Transport and Metabolism of the Essential Vitamin Pantothenic Acid in Human Erythrocytes Infected with the Malaria Parasite Plasmodium falciparum

Abstract

The growth of the human malaria parasite, Plasmodium falciparum, within its host erythrocyte is reliant on the uptake of a number of essential nutrients from the extracellular medium. One of these is pantothenic acid, a water-soluble vitamin that is a precursor of coenzyme A. In this study we show that normal uninfected erythrocytes are impermeable to pantothenate but that the vitamin is taken up rapidly into malaria-infected cells via a transport pathway that has the characteristics (furosemide sensitivity, nonsaturability) of previously characterized, broad specificity permeation pathways induced by the intracellular parasite in the host cell membrane. The transport of pantothenate therefore constitutes a critical physiological role for these pathways. Inside the parasitized cell pantothenate undergoes phosphorylation, the first step in its conversion to coenzyme A. Parasites within saponin-permeabilized erythrocytes were shown to take up and phosphorylate pantothenate, consistent with the intracellular parasite having both a pantothenate transporter and a pantothenate kinase. Comparisons of the rate of phosphorylation of pantothenate by lysates prepared from uninfected and infected erythrocytes revealed that the pantothenate kinase activity of the P. falciparum trophozoite is some 10-fold higher than that of its host cell and that most, if not all, of the phosphorylation of pantothenate within the malaria-infected cell occurs within the intracellular parasite. These results contrast with those of previous studies in which it was proposed that the avian malaria parasite Plasmodium lophurae lacks pantothenate kinase (as well as the other enzymes for the synthesis of coenzyme A) and is reliant upon the uptake of preformed coenzyme A from the host cell cytosol.Malaria is caused by a protozoan parasite (genus Plasmodium) which, during the course of its life cycle, invades the red blood cells of its vertebrate host. This strategy serves to protect the parasite from attack by the immune system of the host; however, it poses significant challenges to the protozoan with regard to obtaining essential nutrients from the plasma.One such compound is pantothenic acid, a water-soluble vitamin (M r 219; Fig. 1) that serves as a precursor of the enzyme cofactor coenzyme A. Early evidence for the importance of exogenous pantothenate in supporting the growth of the intracellular malaria parasite came from the observation that in chickens infected with the avian parasite Plasmodium gallinaceum a dietary deficiency of pantothenate resulted in a marked reduction in the parasitemia (1). Forty years later it was demonstrated in in vitro growth assays that the sustained growth of the human malaria parasite Plasmodium falciparum within human erythrocytes is dependent upon there being pantothenate in the extracellular medium (2). The requirement of the intracellular malaria parasite for an exogenous supply of pantothenate implies that there is a mechanism by which this compound can gain entry into the infected erythrocyte. However, thi...

The Human Malaria Parasite Plasmodium falciparum Is Not Dependent on Host Coenzyme A Biosynthesis

“…In the only study on the subject, Annous and Song (9) reported that although pantothenate is phosphorylated within rat erythrocytes (the first step in CoA biosynthesis), there is no evidence for the subsequent steps of the CoA biosynthesis pathway. Saliba et al (10) confirmed that human erythrocytes similarly phosphorylate pantothenate, but did not investigate whether CoA synthesis also occurs in the cells. A lack of CoA biosynthesis in mammalian erythrocytes would seemingly place the burden of CoA synthesis squarely on malaria parasites that infect mammals (such as P. falciparum), contrary to the situation in birds.…”

“…The cells were washed (four times) and suspended in pantothenate-free medium, pH 7.4. For experiments with parasites isolated from their host erythrocyte, following enrichment to Ͼ95% parasitemia, trophozoite stage P. falciparum-infected erythrocytes in suspension were treated with saponin (0.05% (w/v)) essentially as described previously (10). Following treatment with saponin, which permeabilizes the erythrocyte and parasitophorous vacuole membranes that enclose the parasite, while leaving the parasite plasma membrane intact, the cells were washed (four times) and suspended in pantothenate-free medium, adjusted to pH 7.1 to match the pH in the cytosol of a human erythrocyte infected with a trophozoite stage P. falciparum parasite (13).…”

Transport of nucleosides across the Plasmodium falciparum parasite plasma membrane has characteristics of PfENT1

“…the estimated intracellular concentration relative to the extracellular concentration), rather than amounts of nucleoside taken up, in order to facilitate the assessment of whether the radiolabel equilibrates between the intra-and extracellular solutions or is taken up to levels that exceed those that can be accounted for by equilibrative transport. The concentration of radiolabel inside the cells was calculated by dividing the amount of radiolabel associated with the cells by the intracellular water volume of the pellet, calculated from the cell count using the previously reported value of 28 fl for the cell water volume of parasites isolated by saponin permeabilization of the host cell (Saliba et al, 1998). Distribution ratios were calculated by dividing this intracellular concentration of radiolabel by the concentration of radiolabel in the extracellular solution.…”

“…Parasites were 'isolated' from their host erythrocytes by treating parasitized erythrocytes with saponin as described elsewhere (Saliba et al, 1998). Saponin renders the erythrocyte and parasitophorous vacuole membranes permeable to macromolecules (Ansorge et al, 1996) but leaves the parasite plasma membrane intact and able to maintain transmembrane ion gradients (Saliba and Kirk, 1999;Alleva and Kirk, 2001) as well as a substantial membrane potential (Allen and Kirk, 2004).…”

“…Time points less than 15 s were sampled to the beat of a digital metronome (Model MT40, Wittner GmbH and Co.) to ensure accuracy. The cell pellets beneath the oil layer were processed for scintillation counting as described previously (Saliba et al, 1998).…”