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Parasit Vectors
2015 Jun 25;8:346. doi: 10.1186/s13071-015-0950-y.
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Identification and functional characterization of a novel arginine/ornithine transporter, a member of a cationic amino acid transporter subfamily in the Trypanosoma cruzi genome.
Henriques C
,
Miller MP
,
Catanho M
,
de Carvalho TM
,
Krieger MA
,
Probst CM
,
de Souza W
,
Degrave W
,
Amara SG
.
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Trypanosoma cruzi, the etiological agent of Chagas disease, is auxotrophic for arginine. It obtains this amino acid from the host through transporters expressed on the plasma membrane and on the membranes of intracellular compartments. A few cationic amino acid transporters have been characterized at the molecular level, such as the novel intracellular arginine/ornithine transporter, TcCAT1.1, a member of the TcCAT subfamily that is composed of four almost identical open reading frames in the T. cruzi genome. The functional characterization of the TcCAT1.1 isoform was performed in two heterologous expression systems. TcCAT subfamily expression was evaluated by real-time PCR in polysomal RNA fractions, and the cellular localization of TcCAT1.1 fused to EGFP was performed by confocal and immunoelectron microscopy. In the S. cerevisiae expression system, TcCAT1.1 showed high affinity for arginine (K m = 0.085 ± 0.04 mM) and low affinity for ornithine (K m = 1.7 ± 0.2 mM). Xenopus laevis oocytes expressing TcCAT1.1 showed a 7-fold increase in arginine uptake when they were pre-loaded with arginine, indicating that transport is enhanced by substrates on the trans side of the membrane (trans-stimulation). Oocytes that were pre-loaded with [(3)H]-arginine displayed a 16-fold higher efflux of [(3)H]-arginine compared with that of the control. Analysis of polysomal RNA fractions demonstrated that the expression of members of the arginine transporter TcCAT subfamily is upregulated under nutritional stress and that this upregulation precedes metacyclogenesis. To investigate the cellular localization of the transporter, EGFP was fused to TcCAT1.1, and fluorescence microscopy and immunocytochemistry revealed the intracellular labeling of vesicles in the anterior region, in a network of tubules and vesicles. TcCAT1.1 is a novel arginine/ornithine transporter, an exchanger expressed in intracellular compartments that is physiologically involved in arginine homeostasis throughout the T. cruzi life cycle. The properties and estimated kinetic parameters of TcCAT1.1 can be extended to other members of the TcCAT subfamily.
Fig. 1. TcCAT1.1 sequencing alignment and membrane topology. a Alignment between TcCAT1.1 from T. cruzi and LdAAP3 from L. donovani. b Prediction of TcCAT1.1 membrane topology, as determined using HMMTOP server 2.0
Fig. 2. Substrate saturation curves for [3H]-arginine and [14C]-ornithine in the Saccharomyces cerevisiae expression system. a and b are time course curves in (●) galactose-induced and (○) glucose-repressed yeast cells. c and d depict the concentration dependence of the [3H]-arginine and [14C]-ornithine uptake rates in linear ranges. Data from control assays with glucose-repressed cells were subtracted from those from assays with galactose-induced cells, and a Lineweaver-Burk plot of the data is shown in the inset. The results are presented as the mean ± SD of one experiment performed in triplicate
Fig. 3. Trans-stimulation in Xenopus laevis oocytes expressing TcCAT1.1. Oocytes were pre-loaded overnight with 1 mM arginine. Subsequently, uptake assays were performed by incubating oocytes expressing TcCAT1.1 with 100 μM [3H]-arginine. a TcCAT1.1-injected oocytes that were pre-loaded with arginine (●); TcCAT1.1-injected oocytes that were not pre-loaded with arginine (▼); and water-injected oocytes, used as controls (○). b Efflux of [3H]-arginine or arginine sub-products from oocytes injected with TcCAT1.1 cRNA (●) and from control oocytes injected with water (○). Oocytes were pre-loaded for 6 h with 1 μM [3H]-arginine in ND96 buffer. c Effect of proton uncouplers on [3H]-arginine uptake in pre-loaded oocytes expressing TcCAT1.1
Fig. 4. Differential expression of TcCAT subfamily members as determined by real-time polymerase chain reaction. Quantification of polysome-associated mRNA expression during metacyclogenesis >relative to that in epimastigotes in samples obtained from metacyclogenesis experiment. Ribosomal L9(gray) and histone H2B(black) were used as controls to normalize the expression of polysomal mRNAs encoding TcCAT. EPI, replicating epimastigotes; STR, nutritional stress; 3H, parasites after differentiation for 3 h; 12H, parasites after differentiation for 12 h; 24H, parasites after differentiation for 24 h; and MET, metacyclics
Fig. 5. Localization of EGFP-TcCAT1.1 in Trypanosoma cruzi. Stable transfectants of Dm28c over-expressing EGFP-TcCAT1.1 were cloned and maintained in 500 μg/ml of G418. Expression was evaluated by confocal microscopy. Panel (a) shows the intracellular distribution of EGFP-TcCAT1.1 at the anterior regions of the parasite (arrows). Panel (b) shows additional sites of reduced expression following incubation with anti-GFP, including the epimastigote surface (arrow head). Panels (c) and (d) show endocytosis with transferrin-Alexa Fluor 546 (small arrow) after 1 min in ammonium chloride-treated epimastigotes (c) and after 15 min (d). Panels (e) and (f) show the results of labeling with anti-RAB7 (small arrow) to evaluate co-localization of EGFP-TcCAT1.1 (arrow) with the Golgi complex. The left column shows differential interference contrast microscopy, the central column depicts EGFP-TcCAT1.1 expression (green), and the right column shows DAPI labeling of the nucleus and kinetoplast (blue), anti-RAB7, and transferrin-Alexa Fluor 546 tracer (red). N, nucleus; K, kinetoplast; FP, flagellar pocket
Fig. 6. Ultrastructural localization of EGFP-TcCAT1.1. Stable transfectants of Dm28c epimastigotes over-expressing EGFP-TcCAT1.1 were evaluated by transmission electron microscopy. a Ultrastructural organization of the epimastigote anterior region embedded in epoxy resin. b-d Immunoelectron microscopy to evaluate EGFP-TcCAT1.1 localization in the spongiome region, as observed by gold particle distribution (arrow), after incubation with anti-GFP. N, nucleus; K, kinetoplast; FP, flagellar pocket; Go, Golgi complex; Sp, spongiome; Cy, cytostome
Fig. 7. Time course curve of [3H]-arginine in Trypanosoma cruzi epimastigotes. [3H]-arginine uptake was evaluated in two clones of transgenic Dm28c EGFP-TcCAT1.1 epimastigotes (■,▼), in WT Dm28c (◊), and in transgenic Dm28c-EGFP, used as a control (○)
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