Hexose Transport Research Articles

Involvement of abscisic acid in the coordinated regulation of a stress-inducible hexose transporter (VvHT5) and a cell wall invertase in grapevine in response to biotrophic fungal infection.

Biotrophic fungal and oomycete pathogens alter carbohydrate metabolism in infected host tissues. Symptoms such as elevated soluble carbohydrate concentrations and increased invertase activity suggest that a pathogen-induced carbohydrate sink is established. To identify pathogen-induced regulators of carbohydrate sink strength, quantitative real-time polymerase chain reaction was used to measure transcript levels of invertase and hexose transporter genes in biotrophic pathogen-infected grapevine (Vitis vinifera) leaves. The hexose transporter VvHT5 was highly induced in coordination with the cell wall invertase gene VvcwINV by powdery and downy mildew infection. However, similar responses were also observed in response to wounding, suggesting that this is a generalized response to stress. Analysis of the VvHT5 promoter region indicated the presence of multiple abscisic acid (ABA) response elements, suggesting a role for ABA in the transition from source to sink under stress conditions. ABA treatment of grape leaves was found to reproduce the same gene-specific transcriptional changes as observed under biotic and abiotic stress conditions. Furthermore, the key regulatory ABA biosynthetic gene, VvNCED1, was activated under these same stress conditions. VvHT5 promoter::beta-glucuronidase-directed expression in transgenic Arabidopsis (Arabidopsis thaliana) was activated by infection with powdery mildew and by ABA treatment, and the expression was closely associated with vascular tissue adjacent to infected regions. Unlike VvHT1 and VvHT3, which appear to be predominantly involved in hexose transport in developing leaves and berries, VvHT5 appears to have a specific role in enhancing sink strength under stress conditions, and this is controlled through ABA. Our data suggest a central role for ABA in the regulation of VvcwINV and VvHT5 expression during the transition from source to sink in response to infection by biotrophic pathogens.

Effect of HXT 1 and HXT 7 hexose transporter overexpression on wild-type and lactic acid producing Saccharomyces cerevisiae cells

BackgroundSince about three decades, Saccharomyces cerevisiae can be engineered to efficiently produce proteins and metabolites. Even recognizing that in baker's yeast one determining step for the glucose consumption rate is the sugar uptake, this fact has never been conceived to improve the metabolite(s) productivity.In this work we compared the ethanol and/or the lactic acid production from wild type and metabolically engineered S. cerevisiae cells expressing an additional copy of one hexose transporter.ResultsDifferent S. cerevisiae strains (wild type and metabolically engineered for lactic acid production) were transformed with the HXT1 or the HXT7 gene encoding for hexose transporters.Data obtained suggest that the overexpression of an Hxt transporter may lead to an increase in glucose uptake that could result in an increased ethanol and/or lactic acid productivities. As a consequence of the increased productivity and of the reduced process timing, a higher production was measured.ConclusionMetabolic pathway manipulation for improving the properties and the productivity of microorganisms is a well established concept. A high production relies on a multi-factorial system. We showed that by modulating the first step of the pathway leading to lactic acid accumulation an improvement of about 15% in lactic acid production can be obtained in a yeast strain already developed for industrial application.

Three years in reviews

as we ring in 2010, I am delighted to highlight a few of our accomplishments, and I take the opportunity to congratulate our authors, reviewers, and Associate Editors for their continued excellence and collegiality. It is undeniable that AJP - Endocrinology and Metabolism has played a pivotal role

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

A Plasmodium falciparumhexose transporter (PfHT) has previously been shown to be a facilitative glucose and fructose transporter. Its expression in Xenopus laevisoocytes 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.

