<i>T</i>. <i>b</i>. <i>brucei</i> tolerates the loss of all three <i>AQPs</i>.

<p>(A) The schematic maps indicate the <i>AQP1</i> and <i>AQP2-3</i> regions replaced by selectable markers as also indicated on the right. Δ indicates the regions deleted while the probes used for Southern blotting are shown above the maps. H, <i>Hpa</i>I; S, <i>Sac</i>II. (B) The Southern blots indicate deletion of the <i>AQP1</i> alleles in <i>aqp1</i> and three independent <i>aqp1-2-3</i> strains. Wild-type (WT) is shown for comparison. Genomic DNA was digested with <i>Hpa</i>I. (C) The Southern blots indicate deletion of the <i>AQP2-3</i> alleles in <i>aqp1-2-3</i> strains. WT is shown for comparison. Genomic DNA was digested with <i>Sac</i>II.</p

Glycerol uptake and utilisation is perturbed in <i>aqp</i>-null <i>T</i>. <i>b</i>. <i>brucei</i>.

<p>(A) ATP levels were assessed in the strains indicated after incubation in 5 mM glucose or glycerol. Readings were taken in triplicate and normalised to substrate only. * indicates significantly different (<i>P</i><0.001) to wild-type (WT) using an ANOVA test in GraphPad Prism. Error bars, SD. (B) Radiolabelled glycerol uptake was assessed in the strains indicated. Readings were taken in quadruplicate. * indicates significant difference (<i>P</i><0.05) using a Student’s <i>t</i>-test. Error bars, SD.</p

Respiratory inhibitor-sensitivity in <i>T</i>. <i>b</i>. <i>gambiense</i> isolates and AQP-mediated glycerol transport.

<p>(A) SHAM EC₅₀ values for the <i>T</i>. <i>b gambiense</i> strains are indicated +/- glycerol. The inset shows pentamidine EC₅₀ values. * indicates significantly different (<i>P</i><0.05) to STIB930 using an ANOVA test in GraphPad Prism. All pairwise comparisons +/- 10 mM glycerol also indicated significant (<i>P</i> <0.001) differences using a Student’s <i>t</i>-test. Error bars, SD. (B) Propyl gallate and (C) Ascofuranone EC₅₀ values. Other details as in A. (D) Model for glycerol transport by AQPs in <i>T</i>. <i>b</i>. <i>gambiense</i>. The weight of the arrows indicates relative impact on glycerol utilisation and efflux, with AQP2 being the major contributor; note that transport across both the plasma and glycosomal membranes contributes to glycerol utilisation and efflux, see the text for more details. The right-hand panel indicates the situation in melarsoprol-resistant (reduced melarsoprol uptake) and SHAM-sensitive (reduced glycerol efflux) clinical isolates where a chimeric AQP2/3 replaces AQP2 and AQP3.</p

<i>aqp</i>-null <i>T</i>. <i>b</i>. <i>brucei</i> display defective glycerol-efflux and respiratory inhibitor-sensitivity.

<p>(A) Bloodstream <i>T</i>. <i>brucei</i> express a SHAM-sensitive mitochondrial trypanosome alternative oxidase (TAO). Under aerobic conditions, TAO activity allows ATP production without glycerol production as indicated by the black lines (left-hand blue ‘cell’). SHAM blocks TAO-activity, leading to the anaerobic production of glycerol, which is toxic if not removed, as indicated by the black lines (right-hand blue ‘cell’). SHAM dose-response curves for wild-type (WT) and <i>aqp1-2-3</i> null-cells. EC₅₀ values are indicated. (B) In the presence of SHAM and glycerol, the glycerol inhibits glycerol kinase (GK), also preventing ATP-production by the anaerobic route (blue ‘cell’). SHAM dose-response curves as in A but in the presence of 10 mM glycerol. (C) Propyl gallate and octyl gallate dose-response curves for wild-type (WT) and <i>aqp1-2-3</i> null-cells. EC₅₀ values are indicated. (D) SHAM EC₅₀ values +/- 10 mM glycerol from A-B and also from <i>aqp2</i>, <i>aqp2-3</i> and <i>aqp1-2-3</i> cells re-expressing ^GFPAQP2. * indicates significantly different (<i>P</i><0.01) to WT using an ANOVA test in GraphPad Prism. Pairwise comparisons +/- glycerol, except in the case of the <i>aqp1-2-3</i> null, indicated significant (<i>P</i> <0.001) differences using a Student’s <i>t</i>-test. Error bars, SD. The images to the right show re-expression of ^GFPAQP2 in <i>aqp1-2-3</i> null-cells.</p

Model of the pentamidine binding mode to TbAQP2 and proposed uptake by endocytosis in the flagellar pocket.

<p>(A) Shown are the crystal structure of the prototypical aquaglyceroporin GlpF and a model of TbAQP2. GlpF Arg206 and TbAQP2 Leu264 mark the position of the ar/R selectivity filter. In TbAQP2, the Asp265 sidechain carboxylate binds to an amidine moiety of pentamidine (light blue), whereas in GlpF the space is occupied by the guanidine sidechain of Arg206. The location of the ‘NPA/NPA’ region (white bar) and sequence deviations in TbAQP2 are indicated. (B) Proposed uptake mechanism of pentamidine via high-affinity binding to TbAQP2, endocytosis of the complex, and release of pentamidine in the acidic lysosome due to pH shift or TbAQP2 degradation.</p

Inhibition of TbAQP2 glycerol permeability by pentamidine and derivatives.

<p>(A) Shown are example traces of protoplast light scattering in 300 mM isotonic glycerol gradients in the presence of pentamidine. Red traces indicate uninhibited glycerol influx via TbAQP2 (left) and TbAQP3 (right), effects of pentamidine concentrations from 50 nM to 50 μM are colored light blue, and of 500 μM pentamidine in dark blue. (B) Structure-function evaluation of TbAQP2 inhibition by pentamidine. The chemical structures of the used compounds are shown on the left and respective dose-response curves on the right. (C) Effect of pH titration of TbAQP2 Asp265 and replacement by mutation to alanine on pentamidine inhibition. The dashed lines show the pentamidine inhibition of wild-type TbAQP2 at pH 7.2 (data taken from Fig 2B). The left panel shows dose-response curves of pentamidine pH 3.5 (closed symbols; IC₅₀ 450 nM), and pH 2.5 (open symbols; IC₅₀ 1.1 μM). The effect of the Asp265Ala point mutation (expression confirming Western blot in the inset) on the inhibition by pentamidine is depicted on the right. Data taken at pH 7.2 (IC₅₀ 4.5 μM) are indicated by closed symbols, those at pH 2.5 (IC₅₀ 5.8 μM) by open symbols. Each data point is an average of 5–9 light scattering traces each from at least two independent experiments.</p