A genomic and evolutionary approach reveals non-genetic drug resistance in malaria

Abstract

Background: 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. Results: 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. Conclusions: 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.

Probing Plasmodium falciparum sexual commitment at the single-cell level

“…We used an optimized version of single-cell RNA barcoding and sequencing 6 (SCRB-seq; 7 ), with some modifications to enable high throughput automated liquid handling. Briefly, cells were thawed on ice and each sample was lysed using 1 µl of 1 mg/ml Proteinase K (ThermoFisher Scientific) followed by incubation at 50°C for 15 min, then 95°C for 10 min uncovered to evaporate all liquid.…”

“…The latter, so-called mature gametocytes are required for parasite transmission to mosquitos. Blood-stage parasite isolates from malaria patients, or from resistance selection or in vitro culture adaption experiments, show significant heterogeneity in the transcriptional profile in population-level expression analyses 4– 6 . To date, the transcriptional diversity of such mixed populations has not been captured appropriately, owing to lack of efficient single-cell mRNA profiling methods in Plasmodium .…”

Genetic resistance to purine nucleoside phosphorylase inhibition in Plasmodium falciparum

“…We sought to characterize the genomic response of P. falciparum toward drug selection pressure using DADMe-ImmG. The analysis of genomic amplification in resistant clones characterizes the responses to drug pressure and identifies the molecular markers of resistance (17)(18)(19)(20)(21)(22). This approach has allowed the identification of antimalarial drug resistance Significance Hypoxanthine salvage is essential for nucleic acid synthesis in Plasmodium falciparum but not in humans.…”

Recent advances in the biology and drug targeting of malaria parasite aminoacyl-tRNA synthetases

“…Febrifugine, a Chinese herb derived molecule, and its analogs, especially halofuginone, are highly efficient inhibitors of malaria parasite growth [ 44 , 52 – 54 ]. Halofuginone targets both the asymptomatic liver stage and the blood stages of Plasmodium parasites [ 44 , 52 , 53 ].…”