SUMMARYPathogenic animal trypanosomes affecting livestock have represented a major constraint to agricultural development in Africa for centuries, and their negative economic impact is increasing in South America and Asia. Chemotherapy and chemoprophylaxis represent the main means of control. However, research into new trypanocides has remained inadequate for decades, leading to a situation where the few compounds available are losing efficacy due to the emergence of drug-resistant parasites. In this review, we provide a comprehensive overview of the current options available for the treatment and prophylaxis of the animal trypanosomiases, with a special focus on the problem of resistance. The key issues surrounding the main economically important animal trypanosome species and the diseases they cause are also presented. As new investment becomes available to develop improved tools to control the animal trypanosomiases, we stress that efforts should be directed towards a better understanding of the biology of the relevant parasite species and strains, to identify new drug targets and interrogate resistance mechanisms.

Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.

African trypanosomes cause sleeping sickness in humans, a disease that is typically fatal without chemotherapy. Unfortunately, drug resistance is common and melarsoprol-resistant trypanosomes often display cross-resistance to pentamidine. Although melarsoprol/pentamidine cross-resistance (MPXR) has been an area of intense interest for several decades, our understanding of the underlying mechanisms remains incomplete. Recently, a locus encoding two closely related aquaglyceroporins, AQP2 and AQP3, was linked to MPXR in a high-throughput loss-of-function screen. Here, we show that AQP2 has an unconventional "selectivity filter." AQP2-specific gene knockout generated MPXR trypanosomes but did not affect resistance to a lipophilic arsenical, whereas recombinant AQP2 reversed MPXR in cells lacking native AQP2 and AQP3. AQP2 was also shown to be disrupted in a laboratory-selected MPXR strain. Both AQP2 and AQP3 gained access to the surface plasma membrane in insect life-cycle-stage trypanosomes but, remarkably, AQP2 was specifically restricted to the flagellar pocket in the bloodstream stage. We conclude that the unconventional aquaglyceroporin, AQP2, renders cells sensitive to both melarsoprol and pentamidine and that loss of AQP2 function could explain cases of innate and acquired MPXR.

Leishmania donovani causes visceral leishmaniasis (VL), the second most deadly vector-borne parasitic disease. A recent epidemic in the Indian subcontinent (ISC) caused up to 80% of global VL and over 30,000 deaths per year. Resistance against antimonial drugs has probably been a contributing factor in the persistence of this epidemic. Here we use whole genome sequences from 204 clinical isolates to track the evolution and epidemiology of L. donovani from the ISC. We identify independent radiations that have emerged since a bottleneck coincident with 1960s DDT spraying campaigns. A genetically distinct population frequently resistant to antimonials has a two base-pair insertion in the aquaglyceroporin gene LdAQP1 that prevents the transport of trivalent antimonials. We find evidence of genetic exchange between ISC populations, and show that the mutation in LdAQP1 has spread by recombination. Our results reveal the complexity of L. donovani evolution in the ISC in response to drug treatment.DOI: http://dx.doi.org/10.7554/eLife.12613.001

Treatment of many infectious diseases is under threat from drug resistance. Understanding the mechanisms of resistance is as high a priority as the development of new drugs. We have investigated the basis for cross-resistance between the diamidine and melaminophenyl arsenical classes of drugs in African trypanosomes. We induced high levels of pentamidine resistance in a line without the tbat1 gene that encodes the P2 transporter previously implicated in drug uptake. We isolated independent clones that displayed very considerable crossresistance with melarsen oxide but not phenylarsine oxide and reduced uptake of [ 3 H]pentamidine. In particular, the highaffinity pentamidine transport (HAPT1) activity was absent in the pentamidine-adapted lines, whereas the low affinity pentamidine transport (LAPT1) activity was unchanged. The parental tbat1 Ϫ/Ϫ line was sensitive to lysis by melarsen oxide, and this process was inhibited by low concentrations of pentamidine, indicating the involvement of HAPT1. This pentamidine-inhibitable lysis was absent in the adapted line KO-B48. Likewise, uptake of the fluorescent diamidine 4Ј,6-diamidino-2-phenylindole dihydrochloride was much delayed in live KO-B48 cells and insensitive to competition with up to 10 M pentamidine. No overexpression of the Trypanosoma brucei brucei ATPbinding cassette transporter TbMRPA could be detected in KO-B48. We also show that a laboratory line of Trypanosoma brucei gambiense, adapted to high levels of resistance for the melaminophenyl arsenical drug melarsamine hydrochloride (Cymelarsan), had similarly lost TbAT1 and HAPT1 activity while retaining LAPT1 activity. It seems therefore that selection for resistance to either pentamidine or arsenical drugs can result in a similar phenotype of reduced drug accumulation, explaining the occurrence of cross-resistance.Trypanosoma brucei subspp. are protozoan parasites that cause human African trypanosomiasis (i.e., sleeping sickness) and the corresponding veterinary condition in livestock. Treatment of both the human and livestock diseases depends on a very small set of mostly very old drugs. The first-line treatment for the late stage of both West African and East African human African trypanosomiasis is melarsoprol, an organoarsenic compound of the melaminophenyl arsenical class, introduced in 1949 (Jannin and Cattand, 2004). A similar but water-soluble melaminophenyl arsenical, melarsamine hydrochloride (Cymelarsan), is increasingly used for animal trypanosomiasis. Early-stage West African sleeping sickness is routinely treated with the diamidine drug pentamidine, introduced in 1937 (Jannin and Cattand, 2004). The corresponding widely used veterinary diamidine is diminazene aceturate (Berenil). The only new trypanocide to be developed in recent decades, DB75, is also a diamidine and currently in clinical trials as an orally available prodrug.It has been known for decades that cross-resistance between melaminophenyl arsenicals and diamidine drugs sometimes occurs (Fulton and Grant, 1955;Williamson a...

