pH Regulation in the Intracellular Malaria Parasite, Plasmodium falciparum

Abstract

The mechanism by which the intra-erythrocytic form of the human malaria parasite, Plasmodium falciparum, extrudes H ؉ ions and thereby regulates its cytosolic pH (pH i ), was investigated using saponin-permeabilized parasitized erythrocytes. The parasite was able both to maintain its resting pH i and to recover from an imposed intracellular acidification in the absence of extracellular Na ؉ , thus ruling out the involvement of a Na ؉ /H ؉ exchanger in both processes. Both phenomena were ATPdependent. Amiloride and the related compound ethylisopropylamiloride caused a substantial reduction in the resting pH i of the parasite, whereas EMD 96785, a potent and allegedly selective inhibitor of Na ؉ /H ؉ exchange, had relatively little effect. The resting pH i of the parasite was also reduced by the sulfhydryl reagent N-ethylmaleimide, by the carboxyl group blocker N,N-dicyclohexylcarbodiimide, and by bafilomycin A 1 , a potent inhibitor of V-type H ؉ -ATPases. Bafilomycin A 1 blocked pH i recovery in parasites subjected to an intracellular acidification and reduced the rate of acidification of a weakly buffered solution by parasites under resting conditions. The data are consistent with the hypothesis that the malaria parasite, like other parasitic protozoa, has in its plasma membrane a V-type H ؉ -ATPase, which serves as the major route for the efflux of H ؉ ions.Malaria, one of the most important infectious diseases in the world today, is caused by parasitic protozoa of the genus Plasmodium. These are unicellular, eukaryotic organisms, which, during the course of their complex lifecycle, invade the red blood cells of their vertebrate host. Having entered a red cell, the invading parasite lies dormant for some hours (the ring stage), after which it begins a period of rapid growth (the trophozoite stage) followed by division (schizogony), resulting in the generation of 20 -30 new parasites.The metabolic and biosynthetic activity of the malaria trophozoite is intense. The parasite is wholly reliant on glycolysis as its energy source, and it consumes glucose and produces lactic acid at a rate some 100 times higher than does a normal, uninfected erythrocyte (1, 2). The high metabolic activity of the parasite generates a substantial intracellular acid load. In addition, in the in vivo situation, malaria infection commonly gives rise to a pronounced extracellular acidosis (3). For the parasite to remain viable, it must therefore have an effective means of protecting its intracellular pH (pH i ) 1 from both intraand extracellular acid loads.Eukaryotic cells extrude H ϩ via a variety of different mechanisms. Plant cells, yeast, various protozoa, and a number of invertebrate and vertebrate cell types have in their plasma membrane H ϩ -ATPases that utilize energy derived from the hydrolysis of ATP to pump H ϩ ions from the cell cytosol (4). These are either P-type ATPases (so-called because they form an acyl-phosphate intermediate during their reaction cycle) or V-type ATPases (so-called because they were first described on t...

Antimalarial drugs disrupt ion homeostasis in malarial parasites

“…6). These results showed that chloroquine stimulated a H + extrusion response with an EC 50 of 16 µM in P. chabaudi, using plasma membrane mechanisms to recover the intracellular pH (Saliba & Kirk 1999).…”

“…Fig 5 shows spectrofluorimetric measurements of calcium release by Thg (10 µM) with parasites previously incubated (15 min at 37 o C) with different concentrations of chloroquine and artemisinin (0-80 µM) in a high calcium ex- The results show that previous parasite incubation with chloroquine or artemisinin leads to a dose-dependent reduction of total amount of calcium release by thapsigargin. When parasites are exposed to chloroquine, the cytosolic pH decrease as a result of extrusion of H + from acidic compartments and the parasites (P. falciparum) try to restore the cytosolic pH level using plasma membrane mechanisms (Saliba & Kirk 1999, Marchesini et al 2000, Saliba et al 2003. By using microphysiometry we showed that addition of chloroquine to P. chabaudi at the trophozoite stage led to a dose-dependent increase in the extracellular acidification rate (Fig.…”

“…It has been reported that P. falciparum and P. chabaudi extrude H + to the extracellular medium by a Na + independent mechanism (Saliba & Kirk 1999). Other mechanisms for pH regulation described in malaria parasites may involve the V-H + -ATPase and the V-H + -PPase in acidocalcisomes (Marchesini et al 2000, McIntosh & Vaidya 2002, Saliba et al 2003, Biagini et al 2003.…”

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

“…The malaria parasite is wholly reliant on glycolysis for the generation of ATP (Sherman, 1998) and ATP is depleted rapidly when parasites are incubated in a glucose-free medium (Saliba and Kirk, 1999). Whereas in ATP-replete parasites the uptake of adenosine (10 µ M) reached a distribution ratio of 4-5 within a few seconds and increased more slowly thereafter (as in Fig.…”

pfmdr1 mutations associated with chloroquine resistance incur a fitness cost in Plasmodium falciparum

“…Every 4-6 days, a proportion of the ring-stage parasites not required to propagate the culture were treated with saponin essentially as described previously (except that centrifugation was performed at 12 000 g) (Saliba and Kirk, 1999) in order to remove erythrocyte haemoglobin. Genomic DNA was then extracted from the isolated parasites using a DNeasy Plant Mini Kit (Qiagen), and stored at -20∞C.…”