Fatty acid synthesis and pyruvate metabolism pathways remain active in dihydroartemisinin-induced dormant ring stages of Plasmodium falciparum

Abstract

Artemisinin (ART)-based combination therapy (ACT) is used as the first-line treatment of uncomplicated falciparum malaria worldwide. However, despite high potency and rapid action, there is a high rate of recrudescence associated with ART monotherapy or ACT long before the recent emergence of ART resistance. ART-induced ring-stage dormancy and recovery have been implicated as possible causes of recrudescence; however, little is known about the characteristics of dormant parasites, including whether dormant parasites are metabolically active. We investigated the transcription of 12 genes encoding key enzymes in various metabolic pathways in P. falciparum during dihydroartemisinin (DHA)-induced dormancy and recovery. Transcription analysis showed an immediate downregulation for 10 genes following exposure to DHA but continued transcription of 2 genes encoding apicoplast and mitochondrial proteins. Transcription of several additional genes encoding apicoplast and mitochondrial proteins, particularly of genes encoding enzymes in pyruvate metabolism and fatty acid synthesis pathways, was also maintained. Additions of inhibitors for biotin acetyl-coenzyme A (CoA) carboxylase and enoyl-acyl carrier reductase of the fatty acid synthesis pathways delayed the recovery of dormant parasites by 6 and 4 days, respectively, following DHA treatment. Our results demonstrate that most metabolic pathways are downregulated in DHA-induced dormant parasites. In contrast, fatty acid and pyruvate metabolic pathways remain active. These findings highlight new targets to interrupt recovery of parasites from ART-induced dormancy and to reduce the rate of recrudescence following ART treatment.

FIG 1

Percentages of parasite density (including normal, dormant, and dead parasites) and dormant rings prior to and post-DHA treatment. Data (means with 95% confidence intervals) were obtained from 3 independent experiments.

FIG 2

Transcription levels of 12 genes encoding enzymes of several metabolic pathways in DHA-treated rings relative to pretreatment rings (fold ratio ± 95% confidence intervals). Relative transcription levels measured for each of the 12 genes are represented with different color bars. For each gene, relative transcription levels measured on days 2 (outlined in black), 3, 4, 5, 6, 8, 10, and 12 post-DHA treatment (normalized against overall parasitemia, including dormant, dead, and normal parasites) are grouped together from left to right. The transcription level measured on day 0 (pretreatment) is set as 1 for each gene. Data (means with 95% confidence intervals) were obtained from the results determined for samples in triplicate and three independent experiments. Note: lcfaee could also be active in the endoplasmic reticulum.

FIG 3

LDH activity and cellular ATP level relative to pretreatment levels during DHA-induced dormancy recovery period. Pretreatment levels are set as 1. Data (means and standard errors) were obtained from the results determined for triplicate samples.

FIG 4

Transcription levels of 8 genes encoding enzymes of apicoplast pyruvate metabolism and fatty acid synthesis pathways in DHA-treated rings relative to pretreatment rings (fold ratio ± 95% confidence intervals). Relative transcription levels measured for each of the 8 genes are represented with different color bars. For each gene, relative transcription levels measured on days 2 (outlined in black), 3, 4, 5, 6, 8, 10, and 12 post-DHA treatment are grouped together from left to right. The transcription level measured on day 0 (pretreatment) is set as 1 for each gene. Data (means with 95% confidence intervals) were obtained from the results determined for samples in triplicate and three independent experiments. Note: lcfaee could also be active in the endoplasmic reticulum.

FIG 5

Effect of haloxyfob (A), triclosan (B), and pyrimethamine (C) on the recovery of DHA-induced dormant parasites. Parasitemia of viable parasites on various days is shown in the figure. Data (means with 95% confidence intervals) were obtained from the results determined for samples in triplicate and three independent experiments.

FIG 6

Transcription levels of 5 genes encoding enzymes of the mitochondrial electron transport chain in DHA-treated rings relative to pretreatment rings (ratio ± confidence intervals). Relative transcription levels measured for each of the 5 genes are represented with different color bars. For each gene, relative transcription levels measured on days 2 (outlined in black), 3, 4, 5, 6, 8, 10, and 12 post-DHA treatment are grouped together from left to right. The transcription level measured on day 0 (pretreatment) is set as 1 for each gene. Data (means with 95% confidence intervals) were obtained from the results determined for samples in triplicate and three independent experiments.