Life cycle studies of the hexose transporter of Plasmodium species and genetic validation of their essentiality - PubMed (original) (raw)

Life cycle studies of the hexose transporter of Plasmodium species and genetic validation of their essentiality

Ksenija Slavic et al. Mol Microbiol. 2010 Mar.

Free PMC article

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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Figures

Fig. 1

A. Strategy for disruption of the PfHT gene. Single cross-over homologous recombination of the knockout plasmid and the endogenous gene results with two truncated copies of the gene. The location of PCR primers is indicated by numbered arrows. Restriction sites of enzymes used to digest genomic DNA prior to Southern blotting and expected Southern blot fragments are also indicated. The complementation construct (pCHD-HT) allows pfht expression under the Pfhsp86 promoter. B. Plasmid rescue of episomes from transfected parasites. Lane 1: re-isolated episome from parasites transfected with the knockout construct (pCAM-BSD-HT). Lanes 2 and 4: re-isolated episomes from parasites co-transfected with the knockout and the complementation construct (pCHD-HT). Lane 3: pCAM-BSD-HT plasmid. Lane 5: pCHD-HT plasmid. BamHI-NotI digestion releases a 1.2 kb knockout fragment from pCAM-BSD-HT. BglII-NotI digestion releases the PfHT gene (1.5 kb) and an additional 1.2 kb fragment from pCHD-HT.

Fig. 2

Genotype analysis of wild-type 3D7 parasites and parasites transfected with pCAM-BSD-HT alone or co-transfected with pCAM-BSD-HT and pCHD-HT. A. PCR analysis: lane 1, detection of the wild-type pfht locus 2 kb (primers 1 + 2, see Fig. 1); lane 2, detection of the 5′ integration of pCAM-BSD-HT into the _pfht_locus ∼1.8 kb (primers 1 + 4); lane 3, detection of the 3′ integration of pCAM-BSD-HT ∼1.7 kb (primers 3 + 2); lane 4, detection of the pCAM-BSD-HT episome 1.4 kb (primers 3 + 4). B. Southern blot analysis. Genomic DNA extracted from wild-type 3D7 parasites (lane 1), parasites transfected with pCAM-BSD-HT (lane 2), selected parasites co-transfected with pCAM-BSD-HT and pCHD-HT after blasticidin cycling – complemented parasites (lane 3) and unselected co-transfected parasites prior to blasticidin cycling (lane 4), and plasmid DNA, pCAM-BSD-HT (lane 5) and pCHD-HT (lane 6) were digested with _Swa_I, _Nco_I and _Eco_NI. The blot was probed with the 1.2 kb _pfht_fragment that was used as an insert for the pCAM-BSD-HT plasmid; wild-type locus 3 kb, integration of pCAM-BSD-HT 4.7 kb (5′) and 3.9 kb (3′), pCAM-BSD-HT episome 5.7 kb, pCHD-HT episome 8.3 kb.

Fig. 3

Phenotype analysis of wild-type 3D7 parasites and complemented parasites. A. Effect of compound 3361 on growth of wild-type 3D7 parasites (squares) and complemented parasites (circles). Complemented parasites have disrupted endogenous pfht locus, instead pfht is expressed from pCHD-HT episome under the _pfhsp86_promoter. Growth inhibition was measured by incorporation of 3H-hypoxanthine with five replicates used per inhibitor concentration. The experiment was repeated five times. Result of a single experiment is shown. Obtained 3361 IC50 values for 3D7 and complemented parasites were 44.2 ± 8.7 and 109.5 ± 9.6 µM respectively (P = 0.001 student's _t_-test, unpaired, two-tailed, n = 5). B. Real-time PCR analysis of pfht_expression normalized to β_-tubulin in wild-type 3D7 (white bars) and complemented parasites (black bars) (result of 6 experiments with 3 replicates each, one sample _t_-test, *P = 0.015).

Fig. 5

Tagging of the pbht locus with GFP. A. The strategy for GFP-tagging of the pbht locus; arrows indicate the location of PCR primers. B. PCR analysis of the wild type (WT) and a pyrimethamine-resistant pbht-gfp transfected line. Lane 1, positive control 2.3 kb (primers Pb1 + Pb11); lane 2, detection of the gfp-tagged locus 2.4 kb (Pb1 + gfpr); lane 3, detection of the wild-type locus 3.2 kb (Pb1 + Pb8). C. Southern blot analysis of wild-type (1) and pbht-gfp transfected line (2). gDNA was digested with _BsrG_I, and blot was probed with a pbht fragment used for generation of the tagging construct; wild-type locus 3.9 kb, integration bands 1.8 kb (5′) and 8.1 kb (3′).

Fig. 4

Pbht knockout attempt. A. A double cross-over knockout strategy used to attempt a knockout of pbht and investigate its function; the locations of primers used for PCR analysis of the locus are indicated by arrows. Tg DHFR/TS, Toxoplasma gondii dihydrofolate reductase/thymidylate synthase. B. PCR analysis of the pbht locus in wild type (WT) and parasites transfected with the knockout construct; lane 1, detection of the wild-type locus 1.1 kb (primers Pb7 + Pb9); lane 2, integration detection 1.2 kb (primers p539 + Pb9); lane 3, episome detection 1.1 kb (primers p539 + Pb8).

Fig. 6

Direct fluorescence imaging of pbht-gfp transgenic line. A. A young, blood-stage pbht-gfp P. berghei parasite. B. Two _pbht-gfp P. berghei_trophozoites inside an erythrocyte. C. Zygotes/or female gametes; interestingly a smaller round cell containing surface GFP fluorescence but no P28 staining is also observed. D. Analysis of fluorescence intensities across the ookinete cell. E. Ookinete [C–E: live parasites from 20 to 24 h culture in the ookinete medium, immunostained with a monoclonal antibody against the female gamete/zygote/ookinete marker P28 (red); DAPI was used as a nuclear dye (blue)]. F. A. stephensi midgut 11 days post infection. G. Sporulating midgut oocyst 21 days post infection. H. Sporozoites released from ruptured midgut oocysts. I. Sporozoites released from salivary glands of A. stephensi 21 days post infection.

Fig. 7

Western blot analysis. Ten micrograms of parasite material from asexual blood stages, ookinete-enriched culture and mosquito midguts and salivary glands stages of PbGFPCON (lane 1) and pbht-gfp line (lane 2) was subjected to SDS-PAGE (10% acrylamide), transferred to a nitrocellulose membrane and probed with 1:1000 diluted anti-GFP antibody (Roche) and 1:3000 diluted HRP-conjugated anti-mouse antibody. *Ookinete-enriched culture obtained by incubation of blood sample of an infected mouse in the ookinete medium overnight at 19°C. p.i., post infection.

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Life cycle studies of the hexose transporter of Plasmodium species and genetic validation of their essentiality - PubMed (original) (raw)