Ornithine uptake and the modulation of drug sensitivity in <i>Trypanosoma brucei</i>

Trypanosoma brucei, protozoan parasites that cause human African trypanosomiasis (HAT), depend on ornithine uptake and metabolism by ornithine decarboxylase (ODC) for survival. Indeed, ODC is the target of the WHO “essential medicine” eflornithine, which is antagonistic to another anti-HAT drug, suramin. Thus, ornithine uptake has important consequences in T. brucei, but the transporters have not been identified. We describe these amino acid transporters (AATs). In a heterologous expression system, TbAAT10-1 is selective for ornithine, whereas TbAAT2-4 transports both ornithine and histidine. These AATs are also necessary to maintain intracellular ornithine and polyamine levels in T. brucei, thereby decreasing sensitivity to eflornithine and increasing sensitivity to suramin. Consistent with competition for histidine, high extracellular concentrations of this amino acid phenocopied a TbAAT2-4 genetic defect. Our findings established TbAAT10-1 and TbAAT2-4 as the parasite ornithine transporters, one of which can be modulated by histidine, but both of which affect sensitivity to important anti-HAT drugs.—Macedo, J. P., Currier, R. B., Wirdnam, C., Horn, D., Alsford, S., Rentsch, D. Ornithine uptake and the modulation of drug sensitivity in Trypanosoma brucei

ANK, a Host Cytoplasmic Receptor for the Tobacco mosaic virus Cell-to-Cell Movement Protein, Facilitates Intercellular Transport through Plasmodesmata

Plasmodesma (PD) is a channel structure that spans the cell wall and provides symplastic connection between adjacent cells. Various macromolecules are known to be transported through PD in a highly regulated manner, and plant viruses utilize their movement proteins (MPs) to gate the PD to spread cell-to-cell. The mechanism by which MP modifies PD to enable intercelluar traffic remains obscure, due to the lack of knowledge about the host factors that mediate the process. Here, we describe the functional interaction between Tobacco mosaic virus (TMV) MP and a plant factor, an ankyrin repeat containing protein (ANK), during the viral cell-to-cell movement. We utilized a reverse genetics approach to gain insight into the possible involvement of ANK in viral movement. To this end, ANK overexpressor and suppressor lines were generated, and the movement of MP was tested. MP movement was facilitated in the ANK-overexpressing plants, and reduced in the ANK-suppressing plants, demonstrating that ANK is a host factor that facilitates MP cell-to-cell movement. Also, the TMV local infection was largely delayed in the ANK-suppressing lines, while enhanced in the ANK-overexpressing lines, showing that ANK is crucially involved in the infection process. Importantly, MP interacted with ANK at PD. Finally, simultaneous expression of MP and ANK markedly decreased the PD levels of callose, β-1,3-glucan, which is known to act as a molecular sphincter for PD. Thus, the MP-ANK interaction results in the downregulation of callose and increased cell-to-cell movement of the viral protein. These findings suggest that ANK represents a host cellular receptor exploited by MP to aid viral movement by gating PD through relaxation of their callose sphincters

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

Expression of <i>TbAAT5</i> and <i>TbAAT16</i> members in PCF and BSF <i>T</i>. <i>brucei</i>.

<p><i>TbAAT5</i> and <i>TbAAT16</i> mRNA abundance in PCF and BSF <i>T</i>. <i>brucei</i> was quantified by qRT-PCR using telomerase reverse transcriptase (TERT, Tb927.11.10190) as reference gene. Due to high homology of ORFs and UTRs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168775#pone.0168775.s004" target="_blank">S1 Fig</a>) only Tb427.08.4700 could be discriminated from the other <i>TbAAT5</i> members (i.e. Tb427.08.4710, Tb427.08.4720, Tb427.08.4730, Tb427.08.4740). The two <i>TbAAT16</i> genes (Tb427tmp.01.7500 and Tb427tmp.01.7520) were not differentiated.</p

Localization of cMyc-TbAAT5-1 and cMyc-TbAAT16-1 in PCF <i>T</i>. <i>brucei</i>.

<p>Confocal microscopy of procyclic <i>T</i>. <i>brucei</i> cells over-expressing N-terminally cMyc-tagged versions of TbAAT5-1 and TbAAT16-1. The proteins localized in a bright ring along the periphery of the cell body and in internal membranes, showing partial localization at the plasma membrane as well as in internal membranes of unknown identity. Anti-cMyc (green); DAPI (blue); bright field image (grey); scale bar: 5 μM. The insets show Western blot analyses of extracts from uninduced (-tet) or induced (+tet) cells probed with anti-cMyc antibody. Ponceau (Ponc) staining is shown as loading control.</p