Molecular Identification and Characterization of an Essential Pyruvate Transporter from Trypanosoma brucei*

Background: Pyruvate transport is vital for growth and survival of T. brucei bloodstream form. Results: TbPT0 is a unique monocarboxylate transporter with preference for pyruvate and essential for bloodstream form viability. Conclusion: TbPT0 represents the first molecular identification of essential pyruvate transporters in T. brucei. Significance: Unique T. brucei pyruvate transporters provide a potential drug target against the bloodstream form parasites. Pyruvate export is an essential physiological process for the bloodstream form of Trypanosoma brucei as the parasite would otherwise accumulate this end product of glucose metabolism to toxic levels. In the studies reported here, genetic complementation in Saccharomyces cerevisiae has been employed to identify a gene (TbPT0) that encodes this vital pyruvate transporter from T. brucei. Expression of TbPT0 in S. cerevisiae reveals that TbPT0 is a high affinity pyruvate transporter. TbPT0 belongs to a clustered multigene family consisting of five members, whose expression is up-regulated in the bloodstream form. Interestingly, TbPT family permeases are related to polytopic proteins from plants but not to characterized monocarboxylate transporters from mammals. Remarkably, inhibition of the TbPT gene family expression in bloodstream parasites by RNAi is lethal, confirming the physiological relevance of these transporters. The discovery of TbPT0 reveals for the first time the identity of the essential pyruvate transporter and provides a potential drug target against the mammalian life cycle stage of T. brucei.

Pyruvate export is an essential physiological process for the bloodstream form of Trypanosoma brucei as the parasite would otherwise accumulate this end product of glucose metabolism to toxic levels. In the studies reported here, genetic complementation in Saccharomyces cerevisiae has been employed to identify a gene (TbPT0) that encodes this vital pyruvate transporter from T. brucei. Expression of TbPT0 in S. cerevisiae reveals that TbPT0 is a high affinity pyruvate transporter. TbPT0 belongs to a clustered multigene family consisting of five members, whose expression is up-regulated in the bloodstream form. Interestingly, TbPT family permeases are related to polytopic proteins from plants but not to characterized monocarboxylate transporters from mammals. Remarkably, inhibition of the TbPT gene family expression in bloodstream parasites by RNAi is lethal, confirming the physiological relevance of these transporters. The discovery of TbPT0 reveals for the first time the identity of the essential pyruvate transporter and provides a potential drug target against the mammalian life cycle stage of T. brucei.
Trypanosoma brucei causes African trypanosomiasis in Saharan and sub-Saharan Africa. African trypanosomiasis is transmitted by the bite of the tsetse fly and is fatal if untreated, causing an economical and medical burden for African countries. Moreover, anti-trypanosomal therapy is far from ideal; it is expensive and toxic, and there is increasing incidence of drug resistance (1). Vaccination against these parasites is unlikely to be effective due to extensive antigenic variation of the variant surface glycoproteins (2). Therefore, there is an urgent need for new drug targets to develop more suitable anti-trypanosomal drugs.
In the past two decades, molecular approaches have helped to define targets for drugs in trypanosomes (3,4). Glycolysis has been studied as a potential target for drug development, because glycolysis is the only source of ATP for the bloodstream form of T. brucei (5). Under normal aerobic conditions, glucose is almost completely converted into pyruvate (Ͼ98%) rather than lactate due to the absence of lactate dehydrogenase. Moreover, pyruvate cannot be further metabolized, as BF 2 trypanosomes lack functional mitochondria and the Krebs cycle. The pyruvate is excreted into the bloodstream of the host by specific permeases and would otherwise accumulate to toxic levels inside of the parasite, ultimately causing cell death. This assertion is supported by the observation that inhibition of trypanosome pyruvate transporters by ␣-cyano-␤-(1-phenylindol-3yl)acrylate (UK5099) resulted in retention and concomitant accumulation of pyruvate within the trypanosomes, causing acidification, osmotic destabilization, and trypanosome death (6,7). Consequently, plasma membrane pyruvate transporters are important for detoxification and survival of the parasite in the mammalian host.
