Arginine and Lysine Transporters Are Essential for Trypanosoma brucei

For Trypanosoma brucei arginine and lysine are essential amino acids and therefore have to be imported from the host. Heterologous expression in Saccharomyces cerevisiae mutants identified cationic amino acid transporters among members of the T. brucei AAAP (amino acid/auxin permease) family. TbAAT5-3 showed high affinity arginine uptake (Km 3.6 ± 0.4 μM) and high selectivity for L-arginine. L-arginine transport was reduced by a 10-times excess of L-arginine, homo-arginine, canavanine or arginine-β-naphthylamide, while lysine was inhibitory only at 100-times excess, and histidine or ornithine did not reduce arginine uptake rates significantly. TbAAT16-1 is a high affinity (Km 4.3 ± 0.5 μM) and highly selective L-lysine transporter and of the compounds tested, only L-lysine and thialysine were competing for L-lysine uptake. TbAAT5-3 and TbAAT16-1 are expressed in both procyclic and bloodstream form T. brucei and cMyc-tagged proteins indicate localization at the plasma membrane. RNAi-mediated down-regulation of TbAAT5 and TbAAT16 in bloodstream form trypanosomes resulted in growth arrest, demonstrating that TbAAT5-mediated arginine and TbAAT16-mediated lysine transport are essential for T. brucei. Growth of induced RNAi lines could partially be rescued by supplementing a surplus of arginine or lysine, respectively, while addition of both amino acids was less efficient. Single and double RNAi lines indicate that additional low affinity uptake systems for arginine and lysine are present in T. brucei.


Introduction
Trypanosomes are unicellular, flagellated eukaryotic parasites that cycle between different hosts and cause severe diseases. Trypanosoma brucei subspecies cause human African trypanosomiasis and also affect wild game and, more severely, cattle [1]. Recent estimates from the Food and Agriculture Organization on economic losses of agricultural gross domestic product due to animal trypanosomiasis reaches US$ 4.75 billion per year [2]. T. brucei are transmitted a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 to mammalian hosts through the bite of an infected tsetse fly. The environments change dramatically upon transfer from the insect to the mammalian host or vice versa, and trypanosomes must adapt nutrient uptake accordingly. In the insect host, glucose is limited and therefore the trypanosomes rely on amino acids as energy source, whereas in the vertebrate host, trypanosomes use glucose as energy source and oxidative phosphorylation is absent [3,4]. Amino acid uptake is of fundamental importance for trypanosomes as they are auxotroph for a number of amino acids, including the proteinogenic amino acids arginine and lysine [5].
Increased transport activity for arginine upon amino acid starvation was found in parasites and in mammalian cells. Leishmania arginine transporter LdAAP3 mRNA and protein levels were elevated in promastigotes when starved for arginine. This process looks similar to mammalian cationic amino acid transporter 1 (CAT-1) regulation upon deprivation of amino acids [6]. It has been suggested that LdAAP3 contributes to parasite virulence by increased arginine uptake [6]. The arginine-deprivation response resulting in LdAAP3 up-regulation was recently shown to be mediated through a MAPK2-dependent signaling pathway [7]. While arginine is important for polyamine biosynthesis in Leishmania, the absence of a functional arginase homolog and the essentiality of ornithine decarboxylase (ODC) in T. brucei indicate that the latter rely on ornithine as precursor for polyamine biosynthesis [8][9][10][11], though in T. brucei ornithine synthesis from arginine seems possible when ornithine is limiting [12]. Furthermore in T. cruzi and T. brucei, arginine kinase converts arginine in an ATP-dependent reaction to phosphoarginine, contributing to the energy buffering of cells [13,14].
Arginine and lysine transporters belonging to the AAAP family have been characterized in Leishmania and T. cruzi [6,[22][23][24][25]. Interestingly, most of the functionally characterized L. donovani and T. cruzi arginine and lysine transporters are highly selective for their substrates. Expression of Leishmania LdAAP3 and T. cruzi TcAAAP411 in Saccharomyces cerevisiae mutants showed high selectivity and high affinity for arginine, i.e. K m of 1.9 μM and 32 μM, respectively [22,23], and the affinity of LdAAP7 for lysine was 7.4 μM [24]. Recently the K m of T. cruzi CAT1.1 for arginine was shown to be 85 μM, while the same transporter showed lower affinity for ornithine (K m 1.7 mM) [25].
