ARTICLE
Received 23 Dec 2013 | Accepted 23 Feb 2015 | Published 31 Mar 2015
DOI: 10.1038/ncomms7721
A lactate and formate transporter in the intraerythrocytic malaria parasite, Plasmodium falciparum
Rosa V. Marchetti1, Adele M. Lehane1, Sarah H. Shak1, Markus Winterberg1, Rowena E. Martin1 & Kiaran Kirk1
The intraerythrocytic malaria parasite relies primarily on glycolysis to fuel its rapid growth and reproduction. The major byproduct of this metabolism, lactic acid, is extruded into the external medium. In this study, we show that the human malaria parasite Plasmodium falciparum expresses at its surface a member of the microbial formatenitrite transporter family (PfFNT), which, when expressed in Xenopus laevis oocytes, transports both formate and lactate. The transport characteristics of PfFNT in oocytes (pH-dependence, inhibitor-sensitivity and kinetics) are similar to those of the transport of lactate and formate across the plasma membrane of mature asexual-stage P. falciparum trophozoites, consistent with PfFNT playing a major role in the efux of lactate and hence in the energy metabolism of the intraerythrocytic parasite.
1 Research School of Biology, Australian National University, Canberra, Australian Capital Territory 2601, Australia. Correspondence and requests for materials should be addressed to K.K. (email: mailto:[email protected]
Web End [email protected] ).
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The asexual intraerythrocytic form of the human malaria parasite Plasmodium falciparum relies primarily on glycolysis to meet its energy requirements1. Glucose is
consumed by P. falciparum-infected erythrocytes at a rate some two orders of magnitude higher than by uninfected erythrocytes2. It enters the parasitized erythrocyte via a combination of the host cells endogenous glucose transporter and the parasite-induced New Permeability Pathways3, and is then taken up into the parasite via the parasites plasma membrane hexose transporter46. Within the parasite, glucose is metabolized via glycolysis to lactic acid, which is excreted in large quantities from the parasite2 via a H -coupled transporter79.
The P. falciparum genome contains genes for a number of candidate monocarboxylate transporters10, one of which is encoded by the gene PF3D7_0316600 (formerly PFC0725c). This protein falls within the microbial formatenitrite family of transporters (FNT; TC 1.A.16), at least one other member of which has been shown to transport lactate and other products of anaerobic fermentation11,12.
The protein encoded by PF3D7_0316600, referred to here as PfFNT (P. falciparum FormateNitrite Transporter), was, together with P. falciparum hexose transporter and the nucleo-side/nucleobase transporter PfENT1, one of the very few (putative or proven) transporter proteins detected in a proteomic analysis of a detergent-resistant membrane fraction prepared from mature blood-stage P. falciparum parasites13. This is perhaps indicative of it playing an important housekeeping role in the intraerythrocytic phase of the malaria parasites life cycle. In this study, we have explored the function and subcellular localization of this protein, and here present data implicating it in the transport of lactic acid and other monocarboxylic acids across the plasma membrane of the intraerythrocytic parasite.
ResultsPfFNT has homologues in other apicomplexan parasites. Formate/nitrite transporter homologues are present in a broad range of microbes but have not been identied in the animal or land plant genomes sequenced to date (Supplementary Fig. 1). With the exception of Cryptosporidium species, all sequenced apicomplexan parasites harbour at least one FNT homologue, with the coccidian parasites (for example, Toxoplasma and Eimeria species) each encoding two or three FNT proteins and the haemosporidians and piroplasms (for example, Plasmodium and Theileria species) harbouring a single FNT homologue (Supplementary Fig. 2). FNT homologues were not detected in the sequenced genomes of diplomonads (Giardia) or kinetoplastids (Leishmania and Trypansoma). The apicomplexan FNT homo-logues formed a monophyletic clade (Supplementary Fig. 2), the topology of which is similar to current estimations of apicomplexan evolution14,15. This suggests that the FNT homologue was present early in the evolution of apicomplexans.
Alignment of the sequence of PfFNT with a selection of homologues from other microorganisms revealed a number of conserved features (Supplementary Fig. 3). For example, residues that have been implicated in the gating of the E. coli FocA transporter (T91, K156, E208, H209 and N213; ref. 16) are conserved in PfFNT.