Simple monocarboxylates are substrates for bidirectional facilitated diffusion via monocarboxylate transporters (MCTs) and are co-transported with protons in an equimolar manner by a symport mechanism (8,9). The physiological role of MCTs is primarily to transport the endogenous monocarboxylates such as lactate, pyruvate, acetoacetate, and ␤-hydroxybutytrate. Currently, 14 members of the MCT family have been identified by sequence homology, seven have been functionally characterized in mammals (10,11), and orthologues have been identified in a variety of species such as S. cerevisiae (12).
The presence of pyruvate transporters in the plasma membrane of T. brucei has been established by biochemical studies demonstrating their physiological importance (6,7,13,14). However, the molecular identification of these important transporters has eluded discovery over an extended period of time. In the present study, I report the cloning and functional analysis of a T. brucei gene that encodes a novel pyruvate transporter TbPT0, which belongs to a multigene family of five members arranged in tandem and encoding distinct but very similar proteins. Notably, inhibition of TbPTs expression by RNAi is lethal, underscoring their critical function for survival and their potential as drug targets. Growth and Transfection of T. brucei Cell Lines-BF and PF T. brucei cell lines were grown as described previously (15). For T7 RNA polymerase-independent driven expression (16), BF T. brucei 427 parasites transfected with pHD1313 (plasmid kindly provided by Dr. Christine Clayton) (17) expressing the tetracycline repressor were generated and grown in 2.5 g ml Ϫ1 bleomycin. 5-10 g of linearized plasmid DNA was used to transfect mid-log phase BF parasites as described (18). Inducible expression experiments employing the plasmid p82M3HAdriven expression by the T7 promoter were performed using the transgenic BF single marker (SM) clone as described previously (18).

EXPERIMENTAL PROCEDURES
RNA Isolation and Northern Blot-Total RNA from T. brucei parasites was isolated using RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. Northern blot analysis was performed as described elsewhere (19). For autoradiographic detection the Storm 825 scanner (GE Healthcare) was employed and NIH ImageJ software for densitometry analysis. Adobe Photoshop CS3 and Adobe Illustrator CS3 were used to create figure composition.
Cloning by Genetic Complementation and Isolation of Plasmids-Chemically competent JMY75 cells were transformed with a cDNA library from T. brucei Lister 427 BF parasites cloned in the yeast expression vector p416Met25 (21) kindly provided by Dr. Hosam Shams-Eldin. Transformed yeast were grown at 30°C on YNB-ura agar plates containing 2% ethanol, 10 mM pyruvate and supplemented for auxotrophic requirements. Plasmids from positive yeast clones were isolated using PureLink Quick Plasmid Miniprep kit (Invitrogen) following the manufacturer's instructions. Then, plasmids were transformed into Escherichia coli DH5␣ competent cells (Invitrogen) for amplification and were isolated and sequenced by the Core Facility of the Department of Molecular Microbiology & Immunology at the Oregon Health & Sciences University using an Applied Biosystems 16-capillary 3130xI Automated Sequence Analysis System.
Deduced Amino Acid Sequence Analysis-For DNA sequence analysis of TbPT0 through TbPT5 and amino acid sequence alignments, MacVector software (Intelligenetics) was used. Transmembrane segments were predicted by the TMHMM server (version 2.0). Sequence similarity analysis was performed using the BLAST program (version 2.0).
Uptake Assays in Yeast-JMY75 yeast harboring the TbPT0 cDNA clone was employed for kinetic analysis. Also, the pyruvate transport-deficient yeast 5067 (⌬jen1) was transformed with the TbPT0 ORF subcloned into the constitutive expression vector pYADH (22) for functional experiments. Uptake of [ 14 C]pyruvate was assayed by incubation of 5 ϫ 10 7 cells with radiolabeled substrate followed by centrifugation through a cushion of dibutyl phthalate as described (22). For kinetic analysis, uptake was measured for a range of substrate concentrations over a time course ranging from 0 to 60 s. Initial uptake rates at each concentration were determined by linear regression analysis over the linear portion of the time course (Prisam, version 4, GraphPad Software, Inc.). These data were fitted to the Michaelis-Menten equation by non-linear regression using the Prism 4 (GraphPad Software, Inc.), and this fit was used to determine K m values. Assays for inhibition utilized a 1-min incubation with 26 M [ 14 C]pyruvate in the presence of the indicated competitors. K i values were estimated by fitting the data to one site competition equation (23) employing Prism (version 4, GraphPad Software, Inc.).
