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Identification and Functional Analysis of the Primary Pantothenate Transporter, PfPAT, of the Human Malaria Parasite Plasmodium falciparum*

Open AccessPublished:May 31, 2013DOI:https://doi.org/10.1074/jbc.M113.482992
      The human malaria parasite Plasmodium falciparum is absolutely dependent on the acquisition of host pantothenate for its development within human erythrocytes. Although the biochemical properties of this transport have been characterized, the molecular identity of the parasite-encoded pantothenate transporter remains unknown. Here we report the identification and functional characterization of the first protozoan pantothenate transporter, PfPAT, from P. falciparum. We show using cell biological, biochemical, and genetic analyses that this transporter is localized to the parasite plasma membrane and plays an essential role in parasite intraerythrocytic development. We have targeted PfPAT to the yeast plasma membrane and showed that the transporter complements the growth defect of the yeast fen2Δ pantothenate transporter-deficient mutant and mediates the entry of the fungicide drug, fenpropimorph. Our studies in P. falciparum revealed that fenpropimorph inhibits the intraerythrocytic development of both chloroquine- and pyrimethamine-resistant P. falciparum strains with potency equal or better than that of currently available pantothenate analogs. The essential function of PfPAT and its ability to deliver both pantothenate and fenpropimorph makes it an attractive target for the development and delivery of new classes of antimalarial drugs.
      Background: Pantothenate transport is essential for Plasmodium development. The transporter that mediates entry of pantothenate is unknown.
      Results: PfPAT encodes the primary pantothenate transporter of P. falciparum.
      Conclusion: PfPAT plays an essential function in parasite development and thus is a valid target for antimalarial therapy.
      Significance: PfPAT is the first pantothenate transporter identified and characterized in protozoan parasites and a valid target for therapy.

      Introduction

      Nearly half of the world population is at risk of contracting malaria, a parasitic disease caused by protozoan parasites of the genus Plasmodium (