In contrast to the transporters for lysine or arginine characterized from parasites, cationic amino acids are transported by the same transport system in mammalian cells, i.e. members of SLC7 family, also referred to as the APC (TC 2.A.3) transporter family [26]. While rBAT, a member of the L-type amino acid transporter (LAT) subgroup, mediates exchange of cationic amino acids and cysteine against neutral amino acids, mammalian CAT1 to 3, which are members of the cationic amino acid transporters (CAT) subgroup, mediate facilitated uptake of cationic amino acids and are trans-stimulated to various degrees [26]. Apparent affinities of CAT members for arginine, lysine and ornithine are comparable and in the high affinity (40-450 μM; CAT1, CAT2B, CAT3) or low affinity (2-5 mM; CAT2A) range [27]. Only histidine is recognized differently by these transporters.
In the present work we report the identification and functional characterization of high affinity arginine and lysine permeases from T. brucei and show that in contrast to the mammalian transporters for cationic amino acids, the T. brucei arginine and lysine transporters are highly selective. Furthermore, we show that uptake of both, arginine and lysine, is essential for parasite survival in culture.

Materials and Methods
Animal experiments (in Prof. E. Sigel's lab) were carried out in strict accordance to the Swiss ethical guidelines, and have been approved by the local committee of the Canton Bern Kantonstierarzt, Kantonaler Veterinärdienst Bern (BE85/15). Surgery of female adult Xenopus laevis was done under anesthesia (0.2% tricaine solution). Oocytes were prepared, injected and defolliculated as described previously [28].

Phylogenetic analysis
Annotated amino acid transporters (after [17]) in the genome of T. brucei TREU 927 within TriTrypDB were screened for Pfam families. Most amino acid transporters were part of Pfam family PF01490, Aa_trans (Transmembrane amino acid transporter protein). This family was used to screen the best-curated genomes in trypanosomatids, namely Trypanosoma cruzi CL Brener non-Esmeraldo-like, T. cruzi CL Brener Esmeraldo-like, Leishmania major strain Friedlin and T. brucei TREU 927, for genes that are members of this family. Using the coding sequences of the resulting 109 genes, we generated a codon-based alignment using MUSCLE [29]. Based on this analysis, we grew a phylogenetic tree by the Neighbor-Joining method. Both of these steps were carried out using the MEGA6 software [30]. Based on this tree, a limited tree showing representative genes was generated. The representative genes include: genes studied here, their homologs in the aforementioned genomes, characterized homologs from T. cruzi, L. donovani as well as TbAAT6 [21][22][23][24][25]. The amino acid sequences were used to calculate pairwise global alignment scores using the Needleman-Wunsch algorithm utilizing the BLOSUM62 matrix, a gap-opening penalty of 10 and a gap extension penalty of 0.5.

Saccharomyces cerevisiae transformation
Transformation of S. cerevisiae was performed according to [31] [33]) was grown in minimal medium [MM: 1.7 g L -1 yeast nitrogen base without amino acids and without ammonium sulfate (Difco), and 20 g L -1 glucose] containing 1 g L -1 urea.
For tightly-regulated inducible expression of cMyc-TbAAT5 and cMyc-TbAAT16, the ORFs were cloned into a modified pLEW100 vector [36], in which the phleomycin resistance gene has been replaced by the puromycin resistance gene and the triple cMyc cassette [39] was inserted to allow N-terminal tagging [40].

Transport assays
Transport assays in S. cerevisiae strain 21.983c and 22Δ7AA were performed as described previously [41] with slight modifications. Cells were grown to a density of OD 578 up to 0.6, washed twice with water and resuspended in buffer A (1/10 initial volume; 0.6 M sorbitol and 50 mM potassium phosphate, different pH values). Prior to the transport assay, cells were preincubated at 30˚C for 6 min in the presence of 100 mM glucose. To start the transport assay, cells (100 to 130 μl) were added to an equal volume of buffer with different concentrations of Larginine or L-lysine and 3.7-18. Samples (48 μl) were transferred after 30 sec, 1 min, 2 min, 4 min (and 6 min) to 4 ml icecold buffer A, filtrated on glass fiber filters and washed twice with 4 ml ice-cold buffer A. The uptake of tritium-labeled substrates was determined by liquid scintillation spectrometry. In all experiments, uptake was linear over the time period investigated and transport rates were calculated from at least four different time-points. Uptake rates of S. cerevisiae transformed with 'empty vector' was subtracted as background. Kinetic parameters were calculated using the Michaelis-Menten equation V = V max x [S] x (K m + [S]) -1 .
