Drug sensitivity and drug resistance in Trypanosoma brucei and Leishmania: the aquaporins

Abstract

Abstract Neglected parasitic diseases (NPD) include some of the worst human infections and are caused by pathogens including T. brucei (human African trypanosomiasis or sleeping sickness), T. cruzi (American trypanosomiasis or Chagas disease), Leishmania spp (leishmaniasis), which cause large disease burdens, as well as high mortality and morbidity rates in afflicted countries. In the absence of effective vaccines against any trypanosomatid diseases, chemotherapy is the main mechanism to combat them. Many anti-protozoal drugs are inherently cytotoxic but derive their selectivity from preferential uptake by the pathogen rather than by the host. Conversely, loss of the specific drug transporters is a main cause of drug resistance. Identification of parasite-specific targets and uptake mechanisms is critical for the development of new therapeutic agents. In this project, the aim of the research is to understand the roles of kinetoplastid aquaporins (AQPs) in trypanosomatid parasites with respect to drug resistance and transport. The observed cross-resistance between melarsoprol and pentamidine (MPXR) of the parasite threatens the status of the latter as a viable drug. There is clear consensus that a membrane transporter protein, T. brucei aquaglyceroporin-2 (TbAQP2), is implicated in the uptake of both drugs and therefore appears a likely basis for MPXR emergence. What has remained, unclear is weather the TbAQP2 protein is acting as transmembrane transporter, while situated in the T. brucei flagellar pocket, with pentamidine being a permeant, or if the protein simply acts as a receptor for pentamidine to then be internalised together via receptor-mediated endocytosis or the natural turnover of TbAQP2. Investigations on TbAQP2 were conducted to determine how the structure of TbAQP2 allows it to transport pentamidine. In T. brucei, AQP2 and AQP3 are closely related, but AQP2 has unusual selectivity filter amino acids residues in and around the pore (NSA/NPS/IVLL), compared to the latter (NPA/NPA/WGYR) in AQP3, and it also lacks the “aromatic/arginine (a/R)” motif. Using site-directed mutagenesis approaches, the TbAQP2 and TbAQP3 selectivity filter residues were therefore swapped with the goal of determining the effects of each amino acid on the uptake of drugs by Trypanosoma brucei. The results showed that the selectivity filter differences between TbAQP2 and TbAQP3 are largely responsible for their differences in pentamidine sensitivity and transport rates. Moreover, the TbAQP2 pore width was constricted using amino acids of different sizes, in order to test whether size restrictions at the cytoplasmic end of TbAQP2, i.e. below the selectivity filter, would impact on pentamidine transport. Through a combination of drug sensitivity determinations and uptake assays, the results of the introduction of different-sized amino acids at selected positions showed that it is likely that the effect depended on the residue size at the cytoplasmic end of the TbAQP2 pore. In addition, the potential correlation between the T. brucei endocytosis rate and the rate of pentamidine uptake was investigated in order to distinguish between pentamidine uptake by transporters and via endocytosis. The combined evidence of the observed results strongly suggests that pentamidine is not taken up by endocytosis, and does not induce endocytosis of TbAQP2. To conclude, the obtained results highlight a clear, direct role for the TbAQP2 membrane transporter in pentamidine uptake, with the drug most likely traversing the protein’s channel to enter the cell. The Leishmania major AQP1 was also investigated for the uptake of heavy metals antimony and arsenic. For this aim, LmAQP1 was cloned and expressed into the TbAQP1-3 null and LmAQP1 null cell lines, which were then tested for changes in sensitivity to antimony and arsenic. The results show that LmAQP1 is abl