The tyrosine transporter of Toxoplasma gondii is a member of the newly defined apicomplexan amino acid transporter (ApiAT) family
[['Apicomplexans are intracellular parasites that cause a range of diseases in humans and animals , imposing a major health and economic burden on many countries .', 'In humans , Plasmodium species are the causative agents of malaria [1] , while Cryptosporidium is a major cause of diarrheal disease and death in children in the developing world [2] .', 'Toxoplasma gondii can infect virtually all nucleated cells in warm-blooded animals , and is thought to chronically infect one-third of the world’s human population .', 'T . gondii infections are usually asymptomatic , but infection in immunocompromised patients may lead to life-threatening toxoplasmic encephalitis , and congenital toxoplasmosis may result in severe birth defects or death of the developing fetus [3] .', 'A common feature of parasites is that they rely on their hosts to supply them with the nutrients necessary for their growth and replication , such as sugars , amino acids , nucleosides , and vitamins [4–6] .', 'Transporters are integral membrane proteins that facilitate the transfer of substrates across biological membranes .', 'In apicomplexans , transporters provide the major route for the acquisition of nutrients and the removal of waste products across the plasma membrane [5 , 7] , and these proteins are important for parasite survival and virulence [8 , 9] .', 'Despite this , the transporters responsible for the uptake of many essential nutrients in apicomplexans have not been defined .', 'A family of Novel Putative Transporters ( the NPT family ) was initially identified in Plasmodium falciparum using a bioinformatics approach [10] .', 'The five P . falciparum NPT family proteins were predicted to be polytopic membrane proteins with a secondary structure characteristic of solute transporters , although they have limited sequence similarity to other eukaryotic or prokaryotic transporters .', 'The NPT family protein PbNPT1 localizes to the plasma membrane of the mouse malaria-causing parasite P . berghei , and is essential for gametocyte development in the murine host and subsequent mosquito transmission [8 , 9 , 11] .', 'Other P . berghei NPT family proteins , PbMFR4 and PbMFR5 , are essential for progression through the insect stages of the life cycle , while PbMFR2 and PbMFR3 are important for exflagellation of male gametes and sporozoite formation , respectively , but are not essential for completion of the P . berghei life cycle [8] .', 'A saturation mutagenesis screen revealed that all five NPTs in P . falciparum are dispensable for the growth of asexual blood stages of the parasite under in vitro culture conditions [12] .', 'In T . gondii , TgNPT1 localizes to the plasma membrane and is essential for parasite growth and virulence [9] .', 'Both PbNPT1 and TgNPT1 are cationic amino acid transporters , with PbNPT1 functioning as a general cationic amino acid transporter and TgNPT1 functioning as a selective arginine transporter [9] .', 'The functions of other NPT-family proteins are not known , although one member of the family has been associated with susceptibility to the anti-T .', 'gondii drug sinefungin [13] .', 'In this study , we have demonstrated that the NPTs are phylogenetically related , and broadly distributed within the apicomplexan phylum .', 'We have characterized the NPT family proteins in T . gondii , demonstrating that 10 of the sixteen members of the family are expressed in the disease-causing tachyzoite stage of the parasite , and that the majority of these localize to the parasite periphery .', 'We have demonstrated that at least three of these proteins are important for in vitro growth of the parasite .', 'Using a combination of genetic , physiological and heterologous expression approaches , we have shown that one of the previously uncharacterized T . gondii NPT-family members transports aromatic and large neutral amino acids , and that this transporter is particularly important for the uptake of tyrosine into the parasite .', 'We conclude that NPTs are a family of amino acid transporter proteins found in apicomplexans , and we propose that the family be renamed the Apicomplexan Amino acid Transporter ( ApiAT ) family .'], ['To identify ApiAT-family proteins in the apicomplexan parasites T . gondii , Neospora caninum , Eimeria tenella , P . falciparum , P . berghei , Theileria annulata , Babesia bovis and Cryptosporidium