Inhibition of Gene Expression by RNAi-To determine the role of pyruvate transporters in the survival of the BF parasite, a 428-bp gene fragment corresponding to position 632 to 1059 bp of TbPT0 determined by RNAit software was employed. The TbPT-RNAi construct was linearized and transfected into BF/pHD1313 cell line as described to generate BF/TbPT-RNAi clone. RNAi clones were selected by resistance to 2.5 g ml Ϫ1 of bleomycin and 5 g ml Ϫ1 of blasticidin and hairpin loop RNAi induced by addition of 1 g ml Ϫ1 doxycycline.
Inducible Expression of TbPT0-3HA-COOH SM Cell Line and Immunofluorescence Microscopy-To generate TbPT0-3HA-COOH tagged transporter the TbPT0 ORF was subcloned into the plasmid p82M3HA (18) for inducible expression driven by the T7 promoter upon addition of doxycycline. SM parasites were transfected with 5 g of linear NotI-digested construct as described (18) selected on 2.5 g ml Ϫ1 bleomycin and 2.5 ng ml Ϫ1 doxycycline, and cloned by limiting dilution. To induce TbPT0-3HA-COOH, 1 g ml Ϫ1 doxycycline was added to the cell culture and incubated for 48 h. Induced parasites (5 ϫ 10 6 ) were centrifuged at 700 ϫ g for 4 min and washed twice at room temperature with PBS, pH 7.2 containing 10 mM glucose. The cell pellet was resuspended in 4% paraformaldehyde in PBS, pH 7.2, and incubated for 15 min at room temperature, cells were centrifuged as described above and washed once with 10 mM glycine in PBS and once with PBS, resuspended in 100 l of PBS, spotted onto poly-L-lysine-coated coverslips, and blocked with 2% goat serum, 0.01% sodium azide, 0.01% saponin in PBS (blocking solution) for 1 h at room temperature, rinsed three times with PBS and incubated with a 1:500 dilution of the primary mouse anti-HA monoclonal antibodies (Covance, Inc.) for 1 h at room temperature. Then, cells were rinsed as before and incubated with a 1:1000 dilution of goat anti-mouse IgG Alexa Fluor 495 TM (Molecular Probes, Invitrogen) in blocking solution for 1 h at room temperature in the dark. For detection of ␣-tubulin as subpellicular plasma membrane marker, a second round of immunodetection was performed as mentioned above using 1:1000 dilution of the primary mouse anti ␣-tubulin monoclonal antibodies (Sigma-Aldrich) and 1:1000 dilution of goat anti-mouse IgG Alexa Fluor 580 TM (Molecular Probes, Invitrogen). Coverslips were rinsed three times with PBS and mounted onto slides using ProLong® Gold antifade reagent with DAPI (Invitrogen). Fluorescence images were obtained using a wide field deconvolution system (Applied Precision Instruments, Inc.) consisting of an inverted Nikon TE 200 Eclipse microscope, a Kodak CH350 CCD camera, and the Deltavision operating system. Images were acquired using a 60ϫ objective in a 1024 ϫ 1024 format and deconvolved with nine iterations using SoftWoRx software. Adobe Photoshop CS3 and Adobe Illustrator CS3 (Adobe Systems, Inc.) were used to create image compositions.