Mechanism of SGLT1-Dependent Translocation of GLUT2 to the Apical Membrane of Enterocytes

Introduction: Traditional models of intestinal glucose absorption confine glucose transporter 2 (GLUT2) to the basolateral membrane. However, considerable evidence suggests that GLUT2 is translocated rapidly to the apical membrane when the enterocyte is exposed to high luminal glucose concentrations. We hypothesized that GLUT2 translocates to the apical membrane of enterocytes by a protein kinase C (PKC) signaling mechanism dependent on activity of the hexose transporter, sodium-glucose co-transporter 1 (SGLT1) and the cellular cytostructure. Methods: Transporter-mediated glucose uptake was studied in Lewis rat jejunum using everted sleeves under 7 conditions: Control, SGLT1 inhibition (phlorizin 0.2 mM), GLUT2 inhibition (phloretin 1 mM), both SGLT1 and GLUT2 inhibition, PKC inhibition (calphostin 500 nM, chelerythrine 50 uM), and disruption of cellular cytostructure (nocodazole 10 μM). Each condition was tested in iso-osmotic solutions of 1, 20, or 50 mM glucose for 1 or 5 min incubations (n=6 rats each). Results: Phlorizin inhibited glucose uptake nearly completely in 1, 20, and 50 mM glucose at both 1 and 5 minutes (p≤0.006 each). Phloretin partially inhibited glucose uptake (p≤0.01 each) in all concentrations and time. Phloretin and phlorizin together almost totally inhibited uptake (p=0.004 each). Calphostin and chelerythrine did not inhibit transporter-mediated glucose uptake except in 1 mM glucose at 5 min (p≤0.05 each). Nocodazole inhibited uptake only in 1 mM glucose at 1 and 5 min (p≤0.01 each) (see figure). Conclusion: Inhibition of SGLT1 led to near complete cessation of transporter-mediated glucose uptake. GLUT2 inhibition led to partial inhibition of glucose uptake suggesting constitutive expression of GLUT2 in the apical membrane. Disruption of PKC signaling or cell cytoskeletal integrity partially inhibited carrier-mediated glucose uptake only in 1 mM glucose, suggesting a non-specific effect rather than inhibition of GLUT2 translocation. In this model, it does not appear that GLUT2 is translocated to the apical membrane on the cellular cytostructure in response to PKC signaling from high luminal concentrations of glucose.

Heterologous expression of yeast Hxt2 in Arabidopsis thaliana alters sugar uptake, carbon metabolism and gene expression leading to glucose tolerance of germinating seedlings

The hexose transporter 2 gene (Hxt2) from Saccharomyces cerevisiae was expressed in Arabidopsis thaliana under control of the 35S promoter. Several independent transgenic lines were selected after confirming single gene insertion by southern blot analysis in the T4 generation. Northern blots revealed the presence of heterologous transcript. Radiolabeling experiments revealed an increased rate of incorporation of the non-metabolizable analog 3-O-methyl-[U-14C]-glucose. This confirmed that the yeast Hxt2 transporter was functional in Arabidopsis. No phenotypic changes at the vegetative and reproductive stages could be detected in the transgenic lines when compared to wild type plants. Shortly after germination some differences in development and glucose signaling were observed. Transgenic seedlings cultivated in liquid medium or on solid agar plates were able to grow with 3% glucose (producing bigger plants and longer roots), while development of wild type plants was delayed under those conditions. Metabolite analysis revealed that the Hxt2 transgenic lines had higher rates of sugar utilization. Transcriptional profiling showed that particular genes were significantly up- or down-regulated. Some transcription factors like At1g27000 were repressed, while others, such as At3g58780, were induced. The mRNA from classical sugar signaling genes such as STP1, Hxk1, and ApL3 behaved similarly in transgenic lines and wild type lines. Results suggest that the Hxt2 transgene altered some developmental processes related to the perception of high carbon availability after the germination stage. We conclude that the developmental arrest of wild type plants at 3% glucose not only depends on Hxk1 as the only sugar sensor but might also be influenced by the route of hexose transport across the plasma membrane.

Discovery and characterization of novel d-xylose-specific transporters from Neurospora crassa and Pichia stipitis