parvum , we undertook Basic Local Alignment Search Tool ( BLAST ) searches using each of the five P . falciparum ApiATs as initial query sequences ( www . eupathdb . org; [14] ) We also undertook BLAST searches of the genomes from the chromerids Chromera velia and Vitrella brassicaformis , which are close free-living relatives of apicomplexans [15] ( www . eupathdb . org , www . blast . ncbi . nlm . nih . gov; [14 , 16] ) .', 'In addition to the previously described five Plasmodium ApiAT family proteins , we identified sixteen ApiAT family proteins in both T . gondii and N . caninum , eight in E . tenella , nine in T . annulata , six in B . bovis , and one in C . parvum ( S1 Fig; S1 Table ) .', 'Using this search strategy , we were unable to identify ApiAT family proteins in chromerids .', 'Using PbNPT1 as a search query in profile hidden Markov Model searches , we identified the LAT3 and LAT4 proteins from humans ( http://hmmer . org; [17]; S2 Fig ) .', 'LAT3 and LAT4 are members of the SLC43 family of the major facilitator superfamily of transporters , and mediate the transport of branched chain and other large neutral amino acids [18] .', 'To determine the phylogenetic relationships between ApiAT family proteins , we constructed a multiple sequence alignment ( S1 Fig ) .', 'This revealed the presence of a major facilitator superfamily ( MFS ) signature sequence between transmembrane domains 2 and 3 of most ApiAT family protein ( S2 Fig; [19 , 20] ) .', 'Most ApiAT proteins were predicted to be polytopic membrane proteins containing 12 transmembrane domains ( www . cbs . dtu . dk/services/TMHMM/; [21] ) , and exhibited highest sequence similarity in the regions encompassing these transmembrane domains ( S1 Fig ) .', 'These analyses are consistent with previous studies and protein database annotations placing members of the ApiAT family into the major facilitator superfamily of transporters [9 , 10] .', 'We next performed a maximum likelihood phylogenetic analysis .', 'This revealed the presence of multiple ApiAT subfamilies , which we defined as groupings that had greater than 75% bootstrap support ( Fig 1 ) .', 'Orthologs of the ApiAT2 subfamily were present in all organisms in the study with the exception of Cryptosporidium parvum , although the only C . parvum ApiAT protein that we identified grouped with the ApiAT2 subfamily with weak support ( Fig 1 ) .', 'Members of the ApiAT1 , ApiAT3 , ApiAT5 , ApiAT6 and ApiAT7 subfamilies were restricted to coccidians ( a group of apicomplexans that includes T . gondii and N . caninum ) , the ApiAT4 , ApiAT8 , ApiAT9 and ApiAT10 subfamilies were restricted to the Plasmodium genus , and the ApiAT11 family was restricted to the piroplasms T . annulata and B . bovis ( Fig 1 ) .', 'The ApiAT8 family includes the previously described cationic transporter PbNPT1 [9] , here annotated as PbApiAT8 .', 'Although similar in function to the T . gondii arginine transporter TgApiAT1 ( previously TgNPT1 ) , PbApiAT8 and TgApiAT1 appear not to be orthologous .', 'Previous studies demonstrated that the P . berghei ApiAT8 protein ( previously PbNPT1 ) localized to the periphery of the parasite ( likely to the plasma membrane ) , and that the T . gondii ApiAT1 protein ( previously TgNPT1 ) localized to the plasma membrane [9 , 11] .', 'To determine the expression pattern and localization of ApiAT family proteins in T . gondii , we introduced a hemagglutinin ( HA ) tag into the 3’ end of the open reading frame of the remaining fifteen ApiAT genes ( S3A and S3B Fig ) .', 'Western blotting indicated that TgApiAT2 , TgApiAT3-1 , TgApiAT3-2 , TgApiAT3-3 , TgApiAT5-3 , TgApiAT6-1 , TgApiAT6-2 , TgApiAT6-3 , and TgApiAT7-2 proteins were expressed in tachyzoite stage parasites ( Fig 2A–2E ) .', 'We were unable to detect expression of TgApiAT5-1 , TgApiAT5-2 , TgApiAT5-4 , TgApiAT5-5 , TgApiAT5-6 and TgApiAT7-1 .', 'Of the ApiATs that were expressed in T . gondii tachyzoites , most exhibited a lower mass than predicted ( S1 Table ) .', 'Although we cannot rule out the possibility of N-terminal processing in these proteins , we note that hydrophobic membrane proteins commonly run faster than predicted on sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( SDS-PAGE; [22] ) .', 'Immunofluorescence assays ( IFAs ) demonstrated that TgApiAT2 , TgApiAT3-1 , TgApiAT3-2 , TgApiAT3-3 , TgApiAT5-3 , TgApiAT6-1 ( as reported previously; [23] ) , and TgApiAT6-3 localized to parasite periphery , overlapping with the plasma membrane