Drug resistance in African trypanosomes

African sleeping sickness reached epidemic proportions in the 1990s, with an estimated 500,000 people infected and millions at risk in 36 African countries. The disease is caused by a parasite called a trypanosome, transmitted by bites of the tsetse fly, and is fatal if not treated. Available drugs are very old, introduced in the 1940s. The early stage, with parasites living freely in the blood, can be effectively treated with pentamidine but treatment can have serious side effects and has to be given through multiple injections in hospital. The treatment for the late stage, in which the parasite has entered the brain and causes severe neurological symptoms ending in coma and death, is melarsoprol - a drug based on arsenic and so toxic that it causes brain damage in up to 10% of patients, killing up to half of these. The only alternative treatment, eflornithine, is very expensive and requires four daily intravenous infusions for up to two weeks ? often impossible in rural Africa. A further problem is that the parasite has developed resistance to melarsoprol, leading to high levels of treatment failure, risking arsenic-induced brain damage for nothing. Newer drugs are in development but these are of the same class as pentamidine, known as diamidine drugs, and parasites resistant to melarsoprol are often cross-resistant to diamidines as well.
We have studied melarsoprol and diamidine resistance in these parasites and found that it is caused by the loss of particular proteins, embedded in the parasites surface membrane, that facilitate the uptake of very specific nutrients by the organism. One such protein, P2, is responsible for uptake of the essential compound adenosine but both diamidines and melarsoprol use this conduit to enter the trypanosome as well. Mutations in this transporter lead to mild resistance to both classes of drugs but we recently found that high levels of resistance would require the further loss of a second transport protein, HAPT, while some diamidines can further enter the parasite through yet a third protein, LAPT. The aim of the proposed research project is to identify the genes for these new transporters so that it can be assessed whether mutations in these genes can indeed be linked to resistance. We further want to study the transporters in sufficient detail to allow chemists to make custom-designed drugs that will use all three transporters, making the new generation of medicines more effective and circumventing resistance.

Human African trypanosomiasis, better known as sleeping sickness, continues to be a scourge of rural Africa. By far the highest incidence is caused by tsetse fly-transmitted infection with the protozoan parasite Trypanosoma brucei gambiense, which causes West-African sleeping sickness. This is a chronic disease that is believed to have a near 100% fatality rate unless adequately treated. In the early stage of the disease the parasite is present in the haemo-lymphatic system and this stage is treated with the diamidine drug pentamidine. In the late stage the parasite has penetrated the blood-brain barrier and infiltrated the central nervous system, causing the characteristic neurological symptoms. This stage is usually treated with the arsenic-containing drug melarsoprol, except in some areas with particularly high incidence of treatment failure where difluormethyl ornithine (DFMO) has been introduced as a replacement.
The very high rate of melarsoprol treatment failure, occurring in geographically distant foci, as well as the well-documented phenomenon of arsenical-diamidine cross-resistance is a genuine threat to the control of this disease, particularly since the only new treatment in development, furamidine, is also a diamidine. We have studied diamidine and arsenical resistance in African trypanosomes and found that this is caused by changes in plasma membrane transporters, leading to reduced uptake of the drug by the parasite. Low level resistance to both pentamidine and melarsoprol are observed when the TbAT1 gene, encoding the P2 aminopurine transporter, is deleted. Much higher levels of resistance to both drugs are in evidence upon subsequent loss of a second transporter, the High Affinity Pentamidine Transporter (HAPT). A third diamidine transporter, the Low Affinity Pentamidine Transporter, does not appear to be involved in arsenical uptake.
While the identity of the P2 transporter gene is known and specific mutations have been tentatively linked to melarsoprol resistance, the genes encoding HAPT and LAPT are not known. The primary aim of this proposal is to identify and clone these genes and express them in a suitable system for further characterisation. Identification of the genes will enable us to assess (1) their status in drug resistant laboratory strains and field isolates and (2) their physiological role in the parasite. We further aim to study the substrate selectivity of these transporters in detail, constructing a model for substrate recognition that will inform the synthesis of improved diamidine drugs that are efficiently taken up trough multiple transporters including LAPT and thus avoid cross-resistance with melarsoprol.