A constitutive pan-hexose permease for the Plasmodium life cycle and transgenic models for screening of antimalarial sugar analogs - PubMed (original) (raw)
A constitutive pan-hexose permease for the Plasmodium life cycle and transgenic models for screening of antimalarial sugar analogs
Martin Blume et al. FASEB J. 2011 Apr.
Abstract
Glucose is considered essential for erythrocytic stages of the malaria parasite, Plasmodium falciparum. Importance of sugar and its permease for hepatic and sexual stages of Plasmodium, however, remains elusive. Moreover, increasing global resistance to current antimalarials necessitates the search for novel drugs. Here, we reveal that hexose transporter 1 (HT1) of Plasmodium berghei can transport glucose (K(m)~87 μM), mannose (K(i)~93 μM), fructose (K(i)~0.54 mM), and galactose (K(i)~5 mM) in Leishmania mexicana mutant and Xenopus laevis; and, therefore, is functionally equivalent to HT1 of P. falciparum (Glc, K(m)~175 μM; Man, K(i)~276 μM; Fru, K(i)~1.25 mM; Gal, K(i)~5.86 mM). Notably, a glucose analog, C3361, attenuated hepatic (IC(50)~15 μM) and ookinete development of P. berghei. The PbHT1 could be ablated during intraerythrocytic stages only by concurrent complementation with PbHT1-HA or PfHT1. Together; these results signify that PbHT1 and glucose are required for the entire life cycle of P. berghei. Accordingly, PbHT1 is expressed in the plasma membrane during all parasite stages. To permit a high-throughput screening of PfHT1 inhibitors and their subsequent in vivo assessment, we have generated Saccharomyces cerevisiae mutant expressing codon-optimized PfHT1, and a PfHT1-dependent Δpbht1 parasite strain. This work provides a platform to facilitate the development of drugs against malaria, and it suggests a disease-control aspect by reducing parasite transmission.
Figures
Figure 1.
PbHT1 and _Pf_HT1 proteins can restore the transport of glucose, mannose, fructose and galactose in the L. mexicana null mutant. Uptake of [14C]
d-
glucose, [14C]
d-
mannose, [14C]
d-
fructose, and [14C]
d-
galactose in Δ_lmgt_ promastigotes expressing _Pb_HT1, _Pf_HT1, or _Tg_GT1 was measured as described in Materials and Methods. Vector-transfected cells were used as a negative control. Values are expressed as means ±
sd
.
Figure 2.
Glucose and mannose exhibit high affinity toward _Pb_HT1 and _Pf_HT1, whereas fructose and galactose are low-affinity ligands. A) Substrate saturation curves of stable transgenic _Pb_HT1- and Pf_HT1-expressing L. mexicana parasites. Δ_lmgt promastigotes (2×107) expressing _Pb_HT1 or _Pf_HT1 were incubated with 0–2 mM [3H]
d-
glucose for 30 s at 25°C. B) Inhibition of _Pb_HT1- or _Pf_HT1-mediated uptake of [3H]
d-
glucose (100 μM) with indicated nonlabeled hexoses as inhibitors. C) _K_m and _K_i values for glucose, mannose, fructose, and galactose for _Pb_HT1 and _Pf_HT1. Kinetic data of _Tg_GT1 (present study and ref. 13) are included for comparison. Asterisks indicate _K_i values that are approximate due to incomplete (∼50–60%) inhibition. Values are expressed as means ±
se
.
Figure 3.
PbHT1 is an essential surface-localized permease, expressed in blood stages of P. berghei. A) Linearized _Pb_HT1-HA-harboring knockout and mock constructs in alignment with the PbHT1 locus. Arrows depict crossover-specific primers (_Pb_HT1–5Scr-F/R and _Pb_HT1–3Scr-F/R) and clone-specific primers (_Pb_HT1-Clone-F/_Pb_HT1–3Scr-R) for PCR-based genotyping. B) Crossover-specific genomic PCR yields 5′/3′ bands only with _Pb_HT1-HA-complemented KO parasite (Δ_pbht1-Pb_HT1-HA) but not with conventional KO construct-transfected (Δ_pbht1_-Mock) or from parental strains. PCR with _Pb_HT1 KO plasmid was included as an additional control. C) Genotyping by integration-specific PCR. Clone-specific primers (_Pb_HT1-Clone-F and _Pb_HT1–3Scr-R) amplify a gDNA fragment of 927 bp in the parental strains and of 6729 bp in Δ_pbht1-Pb_HT1-HA strains, and none with the plasmid control. D) RT-PCR using total RNA isolated from parental and Δ_pbht1-Pb_HT1-HA parasites. _Pb_HT1 (_Pb_HT1-F/_Pb_HT1-R) and _Pb_HT1-HA (_Pb_HT1-F/HA-R)-specific primers produce amplicons of 1566 and 1602 bp, respectively. E) Immunofluorescence of Δ_pbht1-Pb_HT1-HA-infected erythrocytes using rabbit anti-HA antibody. Parasite cytosol is stained with anti-HSP70 antibody.