RESULTS
Cloning of a Pyruvate Transporter Gene by Genetic Complementation of Pyruvate-auxotrophic Yeast-Pyruvate transport has been characterized in T. brucei BF parasites by biochemical means employing whole cells (6,13). Those studies also highlighted the importance of pyruvate efflux to maintain optimal physiological conditions for parasite survival. However, the molecular identity of such transport proteins has been evasive, precluding molecular studies on this essential cellular function. To identify a bona fide pyruvate transporter, a T. brucei bloodstream form cDNA library, previously used to clone other trypanosomal genes (21,24,25), was transformed into the pyruvate auxotrophic mutant of S. cerevisiae JMY75 (20). The JMY75 yeast mutant, deficient in pyruvate kinase, malic enzyme, and pyruvate transporter, can use pyruvate as an efficient carbon source only if the pyruvate transport deficiency conferred by the jen1⌬ mutation is complemented by expression of a functional pyruvate permease. Thus, transformants were selected on YNB agar plates (ϩTrp, ϩHis, -ura, 2% ethanol) containing 10 mM pyruvate as the sole energy source. After 4 days, three yeast colonies, called A, B, and C, grew on the selective plates. To confirm the phenotype, these clones and the JMY75 host strain were streaked on selective medium. The three cDNA clones were able to rescue the growth of the JMY75 mutant, whereas negative control non-transformed JMY75 mutant did not (Fig. 1A), confirming the ability of these three clones to rescue the pyruvate auxotrophy. Plasmid DNA from each clone was isolated and sequenced. Clones A and C contained the same cDNA insert including the spliced leader sequence, a 105-bp 5Ј-UTR, an ORF encoding a 595-amino acid protein, a 364-bp 3Ј-UTR, and a poly(A) tail (Fig. 1B). The deduced amino acid sequence obtained from the cDNA ORF is named T. brucei pyruvate transporter 0 (TbPT0) and shown in Fig. 1B. Clone B contained a cDNA insert different from A and C.
A BLAST search of the T. brucei genome database using the TbPT0 amino acid sequence as query revealed significant identity (Table 1)  TbPT1, TbPT2, TbPT3, TbPT4, and TbPT5, respectively. Moreover, a homologous TbPT gene family is present in the T. brucei Lister 427 and other T. brucei species database. All five TbPT genes are clustered together in chromosome 3, and their expression is up-regulated ϳ13-fold in the BF compared with PF as previous transcriptome analysis reported, albeit some members of the family are preferentially up-regulated (26 -29). However, clone B revealed significant identity to pyruvate kinase, PYK1 (high score of 1034 and P (N) 4.9 ϫ 10 Ϫ106 (the probability of a chance alignment occurring with a better score in a database search); Tb927.10.14140), which genetically complemented the deficiency of the pyruvate kinase gene in JMY75 yeast.
Amino Acid Sequence Analysis of TbPT0-TbPT5-In silico analysis also revealed that all of the TbPT family members encode highly conserved proteins with at least 84% identity and different lengths. Analysis of the genomic 5Ј-flanking sequences revealed another ATG upstream of the annotated (TriTrypDB database) starting ATG for TbPT1, TbPT2, TbPT3, and TbPT4 genes, similar to the ORF of TbPT0 cDNA and TbPT5. Multi-alignment of these predicted proteins and TbPT0 demonstrates the high identity among them, beginning from the upstream ATG (Fig. 2). Of note, Siegel et al. (30), in their studies to determine the transcriptional landscape of T. brucei BF and PF employing RNA-seq technology, predicted that this first ATG in TbPT1 through TbPT4 is preferentially used to encode the larger ORF. Moreover, the algorithm TMHMM (version 2.0) predicts that all five transporters encompass 13-14 transmembrane domains (TMs). Most of the amino acid differences between the TbPT family reside in the first transmembrane domain and the first putative extracellular loop or in the distinctive COOH termini for TbPT2 and TbPT4. Interestingly, the TbPT0 gene cloned by functional complementation (from T. brucei Lister 427 cDNA library) is not 100% identical to any of the TbPTs annotated in the T. brucei genome database (including T. brucei Lister 427 and strain 927) consistent with genetic polymorphism among these T. brucei strains, including the Lister 427 isolate employed for the cDNA library versus that used for the genomic sequence. A BLAST search of GeneBank employing TbPT0 as query sequence revealed