Functional analysis of Plasmodium vivax dihydrofolate reductase-thymidylate synthase genes through stable transformation of Plasmodium falciparum

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.

Figure 1. Transcription profile and parasite stages of dhfr mRNA expression over the parasite life cycle.

A) Transcription levels of the pfdhfr relative to that of seryl-tRNA gene. B) % rings over the parasite life cycle. C) % trophozoites over the parasite life cycle. D) % schizonts over the parasite life cycle. E) Transcription levels of pvdhfr relative to that of seryl-tRNA. Note: NF54 and D6 in panel E represent level of pfdhfr transcription as a comparison.

Figure 2. In vitro susceptibility of P. falciparum NF54 and integrated parasite clones stably expressing various pvdhfr mutants to DHFR inhibitors: pyrimethamine (top left panel), cycloguanil (top right panel), clociguanil (bottom left panel), and WR99210 (bottom right panel).

Each color represents a pvdhfr allele and lines of the same color represent different clones of the same expressed pvdhfr allele. The symbols represent the means of triplicate data points.

Figure 3. In vitro susceptibility profiles of P. falciparum NF54, NF54 stably transfected parasites, D6, D6 episomally transfected parasites and TM91c235 to JPC-2067.

The symbols represent the means of triplicate data points.

Figure 4. pXL-BACII-pvdhfr plasmid design for piggyBac transformation of P. falciparum.

The pXL-BacII-DHFR vector was cut with HindIII to remove the 5′ calmodulin and hdhfr fragments from the pXL-BacII vector. The pfdhfr promoter-bsd-cmyc-pvdhfr-ts cassette was excised from the pRSET-C vector as a 3.1 kb HindIII fragment and cloned into pXL-BacII, such that it is flanked by the piggyBac ITR2 and 3′ histidine-rich protein-2 (hrp2). The sizes of the boxes are not proportional to the lengths of the genes. The pfdhfr promoter drives the expression of bsd-cmyc-pvdhfr-ts fused proteins.