PfFNT transports formate and lactate. The functional characteristics of PfFNT were investigated using the X. laevis oocyte expression system. cRNA encoding a carboxy terminal (C-terminal) hemagglutinin (HA)-tagged form of PfFNT (HA-PfFNT) was injected into X. laevis oocytes. Western blot analysis of an oocyte membrane preparation showed a band corresponding to B32 kDa (Fig. 1a), very close to the predicted size of PfFNT
(34 kDa). Immunouorescence assay of oocytes expressing the tagged protein (Fig. 1b) revealed a uorescent band external to the oocytes internal pigment layer, indicating that the protein is present in the oocyte plasma membrane.
Xenopus oocytes injected with cRNA encoding PfFNT showed a signicant increase in the rate of uptake of both formate (Fig. 2a) and lactate (Fig. 2b) relative to non-injected controls.
As is typically the case with the oocyte expression system (in which independent experiments are performed with oocytes from individual, unrelated frogs) there was substantial variability between experiments in the magnitude of the PfFNT-induced increase in the ux of formate and lactate. In oocytes expressing PfFNT, the rate of formate uptake was increased between 1.5- and9.2-fold relative to the non-injected controls, with an average increase of 3.70.3-fold (means.e.m.; n 35; Po0.001, one
way analysis of variance (ANOVA)). The rate of lactate uptake was increased by between 1.1- and 4.4-fold relative to the non-injected controls, with an average increase of 3.10.3-fold (n 13; Po0.001, one-way ANOVA).
In paired experiments, the uptake of [14C]formate into oocytes expressing PfFNT was comparable to that for oocytes expressing the rat monocarboxylate transporter rMCT1, whereas in oocytes expressing the unrelated P. falciparum (nucleoside/nucleobase) transporter PfENT1, formate uptake was similar to that in non-injected controls (Supplementary Fig. 4; P40.05, one-way
ANOVA).
PfFNT-induced transport is pH-dependent and inhibitable. For both formate and lactate, PfFNT-induced uptake (calculated by subtracting uptake measured in non-injected oocytes from that measured in oocytes expressing PfFNT; Supplementary Fig. 5) increased with decreasing pH (Fig. 3a), consistent with (though not proof of) the transport of the two monocarboxylates being coupled to that of H . The PfFNT-induced uptake of [14C]formate was signicantly reduced in the presence of a 10 mM concentration of lactate (Fig. 3b), as well as by a range of other carboxylatespyruvate, acetate and propionateand by the tricarboxylate citrate and the monovalent nitrite ion (each at 10 mM; Supplementary Fig. 6). The uptake of [14C]lactate was similarly reduced in the presence of a 10 mM concentration of formate (Fig. 3b). These data are consistent with
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Figure 1 | Expression and localization of PfFNT in Xenopus laevis oocytes. (a) Western blots of membranes from oocytes expressing C-terminal tagged HA-PfFNT, probed with an anti-HA antibody, show a bandclose to the predicted size of 34 kDa. The band is absent from membranes from non-injected (ni) oocytes. (b) Immunouorescence images of oocytes injected with cRNA encoding C-terminal tagged HA-PfFNT show a clear band of uorescence (corresponding to the plasma membrane) outside the pigment layer. The band is not seen in non-injected oocytes. Scale bar, 100 mm. In radiolabel uptake experiments, oocytes expressing the HA-tagged form of PfFNT accumulated [3H]formate to levels above those measured in non-injected oocytes, consistent with the tagged protein being functional.
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Figure 2 | Time courses for formate and lactate uptake in Xenopus oocytes. Uptake of (a) formate and (b) lactate into PfFNT-expressing (closed circles) and non-injected (open circles) oocytes. Experiments were performed at 27.5 C, pH 6.4, and an extracellular formate or lactate concentration of 1 mM.
The data are averaged from those obtained in 4 independent experiments, each conducted on different days, using eggs from different frogs, and are shown s.e.m.
