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Life cycle studies of the hexose transporter of Plasmodium species and genetic validation of their essentiality
Authors:Ksenija Slavic  Ursula Straschil  Luc Reininger  Christian Doerig  Christophe Morin  Rita Tewari  Sanjeev Krishna
Institution:1. Centre for Infection, Cellular and Molecular Medicine, St. George's University of London, London SW17 0RE, UK.;2. Division of Cell and Molecular Biology, Imperial College London, London, UK.;3. Institute of Genetics, School of Biology, The University of Nottingham, Nottingham, UK.;4. INSERM U609 Wellcome Centre for Molecular Parasitology, University of Glasgow, Glasgow, UK.;5. INSERM U609, Global Health Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), Station 19, CH‐1015 Lausanne, Switzerland.;6. Département de Chimie Moléculaire (UMR 5250, ICMG FR‐2607, CNRS) Université Joseph Fourier, Grenoble Cedex, France.
Abstract:A Plasmodium falciparum hexose transporter (PfHT) has previously been shown to be a facilitative glucose and fructose transporter. Its expression in Xenopus laevis oocytes and the use of a glucose analogue inhibitor permitted chemical validation of PfHT as a novel drug target. Following recent re‐annotations of the P. falciparum genome, other putative sugar transporters have been identified. To investigate further if PfHT is the key supplier of hexose to P. falciparum and to extend studies to different stages of Plasmodium spp., we functionally analysed the hexose transporters of both the human parasite P. falciparum and the rodent parasite Plasmodium berghei using gene targeting strategies. We show here the essential function of pfht for the erythrocytic parasite growth as it was not possible to knockout pfht unless the gene was complemented by an episomal construct. Also, we show that parasites are rescued from the toxic effect of a glucose analogue inhibitor when pfht is overexpressed in these transfectants. We found that the rodent malaria parasite orthologue, P. berghei hexose transporter (PbHT) gene, was similarly refractory to knockout attempts. However, using a single cross‐over transfection strategy, we generated transgenic P. berghei parasites expressing a PbHT–GFP fusion protein suggesting that locus is amenable for gene targeting. Analysis of pbht‐gfp transgenic parasites showed that PbHT is constitutively expressed through all the stages in the mosquito host in addition to asexual stages. These results provide genetic support for prioritizing PfHT as a target for novel antimalarials that can inhibit glucose uptake and kill parasites, as well as unveiling the expression of this hexose transporter in mosquito stages of the parasite, where it is also likely to be critical for survival.
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