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Nadia Saïdani

Exploratory study of galactolipid biosynthesis in Plasmodium falciparum

Published on 5 September 2008

Thesis presented September 05, 2008

Plastids are semi-autonomous organelles that derive from a unique endosymbiotic event between an ancestral eukaryotic cell and a cyanobacteria. The best characterized plastid is the chloroplast from plant cells and green algae. Differing from eukaryotic membrane systems that are naturally phospholipid-rich, plant chloroplasts membranes are composed of >70% galactoglycerolipids (monogalactosyldiacylglycerol, MGDG; digalactosyldiacylglycerol, DGDG). Their synthesis is catalyzed by galactosyltransferases, namely MGDG synthases (EC and DGDG synthases (EC, localized in the organelle limiting envelope membranes. In plant cells, galactolipids are essential to plastid biogenesis as well as for the composition of non-plastidial membranes under specific physiological conditions, such as phosphate deprivation.
Most of the apicomplexan parasites (obligate intracellular protozoan parasites) harbor a non- photosynthetic plastid, containing a circular DNA. This organelle, called the apicoplast, is considered as a relic of an algal plastid, resulting from a secondary endosymbiosis between two eukaryotic cells, with the ingestion of a unicellular red algae by an ancestra​l protozoan, followed by the disappearance of most of the algal subcellular structures. Interestingly, some apicomplexan nuclear gene products are imported into the apicoplast and involved in typical plant biosynthetic pathways such as FASII fatty acids biosynthesis.
Lipids with chromatographic properties similar to those of MGDG and DGDG, have been detected in Plasmodium falciparum and Toxoplasma gondii total extracts, suggesting the existence of a chloroplast-like galactolipid biosynthetic pathway. Using a series of antibodies directed against DGDG, a first aim of this thesis work was to investigate the localization of a chloroplastic digalactolipid-like epitope (DGLE) in P. falciparum and to determine its evolution along the parasitic cell cycle. These studies suggest that a DGLE may be associated to endomembrane systems in the cell periphery, particularly the inner membrane system. Transgenic parasites expressing an exogenous plant MGDG synthase have been generated. The remarkable accumulation of both MGDG and DGDG demonstrate that the exogenous plant enzyme is functional, catalyzing the synthesis of MGDG, and that a P. falciparum glycosyltransferase is subsequently capable of catalyzing large amounts of DGDG. The possible role of this DGDG-producing glycosyltransferase in the synthesis of the DGLE remains to be demonstrated.
A second aim of this thesis consisted in the evaluation of compounds inhibiting the galactolipid synthesis for a therapeutic purpose. An automated high throughput screening of a 24000 molecule- chemolibrary allowed the identification of inhibitors of Arabidopsis thaliana MGDG synthase 1, among which two compounds harbor​ an inhibiting effect in the 10 µM range on MGDG synthase activity. We characterized the effect of these molecules as competitors of diacylglycerol binding. An antiparasitic activity was measured in vitro. In order to improve 1) the herbicidal effect of the lead compounds, 2) the parasite growth inhibition and to develop new antiplasmodial candidates that could be qualified as herbicidal-drugs, a structure-based diversification of the molecules was conducted. The characterization of 250 analogues allowed the optimization of the molecular selectivity with compounds being active in the 200 nM range.
It cannot be excluded that molecules selected for their ability to inhibit a MGDG synthase activity could exert a distinct effect on a non-related Plasmodium falciparum target. We developed an experimental procedure to isolate the protein target(s) of the bioactive compounds by affinity chromatography against a biotinylated analogue, leading to the identification of several proteins by mass spectrometry after trypsic digestion.
The development of drug candidates is a long process from the target validation to the clinical trials. The properties of the novel class of compounds characterized in this thesis work are promising. Future prospects include therefore sustained efforts to improve the antiparasitic properties of this novel class of diacylglycerol competitors.

Plasmodium, MGDG synthase, galactolipids, Apicoplast, antimalarial, DGLE

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