marker P30 ( Fig 2F–2I ) .', 'TgApiAT3-3 showed additional localization to the trans-Golgi network ( Fig 2G ) .', 'Although detectable by western blotting ( Fig 2D and 2E ) , we could not detect TgApiAT6-2 or TgApiAT7-2 by IFA , possibly because the level of expression of these proteins was below the detection limits of IFAs .', 'T . gondii , like many apicomplexans , contains a series of flattened membrane sacs termed alveolae that contribute to the inner membrane complex ( IMC ) that localizes just beneath the plasma membrane of these parasites [24] .', 'The IMC first appears as the ‘buds’ of daughter parasites within the parent cell just before cytokinesis .', 'To compare the localization of an ApiAT protein with the IMC , we co-labelled TgApiAT5-3-HA parasites with anti-HA and anti-IMC antibodies .', 'We found that TgApiAT5-3-HA did not localize to daughter buds ( S4A Fig ) .', 'TgApiAT5-3-HA localization extended along the cell periphery at the apical and basal ends of the parasite , regions from which the IMC marker was absent ( S4A and S4B Fig , arrows ) .', 'Taken together , these data indicate that TgApiAT5-3 is localized primarily to the plasma membrane , although in the absence of higher resolution microscopy approaches , we cannot rule out that some could also be located in IMC .', 'We conclude that ten of the sixteen TgApiAT proteins are expressed in the tachyzoite stage of T . gondii , and that those with detectable expression by IFA all localize to the periphery of the parasite , most likely to the parasite plasma membrane .', 'To determine the importance of TgApiATs for parasite growth , we attempted to genetically disrupt all sixteen T . gondii ApiATs using a CRISPR/Cas9-based approach [25] .', 'With this strategy , we were able to disrupt fifteen of the sixteen TgApiAT genes ( S2 Table ) .', 'The mutant parasites all had frameshift mutations that will lead to the production of truncated ( and likely non-functional ) proteins .', 'We refer to each of these mutants on the basis of which amino acids will be absent from the final protein ( e . g . TgApiAT5-3 encodes a protein of 504 amino acids; the TgApiAT5-3 mutant is truncated from residue 188 and is referred to as apiAT5-3Δ188–504 ) .', 'TgApiAT1 could only be disrupted when parasites were grown in excess arginine , as described previously [9] .', 'We were unable to generate frameshift mutations in TgApiAT6-1 after screening 12 clones from three separate transfections of a guide RNA targeting the TgApiAT6-1 locus .', 'Of these clones , two had a 3 bp insertion and one had a 3 bp deletion , indicating that the guide RNA was capable of targeting the TgApiAT6-1 locus ( S2 Table ) .', 'To determine which TgApiATs were important for parasite growth , we performed plaque assays on each of the genetically disrupted TgApiAT lines grown in human foreskin fibroblasts ( HFFs ) and cultured in Dulbecco’s modified Eagle’s medium ( DMEM ) .', 'Compared to parental wild type ( WT ) controls , we observed greatly reduced plaque sizes in the apiAT2Δ138–588 and apiAT5-3Δ188–504 strains ( Fig 3 ) .', 'No plaques were observed in the apiAT1Δ54–534 strain grown in DMEM ( containing 400 μM L-arginine ) but normal growth of this strain was observed when grown in Roswell Park Memorial Institute 1640 ( RPMI ) medium ( containing 1 . 15 mM L-Arg; Fig 3A ) , mirroring previous results with TgApiAT1 knockouts [9] .', 'By contrast , the remaining 12 mutant apiAT lines exhibited plaques that were similar in size to those observed in WT controls ( Fig 3 ) .', 'Our previous data indicated that some ApiAT family proteins transport amino acids [9] .', 'We tested each of the mutant apiAT strains for whether growth defects were altered in medium containing reduced amino acid concentrations ( Minimal Amino Acid Medium , MAAM ) .', 'The growth defects in the apiAT2Δ138–588 and apiAT5-3Δ188–504 strains were exacerbated in MAAM ( S5 Fig ) , implicating these ApiATs in the uptake of amino acids .', 'By contrast , the apiAT1Δ54–534 strain grew better in MAAM than in DMEM ( S5 Fig ) .', 'Our previous study of the arginine transporter TgApiAT1 found that parasites lacking this transporter could grow in medium containing a high Arg:Lys ratio [9] .', 'In this context , we note that MAAM contains proportionally more L-Arg than L-Lys ( 3:1 Arg:Lys; S3 Table ) than DMEM ( 1:2 Arg:Lys ) .', 'We observed no major growth differences between minimal amino acid and complete growth media for any of the other mutants ( S5 Fig ) .', 'To test whether the growth defect in the apiAT2Δ138–588 strain was due specifically to disruption of the TgApiAT2 locus , we complemented apiAT2Δ138–588 with constitutively expressed TgApiAT2 .', 'This restored parasite growth ( Fig 3B ) .', 'Together , these results indicate that TgApiAT1 , TgApiAT2 and TgApiAT5-3 are required for normal intracellular growth of T . gondii in standard in vitro culture conditions .', 'TgApiAT proteins that are important for parasite growth are likely to have critical roles in nutrient acquisition .', 'In the remainder of this manuscript , we focus on one such protein , TgApiAT5-3 .', 'Our previous study of TgApiAT1 and PbApiAT8-1 indicated a key role for these transporters in cationic amino acid uptake [9] , and we hypothesized that TgApiAT5-3 could also function as an amino acid transporter .', 'To investigate this possibility , we incubated WT and apiAT5-3Δ188–504 parasites in medium containing a mixture of [13C]-labelled amino acids for 15 mins .', 'Polar metabolites were extracted from parasite lysates and analyzed by GC-MS .', 'These analyses were used to quantitate the levels of intracellular amino acids and determine the extent of labeling with exogenous amino acids based on [13C]-enrichment in each of the two strains .', 'Strikingly , both the abundance and fractional labelling of [13C]-L-tyrosine ( L-Tyr ) was reduced significantly in the apiAT5-3Δ188–504 strain ( Fig 4 ) .', 'Abundance and fractional labeling of a number of other [13C]-amino acids were altered significantly in the apiAT5-3Δ188–504 strain , although none to the same extent as L-Tyr ( Fig 4 ) .', 'The levels of several other amino acids were reduced in the apiAT5-3Δ188–504 mutant , although the differences were not statistically significant ( Fig 4A ) , possibly reflecting differences in the growth rate and physiological state of the two lines .', 'In support of this notion , the rate of turnover of these amino acids , as indicated by rate of 13C-enrichment in intracellular pools was unchanged ( Fig 4B ) .', 'These data indicate that TgApiAT5-3 is important for amino acid homeostasis , playing a key role in the uptake of L-Tyr into the parasite .', 'To characterize the substrate specificity of TgApiAT5-3 further , we expressed HA-tagged TgApiAT5-3 in Xenopus laevis oocytes , and confirmed its expression and plasma membrane localization by western blotting ( S6A Fig ) .', 'Given the GC-MS data implicating TgApiAT5-3 in L-Tyr uptake ( Fig 4 ) , we hypothesised that TgApiAT5-3 transports L-Tyr .', 'We compared the uptake of radiolabelled [14C]-tyrosine ( [14C]Tyr ) into oocytes expressing TgApiAT5-3 relative to uninjected oocytes .', 'Under the conditions of the experiment , there was a significant , 7-fold increase in the initial uptake rate of [14C]Tyr into oocytes expressing TgApiAT5-3 compared to uninjected control oocytes ( Fig 5A ) .', 'To investigate the possibility that TgApiAT5-3 has exchange activity , we tested whether the uptake of [14C]Tyr was stimulated by the presence of amino acids on the trans side of the membrane ( i . e . inside the oocyte ) .', 'Following preliminary experiments to optimise the preloading of L-Tyr into oocytes , we measured [14C]Tyr uptake over 2 hr at an extracellular concentration of 1 mM L-Tyr in TgApiAT5-3 cRNA-injected or uninjected oocytes that had been pre-loaded in medium containing 2 . 5 mM unlabelled L-Tyr .', 'The initial rate of [14C]Tyr uptake in TgApiAT5-3-expressing oocytes preloaded with L-Tyr was 3-fold higher than in TgApiAT5-3-expressed oocytes that were not pre-loaded ( Fig 5B ) , indicating that L-Tyr uptake into TgApiAT5-3-expressing oocytes was stimulated by L-Tyr on the trans side of the membrane .', 'We next tested whether L-Tyr efflux was also trans-stimulated .', 'We preloaded TgApiAT5-3-expressing and uninjected oocytes with radiolabelled L-Tyr and measured the efflux and retention of the radiolabel upon the addition of 2 . 5 mM unlabelled L-Tyr to the external medium .', '[14C]Tyr efflux was increased , and [14C]Tyr retention reduced , in TgApiAT5-3-expressing oocytes exposed to 2 . 5 mM L-Tyr compared to those in external medium lacking L-Tyr ( Fig 5C ) .', 'Nevertheless , there was some [14C]Tyr efflux over time in the absence of trans-substrate ( Fig 5C ) , indicating that TgApiAT5-3 can mediate unidirectional flux of L-Tyr .', 'We observed no differences in [14C]Tyr efflux or retention in control uninjected oocytes upon incubation of oocytes in 2 . 