      World Malaria Report (2011) World Health Organization, Geneva,

      ). Five species of Plasmodium infect humans, and cases of infection caused by Plasmodium knowlesi are on the rise (
      • Singh B.
      • Kim Sung L.
      • Matusop A.
      • Radhakrishnan A.
      • Shamsul S.S.
      • Cox-Singh J.
      • Thomas A.
      • Conway D.J.
      A large focus of naturally acquired Plasmodium knowlesi infections in human beings.
      ). These species cause ∼250 million annual cases of clinical malaria and over 1 million deaths (
      • Murray C.J.
      • Rosenfeld L.C.
      • Lim S.S.
      • Andrews K.G.
      • Foreman K.J.
      • Haring D.
      • Fullman N.
      • Naghavi M.
      • Lozano R.
      • Lopez A.D.
      Global malaria mortality between 1980 and 2010. A systematic analysis.
      ). Most deaths occur in Africa and can be ascribed to infection by Plasmodium falciparum. The lack of an effective vaccine and the emergence of drug-resistant P. falciparum strains emphasize the need for better strategies that target new pathways that are unique to the parasite and that have not yet been targeted for antimalarial therapy.
      Plasmodium intraerythrocytic proliferation is fueled by nutrients, such as purine nucleosides and nucleobases, amino acids, sugars, fatty acids, and vitamins, scavenged from the host (
      • Saliba K.J.
      • Kirk K.
      Nutrient acquisition by intracellular apicomplexan parasites. Staying in for dinner.
      ). The uptake of these essential nutrients involves endogenous transporters at the erythrocyte membrane as well as parasite-encoded permeases targeted to red blood cell membrane, parasite plasma membrane, or intracellular organelles (
      • Downie M.J.
      • El Bissati K.
      • Bobenchik A.M.
      • Nic Lochlainn L.
      • Amerik A.
      • Zufferey R.
      • Kirk K.
      • Ben Mamoun C.
      PfNT2, a permease of the equilibrative nucleoside transporter family in the endoplasmic reticulum of Plasmodium falciparum.
      ,
      • Downie M.J.
      • Saliba K.J.
      • Bröer S.
      • Howitt S.M.
      • Kirk K.
      Purine nucleobase transport in the intraerythrocytic malaria parasite.
      ,
      • El Bissati K.
      • Downie M.J.
      • Kim S.K.
      • Horowitz M.
      • Carter N.
      • Ullman B.
      • Ben Mamoun C.
      Genetic evidence for the essential role of PfNT1 in the transport and utilization of xanthine, guanine, guanosine, and adenine by Plasmodium falciparum.
      ,
      • El Bissati K.
      • Zufferey R.
      • Witola W.H.
      • Carter N.S.
      • Ullman B.
      • Ben Mamoun C.
      The plasma membrane permease PfNT1 is essential for purine salvage in the human malaria parasite Plasmodium falciparum.
      ,
      • Kirk K.
      • Saliba K.J.
      Targeting nutrient uptake mechanisms in Plasmodium.
      ,
      • Martin R.E.
      • Ginsburg H.
      • Kirk K.
      Membrane transport proteins of the malaria parasite.
      ,
      • Martin R.E.
      • Henry R.I.
      • Abbey J.L.
      • Clements J.D.
      • Kirk K.
      The “permeome” of the malaria parasite. An overview of the membrane transport proteins of Plasmodium falciparum.
      ,
      • Slavic K.
      • Straschil U.
      • Reininger L.
      • Doerig C.
      • Morin C.
      • Tewari R.
      • Krishna S.
      Life cycle studies of the hexose transporter of Plasmodium species and genetic validation of their essentiality.
      ,
      • Woodrow C.J.
      • Burchmore R.J.
      • Krishna S.
      Hexose permeation pathways in Plasmodium falciparum-infected erythrocytes.
      ,
      • Woodrow C.J.
      • Penny J.I.
      • Krishna S.
      Intraerythrocytic Plasmodium falciparum expresses a high affinity facilitative hexose transporter.
      ). The water-soluble vitamin pantothenic acid (vitamin B5) is a precursor of the important enzyme cofactor CoA, a universal carrier of activated acyl groups involved in 9% of biochemical reactions identified in all living organisms (
      • Spry C.
      • Kirk K.
      • Saliba K.J.
      Coenzyme A biosynthesis. An antimicrobial drug target.
