Mechanisms of drug resistance in T. brucei: beyond the P2 transporter

The principal aim of this project was to investigate mechanisms of drug resistance in Trypanosoma brucei, the causative agent of disease in humans (sleeping sickness) and livestock (nagana), which affects large areas of sub-Saharan Africa. By understanding the mechanisms of resistance, the useful life of current therapies (of which there are only a few) may be extended, diagnostics to identify resistant parasites could be developed and the design of novel therapies aided. We therefore developed parasites with high levels of resistance to the clinically important drug pentamidine, which is the first-line treatment for early stage West African sleeping sickness and is closely related to the main veterinary treatment diminazene aceturate (Berenil). The characterisation of this strain revealed that the resistance phenotype was at least in part due to the loss of the previously characterised high affinity pentamidine transporter (HAPT). To investigate the protein(s) responsible for HAPT activity, and to identify any other proteins contributing to the resistance phenotype, we employed a proteomic approach. The plasma membrane sub-proteome (TbPM) of long slender bloodstream form trypanosomes was defined. A number of interesting observations were made from TbPM, and it will no doubt be of benefit to the greater scientific community. One example is the positive identification of many proteins hitherto designated as putative. A quantitative approach was then employed to analyse the resistant parasites using isotope-coded affinity tagging (I-CAT) and difference gel electrophoresis (DiGE), including a novel combination of DiGE and 16-BAC protein separation technologies. Both the plasma membrane subproteome and the soluble proteome were investigated, and a number of regulated proteins identified. The role of some proteins, with potentially relevant functions, such as a kinase, adenylate cyclase and a protein involved in kinetoplast stability should be further investigated