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Figure 3 | Characteristics of formate and lactate uptake in Xenopus oocytes. The (a) pH-dependence and (b) susceptibility to inhibition of PfFNT-induced formate and lactate uptake. Uptake was measured over 10 min at an extracellular formate or lactate concentration of 1 mM, except where specied otherwise. The data were averaged from those obtained in n independent experiments, each from different days, using eggs from different frogs, where n was: (a) 6 for [14C]formate (black bars) and 4 for [14C]lactate (white bars); (b) 48 for [14C]formate (black bars) and 4 for [14C]lactate (white bars). In b, the extracellular pH was 6.4 and the potential competing substrates (formate and lactate; 10 mM) and inhibitors (100 mM) were added to the oocytes at the same time as the [14C]formate or [14C]lactate. In both panels, uptake is expressed relative to that measured at pH 6.4 in the absence of competing substrate or inhibitors. The asterisks denote statistical signicance of the apparent differences between [14C]formate or [14C]lactate uptake measured under each of the different conditions tested and that measured in the relevant controls: *Po0.05; **Po0.01; ***Po0.001.
formate and lactate competing with one another for the transporter.
A range of pharmacological agents were tested for their effect on PfFNT-induced formate and lactate uptake (Fig. 3b, with the effects on uptake into non-injected oocytes, and oocytes expressing PfFNT shown in Supplementary Fig. 7). The organo-mercurial reagent pCMBS (100 mM), an inhibitor of monocarboxylate transport in some cell-types17, and the sulfhydryl reagent N-ethylmaleimide (100 mM), both caused a modest inhibition of the PfFNT-induced uptake of formate and lactate. By contrast, the broad-specicity anion transport inhibitors niumic acid (100 mM) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 100 mM) caused a marked (B70%; Po0.05, one-way ANOVA) reduction in the
PfFNT-induced uptake of the two monocarboxylates, while having a much lesser effect on the uptake of [14C]formate into non-injected oocytes (Supplementary Fig. 7).
PfFNT-induced transport is saturable. The concentration dependence of the PfFNT-induced uptake of formate and lactate uptake is shown in Fig. 4, with the concentration dependence of uptake into non-injected oocytes and oocytes expressing PfFNT shown separately in the insets. The uptake of formate into non-injected oocytes (Fig. 4a, inset) was a low-afnity process; a least-
squares t of the MichaelisMenten equation to the data obtained over a formate concentration range of 010 mM yielded an apparent Km (255 mM) well above the maximum concentration tested. By contrast, PfFNT-induced formate uptake (Fig. 4a) showed a much higher afnity, with an estimated Km of 5.10.9 mM, and a mean Vmax of 52883 pmol per oocyte per 10 min (n 8).
Similarly, the uptake of lactate into non-injected oocytes was a low-afnity process (Fig. 4b inset), with an apparent Km (4210 mM) well outside the concentration range tested. By contrast, PfFNT-induced lactate uptake (Fig. 4b) had an estimated Km of 7.31.7 mM and a Vmax of 83483 pmol per oocyte per 10 min (n 5).
PfFNT is at the surface of the intraerythrocytic parasite. The localization of PfFNT in asexual-blood-stage P. falciparum parasites was investigated by transfecting parasites with plasmids giving rise to the expression of either amino terminal (N-terminal) or C-terminal tagged HA-PfFNT. Western blots of membrane preparations from the transfected parasites revealed a band corresponding to 34 kDa (Fig. 5a), the predicted size of PfFNT. Immunouorescence assay (using anti-HA antibodies) of erythrocytes infected with the transfectant parasites revealed uorescence both in the vicinity of the parasite surface, consistent
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Figure 4 | Concentration dependence of PfFNT-induced formate and lactate uptake in Xenopus oocytes. The PfFNT-induced uptake of formate (a) and lactate (b) was calculated by subtracting the uptake measured in non-injected oocytes (inset: open symbols) from that measured in oocytes expressing PfFNT (inset: closed symbols). The formate data are averaged from 8 independent experiments and the lactate data from 5 independent experiments, with each experiment done on a different day, using oocytes from different frogs, and shown s.e.m. The tted curves were drawn using the MichaelisMenten equation.