      ). Because P. falciparum cannot synthesize pantothenate de novo, the acquisition of this vitamin from the host is an indispensable nutritional function for the parasite (Fig. 1).
      Figure thumbnail gr1
      FIGURE 1Schematic representation of pantothenate transport and CoA biosynthesis in P. falciparum-infected erythrocytes. Pantothenate is transported across the plasma membrane by the parasite pantothenate transporter and converted into CoA by the enzymes pantothenate kinase (1), phosphopantothenoylcysteine synthetase (2), phosphopantothenoylcysteine decarboxylase (3), phosphopantotheine adenyltransferase (4), and dephospho-CoA kinase (5). PPM, parasite plasma membrane; PVM, parasitophorous vacuolar membrane; RBCM, red blood cells membrane.
      The transporters involved in the uptake of pantothenic acid across the red blood cell membrane or the parasite plasma membrane remain unknown, and no pantothenate transporters have yet been identified in any other protozoa. Biochemical studies have demonstrated that in contrast to the negligible pantothenate uptake in uninfected erythrocytes, pantothenate transport across infected red blood cells is rapid (
      • Saliba K.J.
      • Horner H.A.
      • Kirk K.
      Transport and metabolism of the essential vitamin pantothenic acid in human erythrocytes infected with the malaria parasite Plasmodium falciparum.
      ). This transport activity detected following parasite infection is mediated by the new permeation pathway. Once inside the red blood cell cytoplasm, pantothenate is transported into the parasite via a parasite-specific low affinity permease (Km = ∼23 mm) and converted into CoA via parasite-encoded enzymes (
      • Saliba K.J.
      • Kirk K.
      H+-coupled pantothenate transport in the intracellular malaria parasite.
      ). Unlike mammalian cells, where the transport of pantothenate is entirely dependent on the presence of Na+, the uptake of pantothenate in P. falciparum isolated trophozoites is Na+-independent, markedly dependent on pH (
      • Saliba K.J.
      • Kirk K.
      H+-coupled pantothenate transport in the intracellular malaria parasite.
      ), and electroneutral with pantothenate and H+ entering the parasite with a stoichiometry of 1:1 (
      • Saliba K.J.
      • Kirk K.
      H+-coupled pantothenate transport in the intracellular malaria parasite.
      ).
      In lower eukaryotes, pantothenate transport has been characterized in Saccharomyces cerevisiae and Schizosaccharomyces pombe (
      • Stolz J.
      • Caspari T.
      • Carr A.M.
      • Sauer N.
      Cell division defects of Schizosaccharomyces pombe liz1-mutants are caused by defects in pantothenate uptake.
      ,
      • Stolz J.
      • Sauer N.
      The fenpropimorph resistance gene FEN2 from Saccharomyces cerevisiae encodes a plasma membrane H+-pantothenate symporter.
      ). In S. cerevisiae, pantothenate is transported via a high affinity plasma membrane H+-pantothenate symporter Fen2, which is also responsible for the uptake of the antifungal drug fenpropimorph (
      • Stolz J.
      • Sauer N.
      The fenpropimorph resistance gene FEN2 from Saccharomyces cerevisiae encodes a plasma membrane H+-pantothenate symporter.
      ). Accordingly, fen2Δ knock-out mutants do not transport pantothenate, are unable to grow on media containing physiological concentrations of pantothenate (1–10 μm), and are resistant to fenpropimorph (
      • Stolz J.
      • Sauer N.
      The fenpropimorph resistance gene FEN2 from Saccharomyces cerevisiae encodes a plasma membrane H+-pantothenate symporter.
      ,
      • Marcireau C.
      • Joets J.
      • Pousset D.
      • Guilloton M.
      • Karst F.
      FEN2. A gene implicated in the catabolite repression-mediated regulation of ergosterol biosynthesis in yeast.
      ).
      Here we report the identification and functional characterization of the first protozoan pantothenate transporter, PfPAT, from P. falciparum. PfPAT plays an essential function in parasite intraerythrocytic development, and its expression in fen2Δ yeast mutant complements the growth deficiency on low pantothenate and mediates the entry of fenpropimorph into these cells.