5 mM L-Tyr compared to incubation in buffer lacking L-Tyr ( S6B Fig ) .', 'These data indicate that the transporter operates more effectively under ‘exchange conditions’ than under conditions in which it is mediating a unidirectional flux .', 'To examine the exchange activity of TgApiAT5-3 further , we investigated the kinetic properties of L-Tyr transport in more detail .', 'Steady-state kinetic parameters for exchangers must be conducted at different trans- and cis-substrate concentrations to determine accurate K0 . 5 values [26] .', 'We examined the kinetics for the uptake of [14C]Tyr following the preloading of oocytes with different L-Tyr concentrations ( note that this , and all subsequent , uptake measurements were conducted over 10 min , which falls within the initial , approximately linear , phase of the influx timecourse; Fig 5B ) .', 'Both Michaelis-Menten analysis ( Table 1 ) and Scatchard linear regressions ( Fig 5D ) demonstrated that the apparent K0 . 5 values for L-Tyr uptake into oocytes was unaffected by the cytosolic L-Tyr concentration .', 'The gradients of fitted Scatchard plots are negative reciprocals of the apparent K0 . 5 and , therefore , equivalent slopes reflect similar apparent substrate affinities .', 'K0 . 5 values were consistent for both Scatchard and Michaelis-Menten plots derived from the same data , ranging from 0 . 25 to 0 . 40 μM ( Table 1 ) .', 'As expected for trans-stimulated uptake , maximum rate ( Vmax ) values increased in proportion to the concentration of preloaded L-Tyr ( Fig 5D; Table 1 ) .', 'To assess whether TgApiAT5-3 might function as an ion:amino acid co-transporter , we conducted systematic ion-replacement uptake experiments as described previously [9] .', 'We observed no significant change in [14C]Tyr uptake under any ion-replacement conditions , suggesting that the transporter does not co-transport any of the ions tested ( S6C Fig ) .', 'To determine whether TgApiAT5-3 is an electrogenic transporter ( as is the case for TgApiAT1; [9] ) , we perfused oocytes expressing TgApiAT5-3 with L-Tyr and measured the electrical current across the oocyte plasma membrane using a two-electrode voltage clamp configuration .', 'We observed no net current under any of the conditions tested , including those in which the membrane potential and pH gradient across the membrane were altered ( S6D Fig ) .', 'Together , these data indicate that L-Tyr transport by TgApiAT5-3 does not co-transport any charged species .', 'Incubation of WT and apiAT5-3Δ188–504 parasites in a [13C]-amino acid mix resulted in significant increases or decreases in the 13C-labelling of several amino acids ( Fig 4 ) , suggesting that TgApiAT5-3 may transport a range of amino acids .', 'To investigate the substrate specificity of TgApiAT5-3 further , we measured the uptake of 500 μM [14C]Tyr in oocytes expressing TgApiAT5-3 in the presence of equimolar amounts of unlabelled L-amino acids on the cis side of the membrane .', 'Only unlabelled L-tryptophan ( L-Trp ) caused a significant reduction of L-Tyr uptake under the conditions tested ( S7A Fig ) .', 'To examine the trans-stimulated influx specificity of TgApiAT5-3 , we pre-injected mixtures of L-amino acids and other metabolites into oocytes expressing TgApiAT5-3 , then measured uptake of [14C]Tyr .', 'TgApiAT5-3-mediated [14C]Tyr uptake was trans-stimulated by pre-injected mixtures of L-amino acids , but not amino acid derivatives , D-amino acids , nucleotides , nitrogenous bases , or sugars ( S7B Fig; S4 Table ) .', 'To identify which amino acids might be responsible for this trans-stimulation , we pre-injected oocytes expressing TgApiAT5-3 with 19 of the 20 proteinogenic amino acids , each to a final concentration of 5 mM .', 'The one exception was L-Tyr which , in light of its low solubility , was preloaded to equilibrium instead of being injected .', 'After pre-injecting or preloading of the amino acids , we measured the trans-stimulated uptake of [14C]Tyr .', 'We found that aromatic and large neutral amino acids , but not smaller or charged amino acids , trans-stimulated [14C]Tyr uptake ( Fig 6A ) .', 'We tested the uptake of a range of [14C]-labelled aromatic and large neutral amino acids , including L-Trp , L-histidine ( L-His ) , L-phenylalanine ( L-Phe ) , L-leucine ( L-Leu ) and L-isoleucine ( L-Ile ) , into oocytes expressing TgApiAT5-3 .', 'In the absence of a trans substrate , the rates of uptake of aromatic and large neutral amino acids were significantly increased compared to uninjected oocytes ( 1 . 