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

Abstract

Background: Artemisinin-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.

Results: A 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.

Conclusions: These 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.

Keywords: Artemisinin resistance; Autophagy; Drug resistance; Fitness; atg18.

Fig. 1

PfAtg18 is capable of binding PI3P. a The alignment of the protein sequence of P. falciparum PF3D7_1012900, now annotated as autophagy-related gene 18 (atg18), is similar to mammalian homologues, the WD-repeat protein Interacting with Phosphoinositides (WIPI) proteins. Conserved binding domains are highlighted in yellow. WIPI protein FRRG domain binds PI3P while the double arginine residues bind Atg16. b Image depicts P. falciparum Atg18 protein modeled based on homologous Hsv2 protein in yeast. Putative binding sites for PI3P and Atg16 are highlighted, as well as the location of the T38I SNP (red circles). c The threonine amino acids in PfAtg18 are shown with their likelihood of being phosphorylation sites. The T38 site is shown in orange. The threshold (dotted line) is 0.5. d Binding of Atg18 from Dd2^R539T GFP-tagged Atg18 parasite line to various lipids based on integrated density from an Echelon PIP strip membrane demonstrates binding to PI3P

Fig. 2

PfAtg18 forms puncta following DHA exposure a TEM images of Dd2^R539T GFP-Atg18 parasites that were untreated (1) or exposed to 700 nM DHA for 10 min (2) or 1 h (3). Antibodies to GFP were gold-labelled (dots). Labels that congregate to form puncta are highlighted (circles). b Late-stage Dd2^R539T Atg18-GFP parasites stained with DAPI (blue) and anti-GFP antibody (red). Rows depict images with (i) DAPI only, (ii) DIC only, (iii) anti-GFP only, and (iv) a merged image with all channels. Columns depict parasites that were untreated (1) or exposed to 700 nM DHA for 10 min (2) or 1 h (3)

Fig. 3

Differential growth rates in nutrient-limited settings Growth over 48 h of isogenic lines Dd2 (blue), Dd2^R539T (red), Dd2^R539T/T38I (green) in varying nutrient deprivation conditions with glucose at normal (supra-physiologic) media concentration (a) and glucose at limiting concentration (b)

Fig. 4

An atg18 T38I SNP is associated with higher IC₅₀ values to ART derivatives ex vivo IC₅₀ values for six anti-malarial drugs according to the presence of the atg18 T38I polymorphism and/or the polymorphism associated with resistance for that particular drug. a DHA and b artesunate are synthetic ART derivatives. IC₅₀ values for ART derivatives are displayed against any K13-propeller mutation. c Mefloquine in the presence or absence of mdr copy number variations. d Piperaquine in the presence or absence of plasmepsins 2–3 copy number variations. e Chloroquine in the presence or absence of crt mutations. Given the single value in crt^WT/atg18^MT, it was not possible to statistically test for differences between this and the other groups. SCN denotes single copy number; ICN denotes increased copy number

Fig. 5

Overview of drug screen against atg18 isogenic lines. a NCATS libraries MIPE and NPACT were used to screen 6349 unique compounds. The atg18 T38I SNP was associated with increased susceptibility to 90 compounds. Of these, 8 compounds demonstrated curves in curve classes 1 or 2, demonstrating a shift in susceptibility. On the other hand, this SNP was associated with decreased susceptibility to 137 compounds, 16 of which showed curve classes 1 or 2. b The atg18 T38I SNP was associated with increased susceptibility to 90 compounds, 29 of which have known mechanisms of action. The SNP caused decreased susceptibility to 137 compounds, of which 62 were annotated. Drugs are categorized into antimicrobial (blue), cell growth (red), cell signaling (green), cell surface protein (purple), DNA replication (orange), metabolism (black), physiological homeostasis (navy), transcriptional regulation (crimson), or other (tan). c AUC values for parent (Dd2^R539T) and mutant (Dd2^R539T/T38I) lines organized by superclasses: transcriptional regulation (pink), physiological homeostasis (purple), other (periwinkle blue), metabolism (sky blue), DNA replication (aqua), cell surface protein (green), cell signaling (olive), cell growth (orange), and antimicrobial (coral). These are further broken down into classes by line

Fig. 6

An atg18 SNP does not alter IC₅₀ values for common anti-malarial compounds in isogenic lines Dose response curves from a ART and two derivatives, b DHA, and c artesunate, which are used in Cambodia and elsewhere in Southeast Asia. Also shown are dose response curves for d chloroquine, e mefloquine, and f piperaquine, long-lasting partner drugs which have been recently used in Cambodia and elsewhere in Southeast Asia. Parent displayed in blue, mutant in red. Drug structures are also shown