HAD2 Regulates Central Carbon Metabolism in Malaria Parasite P. falciparum

Abstract Background The malaria parasite Plasmodium falciparum causes nearly half a million deaths each year. Widespread resistance to antimalarials prompts an urgent need for novel drug targets both unique and essential to the parasite. The methylerythritol (MEP) pathway of isoprenoid biosynthesis fits this profile. Previous studies showed that P. falciparum controls substrates available to the MEP pathway via the phosphatase HAD2. A point mutation in the HAD2 gene was found to misdirect metabolites from glycolysis to the MEP pathway. Elucidating the mechanism by which HAD2 controls central carbon metabolism will inform our understanding of essential parasite biology and the potential for resistance to metabolic inhibitors under development. Methods In this work, we have generated a P. falciparum strain in which HAD2 expression is under control of anhydrous tetracycline (aTc), such that growth in the absence of aTc leads to loss of HAD2 expression. We have used this tunable knockdown strain to regulate expression of HAD2 in asexual-stage parasites to test whether HAD2 is required for glycolytic and isoprenoid biosynthesis homeostasis. Asexual parasite growth rate was measured through flow cytometry, and dose-response assays were performed against a competitive small molecule inhibitor of the MEP pathway. Targeted metabolite profiling with liquid chromatography-tandem mass spectrometry (LC-MS/MS) is in process to quantify glycolytic and isoprenoid metabolites in this knockdown strain. Results Knockdown parasites off aTc exhibited reduced growth rates relative to the wild-type strain. These parasites also exhibited a 4-fold increase in resistance to a competitive inhibitor of the MEP pathway compared to the wild-type [0.755 +/- 0.12 µM vs. 3.201 +/- 0.35 µM; p<0.001). Conclusions Reduced expression of HAD2 confers resistance to a competitive inhibitor of the MEP pathway, consistent with the effect of elevated levels of MEP pathway intermediates as a result of disrupted homeostasis. Ongoing work will use this strain to understand the role of HAD2 on parasite metabolism, parasite growth, and drug resistance.