Genome Wide Adaptations of Plasmodium falciparum in Response to Lumefantrine Selective Drug Pressure

Abstract

The combination therapy of the Artemisinin-derivative Artemether (ART) with Lumefantrine (LM) (Coartem®) is an important malaria treatment regimen in many endemic countries. Resistance to Artemisinin has already been reported, and it is feared that LM resistance (LMR) could also evolve quickly. Therefore molecular markers which can be used to track Coartem® efficacy are urgently needed. Often, stable resistance arises from initial, unstable phenotypes that can be identified in vitro. Here we have used the Plasmodium falciparum multidrug resistant reference strain V1S to induce LMR in vitro by culturing the parasite under continuous drug pressure for 16 months. The initial IC50 (inhibitory concentration that kills 50% of the parasite population) was 24 nM. The resulting resistant strain V1SLM, obtained after culture for an estimated 166 cycles under LM pressure, grew steadily in 378 nM of LM, corresponding to 15 times the IC50 of the parental strain. However, after two weeks of culturing V1SLM in drug-free medium, the IC50 returned to that of the initial, parental strain V1S. This transient drug tolerance was associated with major changes in gene expression profiles: using the PFSANGER Affymetrix custom array, we identified 184 differentially expressed genes in V1SLM. Among those are 18 known and putative transporters including the multidrug resistance gene 1 (pfmdr1), the multidrug resistance associated protein and the V-type H+ pumping pyrophosphatase 2 (pfvp2) as well as genes associated with fatty acid metabolism. In addition we detected a clear selective advantage provided by two genomic loci in parasites grown under LM drug pressure, suggesting that all, or some of those genes contribute to development of LM tolerance – they may prove useful as molecular markers to monitor P. falciparum LM susceptibility.

Genetic resistance to purine nucleoside phosphorylase inhibition in Plasmodium falciparum

“…In Vitro Selection of DADMe-ImmG Resistant Parasites. P. falciparum isolates resistant to DADMe-ImmG were selected by continuous in vitro drug pressure (26)(27)(28). The high (370 μM) hypoxanthine usually added to culture media was replaced with a morephysiological level of 10 μM (29).…”

Single nucleotide polymorphisms in Plasmodium falciparum V type H+ pyrophosphatase gene (pfvp2) and their associations with pfcrt and pfmdr1 polymorphisms

“…Amodiaquine resistance is linked to pfcrt 76T just as CQ and allelic exchange experiments have linked pfcrt 76T to reduced susceptibility to mefloquine, artemisinin and QN [150,156,157]. Furthermore, K76 has been linked to reduced susceptibility to lumefantrine [158].…”

“…Pfvp2 is a class of H + pump found in plants and some protozoa [187][188][189][190][191]. Pfvp2 is located in the DV membrane and increased transcription of pfvp2 has been observed in vitro when P. falciparum are exposed to CQ [191] and lumefantrine [158]. Specifically a ten-fold upregulation of pfvp2 was observed when the CQ resistant (pfcrt 76I) P. falciparum 106/1 clone was exposed to CQ but no up-regulation was seen with the CQ sensitive 106/1 (pfcrt 76K) clone.…”

Temporal Changes in Prevalence of Molecular Markers Mediating Antimalarial Drug Resistance in a High Malaria Transmission Setting in Uganda

“…8 In addition, polymorphisms in pfmdr1, which is homologous to proteins that mediate drug sensitivity in mammalian cells, 9 modulate sensitivity of parasites to multiple drugs. 10,11 Considering pfmdr1 mutations that are common in Africa, 86Y and 1246Y are associated with decreased sensitivity to CQ and AQ, but wild-type (WT) sequences at the same alleles mediate decreased sensitivity to lumefantrine, mefloquine, and artemisinins. [12][13][14] Another pfmdr1 polymorphism, Y184F, is common, but of uncertain significance, and two others, S1034C and N1042D, are seen in Asia, but rare in Africa.…”