Saccharomyces cerevisiae is considered one of the most promising organisms for ethanol production from lignocellulosic feedstock. Unfortunately, pentose sugars, which comprise up to 30% of lignocellulose, cannot be utilized by wild type S. cerevisiae. Heterologous pathways were introduced into S. cerevisiae to enable utilization of d-xylose, the most abundant pentose sugar. However, the resulting recombinant S. cerevisiae strains exhibited a slow growth rate and poor sugar utilization efficiency when grown on d-xylose as the sole carbon source. d-xylose uptake is the first step of d-xylose utilization. d-xylose can only enter yeast cells through hexose transporters, which have two orders of magnitude lower affinity towards d-xylose compared to hexoses. It was also shown that inefficient pentose uptake is the limiting step in some d-xylose metabolizing yeast strains. Here we report the cloning and characterization of two novel d-xylose-specific transporters from Neurospora crassa and Pichia stipitis. These two transporters were identified from a total of 18 putative pentose transporters. They were functionally expressed and properly localized in S. cerevisiae as indicated by HPLC analysis and fluorescence confocal microscopy, respectively. Kinetic parameters of the d-xylose-specific transporters were determined using a (14)C-labeled sugar uptake assay. Use of pentose-specific transporters should improve d-xylose consumption and ethanol production in fast d-xylose assimilating strains, thereby lowering the cost of lignocellulosic ethanol production.

Functional Analysis of an Arabidopsis thaliana Abiotic Stress-inducible Facilitated Diffusion Transporter for Monosaccharides

Sugars play indispensable roles in biological reactions and are distributed into various tissues or organelles via transporters in plants. Under abiotic stress conditions, plants accumulate sugars as a means to increase stress tolerance. Here, we report an abiotic stress-inducible transporter for monosaccharides from Arabidopsis thaliana that is termed ESL1 (ERD six-like 1). Expression of ESL1 was induced under drought and high salinity conditions and with exogenous application of abscisic acid. Promoter analyses using beta-glucuronidase and green fluorescent protein reporters revealed that ESL1 is mainly expressed in pericycle and xylem parenchyma cells. The fluorescence of ESL1-green fluorescent protein-fused protein was detected at tonoplast in transgenic Arabidopsis plants and tobacco BY-2 cells. Furthermore, alanine-scanning mutagenesis revealed that an N-terminal LXXXLL motif in ESL1 was essential for its localization at the tonoplast. Transgenic BY-2 cells expressing mutated ESL1, which was localized at the plasma membrane, showed an uptake ability for monosaccharides. Moreover, the value of K(m) for glucose uptake activity of mutated ESL1 in the transgenic BY-2 cells was extraordinarily high, and the transport activity was independent from a proton gradient. These results indicate that ESL1 is a low affinity facilitated diffusion transporter. Finally, we detected that vacuolar invertase activity was increased under abiotic stress conditions, and the expression patterns of vacuolar invertase genes were similar to that of ESL1. Under abiotic stress conditions, ESL1 might function coordinately with the vacuolar invertase to regulate osmotic pressure by affecting the accumulation of sugar in plant cells.

Amino acid residues involved in ligand preference of the Snf3 transporter‐like sensor in Saccharomyces cerevisiae

Snf3 is a plasma membrane protein in Saccharomyces cerevisiae able to sense the presence of glucose. Although the Snf3 protein does not transport sugars, it shares sequence similarity with various glucose transporters from other organisms. We investigated the sugar specificity/preferences of Snf3. The ability of cells to sense sugars in vivo was monitored by following the degradation of the Mth1 protein, an early event in the signal pathway. Our study reveals that Snf3, in addition to glucose, also senses fructose and mannose, as well as the glucose analogues 2-deoxyglucose, 3-O-methylglucoside and 6-deoxyglucose. The signalling proficiency of a non-phosphorylatable analogue strongly supports the notion that sensing through Snf3 does not require sugar phosphorylation. Sequence comparisons of Snf3 to glucose transporters indicated amino acid residues possibly involved in sensing of sugars other than glucose. By site-specific mutagenesis of the structural gene, roles of specific residues in Snf3 could be established. Change of isoleucine-374 to valine in transmembrane segment 7 of Snf3 partially abolished sensing of fructose and mannose, while mutagenesis causing a change of phenylalanine-462 to tyrosine in transmembrane segment 10 of Snf3 abolished sensing of fructose. Neither of these amino acid changes affected the ability of Snf3 to sense glucose, nor did they permit Snf3 to sense galactose. These data indicate a similarity between a ligand binding site of the sensor Snf3 and binding sites used for facilitated hexose transport in the GLUT proteins.