For TbAAT16, three variants were found; the first amino acid sequence corresponded to the database annotation of Tb427tmp.01.7500; the second Tb427tmp.01.7500_V343G, differed from Tb427tmp.01.7500 in seven nucleotides, but only one (nt 1028) resulted in a single amino acid difference, i.e. a valine at position 343 was replaced by glycine; the third, Tb427tmp.01.7520_G343V, differed from the database annotation of Tb427tmp.01.7520 in one amino acid. Since Tb427tmp.01.7520 was not amplified in our analysis, it was generated by site directed mutagenesis of Tb427tmp.01.7520_G343V (S3 Table).
Comparable to predictions for other members of the AAAP family [47], membrane topology prediction platforms (Phobius [48] and HMMTOP [49]) suggested 11 transmembrane domains for the full-length TbAAT5 proteins and 10 or 11 transmembrane domains for TbAAT16 proteins.
None of the ORFs mediated growth on any of the other tested amino acids (i.e. proline, alanine, cysteine, γ-aminobutyric acid, glutamate, aspartate, methionine, phenylalanine, valine, isoleucine, leucine, tryptophan, tyrosine, threonine and citrulline, data not shown). These results indicated that TbAAT5 and TbAAT16 are selective cationic amino acid transporters.
As it is unclear whether the different single nucleotide polymorphisms represent strain differences or may originate from (fusion) PCR events, for further functional analysis we decided to focus on one representative each that corresponded to the database amino acid sequence, i.e. Tb427.08.4720 (TbAAT5-3) and Tb427tmp.01.7500 (TbAAT16-1).
Members of the AAAP family in plants are known to be driven by the proton motive force [50], while mammalian AAAP members may use either proton or sodium [51]. In general, uptake of different substrates in trypanosomes is proton-coupled, e.g. nucleosides [52], choline and myo-inositol [53,54], and amino acids [21]. Between pH 4.5 and 7, and using 50 μM Larginine, the arginine uptake rates were only slightly pH-dependent, indicating that TbAAT5-3 may mediate arginine uptake in different pH environments (Fig 3A). TbAAT16-1 showed highest 50 μM L-lysine transport rates at pH 5.5. At pH 4.5 and 6.5 the relative transport rate was approximately 80% while at pH 7.5, lysine uptake was reduced to 50% (Fig 3B).
To determine the dependence on Na + and K + , substrate-induced currents were analyzed in Xenopus laevis oocytes injected with TbAAT5-3 or TbAAT16-1 cRNA using two-electrode voltage clamping. While no currents could be recorded for TbAAT16-1 when using up to 10 mM lysine, arginine or histidine as substrates (pH 7.4 and pH 5.5), in oocytes injected with TbAAT5-3 cRNA, the addition of 50 μM arginine in Na + -Ringer induced small inward currents in the range of 15 to 90 nA (Fig 4B, V m -80 mV, pH 7.4). Comparable currents were measured when Na + and K + were replaced by choline (choline Ringer, Fig 4C). These findings indicate that arginine transport via TbAAT5-3 does not depend on extracellular Na + and K + .  TbAAT5-3 and TbAAT16-1 mediate high affinity, selective transport of arginine and lysine, respectively Transport assays performed using S. cerevisiae expressing TbAAT5-3 revealed high affinity transport of L-[ 3 H]-arginine, with a K m of 3.6 ± 0.4 μM (strain 21.983c, Fig 5A), while expression of TbAAT16-1 showed high affinity transport of L-[ 3 H]-lysine, with a K m of 4.3 ± 0.5 μM (strain 22Δ7AA, Fig 5B).

TbAAT5 and TbAAT16 are highly expressed and localized at the plasma membrane
TbAAT5 and TbAAT16 transcripts were abundant in both PCF and BSF T. brucei (Fig 6). Due to high sequence homology only Tb427.08.4700, which has a distinct 3'UTR, could be distinguished from the other TbAAT5 members (see S1 Fig). Transcriptome analyses support high transcript levels of TbAAT5 and TbAAT16 in BSF and PCF trypanosomes [45], in line with T. brucei being auxotroph for cationic amino acids [5], and thus, depending on their import in  Trypanosoma brucei Arginine and Lysine Transporters insect and mammalian hosts. While protein synthesis necessitates lysine and arginine import into the cytosol and the mitochondrion, the presence of arginine kinase in the cytosol, glycosome and flagellum [14] indicates the presence of arginine in additional subcellular compartments. Protein sorting motifs are generally better defined for soluble proteins than for  Table 1. Substrate selectivity of TbAAT5-3.

