enzyme? FEBS Lett. 285, 1–5. Mitsuda, N., Enami, K., Nakata, M., Takeyasu, K., Sato, M.H., 2001. Novel type Arabidopsis thaliana H 1-PPase is localized to the Golgi apparatus. FEBS Lett. 488, 29–33. Moreno, S.N.J., Urbina, J.A., Oldfield, E., Bailey, B.N., Rodrigues, C.O., Docampo, R., 2000. 31P NMR spectroscopy of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. Evidence for high levels of condensed inorganic phosphates. J. Biol. Chem. 275, 28356–62. Nakanishi, T., Maeshima, M., 1998. Molecular cloning of vacuolar H 1pyrophosphatase and its developmental expression in growing hypocotyl of mung bean. Plant Physiol. 116, 589–97. Nakanishi, Y., Saijo, T., Wada, Y., Maeshima, M., 2001. Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H 1-pyrophosphatase. J. Biol. Chem. 276, 7654– 60. Nelson, K.E., Clayton, R.A., Gill, S.R., Gwinn, M.L., Dodson, R.J., Haft, D.H., Hickey, E.K., Peterson, J.D., Nelson, W.C., Ketchum, K.A., McDonald, L., Utterback, T.R., Malek, J.A., Linher, D.D., Garrett, M.M., Stewart, A.M., Cotton, M.D., Pratt, M.S., Phillips, C.A., Richardson, D., Heidelberg, J., Sutton, G.G., Fleischmann, R.D., Eisen, J.A., Fraser, C.M., et al., 1999. Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of Thermotoga maritima. Nature 399, 323–9. Obermeyer, G., Sommer, A., Bentrup, F.W., 1996. Potassium and voltage dependence of the inorganic pyrophosphatase of intact vacuoles from Chenopodium rubrum. Biochim. Biophys. Acta 1284, 203–12. Peng, Z.Y., Mansour, J.M., Araujo, F., Ju, J.Y., McKenna, C.E., Mansour, T.E., 1995. Some phosphonic acid analogs as inhibitors of pyrophosphate-dependent phosphofructokinase, a novel target in Toxoplasma gondii. Biochem. Pharmacol. 49, 105–13. Perez-Castineira, J.R., Lopez-Marques, R.L., Losada, M., Serrano, A., 2001. A thermostable K(1)-stimulated vacuolar-type pyrophosphatase from the hyperthermophilic bacterium Thermotoga maritima. FEBS Lett. 496, 6–11. Rassow, J., Pfanner, N., 2000. The protein import machinery of the mitochondrial membranes. Traffic 1, 457–64. Rea, P.A., Kim, Y., Sarafian, V., Poole, R.J., Davies, J.M., Sanders, D., 1992a. Vacuolar H 1-translocating pyrophosphatases: a new category of ion translocase. Trends Biochem. Sci. 17, 348–53. Rea, P.A., Britten, C.J., Jennings, I.R., Calvert, C.M., Skiera, L.A., Leigh, R.A., Sanders, D., 1992b. Regulation of vacuolar H 1-pyrophosphatase by calcium. A reaction kinetic analysis. Plant Physiol. 100, 1706–15. Rea, P.A., Poole, R.J., 1993. Vacuolar H 1-translocating pyrophosphatases. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 157–80. Ridley, R.G., 1999. Planting the seeds of new antimalarial drugs. Science 285, 1502–3.

