Uptake and kinetic properties of choline and ethanolamine in Plasmodium falciparum

Purpose: The asexual proliferation of Plasmodium, inside the erythrocyte, is accompanied by the synthesis of huge quantities of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn). These needful phospholipids for the cytoplasmic membrane of the merozoites are provided by the precursors choline and ethanolamine. PtdCho and PtdEtn are synthesized by the parasite because the erythrocyte is unable to do it. In order to assess the dynamism of the phospholipid pathways, we aimed to investigate the respective shape of the uptake of choline and ethanolamine by Plasmodium falciparum. Method: Time-course experiments and kinetic assays were performed respectively with fixed and ranged concentrations of radioactively-labelled choline and ethanolamine. The labelled-precursors were added in the culture of P. falciparum infected-erythrocytes and the incorporated molecules in phospholipids were measured with a scintigraph counter. Result: The results showed that the incorporation of precursors in the infected-erythrocyte occurred with a Michaelis-Menten’s kinetic shape. According to the maximum rate (Vmax), the pathway of ethanolamine incorporation was faster than that of choline. Similarly, affinity for ethanolamine was greater than that of choline. Conclusion: Although PtdCho is the major phospholipid in the membrane, this study rules out that the influx of ethanolamine in the infected-erythrocyte, in vivo conditions, is more dynamic than choline.


INTRODUCTION
Like many other animal cells, the cytoplasmic membrane of the Plasmodium is made of bulk phospholipid molecules organized in a double layer.The major ones are phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn).After the invasion of the erythrocyte, the parasite starts a schizogonic cycle marked by the production of several merozoites.This asexual proliferation of the parasite inside the erythrocyte is accompanied by the synthesis of huge quantities of needful phospholipids for the membrane of the merozoites.It means a biosynthesis of PtdCho and PtdEtn by the parasite because the erythrocyte is unable to do it on its own.In order to assess the needs of phospholipids for the parasite, we evaluated the uptake of its precursors, choline and ethanolamine.Many studies reveal metabolic differences within the species of Plasmodium .The uptake of choline and ethanolamine by the Plasmodium seem to vary with the species, the stage of the life cycle and the experimental conditions 4 .
In this study, we determined the uptake and apparent kinetic properties of choline and ethanolamine in P. falciparum infestederythrocyte, the most widespread human malarial parasite in West Africa.

In vitro culture of P. falciparum
The 3D7 strain of P. falciparum in infected erythrocytes was incubated in modified RPMI 1640 medium supplemented with 25 mM Hepes (pH 7.6).The RPMI 1640 was free of choline and ethanolamine.The parasitemia was 7 -12 % with a hematocrit of 2.5 % in a total amount of 3.5 x10 6 -6 x10 6 infected cells.The cell suspension was distributed in microwell plate and incubated at 37°C in a 5 % carbon dioxide atmosphere at time 0.

Labelling for time-course studies
To assess the uptake of the phospholipid precursors by the infected-erythrocyte, radioactive isotopes of [1-3 H]ethan-1-ol-2amine and [methyl-3 H]choline, purchased with Amersham™ corp were used They were added, in the respective microwells, at time 60, 120, 180 and 210 min (final concentration of 2.5 µM; specific activity of 2 Ci/mmol).The final volume of the suspension in each microwell was 200 µl.At time 240 min, reactions were stopped at 4°C.Radioactive incorporation in bulk molecules was measured after haemolysis, filtration and transfer onto glass-fiber filter plate using a cell harvester.Radioactivity was measured on a scintigraph counter from Beckman™.Incorporation of the labelled precursors into the infectederythrocyte was expressed in nmol/10 10 cells.

Labelling for kinetic assay
To assess the kinetic features, different specific activity of radioactive choline and ethanolamine were added at indicated concentrations in the suspension of the infected-erythrocytes.The suspension was incubated for 2 hours at 37 °C in a 5 % CO 2 incubator.Reactions were stopped at 4 °C and radioactive incorporation was measured after filtration and transfer on fiber glass plate using a cell harvester.Radioactivity was measured as described above.The blank test was performed with an uninfected-erythrocyte suspension provided by an uninfected human to assess nonspecific uptake by the erythrocyte.

Data analysis
Experiments were performed twice.Blank test was deduced from each result and all of them were means of triplicate assays.Regression curve was drawn and the Michaelis constant (K m ) and V max were calculated with the software GraphPad Prism ® version 4.

Time course of choline and ethanolamine uptake
P. falciparum incorporates choline and ethanolamine in its bulk molecules retained by the cell harvester.For the two precursors, the uptake is constant and linear at least from 60 to 240 minutes.The rate of choline uptake is 0.91 ± 0.03 nmol.10 -10 parasites.h - and 5.01 ± 0.24 nmol.10 -10 parasites.h - for ethanolamine uptake (Figs. 1 and.2).The in vitro conditions show that the uptake of ethanolamine is greater than that of choline.