Competitor added 10x 100x
None 100 100 L-Arginine 9.9 ± 2.8 n.d.  membrane proteins and the fact that none of the sorting motif for mitochondria (using Mito-Prot [55] or TargetP [56]), peroxisomes/glycosomes [57] or flagellum [58] could be identified, does not exclude localization in the membrane of these subcellular compartments. Proteome analyses did not identify these transporters in the flagellum [59,60] or glycosome proteomes [61][62][63]. PCF T. brucei over-expressing cMyc-tagged versions of either TbAAT5-3 or TbAAT16-1 indicated plasma membrane localization of the tagged proteins (Fig 7), which is consistent with TbAAT5 or TbAAT16 mediating import of extracellular arginine or lysine, respectively, in S. cerevisiae. Fluorescence was also detected in unknown intracellular structures, possibly as a result of overexpression of the tagged protein.
TbAAT5 and TbAAT16 are essential for arginine and lysine uptake and growth of bloodstream form trypanosomes To investigate the role of TbAAT5 and TbAAT16 in trypanosomes, expression was down-regulated by RNAi using a tetracycline-inducible system [36]. After one day of induction, mRNA of TbAAT5 and TbAAT16 were significantly reduced. RNAi against TbAAT5 or TbAAT16 substantially reduced growth of BSF parasites after one day of induction (Fig 8B and 8C), thus hampering mRNA isolation at later time points.
Although down-regulation of individual genes was not analyzed, the high homology among the 5 genes of the AAT5 locus (> 98% overall identity on nucleotide level) and between the 2 genes of the AAT16 locus (99% overall identity on nucleotide level) suggests that the RNAi constructs targeted all genes of the TbAAT5 or TbAAT16 locus, respectively. Eflornithine 96.1 ± 6.0 Fmoc-Lysine 57.6 ± 6.9 Methyl-L-lysine 82.9 ± 9.  To test the hypothesis that the growth defect of the induced RNAi lines is due to the inability to import sufficient amounts of arginine or lysine, the medium was supplemented with 5 mM arginine or 10 mM lysine, i.e. approximately 10 times the concentration of arginine or lysine in HMI-9 (482 μM Arg and 1 mM Lys, respectively [64]). Indeed, the addition of arginine or lysine to the medium significantly improved growth of the tetracycline-induced TbAAT5-RNAi (Fig 8B) or TbAAT16-RNAi (Fig 8C) BSF T. brucei, respectively, while growth of non-induced cells remained unchanged (Fig 8A). In contrast, addition of both amino acids improved growth of induced RNAi lines less efficiently, indicating that at least part of the import at high substrate concentrations is mediated by a system that transports both arginine and lysine. Down-regulation of TbAAT5 in PCF parasites also impacts growth indicating that arginine uptake is essential in this life-cycle stage (S2 Fig). In a double-RNAi BSF line , TbAAT16 (C) or both (D) for 5 days in HMI-9. 5 mM arginine and/ or 10 mM lysine was added as indicated above the growth curves. Data points of growth curves correspond to mean values ± SD from three independent experiments. Insets show mRNA levels relative to telomerase reverse transcriptase (TERT, Tb927.11.10190) transcripts after one day of induction as determined by qRT-PCR. Expression in the absence of tetracycline was set as 100%. Data points correspond to mean values ± SD from three independent experiments. doi:10.1371/journal.pone.0168775.g008 (TbAAT5/16-RNAi) with simultaneous down-regulation of TbAAT5 and TbAAT16, reduction of transcript levels was comparable to the single RNAi lines (Fig 8D). Induced TbAAT5/ 16-RNAi parasites stopped growth after one day of tetracycline addition. As expected, supplementing the medium with 5 mM arginine or 10 mM lysine did not prevent the growth defect. Addition of both arginine and lysine resulted in partial rescue, comparable to the addition of both amino acids to single RNAi knockdown lines.