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Figure 5 | Localization of HA-PfFNT in 3D7 P. falciparum trophozoites. (a) Western blot analysis of membrane preparations from P. falciparum trophozoites expressing either N-terminal or C-terminal tagged HA-PfFNT (lanes N and C, respectively), with a protein corresponding to the predicted size of 34 kDa identied in both cases. (b) An image of parasitized erythrocytes expressing C-terminal HA-tagged PfFNT, showing immunouorescence both at the surface of the intraerythrocytic parasite and in the region surrounding the hemozoin crystals. Similar resultswere obtained with N-terminal HA-tagged PfFNT (not shown). (c) Co-localization of C-terminal tagged HA-PfFNT with the parasites plasma membrane nucleoside/nucleobase transporter PfENT1. Scale bar, 5 mm.
with a plasma membrane localization, and in the region surrounding the hemozoin crystals, consistent with the presence of the protein on the membrane of the parasites digestive vacuole (a lysosomal-type compartment; Fig. 5b). The parasite surface localization of the protein was conrmed by co-localizing HA-PfFNT with the plasma membrane transporter PfENT1 (ref. 18; Fig. 5c). Multiple attempts to conrm the localization by introducing an epitope tag through targeting the endogenous PfFNT locus proved unsuccessful.
Parasite formate transport resembles that via PfFNT. Although the transport of lactate across the plasma membrane of the intraerythrocytic malaria parasite has been demonstrated and characterized previously8,9, that of the smaller formate ion has not. Formate was found here to traverse the parasite plasma membrane rapidly; in parasites functionally isolated from their host erythrocytes by saponin-permeabilization of the erythrocyte membrane and suspended at 4 C and an extracellular pH of 6.1, [14C]formate added to the extracellular medium reached a nal distribution ratio (that is, the intracellular concentration relative to the extracellular concentration) of 2.70.1 (means.e.m.;
n 4) within B1 min (Fig. 6a). On raising the extracellular pH
from 6.1 to 7.1, there was a marked decrease in the initial inux rate, as well as an B10-fold decrease in the nal distribution ratio (Fig. 6b). When the extracellular pH was increased further to 8.1, uptake of [14C]formate was negligible (Fig. 6b). The pH-dependence of formate uptake into isolated parasites is consistent with formate uptake occurring via a H -coupled mechanism. This was conrmed by the nding that addition of formate (10 mM) to isolated parasites, preloaded with the uorescent pH-indicator BCECF, resulted in a rapid acidication (Fig. 6c), consistent with H ions entering the parasite together with the formate. The uptake of formate into the isolated parasites was saturable, with a Km of 1.9 mM0.7 and a
Vmax of 9.21.8 pmol per 1012 cells per hour (Fig. 6d).
The uptake of formate and lactate into isolated P. falciparum trophozoites was inhibited to a modest and somewhat variable extent by pCMBS (100 mM) and by N-ethylmaleimide (100 mM)
and, as was seen for the PfFNT-induced uptake of both substrates into oocytes, to a much greater extent by niumic acid (100 mM;
Po0.01) and NPPB (100 mM; Po0.01, one-way ANOVA) (Fig. 6e).
A comparison of the rates of uptake of formate and lactate into 3D7 parasites expressing N-terminal tagged HA-PfFNT with those into wild type 3D7 parasites revealed that, for both substrates, there was no signicant difference between the two parasite lines (Supplementary Fig. 8). Without a quantitative understanding of the relative levels of expression of the endogenous (untagged) FNT protein and the HA-tagged protein, or the relative transport activities of the untagged and tagged proteins, it is not possible to draw a denitive conclusion from these data.
