Formation of catalytically active cross-species heterodimers of thymidylate synthase from Plasmodium falciparum and Plasmodium vivax

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BackgroundThe prevalence of drug resistance amongst the human malaria Plasmodium species has most commonly been associated with genomic mutation within the parasites. This phenomenon necessitates evolutionary predictive studies of possible resistance mutations, which may occur when a new drug is introduced. Therefore, identification of possible new Plasmodium falciparum dihydrofolate reductase (PfDHFR) mutants that confer resistance to antifolate drugs is essential in the process of antifolate anti-malarial drug development.MethodsA system to identify mutations in Pfdhfr gene that confer antifolate drug resistance using an animal Plasmodium parasite model was developed. By using error-prone PCR and Plasmodium transfection technologies, libraries of Pfdhfr mutant were generated and then episomally transfected to Plasmodium berghei parasites, from which pyrimethamine-resistant PfDHFR mutants were selected.ResultsThe principal mutation found from this experiment was S108N, coincident with the first pyrimethamine-resistance mutation isolated from the field. A transgenic P. berghei, in which endogenous Pbdhfr allele was replaced with the mutant PfdhfrS108N, was generated and confirmed to have normal growth rate comparing to parental non-transgenic parasite and also confer resistance to pyrimethamine.ConclusionThis study demonstrated the power of the transgenic P. berghei system to predict drug-resistant Pfdhfr mutations in an in vivo parasite/host setting. The system could be utilized for identification of possible novel drug-resistant mutants that could arise against new antifolate compounds and for prediction the evolution of resistance mutations.

Section: Discussionsupporting

confidence: 91%

Selection of drug resistant mutants from random library of Plasmodium falciparum dihydrofolate reductase in Plasmodium berghei model

Tipsuwan

Srichairatanakool

Kamchonwongpaisan

et al. 2011

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“…The fact that it is possible to replace the endogenous Plasmodium dhfr-ts with a homologue from a different species suggests that dhfr-ts function is conserved among species of this genus. This is in agreement with biochemical evidence that cross-species heterodimers of Plasmodium DHFR-TS are fully functional [34]. While the expression of double mutant Pv DHFR-TS SP21 was not deleterious and can support the growth of the transgenic P. berghei parasites, attempts to obtain P. falciparum stably expressing double mutant Pv DHFR-TS SP21 were not successful (data not shown).…”

Section: Discussionsupporting

confidence: 86%

Transgenic Plasmodium parasites stably expressing Plasmodium vivax dihydrofolate reductase-thymidylate synthase as in vitro and in vivo models for antifolate screening

Somsak

Uthaipibull

Prommana

et al. 2011

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BackgroundPlasmodium vivax is the most prevalent cause of human malaria in tropical regions outside the African continent. The lack of a routine continuous in vitro culture of this parasite makes it difficult to develop specific drugs for this disease. To facilitate the development of anti-P. vivax drugs, bacterial and yeast surrogate models expressing the validated P. vivax target dihydrofolate reductase-thymidylate synthase (DHFR-TS) have been generated; however, they can only be used as primary screening models because of significant differences in enzyme expression level and in vivo drug metabolism between the surrogate models and P. vivax parasites.MethodsPlasmodium falciparum and Plasmodium berghei parasites were transfected with DNA constructs bearing P. vivax dhfr-ts pyrimethamine sensitive (wild-type) and pyrimethamine resistant (mutant) alleles. Double crossover homologous recombination was used to replace the endogenous dhfr-ts of P. falciparum and P. berghei parasites with P. vivax homologous genes. The integration of Pvdhfr-ts genes via allelic replacement was verified by Southern analysis and the transgenic parasites lines validated as models by standard drug screening assays.ResultsTransgenic P. falciparum and P. berghei lines stably expressing PvDHFR-TS replacing the endogenous parasite DHFR-TS were obtained. Anti-malarial drug screening assays showed that transgenic parasites expressing wild-type PvDHFR-TS were pyrimethamine-sensitive, whereas transgenic parasites expressing mutant PvDHFR-TS were pyrimethamine-resistant. The growth and sensitivity to other types of anti-malarial drugs in the transgenic parasites were otherwise indistinguishable from the parental parasites.ConclusionWith the permanent integration of Pvdhfr-ts gene in the genome, the transgenic Plasmodium lines expressing PvDHFR-TS are genetically stable and will be useful for screening anti-P. vivax compounds targeting PvDHFR-TS. A similar approach could be used to generate transgenic models specific for other targets of interest, thus facilitating the development of anti-P. vivax drugs in general.

“…Among the 11 drug resistance genes with positive selection signals, six genes are involved in chloroquine resistance, and three other genes are associated with multidrug resistance. The dihydrofolate reductase gene which confer resistance to pyrimethamine ( dhfr - ts , PVX_089950) was also found [48]. A recent study in Peru found haplotypes in drug-resistance genes including dhfr and mdr , suggesting that resistance mutations have arisen independently and might be directly linked to the widespread use of chloroquine, artemisinin and primaquine [49].…”

Section: Discussionmentioning

confidence: 99%

Genome-wide scans for the identification of Plasmodium vivax genes under positive selection

Shen

Chen

Wang

et al. 2017

BackgroundThe current trend of Plasmodium vivax cases imported from Southeast Asia into China has sharply increased recently, especially from the China–Myanmar border (CMB) area. High recombination rates of P. vivax populations associated with varied transmission intensity might cause distinct local selective pressures. The information on the genetic variability of P. vivax in this area is scant. Hence, this study assessed the genetic diversity of P. vivax genome sequence in CMB area and aimed to provide information on the positive selection of new gene loci.ResultsThis study reports a genome-wide survey of P. vivax in CMB area, using blood samples from local patients to identify population-specific selective processes. The result showed that considerable genetic diversity and mean pair-wise divergence among the sequenced P. vivax isolates were higher in some important gene families. Using the standardized integrated haplotype score (|iHS|) for all SNPs in chromosomal regions with SNPs above the top 1% distribution, it was observed that the top score locus involved 356 genes and most of them are associated with red blood cell invasion and immune evasion. The XP-EHH test was also applied and some important genes associated with anti-malarial drug resistance were observed in high positive scores list. This result suggests that P. vivax in CMB area is facing more pressure to survive than any other region and this has led to the strong positive selection of genes that are associated with host-parasite interactions.ConclusionsThis study suggests that greater genetic diversity in P. vivax from CMB area and positive selection signals in invasion and drug resistance genes are consistent with the history of drug use during malaria elimination programme in CMB area. Furthermore, this result also demonstrates that haplotype-based detecting selection can assist the genome-wide methods to identify the determinants of P. vivax diversity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-017-1882-0) contains supplementary material, which is available to authorized users.