A single nucleotide polymorphism in the Plasmodium falciparum atg18 gene associates with artemisinin resistance and confers enhanced parasite survival under nutrient deprivation

Kimberly F Breglio,Anna Katharina Simon,Rajarshi Guha,Crystal Mcknight,David Roberts,Craig Thomas,Roberto Amato,Richard Eastman,Pharath Lim,Juliana M Sa,Carleen Klumpp-Thomas,Sundar Ganesan,David W Dorward,Rick M Fairhurst

doi:10.1186/s12936-018-2532-x

Abstract

BackgroundArtemisinin-resistant Plasmodium falciparum has been reported throughout the Greater Mekong subregion and threatens to disrupt current malaria control efforts worldwide. Polymorphisms in kelch13 have been associated with clinical and in vitro resistance phenotypes; however, several studies suggest that the genetic determinants of resistance may involve multiple genes. Current proposed mechanisms of resistance conferred by polymorphisms in kelch13 hint at a connection to an autophagy-like pathway in P. falciparum.ResultsA SNP in autophagy-related gene 18 (atg18) was associated with long parasite clearance half-life in patients following artemisinin-based combination therapy. This gene encodes PfAtg18, which is shown to be similar to the mammalian/yeast homologue WIPI/Atg18 in terms of structure, binding abilities, and ability to form puncta in response to stress. To investigate the contribution of this polymorphism, the atg18 gene was edited using CRISPR/Cas9 to introduce a T38I mutation into a k13-edited Dd2 parasite. The presence of this SNP confers a fitness advantage by enabling parasites to grow faster in nutrient-limited settings. The mutant and parent parasites were screened against drug libraries of 6349 unique compounds. While the SNP did not modulate the parasite’s susceptibility to any of the anti-malarial compounds using a 72-h drug pulse, it did alter the parasite’s susceptibility to 227 other compounds.ConclusionsThese results suggest that the atg18 T38I polymorphism may provide additional resistance against artemisinin derivatives, but not partner drugs, even in the absence of kelch13 mutations, and may also be important in parasite survival during nutrient deprivation.

Highlights

Artemisinin-resistant Plasmodium falciparum has been reported throughout the Greater Mekong sub‐ region and threatens to disrupt current malaria control efforts worldwide
A sub-analysis of a previously performed genome-wide association study (GWAS) on 782 isolates from Southeast Asia was performed to determine if any Single nucleotide polymorphisms (SNPs) in genes involved in autophagy were associated with slow parasite clearance rates following ART treatment or partner drug resistance (IC50 values)
A SNP in atg18 is associated with ART‐resistance phenotypes A GWAS of 782 isolates from Southeast Asia significantly associated a nonsynonymous SNP in PF3D7_1012900(encoding a T38I substitution) with slow parasite clearance rate in patients treated with an ART derivative (p = 5.89E−7) and non-significantly associated with chloroquine resistance (p = 0.0002) (Table 1)

Summary

Introduction

Artemisinin-resistant Plasmodium falciparum has been reported throughout the Greater Mekong sub‐ region and threatens to disrupt current malaria control efforts worldwide. The slow parasite clearance rates following ACT suggest resistance to ART derivatives This resistance places increasing selective pressure for variants or traits that confer resistance to ACT partner drugs and has led to the rapid failure of several artemisinin-based combinations, including dihydroartemisinin-piperaquine in Cambodia [1, 2]. Single nucleotide polymorphisms (SNPs) in kelch (k13) have been associated with slow parasite clearance rates in response to ACT [3] and increased in vitro parasite survival in response to ART derivatives [4, 5]. The genetic background specific to some areas in Southeast Asia may be responsible for some of the drug resistance phenotype These background mutations may be important in transmission or confer a survival advantage over parasites without this array of SNPs. overcoming the cellular stress response following ART treatment may underlie the parasite resistance mechanism. Autophagy is one such cellular stress response that may be employed by a parasite and may be modified by genetic variants that promote survival and may represent a potential target pathway for novel anti-malarial compounds

Methods

Results

Conclusion