Mechanism of aquaporin-mediated drug resistance of trypanosomes
Biochemistry
Final Report Abstract
We have investigated the molecular mechanisms of human African sleeping sickness parasites, Trypanosoma brucei, that deal with the uptake of the anti-trypanosomal drug compound pentamidine. Concomitantly, we addressed basic questions on the resistance of the parasite against the drug, because resistance is based on the failure of the drug to be taken up into the parasite cell. There was evidence in the literature that a member of the aquaporin water and neutral solute channel protein family, TbAQP2, facilitates pentamidine uptake and renders the parasites resistant if TbAQP2 is mutated or missing. A hypothesis stated that TbAQP2 acts as a direct transporter for pentamidine into trypanosomes. Since the pentamidine molecule is positively charged at physiological pH conditions and aquaporins typically exclude charged molecules from passing, we studied the transport of TbAQP2 for pentamidine and related, smaller positively charged compounds. We used yeast cells to produce TbAQP2 for growth assays on media containing pentamidine or methylammonium. Both compounds are toxic for the yeast cell and uptake would lead to reduced or absent growth. However, the yeast grew under these conditions indicating that TbAQP2 does not conduct pentamidine or the smaller methylammonium. We confirmed and quantified this result using a biophysical method, which determines the degree of light scattering of a yeast cell suspension. If the cells take up the charged compounds an increase in volume would occur that reduces the light scattering affect. Aquaporins contain a so-called selectivity filter for substrate selection. Here, TbAQP2 exposes an unusual, negatively charged amino acid residue into the transduction path, whereas virtually all other aquaporins have selectivity filters that are positively charged. We found that the negative site in TbAQP2 binds the positively charged pentamidine by electrostatic attraction and unprecedented affinity. We were able to selectively modulate this affinity by manipulating the protonation status of the substrates at different the pH conditions, and by replacing charged and uncharged amino acids in the TbAQP2 selectivity filter region by mutation. In collaboration with David Horn, Dundee, UK, we successfully transferred our findings on TbAQP2 that was produced in the yeast system to trypanosome parasites. The parasites exhibited the same resistance effect as predicted. Our data have changed the view on how pentamidine enters the trypanosome cell. Our new model explains pentamidine uptake by high-affinity binding to TbAQP2, which is subsequently internalized by the process of membrane internalization, i.e. endocytosis. The complex of pentamidine and TbAQP2 dissociates inside the cell for drug action. This concept may be exploited in the future to deliver drugs into trypanosomes.
Publications
- (2015) Challenges and achievements in the therapeutic modulation of aquaporin functionality. Pharmacol. Ther. 155: 22-35
Beitz, E., Golldack, A., Rothert, M., von Bülow, J.
(See online at https://doi.org/10.1016/j.pharmthera.2015.08.002) - (2015) Number and regulation of protozoan aquaporins reflect environmental complexity. Biol. Bull. 229: 38-46
von Bülow, J., Beitz, E.
(See online at https://doi.org/10.1086/BBLv229n1p38) - (2016) Attacking aquaporin water and solute channels of humanpathogenic parasites: new routes for treatment? in Aquaporins in Health and Disease, CRC Press
von Bülow, J., Beitz, E.
- (2016) Pentamidine is not a permeant but a nanomolar inhibitor of the Trypanosoma brucei aquaglyceroporin-2. PLoS Pathog. 12: e1005436
Song, J., Baker, N., Rothert, M., Henke, B., Jeacock, L., Horn, D., Beitz, E.
(See online at https://doi.org/10.1371/journal.ppat.1005436) - (2017) Electrostatic attraction of weak monoacid anions increases probability for protonation and passage through aquaporins. J. Biol. Chem. 292: 9358-9364
Rothert, M., Rönfeldt, D., Beitz E.
(See online at https://doi.org/10.1074/jbc.M117.782516)