Kinetics of uptake
This assay was performed assuming an enzymatic process for the uptake of precursor  and their incorporation in bulk molecules of the parasite.The uptake of choline and ethanolamine in P. falciparum showed an apparent Michaelis-Menten kinetics with the following features: choline uptake had a V max of 15.9 ± 1.2 nmol.10 -10 cells.h - with an apparent K m of 58.2 ± 9.8 µM and ethanolamine uptake had a V max of 63.7 ± 2.4 nmol.10 -10 cells.h - with an apparent K m of 16.7 ± 1.7 µM (Figs 3 and 4).The metabolic pathway of precursor incorporation in the phospholipids of P. falciparum seems to have a greater affinity for ethanolamine than for choline.Beside this, the incorporation of ethanolamine was faster than that of choline.

DISCUSSION
In human infection, signs of clinical morbidity, associated with malaria may occur at a parasitaemia range of about 100 -720,000 per µl (i.e., 0.02 to 15%) 5, 6 . Our experimental conditions were chosen to get an optimal growth of the parasite.It means not too low parasitaemia to get a sufficient measurable metabolic reaction through the radioactivity signal.But not too high parasitaemia to get a dynamic and optimal growth of the parasite and avoid a high amount of lactic acid or other biological wastes that a high parasitaemia can release.It is the same caution that led to a relatively short incubation time (less than 240 min).In human blood, the choline concentration is about 10 -12 µM and ethanolamine is about 2.5 µM .In order to assess the match of choline and the ethanolamine pathways in in vitro conditions, we chose an identical final concentration of precursors (2.5 µM for each).The uninfected-erythrocyte is not expected to incorporate choline or ethanolamine in any metabolic pathways.However, Staines and Kirk showed that in an infected mammalian, the choline enters in uninfected erythrocyte with the same rate than in infected erythrocyte Our experiments show that P. falciparum incorporates choline in a constant way within 60 -240 minutes while P. knowlesi optimal incorporation of choline into PtdCho is less than 180 min.But the rate of choline uptake, supposed to be mainly incorporated in PtdCho, is about 100 fold lesser for P. falciparum than for P. knowlesi 11 .In the same way, Staines and Kirk showed that the rate of choline uptake by P. vinckei is greater than that of P. falciparum one 4 .This correlates with the length of life cycle of P. knowlesi or P. vinckei which is 24 hours while P. falciparum is much longer, 48 hours.
Infestation of the erythrocyte by Plasmodium entails a huge change of phospholipid composition in the erythrocyte membrane and in the whole complex parasite-erythrocyte relationship.
At the schizonte stage phospholipid composition increases to about 500 -700% and is mainly composed of PtdCho and PtdEtn (75 -85%) with predominantly PtdCho 12-14 .However in our condition of experiments, we noticed that the rate of ethanolamine uptake was superior to the rate of choline uptake.The normal mammalian plasma choline concentration is in the range 10 -40 µM 15 .Therefore, we expected the rate of choline uptake to increase and be superior to that of ethanolamine.But on the one hand V max of choline uptake was inferior to V max of ethanolamine uptake.On the other hand, the apparent K m of ethanolamine uptake showed a higher affinity of the parasite for ethanolamine rather than choline.These observations contribute to the hypothesis that PtdCho is mainly issued from ethanolamine according the three steps of N-methylation of phosphoethanolamine to phosphocholine and catalyzed by phosphoethanolamine Nmethyltransferase (EC This apparent feature of Mickaelis-Menten kinetics for the choline and ethanolamine uptakes hide the possibility that these pathways possess any key-steps regulated by a possible allosteric enzyme.

CONCLUSION
Choline and ethanolamine are incorporated in bulk molecules of P. falciparum.The uptake of these precursors by infected-erythrocyte increased when compared with uninfected erythrocyte.Such differences in the apparent kinetic properties of the choline and ethanolamine uptake are easily understandable if we are aware that ethanolamine has to provide both PtdCho and PtdEtn metabolic pathways.On the contrary, choline provides only the PtdCho pathway.These results are in favor for a most dynamic influx of ethanolamine in the P. falciparum phospholipids but may not be the case in in vivo conditions.

7
incubated in a final volume of 200 µl of modified RPMI 1640 supplemented with 25 mM Hepes (pH 7.6).Suspensions were incubated at 37 °C in a 5 % CO2 incubator at time 0. Radioactive choline (2.5 µM, 2 Ci/mmol) was added at time 60, 120, 180 and 210 min.At time 240 min, reaction was stopped at 4 °C and radioactive incorporation in phosphatidylcholine was measured after filtration using a cell harvester.Incorporation of the labelled precursors into phosphatidylcholine is expressed in nmol/10 10 cells and is mean of triplicate values ± SEM.