DiscussionThe characteristics of formate and lactate transport across the plasma membrane of isolated parasites are very similar to those of the PfFNT-induced transport of the two monocarboxylates in Xenopus oocytes. Both appear saturable with a Km in the low mM range; for formate the Km was 1.9 mM in the parasite (measured at 4 C) and 5.1 mM in the oocyte (measured at 27.5 C), while for lactate the Km (measured at 5 C in a previous study8) was3.8 mM in the parasite and (as measured in this study at 27.5 C)7.3 mM in the oocyte. Both show similar pH-dependence, consistent with the transport being H -coupled, and both were effectively inhibited by 100 mM concentrations of the anion transport blockers niumic acid and NPPB. None of the inhibitors used in this study are specic; in particular, both
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Figure 6 | Formate and lactate uptake into isolated 3D7 P. falciparum trophozoites. Unless specied otherwise, the extracellular pH was 6.1 and the extracellular formate concentration was 3.6 mM. (a) Time courses for [14C]formate uptake, shown as a distribution ratio (that is, the intracellular concentration of radiolabel relative to that in the extracellular solution) and tted to a rst-order exponential equation. (b) pH-dependence of formate uptake. The parasites were suspended at an extracellular pH of 6.1 (black symbols), 7.1 (white symbols) or 8.1 (grey symbols) 10 min before the addition of [14C]formate. (c) Acidication of the cytosol of BCECF-loaded isolated parasites on addition of 10 mM formate to the extracellular medium (pH 7.1). (d) Concentration dependence of the uptake of formate (measured over 5 s), tted to the MichaelisMenten equation. (e) Effect of competing substrates (10 mM) and pharmacological agents (100 mM) on the uptake of formate (black bars) and lactate (external concentration 0.8 mM; white bars). The competing substrates and pharmacological agents were, in each case, added to the parasite suspension at the same time as the radiolabelled formate or lactate. Uptake was measured over either 5 or 20 s (both times fall within the initial, approximately linear phase of the uptake time course). In c, data from a single representative experiment are shown. In the case of the other panels, the data were averaged from those obtained in n independent experiments, each from different days, where n was 4 in a; 3 in b for the pH 6.1 and 7.1 data and 2 for the pH 8.1 data; 5 in d; 36 in e. The data are shown s.e.m (or, in the case of the pH 8.1 data in b, range/2). In e, the asterisks denote the statistical signicance of the apparent differences between [14C]formate or [14C]lactate uptake measured under each of the different conditions tested and that measured in the relevant ([14C]formate or [14C]lactate) control: *Po0.05; **Po0.01; ***Po0.001.
niumic acid and NPPB inhibit a range of anion transport pathways. Nevertheless, the fact that for both compounds the effect on formate/lactate transport in parasites is similar to that on PfFNT-induced transport in oocytes is at least consistent with PfFNT playing a role in the transport of monocarboxylates across the parasite plasma membrane.
The marked similarities between the monocarboxylate transport characteristics observed in PfFNT-expressing Xenopus oocytes and in isolated parasites, together with the nding that PfFNT is localized to the parasite surface, are consistent with the hypothesis that PfFNT plays a major role in the ux of monocarboxylates across the parasite plasma membrane. From a physiological perspective, the primary monocarboxylate is lactate, excreted from the infected erythrocyte in large quantities as the end-product of the parasites anaerobic metabolism2. Formate is also present in signicant quantities in asexual-stageP. falciparum parasites19. A recent study has highlighted the ability of a formatenitrite transporter family member (FocA) from the enterobacterium Salmonella typhimurium to facilitate the export of the major end-products of anaerobic mixed-acid fermentation11. Such a role has not previously been demonstrated
for eukaryote formatenitrite transporter family members. However the ndings here, consistent with PfFNT providing the major route for the transport of lactate across the plasma membrane of the intraerythrocytic (eukaryotic) malaria parasite, indicate that it may be more common than has previously been recognized.
The formatenitrite transporter family is unrelated to the monocarboxylate transporter family, members of which play the major role in the export of lactate from human cells. With the malaria parasite now showing resistance to most (if not all) antimalarials currently available, the revelation of such a fundamental biochemical difference between the parasite and its host, and the consequent potential of the PfFNT protein as an antimalarial drug target, is of obvious interest.
A new paper by Beitz and colleagues reports the functional expression of PfFNT in yeast20. The authors present evidence that PfFNT transports lactate, formate and a number of other monocarboxylates via a coupled lactate:H symport mechanism, and is localized to the parasite surface. The results of the study by
Beitz and colleagues, obtained using a different expression system, are highly complementary to those presented here.
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Methods
Phylogenetic analysis of PfFNT. FNT-family proteins were retrieved from the databases accessed at http://blast.ncbi.nlm.nih.gov/Blast.cgi
Web End =blast.ncbi.nlm.nih.gov/Blast.cgi , http://www.sanger.ac.uk/resources/downloads/protozoa/
Web End =www.sanger.ac.uk/resources/ http://www.sanger.ac.uk/resources/downloads/protozoa/
Web End =downloads/protozoa/ , and http://www.eupathdb.org/eupathdb/
Web End =www.eupathdb.org/eupathdb/ using a Basic Local Alignment Search Tool (BLAST) search and PfFNT as the query sequence.
