Trypanosoma brucei subspecies cause Human African Trypanosomiasis, which is fatal unless treated. T. brucei Aquaglyceroporin 2 (TbAQP2) is required for the uptake of two of the drugs used clinically, pentamidine and melarsoprol. TbAQP2 mutations or the formation of a chimeric gene with TbAQP3 (TbAQP2X3) cause pentamidine-melarsoprol cross-resistance (PMXR). TbAQP2 is an aquaporin, a type of water permeable pore forming integral membrane protein, which typically exclude large or positively charged molecules. Melarsoprol (398 Da) and pentamidine (340 Da) are significantly larger than any other known permeants for aquaporins and pentamidine is double positively charged under physiological conditions. The molecular mechanism of the TbAQP2-linked drug uptake in trypanosomes is controversial. Suggested mechanisms include transport through the unconventional TbAQP2 pore and indirect uptake via endocytosis of the TbAQP2-drug complexes. The physiological role of trypanosome aquaporins (TbAQP1-2-3) remains enigmatic, since TbAQP1-2-3 knockout trypanosomes are viable in vitro.

This thesis describes work towards answers to the following questions: what is the molecular mechanism of solute transport by TbAQP2? What are the TbAQP2-linked pentamidine/melarsoprol uptake mechanisms? What are the structural differences between TbAQP2 and TbAQP2X3? Finally, what is the physiological role of TbAQP2 and TbAQP3?

These questions were addressed in structural, functional and physiological experiments. A protocol for heterologous expression in insect cells and for the purification of TbAQP2 and TbAQP2X3 in DDM micelles was established and optimised to obtain pure, monodisperse, homogenous and tetrameric proteins. Subsequent reconstitution into saposin A-lipid nanoparticles was essential to stabilise TbAQP2 and TbAQP2X3 sufficiently in a buffer compatible with cryo-EM structural studies. Cryo-EM sample preparation of TbAQP2, TbAQP2X3 and the TbAQP2-drug complexes was optimised and suitable particles could be validated for all samples in cryo-EM screenings. Data sets were collected and initial image processing enabled refinement of a 3D map of WT TbAQP2 to 8.9 Å resolution. This map validated the tetrameric structure of TbAQP2, but the resolution was not high enough to elucidate its molecular mechanism. Comparison of models for the structure of TbAQP2 and TbAQP2 variants causing the PMXR phenotype, showed differences in the selectivity filter and/or other locations including loop E and transmembrane helices 4, 5 or 6.

The functional state of the purified TbAQP2 reconstituted into liposomes was validated in glycerol uptake/water efflux assays. Radiolabelled pentamidine uptake/binding assays using functional TbAQP2 reconstituted liposomes were developed but did not enable clarification of the drug uptake mechanism.

Analysis of the evolutionary appearance of the T. brucei aquaporins indicated that TbAQP2 and TbAQP3 are the products of a recent gene duplication. The physiological role of TbAQP2 and TbAQP3 was addressed by making pleomorphic TbAQP2-3 knockout trypanosome cell lines. These were viable in vitro and could be differentiated from long slender bloodstream form to dividing procyclic form in vitro, indicating that TbAQP2 and TbAQP3 are not required for this developmental transition.

Together, the experiments in this thesis provide the ground work to elucidate the molecular mechanism of TbAQP2 and how alterations in TbAQP2 mediate drug resistance.