      DISCUSSION

      Molecular characterization of nutrient transporters from P. falciparum has been limited to only few transporters such as the purine transporters PfNT1 and PfNT2, the hexose transporter PfHT1, and the potassium channel PfK1 (
      • Downie M.J.
      • El Bissati K.
      • Bobenchik A.M.
      • Nic Lochlainn L.
      • Amerik A.
      • Zufferey R.
      • Kirk K.
      • Ben Mamoun C.
      PfNT2, a permease of the equilibrative nucleoside transporter family in the endoplasmic reticulum of Plasmodium falciparum.
      ,
      • El Bissati K.
      • Zufferey R.
      • Witola W.H.
      • Carter N.S.
      • Ullman B.
      • Ben Mamoun C.
      The plasma membrane permease PfNT1 is essential for purine salvage in the human malaria parasite Plasmodium falciparum.
      ,
      • Waller K.L.
      • McBride S.M.
      • Kim K.
      • McDonald T.V.
      Characterization of two putative potassium channels in Plasmodium falciparum.
      ). For most candidate permeases, however, this remains a difficult task because of poor expression, mislocalization, or inadequate membrane orientation of the transporter in the heterologous system used. Furthermore, because of the high A+T content of P. falciparum genes, further codon optimization or harmonization is needed to increase the expression efficiency and prevent early transcription termination (
      • Downie M.J.
      • El Bissati K.
      • Bobenchik A.M.
      • Nic Lochlainn L.
      • Amerik A.
      • Zufferey R.
      • Kirk K.
      • Ben Mamoun C.
      PfNT2, a permease of the equilibrative nucleoside transporter family in the endoplasmic reticulum of Plasmodium falciparum.
      ,
      • Santiago T.C.
      • Zufferey R.
      • Mehra R.S.
      • Coleman R.A.
      • Mamoun C.B.
      The Plasmodium falciparum PfGatp is an endoplasmic reticulum membrane protein important for the initial step of malarial glycerolipid synthesis.
      ,
      • Reynolds J.M.
      • Takebe S.
      • Choi J.Y.
      • El Bissati K.
      • Witola W.H.
      • Bobenchik A.M.
      • Hoch J.C.
      • Voelker D.R.
      • Mamoun C.B.
      Biochemical and genetic analysis of the phosphoethanolamine methyltransferase of the human malaria parasite Plasmodium falciparum.
      ,
      • Baro N.K.
      • Pooput C.
      • Roepe P.D.
      Analysis of chloroquine resistance transporter (CRT) isoforms and orthologues in S. cerevisiae yeast.
      ). In this study, we attempted to identify the primary pantothenate transporter of Plasmodium by genetic complementation in yeast using a P. knowlesi cDNA library made in a yeast expression vector under a constitutive promoter. This approach, however, failed to identify such a transporter. Further analysis of the library using specific PCR primers showed that the cDNA encoding the full-length P. knowlesi PAT ortholog is not represented in the library. PfPAT was thus found in silico based on sequence similarity with the S. cerevisiae Fen2. The best homologues of PfPAT are found in apicomplexa and Viridiplantae (green algae and land plants). Because pantothenate transport has not yet been characterized in plants, the finding of several plant orthologs likely resulting from duplication events (supplemental Fig. S2), suggests that this process might be critical in plant physiology.
      So far, attempts to target PfPAT to the plasma membrane of mammalian cells to measure its transport activity have not been successful (Fig. 6). Future efforts aimed to create chimeric proteins similar to those generated in yeast could make it possible to measure pantothenate uptake in heterologous systems and determine its kinetics parameters and inhibition profile.
      The successful localization of a chimeric form of PfPAT to the yeast plasma membrane following addition of the N-terminal domain of Fen2 allowed us to perform complementation assays in yeast to demonstrate the function of PfPAT in pantothenate transport. PfPAT complemented the growth defect of the yeast fen2Δ strain on media containing low pantothenate concentrations, thus providing the first evidence that PfPAT functions in pantothenate uptake. Our genetic studies in P. falciparum provided strong evidence that the PfPAT gene plays an essential function in parasite development. Unlike PfNT1, whose deletion could be complemented by the addition of high concentrations of exogenous purine nucleosides and nucleobases (
      • El Bissati K.
      • Zufferey R.
      • Witola W.H.
      • Carter N.S.
      • Ullman B.
      • Ben Mamoun C.
      The plasma membrane permease PfNT1 is essential for purine salvage in the human malaria parasite Plasmodium falciparum.
      ), we were unable to generate a genetically null mutant for PfPAT even in the presence of pantothenate at a concentration 400-fold higher than its physiological concentration. Furthermore, targeting PfPAT mRNA degradation using specific PMO conjugates resulted in parasite death. Together, these results suggest that PfPAT is essential for P. falciparum intraerythrocytic development, and no alternative mode of pantothenate entry exists in this parasite. PfPAT does not share homology with the human multivitamin transporter, hSMVT, suggesting that this transporter might be a good target for the development of selective antimalarial drugs.
      Expression of PfPATf in yeast renders fen2Δ more susceptible to the fungicidal drug fenpropimorph, suggesting that PfPAT may transport both pantothenate and fenpropimorph. This finding led us to investigate whether the drug can inhibit P. falciparum intraerythrocytic development. Interestingly, we found that fenpropimorph inhibits the growth of chloroquine and pyrimethamine-sensitive and -resistant strains with IC50 values below 40 μm. Fenpropimorph's antimalarial potency is equal or better than most of the pantothenate analogs discovered so far (
      • Spry C.
      • Chai C.L.
      • Kirk K.
      • Saliba K.J.
      A class of pantothenic acid analogs inhibits Plasmodium falciparum pantothenate kinase and represses the proliferation of malaria parasites.
      ). Although it is unclear whether fenpropimorph exerts its antimalarial activity by inhibiting pantothenate transport, CoA biosynthesis, or another metabolic pathway, the finding that it requires PfPAT for entry suggests that the transporter may also serve to selectively deliver antimalarial drugs into the parasite. Safer analogs of fenpropimorph or chimera of fenpropimorph analogs and other drugs might define new classes of antimalarials, which have not been used in malaria therapy.
      Our in silico analyses identified orthologs of PfPAT in other protozoa (supplemental Fig. S2 and Table S2). Biochemical and genetic characterization of these candidate transporters might help advance our understanding of pantothenate transport during the entire life cycle of protozoan parasites in the arthropod vector and mammalian host. These studies may also help identify new classes of chemicals to treat and prevent parasitic diseases.

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