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Robibaro, B., Hoppe, H.C., Yang, M., Coppens, I., Ngo, H.M., Stedman, T.T., Paprotka, K., Joiner, K.A., 2001. Endocytosis in different lifestyles of protozoan parasitism: role in nutrient uptake with special reference to Toxoplasma gondii. Int. J. Parasitol. 31, 1343–53. Rocha Facanha, A., de Meis, L., 1998. Reversibility of H 1-ATPase and H 1pyrophosphatase in tonoplast vesicles from maize coleoptiles and seeds. Plant Physiol. 116, 1487–95. Rodrigues, C.O., Scott, D.A., Docampo, R., 1999a. Presence of a vacuolar H 1-pyrophosphatase in promastigotes of Leishmania donovani and its localization to a different compartment from the vacuolar H 1-ATPase. Biochem. J. 340, 759–66. Rodrigues, C.O., Scott, D.A., Docampo, R., 1999b. Characterization of a vacuolar pyrophosphatase in Trypanosoma brucei and its localization to acidocalcisomes. Mol. Cell Biol. 19, 7712–23. Rodrigues, C.O., Scott, D.A., Bailey, B.N., De Souza, W., Benchimol, M., Moreno, B., Urbina, J.A., Oldfield, E., Moreno, S.N.J., 2000. Vacuolar proton pyrophosphatase activity and pyrophosphate (PPi) in Toxoplasma gondii as possible chemotherapeutic targets. Biochem. J. 349, 737–45. Rogers, M.J., Watts, D.J., Russell, R.G., Ji, X., Xiong, X., Blackburn, G.M., Bayless, A.V., Ebetino, F.H., 1994. Inhibitory effects of bisphosphonates on growth of amoebae of the cellular slime mold Dictyostelium discoideum. J. Bone Miner. Res. 9, 1029–39. Rogers, M.J., Ji, X., Russell, R.G., Blackburn, G.M., Williamson, M.P., Bayless, A.V., Ebetino, F.H., Watts, D.J., 1994. Incorporation of bisphosphonates into adenine nucleotides by amoebae of the cellular slime mould Dictyostelium discoideum. Biochem. J. 303, 303–11. Rogers, M.J., Brown, R.J., Hodkin, V., Blackburn, G.M., Russell, R.G., Watts, D.J., 1996. Bisphosphonates are incorporated into adenine nucleotides by human aminoacyl-tRNA synthetase enzymes. Biochem. Biophys. Res. Commun. 224, 863–9. Rogers, M.J., Xiong, X., Ji, X., Monkkonen, J., Russell, R.G., Williamson, M.P., Ebetino, F.H., Watts, D.J., 1997. Inhibition of growth of Dictyostelium discoideum amoebae by bisphosphonate drugs is dependent on cellular uptake. Pharm. Res. 14, 625–30. Rogers, M.J., Gordon, S., Benford, H.L., Coxon, F.P., Luckman, S.P., Monkkonen, J., Frith, J.C., 2000. Cellular and molecular mechanisms of action of bisphosphonates. Cancer 88, 2961–78. Ros, R., Romieu, C., Gibrat, R., Grignon, C., 1995. The plant inorganic pyrophosphatase does not transport K 1 in vacuole membrane vesicles multilabeled with fluorescent probes for H 1, K 1, and membrane potential. J. Biol. Chem. 270, 4368–74. Ruiz, F.A., Rodriguez, C.O., Docampo, R., 2001. Rapid changes in polyphosphate content within acidocalcisomes in response to cell growth, differentiation and environmental stress in Trypanosoma cruzi. J. Biol. Chem. (in press). Russell, R.G., Rogers, M.J., 1999. Bisphosphonates: from the laboratory to the clinic and back again. Bone 25, 97–106. Sakakibara, Y., Kobayashi, H., Kasamo, K., 1996. Isolation and characterization of cDNAs encoding vacuolar H 1-pyrophosphatase isoforms from rice (Oryza sativa). Plant Mol. Biol. 31, 1029–38. Sato, M.H., Kasahara, M., Ishii, N., Homareda, H., Matsui, H., Yoshida, M., 1994. Purified vacuolar inorganic pyrophosphatase consisting of a 75kDa polypeptide can pump H 1 into reconstituted proteoliposomes. J. Biol. Chem. 269, 6725–8. Sarafian, V., Kim, Y., Poole, R.J., Rea, P.A., 1992. Molecular cloning and sequence of cDNA encoding the pyrophosphate-energized vacuolar membrane proton pump of Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 89, 1775–9. Scott, D.A., de Souza, W., Benchimol, M., Zhong, L., Lu, H.-G., Moreno, S.N.J., Docampo, R., 1998. Presence of a plant-like proton-pumping pyrophosphatase in acidocalcisomes of Tryponosoma cruzi. J. Biol. Chem. 273, 22151–8. Scott, D.A., Docampo, R., 2000. Characterization of isolated acidocalcisomes of Trypanosoma cruzi. J. Biol. Chem. 275, 24215–21. Sherman, I.W., 1998. Carbohydrate metabolism of asexual stages. In: Sher-

14

M.T. McIntosh, A.B. Vaidya / International Journal for Parasitology 32 (2002) 1–14

man, I.W. (Ed.). Malaria Parasite Biology, Pathogenesis, and Protection, American Society for Microbiology Press, pp. 135–44. Soldati, D., Dubremetz, J.F., Lebrun, M., 2001. Microneme proteins: structural and functional requirements to promote adhesion and invasion by the apicomplexan parasite Toxoplasma gondii. Int. J. Parasitol. 31, 1293–302. Thompson, J.D., Higgins, D.G., Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–80. Urbina, J.A., Moreno, B., Vierkotter, S., Oldfield, E., Payares, G., Sanoja, C., Bailey, B.N., Yan, W., Scott, D.A., Moreno, S.N.J., Docampo, R., 1999. Trypanosoma cruzi contains major pyrophosphate stores, and its growth in vitro and in vivo is blocked by pyrophosphate analogs. J. Biol. Chem. 274, 33609–15. Vercesi, A.E., Moreno, S.N., Docampo, R., 1994. Ca21/H1 exchange in acidic vacuoles of Trypanosoma brucei. Biochem. J. 304, 227–33. von Heijne, G., 1986. Mitochondrial targeting sequences may form amphiphilic helices. EMBO J. 5, 1335–42.

White, J.S., Tobin, J.M., Cooney, J.J., 1999. Organotin compounds and their interactions with microorganisms. Can. J. Microbiol. 45, 541–54. Zancani, M., Macri, F., Dal Belin Peruffo, A., Vianello, A., 1995. Isolation of the catalytic subunit of a membrane-bound H(1)-pyrophosphatase from pea stem mitochondria. J. Biochem. 228, 138–43. Zhen, R.-G., Kim, E.J., Rea, P.A., 1994a. Localization of cytosolically oriented maleimide-reactive domain of vacuolar H 1-pyrophosphatase. J. Biol. Chem. 269, 23342–50. Zhen, R.-G., Baykov, A.A., Bakuleva, N.P., Rea, P.A., 1994b. Aminomethylene diphosphonate as a potent type-specific inhibitor of both plant and phototrophic bacterial H 1-pyrophosphatases. Plant Physiol. 104, 153–9. Zhen, R.-G., Kim, E.J., Rea, P.A., 1997a. The molecular and biochemical basis of pyrophosphate-energized proton translocation at the vacuolar membrane. Adv. Bot. Res. 25, 297–337. Zhen, R.-G., Kim, E.J., Rea, P.A., 1997b. Acidic residues necessary for pyrophosphate-energized pumping and inhibition of the vacuolar H 1pyrophosphatase by N,N 0 -dicyclohexylcarbodiimide. J. Biol. Chem. 272, 22340–8.