Chemical and genetic interrogation of Plasmodium falciparum mitochondrial pyruvate carriers

Abstract

Despite efforts to reduce the global spread and severity of malaria infection, resistance towards current frontline antimalarials has halted malaria elimination progress. Thus, malaria parasite biology should be continually investigated in hopes of finding resistance mechanisms and targetable biology to develop new compounds. Plasmodium falciparum, the most clinically important malaria-causing parasite, employs energy metabolism pathways differently from one intraerythrocytic form to the next. The proliferative asexual stage, the disease-causing form, fulfils its high energy demand through anaerobic glycolysis. However, the slow-maturing gametocyte, the transmissible form, relies on a canonical TCA cycle and oxidative phosphorylation for energy production. Membrane transport proteins crucially maintain the metabolite link between these pathways in both stages of parasite development. For instance, pyruvate production, from extracellularly sourced glucose, is important for glycolysis and the TCA cycle, yet its translocation mechanism across the inner mitochondrial membrane is unknown. In the last decade, the mitochondrial pyruvate carrier (MPC) heterocomplex which consists of MPC1 and MPC2 was identified as the transport complex responsible for pyruvate translocation in humans, yeast, and other eukaryotic organisms. In P. falciparum, two mpc genes, mpc1 (PF3D7_1340800) and mpc2 (PF3D7_1470400) are putatively annotated but have not yet been characterised. Here, we aimed to develop both chemical and genetic mechanisms to characterise the function of the putative MPCs within P. falciparum parasites. A known MPC inhibitor, UK 5099, was used to chemically investigate the asexual parasite and gametocytes’ physiological response to the inhibition of these putative MPCs. Additionally, this study developed three approaches to genetically interrogate MPC function in transgenic P. falciparum lines. In the first instance, genetic disruption and conditional knockdown of these mpc genes were investigated. Low native promoter activity complicated the production of pure integrant lines for these systems. Therefore, an alternative system was investigated in which the two mpc genes would be ectopically expressed under a constitutive promoter to produce a functional MPC heterocomplex for later investigations of the importance of this transporter to parasite development. In the future, a greater catalogue of MPC inhibitors should be screened alongside the characterisation of the ectopic mpc expression line to characterise the MPC heterocomplex to expand our understanding of how the overexpressed MPC heterocomplex phenotype affects parasite biology and the response to MPC inhibitors.