Amino acid efflux by asexual blood-stage Plasmodium falciparum and its utility in interrogating the kinetics of hemoglobin endocytosis and catabolism in vivo

Abstract

The endocytosis and catabolism of large quantities of host cell hemoglobin is a hallmark of the intraerythrocytic asexual stage of the human malaria parasite Plasmodium falciparum. It is known that the parasite's production of amino acids from hemoglobin far exceeds its metabolic needs. Here, we show that P. falciparum effluxes large quantities of certain non-polar (Ala, Leu, Val, Pro, Phe, Gly) and polar (Ser, Thr, His) amino acids to the external medium. That these amino acids originate from hemoglobin catabolism is indicated by the strong correlation between individual amino acid efflux rates and their abundances in hemoglobin, and the ability of the food vacuole falcipain inhibitor E-64d to greatly suppress efflux rates. We then developed a rapid, sensitive and precise method for quantifying flux through the hemoglobin endocytic-catabolic pathway that is based on leucine efflux. Optimization of the method involved the generation of a novel amino acid-restricted RPMI formulation as well as the validation of D-norvaline as an internal standard. The utility of this method was demonstrated by characterizing the effects of the phosphatidylinositol-3-kinase inhibitors wortmannin and dihydroartemisinin on the kinetics of Leu efflux. Both compounds rapidly inhibited Leu efflux, which is consistent with a role for phosphtidylinositol-3-phosphate production in the delivery of hemoglobin to the food vacuole; however, wortmannin inhibition was transient, which was likely due to the instability of this compound in culture medium. The simplicity, convenience and non-invasive nature of the Leu efflux assay described here makes it ideal for characterizing the in vivo kinetics of hemoglobin endocytosis and catabolism, for inhibitor target validation studies, and for medium-throughput screens to identify novel inhibitors of cytostomal endocytosis.