7-fold increase for L-Trp , 3 . 1-fold increase for L-His , 5 . 5-fold increase for L-Phe , 2 . 2-fold increase for L-Leu , and 2 . 4-fold increase for L-Ile; S7C Fig ) .', 'The uptake of the cationic amino acid L-Arg did not differ between TgApiAT5-3 injected and uninjected oocytes ( S7C Fig ) .', 'As was seen for L-Tyr , uptake of the aromatic and large neutral amino acids tested were trans-stimulated by aromatic and large neutral amino acids , and not by smaller or charged L-amino acids ( Fig 6A ) .', 'The uptake of L-Arg was not trans-stimulated by any of the amino acids tested ( Fig 6A ) .', 'We next measured efflux of preloaded [14C]-labelled L-Tyr , L-Trp , L-His , L-Phe and L-Leu in the presence of all 20 proteinogenic amino acids in the extracellular medium .', 'We observed the same substrate specificity , with aromatic and large neutral L-amino acids stimulating the efflux of the [14C]-labelled substrates tested ( Fig 6B ) .', 'From the experiments conducted in this section , we conclude that TgApiAT5-3 mediates the transport of aromatic and large neutral amino acids .', 'The transporter can function as a uniporter , but has a strong propensity for exchange , implying a role for TgApiAT5-3 in the homeostasis of a range of aromatic and large neutral amino acids .', 'The [13C] amino acid uptake data and oocyte experiments indicate a role for TgApiAT5-3 in L-Tyr uptake into the parasite ( Fig 4; Fig 5 ) .', 'We set out to test the importance of TgApiAT5-3 for the uptake of L-Tyr and other amino acids and , subsequently , its importance for parasite growth and virulence .', 'Preliminary experiments indicated that the parental strain used to make the original apiAT5-3Δ188–504 strain was avirulent in mice , precluding its use for virulence studies .', 'We therefore remade the apiAT5-3Δ188–504 mutant in virulent RHΔhxgprt/Tomato strain parasites ( S2 Table; [27] ) , and performed all subsequent experiments with this strain .', 'To test the importance of TgApiAT5-3 for L-Tyr uptake in T . gondii , we measured the kinetics of [14C]Tyr uptake in WT ( RHΔhxgprt/Tomato ) and apiAT5-3Δ188–504 parasites .', 'The initial rate of [14C]Tyr uptake in apiAT5-3Δ188–504 parasites was decreased by 4 . 7-fold compared to that in WT parasites ( Fig 7A; S8A Fig ) .', 'Both [14C]Tyr uptake and parasite growth were restored upon complementation of the apiAT5-3Δ188–504 mutant with a constitutively expressed copy of TgApiAT5-3 ( apiAT5-3Δ188-504/cTgApiAT5-3; Fig 7A; S9 Fig ) .', 'Our previous data indicated that TgApiAT5-3 can transport aromatic amino acids other than L-Tyr ( Fig 6 , S7A Fig ) .', 'We measured uptake of [14C]Phe in WT and apiAT5-3Δ188–504 parasites .', 'We observed a significant , 2 . 8-fold decrease in the initial rate of [14C]Phe uptake in parasites lacking TgApiAT5-3 , and this was restored upon TgApiAT5-3 complementation ( Fig 7B; S8B Fig ) .', 'We were unable to detect robust levels of uptake of [14C]Trp in either WT or apiAT5-3Δ188–504 parasites , precluding analysis of the role of TgApiAT5-3 in uptake of this amino acid into parasites .', 'As a control , we measured uptake of [14C]Arg , which is not transported by TgApiAT5-3 ( Fig 6; S7A Fig ) , in WT , apiAT5-3Δ188–504 and apiAT5-3Δ188-504/cTgApiAT5-3 strain parasites .', 'The rate of [14C]Arg uptake did not differ significantly between these parasite lines ( Fig 7C; S8C Fig ) , indicating that the defect we observed in the uptake of aromatic amino acids is specific to this class of amino acids , and does not represent a general defect in amino acid uptake .', 'We next investigated the dependence of the growth of apiAT5-3Δ188–504 parasites on the concentration of L-Tyr in the culture medium .', 'WT , apiAT5-3Δ188–504 and complemented ( apiAT5-3Δ188-504/cTgApiAT5-3 ) parasites were grown in DMEM containing 0–2 . 5 mM L-Tyr .', 'Growth of all three parasite strains in the absence of L-Tyr was significantly impaired ( S10A Fig ) , consistent with a previous study that indicated T . gondii parasites are auxotrophic for this amino acid [28] .', 'WT and complemented parasites grew normally in [L-Tyr] as low as 39 μM ( Fig 8A ) .', 'By comparison , growth of apiAT5-3Δ188–504 parasites was negligible at [L-Tyr] of 156 μM and below , and severely impaired at concentrations below 1 mM ( Fig 8A ) .', 'We also measured growth of WT , apiAT5-3Δ188–504 and complemented parasites grown in DMEM vs DMEM containing 2 . 