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Abstract
During the blood stages of its life cycle, P. falciparum relies on glycolysis as its major source of ATP rather than oxidative phosphorylation. Yet, we have been unable to disrupt the genes encoding the β and γ subunits of the ATP synthase complex in P. falciparum blood stages. To address the essential nature of ATP synthase we generated a merodiploid line expressing a tagged mutated β subunit from an ectopic site. This should reduce the overall ATP synthase activity. Although the mutant subunit was correctly trafficked to the mitochondrion, the transgenic parasites grew at the same rate as the parental lines, indicating that the ectopically expressed mutant subunits did not have a dominant negative effect. Furthermore, we generated a transgenic parasite line that permitted conditional knockdown of the β subunit through the use of a regulated ribozyme. Knockdown of the β subunit showed no growth phenotype, indicating >80% reduction in its expression was not detrimental to the parasites. These knockdown parasite lines allowed us to revisit a hypothesis regarding the target of proguanil, a drug shown to work synergistically with atovaquone to kill the parasite, and which is a component of the registered antimalarial drug combination Malarone. Earlier work from our laboratory led to a hypothesis that proguanil targets a secondary pathway for generating mitochondrial membrane potential (ΔΨm), and that this pathway becomes essential when the primary pathway of mitochondrial electron transport chain (mtETC) is inhibited by atovaquone. We monitored the effects of the merodiploid catalytic knockdown line and the ribozyme knockdown line for their sensitivity to atovaquone as well as another mtETC inhibitor, ELQ300. We also reengineered the ribozyme conditional knockdown line to become mtETC independent and thus resistant to atovaquone. This provided a means to examine the secondary pathway of ΔΨm generation in absence of mtETC. Through the use of these transgenic parasite lines we were able to provide strong evidence that the alternate pathway to generate the ΔΨm is likely to be the ATP hydrolase activity of the parasite ATP synthase. These findings now solve a decades long mystery regarding the nature of proguanil synergy with atovaquone.