Discussion
In mammalian cells the cationic amino acids arginine and lysine are transported by the same transport systems that mediate exchange of cationic amino acids and cysteine against intracellular neutral amino acids (rBAT), or facilitated uptake of cationic amino acids (CAT) [26,65]. CAT members have similar affinities for arginine, lysine and ornithine (K m 40-450 μM for CAT1, CAT2B and CAT3; 2-5 mM for CAT2A) [27] while histidine recognition varies. In contrast, the cationic amino acid transporters characterized from the parasitic protozoa L. donovani and T. cruzi are selective for either arginine (LdAAP3, TcAAAP411) or lysine (LdAAP7, TcAAP7) and belong to a different gene family [22][23][24]. Only TcCAT1.1 has recently been described as arginine/ornithine transporter [25], but the huge difference in affinity for the two substrates indicates that the latter may not be the preferred substrate in vivo.
Our results show that similar to the trypanosomatid transporters mentioned above, uptake of cationic amino acids in T. brucei is mediated by selective and high affinity transporters.
Despite the similarities among trypanosomatid arginine and lysine transporters, subtle differences were detected. At saturating arginine concentrations, L-arginine transport mediated by TbAAT5-3 was not affected by the presence of a 10 times excess of the cationic amino acids histidine, ornithine, and lysine, with the latter partially reducing arginine transport only at 100 times excess, indicating high selectivity, comparable to LdAAP3 and TcCAT1.1 [22,25]. Furthermore, like the arginine transporter TcAAAP411 [23], TbAAT5-3 is stereoselective since D-arginine has no effect on arginine transport. The arginine analogs canavanine and homoarginine were inhibitors of TbAAT5-mediated arginine transport, suggesting that the aliphatic chain between the guanidino group and the amino-and the carboxylic acid-group is less important for substrate recognition. Canavanine was also found to be a good competitor for TcCAT1.1-mediated arginine uptake, while homoarginine was less efficient [25]. In contrast, in TcAAAP411-expressing S. cerevisiae cells, canavanine was competing only at 50-fold excess, and for LdAAP3 homoarginine at 5-fold excess did not inhibit arginine uptake [22,23], revealing slight preferences in substrate recognition. The arginine analogs homoarginine and canavanine were not only competitors in heterologous expression systems, but were also the best competitors for arginine uptake in T. cruzi epimastigotes [66]. Interestingly in T. cruzi the arginine analogs agmatine, homoarginine and canavanine were shown to inhibit arginine kinase and the latter two compounds were also able to reduce T. cruzi epimastigote growth in culture [67].
Agmatine neither interfered with TbAAT5-3 nor with LdAAP3-or TcCAT1.1-mediated arginine transport indicating that the negatively charged carboxylic acid is essential for substrate recognition [22,23]. Furthermore, alkylation of the carboxylic acid moiety of arginine also decreased competition for arginine uptake by TbAAT5-3 as indicated by the much smaller reduction of arginine transport rates by arginine ethyl ester and arginine methyl ester that diminished arginine transport rates to about 70% (at 100 times excess). In addition to the lack of a negative charge in these compounds, steric constraints might explain reduced interaction of arginine derivatives esterified at the carboxylic acid group. In contrast, arginine β-naphthylamide reduced arginine transport rates, though it was not tested whether the amphipathic compound may reduce transport through an unspecific effect.
Methylation at the guanidine group of arginine lowered recognition by TbAAT5-3 substantially as seen for N G -monomethylarginine (L-NMMA). A second methylation of the same amino group in N G ,N G -dimethylarginine (ADMA) completely impaired recognition by TbAAT5-3. And finally, a guanidine group is preferred to an amine group as lysine only decreases arginine transport if present at 100 times excess. Comparably, the affinity of Leishmania LdAAP3 for lysine was about 500 times lower than for arginine [22].
