Genetic dissection of drug resistance in trypanosomes

Abstract

The trypanosomes cause two neglected tropical diseases, Chagas disease in the Americas and African trypanosomiasis in sub-Saharan Africa. Over recent years a raft of molecular tools have been developed enabling the genetic dissection of many aspects of trypanosome biology, including the mechanisms underlying resistance to some of the current clinical and veterinary drugs. This has led to the identification and characterization of key resistance determinants, including transporters for the anti-Trypanosoma brucei drugs, melarsoprol, pentamidine and eflornithine, and the activator of nifurtimox-benznidazole, the anti-Trypanosoma cruzi drugs. More recently, advances in sequencing technology, combined with the development of RNA interference libraries in the clinically relevant bloodstream form of T. brucei have led to an exponential increase in the number of proteins known to interact either directly or indirectly with the anti-trypanosomal drugs. In this review, we discuss these findings and the technological developments that are set to further revolutionise our understanding of drug-trypanosome interactions. The new knowledge gained should inform the development of novel interventions against the devastating diseases caused by these parasites.

Fig. 1.

Structures of the nitroheterocyclic drugs used to treat T. cruzi infections and their toxic metabolites. The highlighted regions of nifurtimox and benznidazole correspond to the 5-nitrofuran and the 2-nitroimidazole groups, respectively. The structures of the toxic unsaturated open chain nitrile and glyoxal metabolites (Hall et al. ; Hall and Wilkinson, 2012) are shown below the corresponding drugs.

Fig. 2.

A comparison of low and high throughput approaches to understanding drug resistance in trypanosomes.

Fig. 3.

Developments in T. brucei molecular genetics and advances in the understanding of drug resistance in trypanosomes since 1990. (A) The development of the bloodstream form T. brucei RNAi library and the RIT-seq methodology was dependent upon a number of earlier methodological advances. Although concurrent progress was made in dissecting drug resistance mechanisms in trypanosomes, the establishment of a high throughput approach rapidly led to a significant increase in our understanding of both potential resistance mechanisms and the networks of proteins that influence drug efficacy. See text for abbreviations and references. (B) Drug selection of the tetracycline induced RNAi library can rapidly generate a resistant population. The RNAi targets, whose expression confers drug resistance, are sequenced using RNAi plasmid-specific primers (grey bars) on a next generation sequencing platform, such as Illumina, and mapped to the reference genome to identify candidate drug efficacy determinants.