A high-affinity putrescine-cadaverine transporter from Trypanosoma cruzi

Abstract

Whereas mammalian cells and most other organisms can synthesize polyamines from basic amino acids, the protozoan parasite Trypanosoma cruzi is incapable of polyamine biosynthesis de novo and therefore obligatorily relies upon putrescine acquisition from the host to meet its nutritional requirements. The cell surface proteins that mediate polyamine transport into T. cruzi, as well as most eukaryotes, however, have by-in-large eluded discovery at the molecular level. Here we report the identification and functional characterization of two polyamine transporters, TcPOT1.1 and TcPOT1.2, encoded by alleles from two T. cruzi haplotypes. Overexpression of the TcPOT1.1 and TcPOT1.2 genes in T. cruzi epimastigotes revealed that TcPOT1.1 and TcPOT1.2 were high-affinity transporters that recognized both putrescine and cadaverine but not spermidine or spermine. Furthermore, the activities and subcellular locations of both TcPOT1.1 and TcPOT1.2 in intact parasites were profoundly influenced by extracellular putrescine availability. These results establish TcPOT1.1 and TcPOT1.2 as key components of the T. cruzi polyamine transport pathway, an indispensable nutritional function for the parasite that may be amenable to therapeutic manipulation.

Fig. 1

Functional characterization of TcPOT1.1 and TcPOT1.2 in T. cruzi epimastigotes. A. Comparison of the abilities of T. cruzi epimastigotes expressing either pTEX-TcPOT1.1 (TcPOT1.1), pTEX-TcPOT1.1::GFP (TcPOT1.1::GFP) or pTEX-TcGRASP::GFP (control) to take up 417 nM [³H]putrescine. Each value represents the mean ± SD of three independent experiments. B. Comparison of the abilities of T. cruzi epimastigotes expressing either pTEX-TcPOT1.2 (TcPOT1.2), pTEX-TcPOT1.2::GFP (TcPOT1.2::GFP) or pTEX-GFP (control) to take up 484 nM [3H]putrescine. Each value represents the mean ± SD of three independent experiments. C. Michaelis-Menten formulation of putrescine uptake rates obtained for T. cruzi epimastigotes expressing either pTEX-TcPOT1.1::GFP (●) or pTEX-TcPOT1.2::GFP () that had been grown in SDM-79 containing 10% FBS and 200 μM putrescine. Values for endogenous transport in control pTEX-TcGRASP::GFP- or pTEX-GFP-transfected parasites were subtracted from the experimental rates. D. Inhibition profile for TcPOT1.1- and TcPOT1.2-mediated putrescine transport. The ability of the pTEX-TcPOT1.1::GFP and pTEX-TcPOT1.2::GFP transfectants to take up 0.5 μM [3H]putrescine was evaluated in the presence of a variety of structurally related non-radiolabelled compounds, each at a concentration of 50 μM. Results are plotted as a percentage of putrescine uptake obtained without inhibitor (NI). Each value represents the mean ± SD of three independent experiments. E. Abilities of epimastigotes transfected with either pTEX-TcPOT1.1::GFP or pTEX-TcPOT1.2::GFP and grown in putrescine-supplemented SDM-79 medium to take up [¹⁴C]cadaverine over a range of cadaverine concentrations. Uptake rates obtained with the control pTEX-TcGRASP::GFP transfectant were subtracted from experimental data points. Data were fitted to a Michaelis-Menten algorithm to determine apparent K_m and V_max values. F. Michaelis-Menten plot of putrescine uptake rates obtained for T. cruzi epimastigotes expressing pTEX-TcPOT1.1::GFP that had been grown either in SDM-79 containing 10% FBS in the absence (○) or presence (●) of 200 μM putrescine for 48 h. The pTEX-TcGRASP::GFP expressing transfectant served as the control for endogenous transport, and control rates were subtracted from the experimental rates. G. Michaelis-Menten analysis of putrescine uptake rates obtained for T. cruzi epimastigotes expressing pTEX-TcPOT1.2::GFP that had been grown either in SDM-79 containing 10% FBS in the absence (○) or presence (●) of 200 μM putrescine for 48 h. The pTEX-GFP expressing transfectant served as the control for endogenous transport and control rates were subtracted from the experimental rates.

Fig. 2

Localization of TcPOT1.1 and TcPOT1.2 in T. cruzi epimastigotes grown in putrescine-replete media. A. and B. Fluorescence images of fixed T. cruzi epimastigotes expressing either a GFP tag fused to the C-terminus of TcPOT1.1 and TcPOT1.2 (TcPOT1.1-GFP and TcPOT1.2-GFP) or an HA tag fused to the NH₂-terminus of TcPOT1.1 and TcPOT1.2 (HA-TcPOT1.1 and HA-TcPOT1.2). Parasites were propagated in SDM-79 supplemented with 200 μM putrescine. C. Schematic representation of an epimastigote form of T. cruzi adapted from a drawing by Docampo et al. (2005). D. Fluorescence images of T. cruzi epimastigotes demonstrating the absence of colocalization of calmodulin, a presumed contractile vacuolar marker (Rohloff et al., 2004) with TcPOT1.1-GFP and HA-TcPOT1.2 implying that the two permeases are not located at the contractile vacuole. Fluorescence for TcPOT1.1-GFP and TcPOT1.2-GFP (green), TRITC-concanavalin A (red), anti-calmodulin (red) and DAPI (blue) were determined as indicated by the labels in the figure. Differential interference contrast (DIC) images depict the same parasites shown in the fluorescent images.

Fig. 3

Ultrastructural detection of TcPOT1.1 and TcPOT1.2 in transgenic T. cruzi. Immunogold labelling of HA-TcPOT1.1 (A–E) and HA-TcPOT1.2 (F–I) with anti-HA antibodies. Both transporters were observed in the Golgi apparatus (A–C and F) and the contractile vacuole system close to the flagellar pocket (A–D and F–G), while TcPOT1.1 was also detected in a proximal ‘spongy’ structure of unknown identity (E). CV, contractile vacuole; FP, flagellar pocket; Go, Golgi apparatus; k, kinetoplast; n, nucleus; s, spongiosome. Scale bars are 200 nm.

Fig. 4

Localization of TcPOT1.1 and TcPOT1.2 in T. cruzi epimastigotes grown in putrescine-deficient medium. Parasites were grown in SDM-79 supplemented with 10% FBS but lacking putrescine. Fluorescence for TcPOT1.1-GFP and TcPOT1.2-GFP (green), TRITC-concanavalin A (red) and DAPI (blue) were ascertained as specified in the figure. Differential interference contrast (DIC) images depict the same parasites shown in the fluorescent images.