Genetic Investigation of Tricarboxylic Acid Metabolism during the Plasmodium falciparum Life Cycle

Abstract

SUMMARY New antimalarial drugs are urgently needed to control drug resistant forms of the malaria parasite, Plasmodium falciparum. Mitochondrial electron transport is the target of both existing and new antimalarials. Herein, we describe 11 genetic knockout (KO) lines that delete six of the eight mitochondrial tricarboxylic acid (TCA) cycle enzymes. Although all TCA KOs grew normally in asexual blood stages, these metabolic deficiencies halted lifecycle progression in later stages. Specifically, aconitase KO parasites arrested as late gametocytes, whereas α-ketoglutarate dehydrogenase deficient parasites failed to develop oocysts in the mosquitoes. Mass spectrometry analysis of 13C isotope-labeled TCA mutant parasites showed that P. falciparum has significant flexibility in TCA metabolism. This flexibility manifested itself through changes in pathway fluxes and through altered exchange of substrates between cytosolic and mitochondrial pools. Our findings suggest that mitochondrial metabolic plasticity is essential for parasite development.

Apicomplexan Energy Metabolism: Carbon Source Promiscuity and the Quiescence Hyperbole

“…do require mitochondrial metabolism because they are susceptible to drugs targeting the ETC, such as atovaquone[ 1 2 _ T D $ D I F F ] , that inhibits complex III [32][33][34][35][36][37]. Evidently this is not for ATP synthesis because inhibition of the ETC has little effect on cellular ATP levels [38][39][40], and deletion of the functional b-subunit of ATP synthase can be tolerated in blood stages of P. berghei [31].…”

“…the ETC (NADH dehydrogenase and succinate dehydrogenase) are dispensable in asexual Plasmodium [33,36,43,44].…”

“…Counter-intuitively, genetic ablation of two enzymes of the P. falciparum TCA is not tolerated in blood stages, namely fumarate hydratase (FH, converting fumarate to malate) and malatequinone oxidoreductase (MQO, the sole enzyme responsible for mitochondrial malate turnover) [36][ 1 3 _ T D $ D I F F ] (Figure 1). Furthermore, the b-subunit of ATP synthase is also required in blood stages of P. falciparum, although this is not the case in P. berghei [38][ 1 4 _ T D $ D I F F ] (Figure 1).…”

“…Furthermore, the b-subunit of ATP synthase is also required in blood stages of P. falciparum, although this is not the case in P. berghei [38][ 1 4 _ T D $ D I F F ] (Figure 1). It is possible that the classic 'primary' roles of these enzymes are not responsible for these phenotypes, but rather their roles in cytosolic purine salvage (FH [45]) and, perhaps, mitochondrial structure (MQO and ATP synthase [36,38]). …”

“…Isocitrate Dehydrogenase (IDH) The apicomplexan mitochondria have lost NAD + -dependent IDH and instead rely on the retained NADP + -dependent form of IDH for oxidation of isocitrate to /-ketoglutarate (Figure 2) [4,26,36,53,54]. The generated NADPH may contribute to defense against ROS damage, NADPH-dependent enzyme activity, and the electrochemical gradient through conversion to NADH by NAD(P) + transhydrogenase (Figure 2) [9,55], although the localization of the latter is as yet unclear.…”