A representative selection of known and putative FNT homologues from a range of microorganisms were aligned using ClustalW21 and the resulting alignments were manually adjusted in MacVector 12.7.5. Two proteins that are distantly related to the FNT family (from the agellate protozoan Trichomonas vaginalis; gi accession codes 123454345 and123479995) were included as an outgroup. Regions of the alignment that contained gaps or which could not be aligned unambiguously were excluded before analysis. Phylogenetic trees were estimated using the Neighbor-Joining method22 and uncorrected amino acid distances in MacVector 12.7.5. Ties in the tree were resolved randomly and a bootstrap analysis23 was performed with 1,000 pseudoreplicates.
Cloning of PfFNT. The gene encoding PfFNT was amplied from cDNA prepared from 3D7 parasites and inserted into the oocyte expression vector pGem-He-Juel24. The forward primer (50-CCCCCGGGCCACCATGCCACCAAATAATTCC)-30 included an AvaI site (underlined) to enable further cloning as well as a short synthetic Kozak sequence (italicized) before the start codon (bold). The reverse primer (50-GGAATTCTCAATTTCGTAATTCTATAGAT)-30 included an EcoRI site (underlined) and a stop codon (bold). C-terminally HA-tagged PfFNT was generated by PCR using the forward primer above and the reverse primer 50-GGA
ATTCTTAAACAGCGTAATCTGGAACATCATATGGGTATGCAACAGCAT TTCTGAATTCTATAGAT-30. The restriction enzyme site (underlined) was included and the HA-tag is shown in bold. All coding sequences were veried by sequencing.
Expression of PfFNT and uptake of radiolabeled solutes in X. laevis oocytes.
In vitro transcription, oocyte preparation and cRNA injection (30 ng per oocyte) were performed as described elsewhere25. The mMessage mMachine T7 transcription kit and MEGAclear kit (Ambion) were used to produce cRNA. Oocytes were surgically removed from female X. laevis frogs and then collagen digested using collagenase D (Roche). Stage V & VI oocytes were manually selected for injection. The uptake into oocytes of [14C]formate and [14C]lactate was measured (46 days post injection) at 27.5 C in media, which, unless specied otherwise, contained 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2,10 mM MES and 10 mM Tris-base, pH 6.4. Experiments were repeated using oocytes from different frogs (numbers are specied in the relevant gure legends), with each experiment using 810 oocytes for each condition tested.
Western blotting and immunouorescence in oocytes. Membranes were isolated from twenty HA-tagged PfFNT-expressing oocytes as described elsewhere25. Briey, 20 oocytes were homogenized in a homogenization buffer (10 mM NaCl, 1% Triton X-100 and 20 mM Tris-HCl), then centrifuged for 15 min, 15,000g at 4 C. A sample of the supernatant was diluted 1:12 then added to LDS NuPage buffer (Life Technologies) supplemented with 40 mM DTT and b-mercaptoethanol (1% v/v). Samples were separated on a 414% bis-Tris SDS/polyacrylamide gel (Life Technologies) and transferred onto nitrocellulose membranes. Western blots were carried out using a mouse anti-HA antibody (1:1,000) followed by horseradish peroxidase-conjugated goat anti-mouse antibody (1:10,000).
Immunouorescence analyses were performed on oocytes 5 days post injection using a protocol described elsewhere25. Mouse anti-HA antibodies (a gift fromF. Shannon, Australian National University) (1:100), followed by Alexa Fluor 488 goat anti-mouse antibodies (Molecular Probes; 1:500) were applied to xed oocytes. Oocytes were embedded in resin using the Technovit 7100 plastic embedding system (Kulzer) as described in detail elsewhere26 and images of 4 mm slices were obtained on a Leica confocal microscope. For each condition, slices were viewed from at least three different oocytes, from at least two separate experiments using eggs from different frogs.
Parasite culture. P. falciparum parasites (3D7 strain27) were maintained in continuous culture in Group O, Rh erythrocytes, as described previously28,
with some modications29, and were synchronized by sorbitol treatment30.
Parasite transfection. The MultiSite Gateway system (Life Technologies) was used to generate a transfection vector encoding a HA-tagged PfFNT protein.