Figure 4.
A glucose analog, C3361, can inhibit glucose transport by _Pb_HT1 and reduces ookinete development and transmission of P. berghei. A) Surface expression of _Pb_HT1-HA protein during the sexual stages of P. berghei. Immunofluorescence assay was performed with the Δ_pbht1-Pb_HT1-HA parasites using rabbit anti-HA antibody. All samples were fixed with 4% paraformaldehyde. B) C3361-mediated inhibition of [3H]
d-
glucose transport (100 μM, 30 s) by Δ_lmgt_ promastigotes (2×107) expressing _Pb_HT1, _Pf_HT1, and _Tg_GT1 permeases. Unlike _Tg_GT1, Plasmodium transporters display similar inhibition kinetics. C) Effect of C3361 and DMSO treatment on in vitro formation of ookinetes. Blood samples from P. berghei-infected mice were treated with C3361 (∼100 μM) or DMSO during in vitro differentiation of gametocytes into ookinetes for 18–20 h. Ookinetes were bead-purified and counted. D) Sexual development and transmission of P. berghei, as depicted by representative image of GFP-expressing oocysts in mosquito midgut following treatment of asexual host with C3361 (2 mg/kg body weight) or DMSO. Mice were intravenously infected with 105 parasite-infected red blood cells, and then treated with C3361 or DMSO for 3 d prior to a 30-min blood-meal on anesthetized animals. E) Total oocyst numbers were counted following dissection of mosquitoes on d 10. Graph represents the oocyst output/infected insect from 2 assays. Statistics were performed by 2-tailed Mann-Whitney test.
Figure 5.
_Pb_HT1 is expressed during liver stages, and C3361 attenuates the hepatic development of P. berghei. A) Huh7 hepatoma cells were infected with sporozoites isolated from the mosquito salivary glands at an MOI of 3. Immunofluorescence assay was performed with the Δ_pbht1-Pb_HT1-HA parasites using rabbit anti-HA antibody. Samples were fixed with methanol and processed at the indicated time points. Scale bars = 5 μm. B) Huh7 cells were infected with sporozoites at an MOI of 3 and fixed with methanol after 68 h before staining with anti-HSP70 and anti-ACP antibodies. Area of the liver schizonts decreased with increasing concentrations of C3361. Apicoplast development and segregation were also inhibited. C) Mean area of ≥20 liver schizonts from 3 experiments was measured using ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA). Statistics were performed by 2-tailed Mann-Whitney test. Values are expressed as means ±
se
.
Figure 6.
Transgenic P. berghei as a model for the in vivo assessment of _Pf_HT1 inhibitors. A) Double-crossover-mediated deletion of PbHT1 by _Pf_HT1-dependent rescue. The PbHT1 gene locus and _Pf_HT1-harboring knockout construct are aligned; arrows indicate primers. B) The 5′ and 3′ recombination-specific (_Pb_HT1–5Scr-F/R and _Pb_HT1–3Scr-F/R) primer sets amplify genomic fragments from Δ_pbht1-Pf_HT1 but not in parental strain or plasmid. C) _Pf_HT1 can complement the absence of _Pb_HT1 in the transgenic Δ_pbht1-Pf_HT1 parasite line. PCR-based genotyping of the Δ_pbht1-Pf_HT1 strain using _Pb_HT1- or _Pf_HT1-specific primers. _Pb_HT1 is detectable in the parental strain but not in Δ_pbht1-Pf_HT1. Likewise, _Pf_HT1 can only be amplified from transgenic gDNA or cDNA. D, E) In vivo test of C3361 efficacy in mice intravenously injected with 105 parental- or Δ_pbht1-Pf_HT1-infected red blood cells. Following a detectable parasitemia on d 3, mice were treated with C3361 (2 mg/kg body wt) or DMSO. Graphs show parasitemia (D) and animal survival curves (E) from two assays. Values are expressed as means ±
se
.
Figure 7.
Yeast-optimized _Pf_HT1 can rescue the growth of the S. cerevisiae mutant on glucose and mannose and confers a model for high-throughput screening of analog-based _Pf_HT1 inhibitors. A) Complementation of S. cerevisiae EBY4000 mutant with the illustrated expression constructs. Yeast cells (2 μl) were spotted on uracil-free synthetic medium supplemented with 0.06% glucose or 2% mannose and incubated at 30°C for 3 or 4 d before imaging. B) Immunofluorescence assay of S. cerevisiae expressing the indicated his-tagged proteins. Zymolase-treated transgenic cells from log-phase liquid cultures were fixed with 4% PFA overnight at 4°C, then processed for IFA using mouse anti-His antibody at 1:200 dilution. C) C3361-mediated inhibition of _Pf_HT1 transport activity on 0.06% glucose or mannose. Cells were spotted on agar plates supplemented with C3361 (100 μM) or DMSO and incubated at 30°C for 4 d before imaging. Stability of the compound C3361 is unknown.
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