5 mM L-Tyr , by plaque assay .', 'Both WT and complemented parasites grew normally in both media ( S9 Fig ) .', 'Consistent with the results of the fluorescence growth assays , the growth of apiAT5-3Δ188–504 parasites was restored to wild type levels by growth in 2 . 5 mM L-Tyr ( S9 Fig ) .', 'These data indicate that uptake of L-Tyr via TgApiAT5-3 is important for the growth of T . gondii parasites in standard in vitro conditions .', 'They also point to the existence of an alternative L-Tyr uptake pathway that can mediate sufficient L-Tyr uptake to rescue growth of apiAT5-3Δ188–504 parasites if the culture medium contains ≥ 1 mM L-Tyr .', 'To assess the physiological importance of TgApiAT5-3-mediated L-Phe uptake for parasite growth , WT , apiAT5-3Δ188–504 and complemented parasites were cultivated in medium containing 0 to 10 mM L-Phe ( at a constant [L-Tyr] of 2 . 5 mM ) .', 'Both WT and complemented parasites showed minimal growth in the absence of L-Phe ( S10B Fig ) , indicating that T . gondii is auxotrophic for this amino acid , but both lines grew normally at [L-Phe] of 39 μM and above ( Fig 8B ) .', 'By contrast , growth of apiAT5-3Δ188–504 parasites was impaired at 39 μM [L-Phe] ( Fig 8B ) .', 'apiAT5-3Δ188–504 parasites grew optimally at [L-Phe] between 78 μM and 625 μM , but parasite growth decreased at [L-Phe] of 1 . 25 mM and above ( Fig 8B ) .', 'These data indicate that TgApiAT5-3 is required for parasite growth at both low and high exogenous concentrations of L-Phe , but not at intermediate concentrations .', 'This points to the existence of other L-Phe uptake pathways in the parasite .', 'We next measured the growth of WT , apiAT5-3Δ188–504 and complemented parasites in medium containing 0–2 mM L-Trp ( and a constant [L-Tyr] of 2 . 5 mM ) .', 'WT and complemented parasites exhibited minimal growth in the absence of L-Trp ( S10C Fig ) , consistent with a previous study that indicated T . gondii is auxotrophic for this amino acid [29] .', 'WT and complemented parasites grew optimally at [L-Trp] above approximately 0 . 5 mM , and exhibited decreased growth in [L-Trp] below 0 . 5 mM ( Fig 8C ) .', 'By contrast , apiAT5-3Δ188–504 parasites grew optimally at 31–125 μM L-Trp ( Fig 8C ) .', 'Growth of apiAT5-3Δ188–504 parasites was negligible below 8 μM L-Trp , and decreased in [L-Trp] of 250 μM and above ( Fig 8C ) .', 'Together , these data reveal that TgApiAT5-3 is required for parasite growth at high exogenous L-Phe and L-Trp concentrations , consistent with high concentrations of L-Phe or L-Trp competitively inhibiting uptake of L-Tyr via an alternative , non-TgApiAT5-3 L-Tyr uptake pathway ( s ) .', 'This is considered further in the Discussion .', 'Standard media formulations used to culture parasite in vitro do not necessarily reflect the amino acid concentrations that parasites encounter in vivo .', 'Since the importance of TgApiAT5-3 for parasite growth in vitro varied with the concentrations of aromatic amino acids in the growth medium ( Fig 8A–8C ) , we investigated the importance of TgApiAT5-3 for parasite virulence in a mouse infection model .', 'We infected BALB/c mice intraperitoneally with 103 WT ( RHΔhxgprt/Tomato ) , apiAT5-3Δ188–504 or complemented ( apiAT5-3Δ188-504/cTgApiAT5-3 ) parasites and monitored disease progression .', 'Mice infected with WT and complemented parasites exhibited symptoms of toxoplasmosis and were euthanized 5–7 days post-infection ( Fig 8D ) .', 'By contrast , mice infected with apiAT5-3Δ188–504 parasites exhibited no symptoms of toxoplasmosis across the entire 45 days of the experiments ( Fig 8D ) , indicating that TgApiAT5-3 is essential for parasite virulence .'], ['Parasites must scavenge essential amino acids from their environment , although , in the case of apicomplexan parasites , how they do so is poorly understood .', 'We showed recently that uptake of cationic amino acids in T . gondii and P . berghei is mediated by members of a family of polytopic membrane transporters [9] .', 'Here , we have investigated the function of other members of this family and show that one of these proteins , TgApiAT5-3 functions is an aromatic amino acid transporter .', 'Based in these findings , we have renamed this family of proteins Apicomplexan Amino acid Transporters ( ApiATs ) .', 'The ApiAT protein family is found in all apicomplexan species that we analysed .', 'The similarity of ApiATs to mammalian LAT3/4-type amino acid transporters suggests that the ancestral function of ApiATs was amino acid transport .', 'The ApiATs appear to have undergone