The lysine transporter, Tb427tmp.01.7500, is also highly selective. Similar to LdAAP7-mediated lysine transport and lysine uptake into T. cruzi epimastigotes and L. donovani promastigotes, none of the proteinogenic amino acids tested competed for L-lysine uptake [24]. Transport was stereospecific and modifications of the amino group (N-α-acetyl-L-lysine methyl ester), shortening the length of the carbon scaffold as in ornithine and eflornithine (αdifluoromethylornithin) or extending it at the ε-amino residue (N-ε-methyl-L-lysine) impaired recognition as substrates. Also the dipeptide lysyllysine was not recognized. While hydroxylation of the carbon backbone (5-hydroxy-L-lysine) was not tolerated, thialysine (S-(2-aminoethyl)-L-cysteine) in which C2 is replaced by a sulphur was the only compound competing for lysine uptake and, to a lesser extend, Fmoc-lysine (N-α-(9-fluorenylmethyloxycarbonyl)-L-lysine) also reduced L-lysine uptake rates. The various compounds were tested to evaluate the requirements for a substrate to be recognized by TbAAT5 and TbAAT16. In the future, structural models of the transporters may allow identification of the residues involved in substrate binding and selectivity.
The apparent affinities of TbAAT5-3 (K m 3.6 μM) and TbAAT16-1 (K m 4.3 μM) for arginine and lysine, respectively, indicate that the transporters are likely to be saturated and working at maximal rates in mammalian blood where concentrations of 20-218 μM arginine and 164-434 μM lysine have been reported (Human metabolome database, www.hmdb.ca [68]). In human cerebrospinal fluid, 9-25 μM arginine and 22-32 μM lysine were found [68], suggesting that the transporters also mediate uptake of these amino acids in T. brucei parasites in the central nervous system. Although TbAAT5 is more closely related to TcAAAP411 and TcCAT1.1 (Fig 1), the apparent affinity of TbAAT5 for arginine determined in S. cerevisiae is close to 10 times (TcAAAP4111) and more than 20 times (TcCAT1.1) higher and comparable to the K m determined for the arginine-selective transport system AAP3 from L. donovani [22,23,25]. All trypanosomatid lysine transporters characterized so far are highly homologous, and also the K m values were in a comparable range [24]. While transport of arginine and lysine into T. brucei parasites has not been determined, the affinity of lysine and arginine uptake in L. donovani promastigotes (K m values of 3 μM and 14 μM, respectively) are in a range comparable to the K m value determined in S. cerevisiae expressing LdAAP7 (7.4 ± 3.6 μM) or LdAAP3 (1.9 ± 0.1 μM) [22, 24,69] and similar to the affinity of the T. brucei lysine and arginine transporters characterized in our study. Lysine transport assessed in T. cruzi epimastigotes revealed a slightly lower affinity of 23.4 ± 2.3 μM, while kinetic data of TcAAP7 expressed in S. cerevisiae were not determined [24].
Down-regulation of TbAAT5 and TbAAT16 resulted in impaired growth of T. brucei parasites, confirming their importance for arginine and lysine uptake. The growth defect could only be rescued at very high, i.e. non-physiological concentrations of arginine or lysine, respectively. Initially, growth phenotypes of the S. cerevisiae mutants expressing TbAAT5 or TbAAT16 indicated that the transporters recognize both arginine and lysine. However, the competition experiments showed that with a 10-fold excess there is no reduction of transport rates, and thus in vivo, with concentrations of arginine and lysine differing~2-fold, transport of both amino acids by the same transporters is very unlikely. Furthermore, the reduction of growth of TbAAT5 RNAi lines in the presence of high arginine and high lysine compared to high arginine indicated that uptake is mediated partially by a system that also recognizes lysine. This transport system is, however, unlikely to be TbAAT16, because lysine concentrations in HMI9 medium are at saturation for TbAAT16 and a 10-fold excess of arginine is not expected to be transported at high rates. The same line of arguments can be used for the TbAAT16 RNAi lines grown in the presence of lysine or lysine plus arginine. Single and double RNAi lines therefore indicate that additionally one or several low affinity transport systems are present and that at least one of them recognizes both arginine and lysine. Low affinity arginine transport has so far only been described in T. cruzi [70], but the corresponding gene has not been identified and low affinity lysine or arginine transport has not been determined in T. brucei.
Dependence of parasites on these high affinity lysine and arginine transport systems is likely to be more important in vivo since-as indicated above-the concentrations of cationic amino acids in the blood are 5-6 times lower and in the cerebrospinal fluid 30-70-times lower than in standard HMI-9 medium [64,68]. Understanding the multiple mechanisms by which the parasites acquire nutrients remains of major importance to comprehend parasite physiology and may help developing new trypanocidal compounds. TbAAT5 and TbAAT16 may be promising candidates as drug delivery systems or targets, because they are essential and not related to human cationic amino acid uptake systems.