The coding sequence was amplied using the forward primer 50-GGGGACACGT
TTGTACAAAAAAGCAGGCTAAAATCCAATGCCACCAAATAATTCC-30,
which contained the att1 site (underlined) and a start codon (bold). The reverse primer (50-GGGGACCACTTTGTACAAGAAAGCTGGGTAACAGTATTTCG
TAATTCTATAGAT-30) contained the att2 site (underlined). The resulting sequence was inserted into the vector pDONR221 (Life Technologies) using a BP clonase reaction (Life Technologies). The Gateway LR clonase reaction (Life Technologies) was then used to recombine the PfFNT clone with a 50 region from the gene encoding PfCRT (P. falciparum chloroquine resistance transporter) and with a construct encoding a 3 HA sequence. This generated a transfection vector
that contained the gene encoding PfFNT, the PfCRT gene promoter and a sequence encoding the 3 HA epitope tag (to be on either the N or C terminus of the
recombinant protein). Constructs were transfected into 3D7 P. falciparum parasites using eletroporation, as described previously, with WR99210 (5 nM) used to select for transfectants31.
Western blotting and immunouorescence in parasites. For immunouorescence assays, xed parasites were prepared using 4% w/v formaldehyde and0.0075% w/v gluteraldehyde, as described elsewhere31. Fixed cells were blocked using 3% w/v BSA and mounted using SlowFade gold (Molecular Probes). Mouse anti-HA antibodies (a gift from F. Shannon, Australian National University) and rabbit PfENT1 antibodies (a gift from B. Ullman, Oregon Health and Science University) were used at a concentration of 1:200, followed by Alexa Fluor 488 goat anti-mouse antibodies (Molecular Probes) and Alexa Fluor 633 goat anti-rabbit antibodies (Life Technologies), respectively, each at a concentration of 1:1,000. Parasites were viewed on a Leica confocal microscope.
For western blot analysis, a 10 ml parasitized erythrocyte suspension (15% parasitemia; 23% hematocrit) was centrifuged (3,000g, 5 min) and the pellet resuspended in 1 ml of phosphate-buffered saline (PBS) supplemented with Complete-mini EDTA-free protease inhibitor cocktail. Saponin was added (to a nal concentration of 0.09% w/v) to lyse the red blood cells and the sample was centrifuged (15,800g, 5 min). The pellet was washed twice with PBS supplemented with the protease inhibitor cocktail and then triturated 15 times through a 25G syringe. A 15 ml aliquot was combined with 147.5 ml of PBS supplemented with protease inhibitor cocktail, 25 ml of b-mercaptoethanol (giving a nal concentration of 10% v/v) and 62.5 ml NuPage sample loading buffer (giving a nal concentration of 25% v/v). Western blot analysis was performed as described for the preparations of oocyte membrane protein.
[14C]Formate and [14C]lactate uptake into isolated parasites. Mature trophozoite-stage parasites (3D7 strain, B3640 h post invasion) were isolated from the contents of their host erythrocytes by brief exposure of the parasitized cells to saponin (nal concentration 0.05% w/v, yielding a 0.005% w/v concentration of the active agent sapogenin), as described elsewhere3234. Saponin renders the erythrocyte and parasitophorous vacuole membranes permeable to macromolecules35 but leaves the parasite plasma membrane intact and able to maintain transmembrane ion gradients3234, as well as a substantial membrane potential36. Following saponin treatment, the isolated parasites were washed (43 times) and resuspended in a HEPES-buffered saline (125 mM NaCl, 5 mM
KCl, 1 mM MgCl2, 20 mM glucose and 25 mM HEPES, pH 7.1).
The uptake of [14C]formate (and, in one series of experiments, [14C]lactate) into isolated P. falciparum trophozoites was measured as described previously for nucleosides37. Briey, uptake of [14C]formate (or [14C]lactate) was initiated by the addition of a 200 ml volume of a suspension of isolated parasites to an equal volume of radiolabeled substrate solution (at twice the intended nal concentration) layered over a 200 ml dibutyl phthalate/dioctyl phthalate (5:4, v/v) oil mix. The nal cell concentration was 0.51 108 cells ml 1. All such experiments were carried out at
4 C (in a cold room) to slow the rate of uptake. Uptake was stopped at predetermined times by sedimenting the parasites below the oil using centrifugation (15,800g, 2 min) in a rapid-acceleration microcentrifuge (Beckman Microfuge E). Time points o15 s were sampled to the beat of a digital metronome. Where inhibitors or other substrates were used, these were added to the radiolabeled substrate solution before adding to the parasite suspension. The cell pellets were lysed using 0.1% (v/v) Triton X-100 and deproteinized with 5% w/v trichloroacetic acid before measuring the radioactivity in each sample using a scintillation counter37.
