Drug resistance; Trypanosoma brucei; Genomics; Molecular mechanism; Diamidine; Arsenical; Oxaborole
Graf Fabrice E, Ludin Philipp, Arquint Christian, Schmidt Remo S, Schaub Nadia, Kunz Renggli Christina, Munday Jane C, Krezdorn Jessica, Baker Nicola, Horn David, Balmer Oliver, Caccone Adalgisa, de Koning Harry P, Mäser Pascal (2016), Comparative genomics of drug resistance in Trypanosoma brucei rhodesiense., in Cellular and molecular life sciences : CMLS
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Sleeping sickness is a fatal disease of sub-Saharan Africa, caused by Trypanosoma brucei ssp. and transmitted by the Tsetse fly (Glossina spp.). The treatment of sleeping sickness relies on the drugs pentamidine and suramin for the first, haemolymphatic stage of the disease, respectively melarsoprol, eflornithine and nifurtimox-eflornithine combination therapy for the second stage, when the trypanosomes have invaded the cerebrospinal fluid. These drugs are outdated and suffer from severe side-effects. Yet even the most optimistic scenario admits several years until better drugs such as fexinidazole or oxaboroles will be available for the treatment of sleeping sickness. Sustainable use of the current drugs requires an understanding of the molecular mechanisms of drug resistance, allowing for DNA-based diagnostic tests to monitor the spread of resistant parasites. Cross-resistance between pentamidine and melarsoprol is a well known phenomenon and has been attributed to loss of the gene TbAT1, which encodes an adenosine transporter that also mediates cellular uptake of diamidines and melamine-based arsenicals. However, we have selected cross-resistant T. b. rhodesiense lines in vitro with resistance factors to pentamidine and melarsoprol an order of magnitude above those of tbat1 null trypanosomes, demonstrating that additional mutations must be involved. Here we propose to identify these mutations by comparative genomics and transcriptomics using deep sequencing technologies. The genomes of the two resistant T. b. rhodesiense lines and their drug-sensitive parent will be sequenced on the Roche 454 system (possible thanks to a Roche 10Gb Award), and comparative transcriptomics will be performed on the Illumina reader. Candidate mutations for drug resistance will be validated by reverse genetics in wildtype as well as tbat1 null T. b. brucei. In addition, we are selecting for oxaborole-resistant T. b. rhodesiense lines which, once obtained, will be characterized using the same approach. The results of the proposed research have the potential to be directly translated to the improvement of sleeping sickness therapy and the confinement of drug resistance.