RNA editing in trypanosomes: a tale of two ligases

Uridylyl insertion/deletion mRNA editing is essential for mitochondrial gene expression in Trypanosoma brucei and governed by multi-protein complexes called editosomes. The final step in each cycle of this post-transcriptional process is that of re-ligating the edited mRNA fragments. The ~20S RNA editing core complex contains two RNA editing ligases, REL1 and REL2, located, respectively, in a deletion and an insertion subcomplex. While REL1 is clearly essential for RNA editing, REL2 knockdown by RNAi has not resulted in a detectable phenotype. To explain these findings, alternative scenarios have been suggested: (a) REL2 is not functional in vivo; (b) REL1 can function in both insertion and deletion editing, whereas REL2 can only function in insertion editing; (c) REL1 has an additional role in repairing erroneously cleaved mRNAs. To further investigate respective functions of the two RELs this study used three complimentary approaches: (i) genetic complementation with chimeric ligase enzymes, (ii) deep sequencing of RNA editing intermediates after ligase inactivation, and (iii) evolutionary analysis. In vivo expression of two chimeric ligases, providing a REL2 catalytic domain at REL1’s position in the deletion subcomplex and a REL1 catalytic domain at REL2’s position in the insertion subcomplex, did not rescue the growth defect caused by REL1 ablation. Although the results were not fully conclusive they suggest that it is the specific catalytic properties of REL1 rather than its position within the deletion subcomplex that makes it essential. In order to identify in vivo substrates of REL1, specific editing intermediates that accumulated after genetic knockdown of REL1 expression were captured by 5’ linker and deep sequenced using Ion Torrent and Illumina technology. Analyses of such unligated editing intermediates with bespoke bioinformatics tools suggest that REL1 functions in deletion editing as expected, but also in the repair of miscleaved mRNAs, implying a novel role for this ligase. Neither role can be fulfilled by REL2, at least not with sufficient efficiency. Sequencing data also suggest that either REL1 is not involved in ligation of addition editing substrates, or that REL2 in this case can fully compensate for loss of REL1. REL1, REL2 and KREPA3 sequences were subjected to analysis using MEGA5 and the HyPhy package available on the Datamonkey adaptive evolution server. Results indicated that all three editosome genes are under much stronger purifying than diversifying selective forces. In general this selection pressure to conserve protein sequence increased from KREPA3 to REL2 to REL1, suggesting a requirement to maintain catalytic function for both ligases. Taken together, these experiments reveal a novel function for REL1 during RNA editing, providing a rationale for its essentiality. Deductively, the results also suggest REL2, which was previously thought to be non-essential, may still be required by the cell at its position in the addition subcomplex. Evolutionary analysis suggests that the RELs and KREPA3 are under the same evolutionary forces to maintain their respective functions in RNA editing

Repositioning of a diaminothiazole series confirmed to target the cyclin-dependent kinase CRK12 for use in the treatment of African animal trypanosomiasis

African animal trypanosomiasis or nagana, caused principally by infection of the protozoan parasites Trypanosoma congolense and Trypanosoma vivax, is a major problem in cattle and other livestocks in sub-Saharan Africa. Current treatments are threatened by the emergence of drug resistance and there is an urgent need for new, effective drugs. Here, we report the repositioning of a compound series initially developed for the treatment of human African trypanosomiasis. A medicinal chemistry program, focused on deriving more soluble analogues, led to development of a lead compound capable of curing cattle infected with both T. congolense and T. vivax via intravenous dosing. Further optimization has the potential to yield a single-dose intramuscular treatment for this disease. Comprehensive mode of action studies revealed that the molecular target of this promising compound and related analogues is the cyclin-dependent kinase CRK12

Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2

The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis

Aquaglyceroporin-null trypanosomes display glycerol transport defects and respiratory-inhibitor sensitivity

Aquaglyceroporins (AQPs) transport water and glycerol and play important roles in drug-uptake in pathogenic trypanosomatids. For example, AQP2 in the human-infectious African trypanosome, Trypanosoma brucei gambiense, is responsible for melarsoprol and pentamidine-uptake, and melarsoprol treatment-failure has been found to be due to AQP2-defects in these parasites. To further probe the roles of these transporters, we assembled a T. b. brucei strain lacking all three AQP-genes. Triple-null aqp1-2-3 T. b. brucei displayed only a very moderate growth defect in vitro, established infections in mice and recovered effectively from hypotonic-shock. The aqp1-2-3 trypanosomes did, however, display glycerol uptake and efflux defects. They failed to accumulate glycerol or to utilise glycerol as a carbon-source and displayed increased sensitivity to salicylhydroxamic acid (SHAM), octyl gallate or propyl gallate; these inhibitors of trypanosome alternative oxidase (TAO) can increase intracellular glycerol to toxic levels. Notably, disruption of AQP2 alone generated cells with glycerol transport defects. Consistent with these findings, AQP2-defective, melarsoprol-resistant clinical isolates were sensitive to the TAO inhibitors, SHAM, propyl gallate and ascofuranone, relative to melarsoprol-sensitive reference strains. We conclude that African trypanosome AQPs are dispensable for viability and osmoregulation but they make important contributions to drug-uptake, glycerol-transport and respiratory-inhibitor sensitivity. We also discuss how the AQP-dependent inverse sensitivity to melarsoprol and respiratory inhibitors described here might be exploited

<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