Purine salvage is an essential function for all obligate parasitic protozoa studied to date and most are also capable of efficient uptake of preformed pyrimidines. Much progress has been made in the identification and characterisation of protozoan purine and pyrimidine transporters. While the genes encoding protozoan or metazoan pyrimidine transporters have yet to be identified, numerous purine transporters have now been cloned. All protozoan purine transporter-encoding genes characterised to date have been of the Equilibrative Nucleoside Transporter family conserved in a great variety of eukaryote organisms. However, these protozoan transporters have been shown to be sufficiently different from mammalian transporters to mediate selective uptake of therapeutic agents. Recent studies are increasingly addressing the structure and substrate recognition mechanisms of these vital transport proteins.

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Melarsoprol and pentamidine represent the two main classes of drugs, the arsenicals and diamidines, historically used to treat the diseases caused by African trypanosomes: sleeping sickness in humans and Nagana in livestock. Cross-resistance to these drugs was first observed over sixty years ago and remains the only example of cross-resistance among sleeping sickness therapies. A Trypanosoma brucei adenosine transporter is well-known for its role in the uptake of both drugs. More recently, aquaglyceroporin 2 (AQP2) loss-of-function was linked to melarsoprol-pentamidine cross-resistance. AQP2, a channel that appears to facilitate drug accumulation, may also be linked to clinical cases of resistance. Here, we review these findings and consider some new questions as well as future prospects for tackling the devastating diseases caused by these parasites. KeywordsAT1; AQP2; MRPA; drug-resistance; MIP; Trypanosoma brucei Chemotherapy against African trypanosomiasis"Cellular therapy is a consequence of cellular nutrition, for only those compounds can affect the cell that are actually eaten by it. " Paul Ehrlich, 1907 [1] African trypanosomes are parasitic protists that circulate in the bloodstream and tissue fluids of their mammalian hosts. Transmitted by tsetse flies, they cause important human and animal diseases. Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense cause Human African Trypanosomiasis (HAT), also known as sleeping sickness, which is typically fatal without chemotherapy, while the closely related, but human-serum sensitive, T. b. brucei, Trypanosoma congolense and Trypanosoma vivax cause Nagana, an important veterinary disease. HAT affects 8.7 million km 2 of Sub-Saharan Africa, areas where the climate and environment are suitable for the tsetse fly [2]. T. b. gambiense is endemic in many areas of West and central Africa and is currently responsible for the vast majority (>90%) of HAT cases. § To whom correspondence should be addressed. david.horn@lshtm.ac.uk. in approximately 10% of patients [7], is currently the only drug active against both advanced T. b. rhodesiense and T. b. gambiense infections. Melarsoprol and pentamidine are also the most potent drugs used to treat HAT, both displaying low nanomolar 50% effective growthinhibitory concentrations (EC 50 ). Europe PMC Funders GroupThere have been three major epidemics of sleeping sickness recorded since the late 19 th century. Tsetse control, the systematic screening for patients in at-risk populations followed by chemotherapy, and the introduction of nifurtimox-eflornithine combination therapy (NECT) have all contributed to the recent successful reduction in cases [8]. However, the WHO recently warned against neglect and complacency if further epidemics are to be avoided [9]. With no vaccine available and limited therapeutic alternatives, the emergence of drug resistance is a major threat in this regard [10], especially since loss of a single, nonessential transporter can result in eflornithine resistance [11]. In fact, th...