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

Primaerenergieverbrauch in der Bundesrepublik Deutschland 1. Halbjahr 1990/1991

SIGLECopy held by FIZ Karlsruhe; available from UB/TIB Hannover / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman

TbKIFC1 depletion complements NHS sensitivity of <i>12940</i> null <i>T</i>. <i>b</i>. <i>brucei</i>, but has no additive effect on apoL1 sensitivity or suramin efficacy.

<p>Representative EC₅₀ assays carried out in quadruplicate showing the impact of TbKIFC1 RNAi knockdown in wild type (left hand panels) and <i>12940</i> null (middle panels) <i>T</i>. <i>b</i>. <i>brucei</i> on (A) apoL1, (B) NHS and (C) suramin sensitivity. Right hand panels, pooled EC₅₀ data for at least three independent cell lines. Error bars, standard deviation.</p

Evidence for interdependence between Tb927.9.8000 and Tb927.10.12940.

<p>Representative EC₅₀ assays carried out in quadruplicate showing the impact of Tb927.9.8000 RNAi knockdown in wild type (left hand panels) and <i>12940</i> null (middle panels) <i>T</i>. <i>b</i>. <i>brucei</i> on (A) apoL1 and (B) NHS sensitivity. Right hand panels, pooled EC₅₀ data for at least three independent cell lines. Error bars, standard deviation.</p

Generation and complementation of <i>12940</i> null <i>T</i>. <i>b</i>. <i>brucei</i>.

<p>(A) Southern blot confirming <i>Tb927</i>.<i>10</i>.<i>12940</i> deletion; <i>BSD</i>, blasticidin-S-deaminase; <i>NPT</i>, neomycin phosphotransferase. Schematic showing segment deleted from the <i>Tb927</i>.<i>10</i>.<i>12940</i> locus (open triangle); positions of <i>Sal</i>I restriction sites (arrowheads), deletion (12940) and flanking (DS) probes highlighted. (B) Cumulative growth of wild type and three independent <i>12940</i> null <i>T</i>. <i>b</i>. <i>brucei</i> cell lines; error bars showing standard deviation are smaller than the plot symbols. (C, D) Representative EC₅₀ assays carried out in quadruplicate showing the impact of (C) <i>Tb927</i>.<i>10</i>.<i>12940</i> deletion and (D) re-expression on apoL1 sensitivity. Insets, pooled EC₅₀ data for at least two independent cell lines. (E, F) Kinetic analyses of parasite killing in 10 μg.ml^-1 apoL1 following (E) <i>Tb927</i>.<i>10</i>.<i>12940</i> deletion and (F) re-expression; each assay was carried out using three independent cell lines, re-expression was induced in 1 μg.ml^-1 tetracycline for 24 hours prior to exposure to apoL1. Error bars, standard deviation; <i>P</i>-values derived from paired students t-test.</p

RIT-seq profiles of candidate novel apoL1-sensitivity determinants.

<p>ApoL1 sensitivity determinants include (A) putative ubiquitin modifiers and (B) putative membrane trafficking proteins. RIT-seq profiles show predicted transcripts (open reading frames and untranslated regions of interest in black) and the RNAi targeting fragment reads mapped; total reads (red) and tagged reads (blue; containing the 14-base RNAi construct barcode sequence) mapped to each predicted transcript are shown in the top right corner of each panel. Known or putative annotations based on domain organisation are included beneath each accession number and derived from GeneDB (<a href="http://www.genedb.org/Homepage/Tbruceibrucei927" target="_blank">http://www.genedb.org/Homepage/Tbruceibrucei927</a>). Ub, ubiquitin; DUB, deubiquitinase; VAMP, vesicle-associated membrane protein; VAP, VAMP-associated protein.</p

Selecting a genome-scale <i>T</i>. <i>b</i>. <i>brucei</i> RNAi library identifies parasite determinants of apoL1 sensitivity.

