Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Mol Microbiol
2010 Mar 01;756:1402-13. doi: 10.1111/j.1365-2958.2010.07060.x.
Show Gene links
Show Anatomy links
Life cycle studies of the hexose transporter of Plasmodium species and genetic validation of their essentiality.
Slavic K
,
Straschil U
,
Reininger L
,
Doerig C
,
Morin C
,
Tewari R
,
Krishna S
.
???displayArticle.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.
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 5â² integration of pCAM-BSD-HT into the pfhtlocus â¼1.8 kb (primers 1 + 4); lane 3, detection of the 3â² integration of pCAM-BSD-HT â¼1.7 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 â 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 SwaI, NcoI and EcoNI. The blot was probed with the 1.2 kb pfhtfragment 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 (5â²) and 3.9 kb (3â²), 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 pfhsp86promoter. 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 pfhtexpression 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 BsrGI, 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 (5â²) and 8.1 kb (3â²).
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. bergheitrophozoites 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 [CâE: 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.
Barrett,
Trypanosome glucose transporters.
1998,
Pubmed
Burchmore,
Genetic characterization of glucose transporter function in Leishmania mexicana.
2003,
Pubmed
Cowman,
Functional genomics: identifying drug targets for parasitic diseases.
2003,
Pubmed
Daily,
Distinct physiological states of Plasmodium falciparum in malaria-infected patients.
2007,
Pubmed
Dessens,
CTRP is essential for mosquito infection by malaria ookinetes.
1999,
Pubmed
Dondorp,
Artemisinin resistance in Plasmodium falciparum malaria.
2009,
Pubmed
Dorin-Semblat,
Functional characterization of both MAP kinases of the human malaria parasite Plasmodium falciparum by reverse genetics.
2007,
Pubmed
Florens,
A proteomic view of the Plasmodium falciparum life cycle.
2002,
Pubmed
Franke-Fayard,
A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle.
2004,
Pubmed
Gardner,
Genome sequence of the human malaria parasite Plasmodium falciparum.
2002,
Pubmed
Hall,
A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses.
2005,
Pubmed
Ikekawa,
Studies on synthesis of 3-O-alkyl-D-glucose and 3-O-alkyl-D-allose derivatives and their biological activities.
1987,
Pubmed
Imming,
Drugs, their targets and the nature and number of drug targets.
2006,
Pubmed
Janse,
High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei.
2006,
Pubmed
Joet,
Validation of the hexose transporter of Plasmodium falciparum as a novel drug target.
2003,
Pubmed
,
Xenbase
Joët,
Comparative characterization of hexose transporters of Plasmodium knowlesi, Plasmodium yoelii and Toxoplasma gondii highlights functional differences within the apicomplexan family.
2002,
Pubmed
,
Xenbase
Khan,
Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology.
2005,
Pubmed
Lasonder,
Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity.
2008,
Pubmed
Lasonder,
Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry.
2002,
Pubmed
Liu,
The conserved plant sterility gene HAP2 functions after attachment of fusogenic membranes in Chlamydomonas and Plasmodium gametes.
2008,
Pubmed
Martin,
The 'permeome' of the malaria parasite: an overview of the membrane transport proteins of Plasmodium falciparum.
2005,
Pubmed
Noedl,
Evidence of artemisinin-resistant malaria in western Cambodia.
2008,
Pubmed
Price,
Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number.
,
Pubmed
Reininger,
An essential role for the Plasmodium Nek-2 Nima-related protein kinase in the sexual development of malaria parasites.
2009,
Pubmed
Sidhu,
pfmdr1 mutations contribute to quinine resistance and enhance mefloquine and artemisinin sensitivity in Plasmodium falciparum.
2005,
Pubmed
Sinden,
Maintenance of the Plasmodium berghei life cycle.
2002,
Pubmed
Tonkin,
Localization of organellar proteins in Plasmodium falciparum using a novel set of transfection vectors and a new immunofluorescence fixation method.
2004,
Pubmed
Trager,
Human malaria parasites in continuous culture.
1976,
Pubmed
Woodrow,
Intraerythrocytic Plasmodium falciparum expresses a high affinity facilitative hexose transporter.
1999,
Pubmed
,
Xenbase