Estimates of the amount of radioactivity present in the extracellular uid trapped around the cell pellet was obtained by performing a short time course of radiolabelled uptake and extrapolating the time course data back to time-zero37. The radioactivity in the extracellular uid was subtracted from the total radioactivity in the pellet to provide an estimate of the amount of radiolabel within the cells. The concentration of radiolabel inside the cells was calculated by dividing the amount of radiolabel within the cells by the intracellular water volume of the pellet, calculated from the cell count using the previously reported value of 28 fL for the cell water volume of parasites isolated by saponin permeabilization of the host cell38. Time courses are presented in terms of distribution ratios (that is, the estimated intracellular [14C]formate concentration relative to the extracellular [14C]formate concentration).
pH measurements on isolated parasites. The cytosolic pH (pHi) of saponin-isolated trophozoite-stage parasites was measured (in parasites maintained at
B10 C) using the ratiometric pH-sensitive uorescent dye BCECF (Molecular Probes, Life Technologies), as described previously33.
Statistics. All errors cited and shown in the gures are s.e.m. All P values were obtained from one-way ANOVAs, using the pre-normalized uptake data (not the % of control data). In the case of experiments with parasites, the ANOVA was followed by a post hoc Tukey test. In experiments with Xenopus oocytes, there was considerable variation in formate/lactate uptake in oocytes from different frogs (that is, considerable variation between independent experiments). To prevent the inter-experimental variation from eroding the precision of the test, we nominated
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experiment as a blocking factor in our one-way ANOVAs. When the ANOVA resulted in a P value o0.05, post hoc comparisons were performed using the least signicant difference test.
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Acknowledgements
This work was supported by grants from the Australian National Health and Medical Research Council (NHMRC; 316933 and 525428 to K.K. and 1007035 to R.E.M.), and by the LOral Australia For Women in Science programme (R.E.M.). A.M.L. was supported by an NHMRC Overseas Biomedical Fellowship (585519) and R.E.M. was supported by NHMRC Australian Biomedical Fellowships (520320 and 1053082). We are grateful to the Canberra Branch of the Australian Red Cross Blood Service for the provision of blood, to Stefan Brer for providing the plasmid containing rMCT1, and to Terry Neeman for statistical advice.
Author contributions
R.V.M. designed and performed the immunolocalization experiments (in oocytes and parasites) and the majority of the transport experiments (in both oocytes and parasites). S.H.S. carried out a number of oocyte transport experiments and M.W. a number of parasite transport experiments. R.E.M. supervised the oocyte experiments and K.K. the parasite experiments. A.M.L. performed parasite transfection experiments and R.E.M. carried out the bioinformatics analyses. R.V.M., A.M.L., R.E.M. and K.K. wrote the manuscript.
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How to cite this article: Marchetti, R. V. et al. A lactate and formate transporter in the intraerythrocytic malaria parasite, Plasmodium falciparum. Nat. Commun. 6:6721doi: 10.1038/ncomms7721 (2015).
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Abstract
The intraerythrocytic malaria parasite relies primarily on glycolysis to fuel its rapid growth and reproduction. The major byproduct of this metabolism, lactic acid, is extruded into the external medium. In this study, we show that the human malaria parasite Plasmodium falciparum expresses at its surface a member of the microbial formate-nitrite transporter family (PfFNT), which, when expressed in Xenopus laevis oocytes, transports both formate and lactate. The transport characteristics of PfFNT in oocytes (pH-dependence, inhibitor-sensitivity and kinetics) are similar to those of the transport of lactate and formate across the plasma membrane of mature asexual-stage P. falciparum trophozoites, consistent with PfFNT playing a major role in the efflux of lactate and hence in the energy metabolism of the intraerythrocytic parasite.
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