significant similarity to hypothetical nodulin-like proteins from plants, which also predict to be members of the major facilitator superfamily of transporters with the highest scoring for the protein with accession no. EAY83124.1 (85.2 and P (N) 3 ϫ 10 Ϫ14 (the probability of a chance alignment occurring with a better score in a database search)) from Oryza sativa. However, no similarity was found to any known mammalian monocarboxylate transporter. A multi-alignment of 17 protein sequences, including 11 among the highest scores based on the BLAST results plus the rat monocarboxylate transporter (MCT1), yeast lactate transporter (JEN1), two nodulin proteins from Arabidopsis thaliana (NP2.1 lactate transporter and Nod26 aquaporin), betaine transporter from rabbit, and TbPT0 was performed using ClustalW software, and an evolutionary tree was generated (Fig. 3), suggesting a common ancestor for nodulin-like proteins from plants and trypanosome pyruvate transporters. Interestingly, NP2.1, a lactate transporter, which is up-regulated by oxygen deprivation in A. thaliana (31), also belongs to the generic nodulin-like proteins albeit with only 6 TMs, whereas all of the putative proteins in the TbPT0 clade predict 12-14 TMs. Of interest, a signature sequence localized in the TM13-TM14 loop composed of six amino acids, MYDAEA, is present in all the TbPTs (Fig. 2, double underlining) and also is highly conserved in all the proteins of the TbPT0 clade (Fig. 3). However, this sequence signature is not present in MCT1, JEN1, betaine transporter, Nod26, and NP2.1 lactate transporter. Moreover, BLAST searches employing TbPT0 sequence revealed the presence of highly similar predicted proteins in Trypanosoma cruzi and Leishmania major but not in Plasmodium falciparum or Toxoplasma gondii (Table 1), suggesting that those orthologues might exert similar biochemical functions in those kinetoplastids.

TABLE 1 TbPT0 homologs in T. brucei and orthologs in T. cruzi and L. major
The alignment (score) and probability (P(N)) scores are indicated for a BLASTP search employing TbPT0 as query.

M R M L M A G V Y A G L G V S K A Y C F S I F T P F L R S K F N M T Q G D I T T I S T V G T C M L Y M R M L M S G M Y A E L G V S K A Y C F S I F T P F L K K K L N L T Q T E V T T I S T V G N C I M Y M L M A G V Y A G L V V S S T Y G F S V F A A H L R E K Y R L S Q S D I T T I S T V G N C I G Y M L M A G V Y A G L V V S S T Y G F S L F S V Y M R K Q Y R L S Q S E I T T I S T V G N C I G Y M R M L M A G V Y A G L G V S K A Y C F S I F T P F L R S K F N M T Q G D I T T I S T V G T C M L Y M Y I I D D L A R V R M L A S G V Y T M A S T A A P Y C F T L M A V L L R K K Y R L S Q S E I A T I S T V G N
TbPT Gene Family Is Essential for Survival of BF Parasites-To determine the physiological relevance of TbPTs, the expression of the TbPT gene family was inhibited using a hairpin RNAi construct. The 428-bp DNA fragment used for RNAi experiments exhibits Ͼ90% identity between each of the TbPT family members, allowing simultaneous knock down of all five genes. Initially, a transgenic BF clone expressing the tetracycline repressor was generated by transfection of BF parasites with a linear pHD1313 that harbors two copies of this gene separated by the bleomycinresistant marker (17) and integrated in the tubulin locus under expression of the endogenous promoter. This transgenic BF/1313 clone was used as host cell for subsequent experiments. Then, BF/TbPT0-RNAi cells were generated by transfection of BF/pHD1313 clone with the TbPT0-RNAi construct employing the p3666 vector (16) that transcribes the hairpin loop RNAi unit driven by the EP procyclin promoter fused to four tetracycline operators. Doxycycline was added to induce synthesis of hairpin loop RNA, and the cell number was quantified over time (Fig. 6A,  filled circles). Upon induction of RNAi, parasite growth was severely compromised, causing growth arrest and eventually cell death after 2 weeks. In contrast, parasites growing in the absence of doxycycline grew robustly (Fig. 6A, open circles). Northern blot analysis demonstrates that TbPT gene family mRNA level is reduced ϳ7-fold after 48 h of RNAi induction (Fig. B, upper left  panel), whereas tubulin RNA level remains unchanged (Fig. 6B, lower left panel). Similar analysis employing the transgenic BF/1313 clone as control were performed revealing that both TbPT family mRNA and tubulin mRNA levels did not change (Fig.  6B, upper right