expansion in various apicomplexan lineages , including Plasmodium spp , T . gondii and piroplasms such as Babesia spp and Theileria spp , whereas only a single representative is present in Cryptosporidium spp , an early-diverging lineage of apicomplexans [30] .', 'These observations are consistent with ancestral apicomplexans having a single ApiAT protein that diversified in various lineages of the phylum to encompass new and/or more discriminating amino acid substrate selectivities .', 'A similar expansion has been observed in the amino acid/auxin permease ( AAAP ) family of trypanosomatid parasites , in which fourteen AAAP genes arose from a single AAAP gene locus through a series of gene duplication events in ancestral trypanosomatids [31] .', 'AAAP expansion is likely to reflect an early parasitic innovation that contributed to establishing parasite dependency on the host organism , and thereby facilitating the evolution of parasitism in trypanosomatids [31 , 32] .', 'By contrast , much of the expansion in the ApiAT family appears to have occurred subsequent to the diversification of the major lineages in the phylum .', 'Of the eleven ApiAT subfamilies that we define , only the ApiAT2 subfamily is b
['Introduction', 'Results', 'Discussion', 'Materials and methods']
['Apicomplexan parasites are auxotrophic for a range of amino acids which must be salvaged from their host cells , either through direct uptake or degradation of host proteins .', 'Here , we describe a family of plasma membrane-localized amino acid transporters , termed the Apicomplexan Amino acid Transporters ( ApiATs ) , that are ubiquitous in apicomplexan parasites .', 'Functional characterization of the ApiATs of Toxoplasma gondii indicate that several of these transporters are important for intracellular growth of the tachyzoite stage of the parasite , which is responsible for acute infections .', 'We demonstrate that the ApiAT protein TgApiAT5-3 is an exchanger for aromatic and large neutral amino acids , with particular importance for L-tyrosine scavenging and amino acid homeostasis , and that TgApiAT5-3 is critical for parasite virulence .', 'Our data indicate that T . gondii expresses additional proteins involved in the uptake of aromatic amino acids , and we present a model for the uptake and homeostasis of these amino acids .', 'Our findings identify a family of amino acid transporters in apicomplexans , and highlight the importance of amino acid scavenging for the biology of this important phylum of intracellular parasites .']
['The Apicomplexa comprise a large number of parasitic protozoa that have obligate intracellular lifestyles and cause significant human and animal diseases , including malaria , cryptosporidiosis , toxoplasmosis , coccidiosis in poultry , and various cattle fevers .', 'Apicomplexans must scavenge essential nutrients from their hosts in order to proliferate and cause disease , including a range of amino acids .', 'The direct uptake of these nutrients is presumed to be mediated by transporter proteins located in the plasma membrane of intracellular stages , although the identities of these proteins are poorly defined .', 'Using a combination of bioinformatic , genetic , cell biological , and physiological approaches , we have characterized an apicomplexan-specific family of plasma membrane-localized transporter proteins that we have called the Apicomplexan Amino acid Transporters ( ApiATs ) .', 'We show that TgApiAT5-3 , a member of the family in the apicomplexan Toxoplasma gondii , is an exchanger for aromatic and large neutral amino acids .', 'In particular , it is critical for uptake of tyrosine , and for parasite virulence in a mouse infection model .', 'We conclude that ApiATs are a family of plasma membrane transporters that play crucial roles in amino acid scavenging by apicomplexan parasites .']
['parasite groups', 'medicine and health sciences', 'toxoplasma gondii', 'vertebrates', 'parasitic diseases', 'parasitic protozoans', 'animals', 'parasitology', 'xenopus', 'germ cells', 'membrane proteins', 'animal models', 'developmental biology', 'oocytes', 'apicomplexa', 'model organisms', 'amphibians', 'protozoans', 'toxoplasma', 'experimental organism systems', 'cellular structures and organelles', 'research and analysis methods', 'animal cells', 'animal studies', 'life cycles', 'cell membranes', 'xenopus oocytes', 'eukaryota', 'cell biology', 'ova', 'biology and life sciences', 'cellular types', 'frogs', 'organisms', 'parasitic life cycles']