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

Abstract

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.

Author summary

Protein channels in cell membranes transport specific molecules in and out of cells, and can also facilitate drug-uptake. One such protein, known as an aquaglyceroporin (AQP), allows parasitic African trypanosomes, the cause of lethal diseases in humans and livestock, to accumulate an arsenic-based drug known as melarsoprol. Unfortunately, parasites with a mutated AQP have resisted this drug and have spread, leading to treatment-failure in >50% of patients in some areas. The functions of this particular AQP, and two other similar AQPs normally expressed by these parasites, remain to be fully characterised in trypanosomes. We therefore generated and characterised parasites lacking all three AQPs. The cells grow well and, to our surprise, continue to effectively allow water to flow in and out of the cell. Glycerol uptake and efflux are both perturbed, however. As a consequence, drugs that cause these parasites to produce toxic quantities of glycerol are more effective against parasites lacking the AQPs. Indeed, even the melarsoprol-resistant, patient-derived parasites described above are more sensitive to these drugs. Our findings not only reveal the relative contributions of the AQPs to glycerol transport, they also point to therapies that could be more effective in the many patients infected by melarsoprol-resistant parasites.