and lower right panels). Also, the BF/1313 host did not show any phenotypic change growing in the presence or absence of doxycycline (not shown). These results demonstrate that knocking down TbPT gene family expression caused cell death, presumably because these parasites were unable to export pyruvate. Moreover, RNA interference target sequencing analysis by Alsford et al. (33) revealed that at least two members of the TbPT gene family are essential in BF, and at least one of them is crucial in PF trypanosomes. All of these results emphasize the essential physiological role of these transporters in BF parasites. Subcellular Localization of TbPT0-All of the members of this gene family encode proteins with 13-14 predicted TMs, strongly suggesting membrane localization. To determine subcellular localization of TbPT0, a triple hemagglutunin epitope fused to its COOH terminus was constructed, and the tagged protein was expressed in BF parasites from the inducible expression vector. TbPT0-3HA fluorescence signal was detected primarily in the plasma membrane (Fig. 7A), as indicated by the overlap with ␣-tubulin signal from the subpellicular microtubules (Fig. 7, B and C).

DISCUSSION
For more than two decades, it has been known that in the mammalian infective form of T. brucei, BF slender form, pyruvate is not reduced to lactate or oxidized to CO 2 because of the lack of lactate dehydrogenase and a functional mitochondrion (6,7,13,34,35). Pyruvate is the final product of glycolysis and has to be extruded to the extracellular milieu by specific transporters localized in the plasma membrane of the parasite to maintain the integrity and viability of the cell. In 1995, Weiner et al. (7) demonstrated that inhibition of pyruvate transport by UK5099, a universal pyruvate transport inhibitor, causes severe physiological alterations and ultimately leads to cell death,  whereas Barnard et al. (6) showed that the subversive pyruvate analogue, bromopyruvate, enters the trypanosomes via pyruvate permeases targeting GAPDH, consequently affecting cell motility and pyruvate efflux and causing death of the parasites. Together, those previous findings highlighted the physiological importance and the pharmacological relevance of pyruvate transporters. However, the molecular identity of pyruvate transporters has been elusive, hampering their thorough molecular and biological analysis that would define these permeases as promising drug targets. In the present study, I report the cloning, functional expression, and characterization of TbPT0 that encodes a unique pyruvate transporter of T. brucei. Due to its bidirectional transport ability, TbPT0 is able to rescue the mutant phenotype of the pyruvate auxotrophic JMY75 yeast clone (Fig. 1). Thus, TbPT0 is a bona fide pyruvate transporter. Genetic complementation in yeast is a powerful technique that has been applied to the cloning of other trypanosomal proteins (21,24,25). For instance, Mäser et al. (24) cloned the gene coding for the TbAT1 adenosine/adenine/ pentamidine transporter using a yeast mutant deficient in adenine synthesis. Functional expression of TbPT0 in S. cerevisiae revealed that TbPT0 is a reasonably high affinity pyruvate transporter (Fig. 4A, 4B), supporting its important physiological function in efflux of pyruvate from the BF cytosol. Also, specific inhibition of pyruvate transport mediated by TbPT0 upon addition of UK5099, a proven trypanosomal pyruvate transport inhibitor (Fig. 4D) (7), supports the identity of TbPT0 as a critical pyruvate transporter. In addition, the ability of TbPT0 to transport acetate (Fig. 5A) suggests another potentially important physiological function mediated by the TbPT family. Recently, Tielens et al. (36) reported that short stumpy BF parasites (as opposed to long slender BF) generate acetate and express the mitochondrial enzyme acetate:succinate CoA transferase, demonstrating that this life cycle stage also uses in addition to glycolysis a mitochondrial pathway for catabolism of glucose and ATP generation, yielding acetate and pyruvate as end products of glucose metabolism. Possibly, TbPT family members are involved in the efflux of pyruvate and acetate generated by the short stumpy BF parasites. Moreover, the relatively low affinity exhibited by TbPT0 for lactate, as well as the absence of a trypanosome LDH that could generate lactate, suggests that this lactate transport activity is not physiologically relevant.
