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.

Evidence for Regulation of Hemoglobin Metabolism and Intracellular Ionic Flux by the Plasmodium falciparum Chloroquine Resistance Transporter

“…To determine whether the L272F mutation impairs the ability of parasites to secure essential amino acids from digested Hb, we profiled the amino acid requirements in the L272F mutant. We focused our analysis on amino acids that appear to not be effluxed from the parasite in substantial quantities, namely Methionine (M), Cysteine (C; supplemented as the oxidized dimer Cystine that is presumably reduced in the host RBC), Aspartate (D), Asparagine (N), Glutamate (E), Glutamine (Q), Lysine (K) and Arginine (R) 56 . Growth conditions were profiled in media containing amino acid-rich media (RPMI-VLA), which has all amino acids other than valine, leucine, and alanine and which allows unhindered parasite growth.…”

“…Growth conditions were profiled in media containing amino acid-rich media (RPMI-VLA), which has all amino acids other than valine, leucine, and alanine and which allows unhindered parasite growth. Growth in that media was compared with growth in minimal (amino-acid deficient) media that contained only amino acids added separately or as restricted combinations (minimal media +I, +IMC, +IM, +IC, +IKR, or +IDENQ) 56 . Dd2 Dd2 showed good growth in all media conditions (Fig.…”

“…Asynchronous Dd2 Dd2 and Dd2 Dd2 L272F parasite cultures were washed with a variety of media formulations. Our control RPMI media, denoted “RPMI-VLA”, contained all amino acids except for valine, leucine, and alanine, as described 56 . This reconstituted medium was earlier observed to enable parasites to proliferate at the same rate as with RPMI culture medium containing all amino acids 56 .…”

“…These peptidases generate short peptides and amino acids, which can be transported out of the DV 48 – 52 . During one 48-hour intra-erythrocytic development cycle, parasites degrade ~75% of the Hb content of infected RBCs (iRBCs) 53 – 55 , while only metabolizing ~16% as a nutrient source 54 and releasing the majority of the remainder into host plasma 56 .…”

NovelPlasmodium falciparummetabolic network reconstruction identifies shifts associated with clinical antimalarial resistance

“…Similarly, hemoglobin degradation is essential for the blood-stage parasite to produce free amino acids. Parasites can also import and synthesize some amino acids, but the breakdown of hemoglobin (and subsequent production of its byproduct, hemozoin) is necessary for growth [23, 63, 64]. Thus, by requiring hemozoin export, we force the in silico parasite to degrade hemoglobin as the primary pathway for amino acid production.…”