<p>(A) Genome-wide human apoL1 RIT-seq profile representing 7,398 non-redundant predicted transcripts, showing those targeted by 100 or more reads per kilobase containing the 14-base RNAi construct barcode sequence. ‘Hits’ targeting V-ATPase subunits and the kinesin, TbKIFC1, are highlighted in green and red, respectively; novel high confidence hits corresponding to six putative ubiquitin modifiers and four putative membrane transporters are highlighted in black and blue, respectively (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006855#ppat.1006855.g004" target="_blank">Fig 4</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006855#ppat.1006855.s007" target="_blank">S1 Table</a> for further details). (B) RNAi library selection with human or <i>P</i>. <i>papio</i> apoL1 identified similar sets of sensitivity determinants (r² = 0.78); key hits are coloured as in (A), and are similarly abundant in both selected libraries. Data presented as RPKM (plus 0.1; <u>r</u>eads <u>p</u>er <u>k</u>ilobase of transcript per <u>m</u>illion mapped reads) to correct for variations in read depth between the respective RNAi library screens (and to enable plotting of zero read outputs on log₁₀ scales). Dashed lines represent the 100-read stringency criterion converted to RPKM for each RNAi library screen (human apoL1, 158; <i>P</i>. <i>papio</i> apoL1, 132). (C) RIT-seq profiles for three of the V-ATPase subunits and for TbKIFC1; predicted transcripts (open reading frames and untranslated regions) are coloured as in (A); total reads (red) and tagged reads (blue; containing the 14-base RNAi construct barcode sequence) mapped to each predicted transcript are shown in the top right corner of each panel.</p

Tb927.10.12940 has opposing effects on apoL1 and NHS sensitivity, and localises to the lysosome.

<p>Representative EC₅₀ assays carried out in quadruplicate showing the impact of Tb927.10.12940 RNAi knockdown on <i>T</i>. <i>b</i>. <i>brucei</i> sensitivity to (A) apoL1 and (B) NHS. Inset charts show pooled EC₅₀ data for three independent cell lines. Error bars, standard deviation; <i>P</i>-values derived from paired students t-test. (C) Western blot showing tetracycline (tet)-inducible expression of Tb927.10.12940^GFP. (D) Immunofluorescence localisation of Tb927.10.12940^GFP and the lysosomal membrane protein, p67; counter-staining with the DNA intercalating dye, DAPI, reveals the kinetoplast (k) and nucleus (n). A processed merged image of the region of interest shows the co-localisation of Tb927.10.12940^GFP and p67. Scale bar, 5 μm.</p

Distinct sets of <i>T</i>. <i>b</i>. <i>brucei</i> proteins determine parasite sensitivity to apoL1 and NHS.

<p>(A) Reads containing the 14-base RNAi construct-specific barcode identified following RNAi library selection in human apoL1 (this study) or NHS [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006855#ppat.1006855.ref032" target="_blank">32</a>] plotted as RPKM (plus 0.1) to correct for variations in read depth between the respective RNAi library screens (and to enable plotting of zero read outputs on log₁₀ scales); r² = 0.016. Dashed lines represent the 100-read stringency criterion converted to RPKM for each RNAi library screen (human apoL1, 158; NHS, 404); apoL1 (purple) and NHS (orange)-specific RNAi targets are highlighted and the top hits listed (gene ID and functional annotation; see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006855#ppat.1006855.s008" target="_blank">S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006855#ppat.1006855.s009" target="_blank">S3</a> Tables for further details). (B-D) Representative EC₅₀ assays carried out in quadruplicate confirming that (B) p67 knockdown, (C) <i>ICP</i> deletion and (D) TbCATL depletion does not affect <i>T</i>. <i>b</i>. <i>brucei</i> sensitivity to apoL1. Insets, representative EC₅₀ assays carried out in quadruplicate showing the known impact of the corresponding manipulations on sensitivity to NHS. Error bars, standard deviation.</p