TbPT0 and all of the members of the TbPT family are phylogenetically more related to nodulin-like proteins from plants then to mammalian monocarboxylate transporters (Fig. 3). Analysis of the alignments between TbPTs and the protein sequences with the highest similarity (data not shown) identifies a shared signature sequence, MYDAEA, in the last predicted extracellular loop, whereas neither MCT1 (37) nor NP2.1, an A. thaliana lactate transporter (31) encompassed this motif. Such a conserved signature sequence might play a significant role in the transport specificity or kinetics and perhaps be relevant for structure function studies, which will be addressed in the future. Members of the same family of transporters sometimes have distinct biochemical functions despite their high identity (15). A complete biochemical analysis of all of the members of the TbPT family will determine whether all of these have the same substrate specificities or whether some permeases have additional or alternative substrates.
Similarities between trypanosomal and plant proteins have been documented previously by Hannaert et al. (38,39), suggesting that horizontal genetic transfer occurred during evolution impacting mostly proteins involved in trypanosome glucose metabolism. Recently, Matthews and co-workers (40) identified a plasma membrane protein from T. brucei involved in sensing cis-acconitate, which triggers differentiation from BF to PF parasites, and also showing high similarity to plant permeases. Therefore, TbPTs are trypanosomal proteins with a possible plant origin that significantly differ from their mammalian counterparts (41) and perform an essential physiological function. The uniqueness of TbPTs makes them attractive A B C FIGURE 7. Localization of epitope tagged TbPT0 in BF trypanosomes. Trypanosomes expressing TbPT0 fused to a triple hemagglutinin tag at its COOH terminus (TbPT0-3HA) were immunostained with anti-HA tag antibody (A) and anti-␣-tubulin antibody (B); the merged images are shown in C.
for pharmacological intervention because the absence of human orthologues to TbPTs may facilitate the discovery of selective inhibitors. In addition, the yeast expression system described here could be employed to screen for potent inhibitors of TbPTs.
The expression of the TbPT gene family is up-regulated at the RNA level about 13-fold in the BF parasites as demonstrated by previous published studies on transcriptomes of T. brucei strains comparing BF with PF parasites (26 -28, 30). In addition recent publications by Urbaniak et al. (42) and Butter et al. (43) have shown using genome-wide proteomic analysis that members of the TbPT family of transporters are significantly up-regulated in BF parasites. Moreover, 3ϫ HA-tagged TbPT0 targets to the plasma membrane of the BF parasites (Fig. 7, A and C), consistent with its essential pyruvate efflux function. Also experiments by Barnard et al. (6,34) determined that pyruvate efflux in PF parasites is ϳ100-fold lower compared with the BF parasites mainly due to a down-regulation of glycolytic enzymes, particularly PK which is decreased 25-fold, in addition to further metabolism of pyruvate yielding low levels of cytosolic pyruvate.
Down-regulation of the expression of the TbPT gene family by RNAi (Fig. 6, A and B) resulted in a lethal phenotype in BF trypanosomes. The high sequence identity between members of this family makes it difficult to assess, by RNAi, the relative contribution of each family member to this lethal phenotype. However, the most conservative interpretation at present would be that all TbPT isoforms likely contribute to pyruvate efflux and that coordinate knockdown of all five transcripts probably contributes to cell death. Cell growth arrest was initially observed followed by parasite death after 2 weeks, highlighting an essential role of the TbPT gene family. This result implies that either the absence or a reduced level of TbPTs in the BF plasma membrane impairs pyruvate efflux, causing a cascade of negative physiological effects and ultimately cell death. The definition of these pyruvate transporters presents a potential target for pharmacological intervention in human African trypanosomiasis.