Abstract
Mechanisms of drug resistance in Plasmodium vivax have been difficult to study partially because of the difficulties in culturing the parasite in vitro. This hampers monitoring drug resistance and research to develop or evaluate new drugs. There is an urgent need for a novel method to study mechanisms of P. vivax drug resistance. In this paper we report the development and application of the first Plasmodium falciparum expression system to stably express P. vivax dhfr-ts alleles. We used the piggyBac transposition system for the rapid integration of wild-type, single mutant (117N) and quadruple mutant (57L/58R/61M/117T) pvdhfr-ts alleles into the P. falciparum genome. The majority (81%) of the integrations occurred in non-coding regions of the genome; however, the levels of pvdhfr transcription driven by the P. falciparum dhfr promoter were not different between integrants of non-coding and coding regions. The integrated quadruple pvdhfr mutant allele was much less susceptible to antifolates than the wild-type and single mutant pvdhfr alleles. The resistance phenotype was stable without drug pressure. All the integrated clones were susceptible to the novel antifolate JPC-2067. Therefore, the piggyBac expression system provides a novel and important tool to investigate drug resistance mechanisms and gene functions in P. vivax.
Highlights
Plasmodium vivax is the most widely distributed of the five known human malaria causing parasites and accounts for 80–391 million cases of malaria annually [1,2,3]
In this article we report the development of the piggyBac transposition system for the integration of pvdhfr into the P. falciparum genome, the transcription of the pvdhfr gene, and the response of the pvdhfr alleles to conventional antifolate drugs as a proof of concept for the utility of the system
Southern blot hybridization analysis performed on these clones, revealed that a single piggyBac insertion occurred in each clone (Table 1) and that none of the clones retained the piggyBac plasmid as episomes indicating highly efficient transposition events
Summary
Plasmodium vivax is the most widely distributed of the five known human malaria causing parasites and accounts for 80–391 million cases of malaria annually [1,2,3]. Recent reports revealed that severe complications and death caused by P. vivax are not uncommon [2,4,5,6]. Appropriate and timely treatment is the key to prevent morbidity and severe complications. Over the last twenty years there have been many reports that highlight the significant increase of resistance of P. vivax to CQ [7,8,9,10,11,12,13,14,15,16,17,18,19,20] and to the sulfa/antifolate combination sulfadoxine/pyrimethamine (SP) [7,21,22,23,24,25]. The spread of drug resistance in P. vivax makes the control and elimination of this species much more difficult
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