Abstract
Drug resistance remains a major public health challenge for malaria treatment and eradication. Individual loci associated with drug resistance to many antimalarials have been identified, but their epistasis with other resistance mechanisms has not yet been elucidated. We previously described two mutations in the cytoplasmic prolyl-tRNA synthetase (cPRS) gene that confer resistance to halofuginone. We describe here the evolutionary trajectory of halofuginone resistance of two independent drug resistance selections in Plasmodium falciparum. Using this novel methodology, we discover an unexpected non-genetic drug resistance mechanism that P. falciparum utilizes before genetic modification of the cPRS. P. falciparum first upregulates its proline amino acid homeostasis in response to halofuginone pressure. We show that this non-genetic adaptation to halofuginone is not likely mediated by differential RNA expression and precedes mutation or amplification of the cPRS gene. By tracking the evolution of the two drug resistance selections with whole genome sequencing, we further demonstrate that the cPRS locus accounts for the majority of genetic adaptation to halofuginone in P. falciparum. We further validate that copy-number variations at the cPRS locus also contribute to halofuginone resistance. We provide a three-step model for multi-locus evolution of halofuginone drug resistance in P. falciparum. Informed by genomic approaches, our results provide the first comprehensive view of the evolutionary trajectory malaria parasites take to achieve drug resistance. Our understanding of the multiple genetic and non-genetic mechanisms of drug resistance informs how we will design and pair future anti-malarials for clinical use.
Highlights
Drug resistance remains a major public health challenge for malaria treatment and eradication
Selections began with 10× the parental EC50 for halofuginone (7 nM) and were increased stepwise upon growth tolerance (Additional files 1, 2, and 3). Both halofuginone resistance selected line II (HFGRII) and halofuginone resistance selected line II (HFGRIII) grew tolerant of 7 nM halofuginone in 18 generations, 21 nM in 9 generations, 42 nM in 7 and 9 generations, respectively, and 140 nM in 16 and 22.5 generations, respectively
We found that the cytoplasmic prolyl-tRNA synthetase (cPRS) was part of larger amplifications at later time points in HFGRII and HFGRIII (Figure 2A-C)
Summary
Drug resistance remains a major public health challenge for malaria treatment and eradication. The targets of more than 12 families of small molecules (reviewed in [10]) have been identified in Plasmodia through in vitro selection and genomic characterization of the end-points of these selections. This approach inherently assumes a single mechanism of drug resistance and overlooks the temporality of genetic and non-genetic epistasis involved in the complex evolution of drug resistance in a eukaryotic parasite with a genome of approximately 23 megabases and roughly 5,500 expressed proteins in the parasites’ life cycles [11]
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