332 GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) ENHANCES CUMULUS CELLS EXPANSION OF IN VITRO-MATURED BOVINE CUMULUS - OOCYTE COMPLEXES IN A CHEMICALLY DEFINED MEDIUM

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine that stimulates proliferation, differentiation and function in different cells types. We have previously demonstrated (Bücher DD et al. 2008 Reprod. Dom. Anim. 43 (Suppl. 3), 146 abst.) that both subunits of GM-CSF receptor are expressed in granulosa cells from antral follicles in bovine ovaries. Also, we determined that the cytokine enhances glucose uptake through facilitative hexose transporters in granulosa cells in primary culture. The goals of the present study were to characterize the expression of GM-CSF receptor in cumulus cells and oocytes from bovine antral follicles and to determine its effects on in vitro-matured bovine COCs in a chemically defined medium. To determine the presence of a and |5 subunits of GM-CSF receptor, COCs were aspirated from follicles <8 mm in diameter, fixed, and submitted to immunocytochemistry. To study the effect of GM-CSF on in vitro maturation of oocytes, COCs (n =481) were cultured using serum-free medium (SOF) containing 0, 1, 10, and 100 ng mL-1 of human recombinant GM-CSF (R&D Systems, Inc., Minneapolis, MN, USA) for 22 h at 39°C, 5% CO2 in humidified air. Nuclear stage, cumulus expansion, cumulus cell number, and viability were analyzed after in vitro maturation. Cumulus expansion was assessed using the cumulus expansion index (CEI) (Fagbohun C and Down S 1990 Biol. Reprod. 42, 413-423). Nuclear stage was evaluated using aceto-orcein stain. To determine cumulus cell viability and number, COCs (n = 10-12 per group) were transferred into an Eppendorf tube and cumulus cells were removed by vortexing for 3 min, stained with trypan blue and counted with a hemocytometer. The study was conducted in 6 replicates. Data from cumulus expansion and cell number were analyzed by Kruskal-Wallis analysis. Data for nuclear stage and cell viability were analyzed by chi-square analysis and one way ANOVA, respectively. Both receptor subunits were present in cumulus cells and oocytes from COCs. COCs cultured in 10 and 100 ng mL-1 GM-CSF had CEI scores (0.8 and 1.22, respectively) greater (P < 0.01) than controls (0.2), but the proportion of COCs displaying second metaphase did not differ (P = 0.5) among treatment groups. GM-CSF at a concentration of 100 ng mL-1 increased (P < 0.01) cumulus cell viability by more than 20% compared to the control group. Similarly, GM-CSF at concentrations of 10 and 100 ng mL-1 increased (P < 0.05) cumulus cell number by more than 20% and 45%, respectively, from the control group. The use of a specific inhibitor of PI3 kinase (Ly294002; 10 and 100 μM) blocked the stimulatory effect of GM-CSF on cumulus expansion, cell viability, and cell number. In conclusion, the results of the study suggest a plausible modulator role of GM-CSF in the metabolism and function of cumulus cells and oocytes during in vitro maturation. Funding from Faculty of Veterinary Sciences, Universidad Austral de Chile, MECESUP AUS-0005, AUS-0601, and DID D-2006-24 and from Universidad Católica de Temuco, research grant 2007 DGI-CDA-04.

Tomato yellow leaf curl virus infection of a resistant tomato line with a silenced sucrose transporter gene LeHT1 results in inhibition of growth, enhanced virus spread, and necrosis

To identify genes involved in resistance of tomato to Tomato yellow leaf curl virus (TYLCV), cDNA libraries from lines resistant (R) and susceptible (S) to the virus were compared. The hexose transporter LeHT1 was found to be expressed preferentially in R tomato plants. The role of LeHT1 in the establishment of TYLCV resistance was studied in R plants where LeHT1 has been silenced using Tobacco rattle virus-induced gene silencing (TRV VIGS). Following TYLCV inoculation, LeHT1-silenced R plants showed inhibition of growth and enhanced virus accumulation and spread. In addition, a necrotic response was observed along the stem and petioles of infected LeHT1-silenced R plants, but not on infected not-silenced R plants. This response was specific of R plants since it was absent in infected LeHT1-silenced S plants. Necrosis had several characteristics of programmed cell death (PCD): DNA from necrotic tissues presented a PCD-characteristic ladder pattern, the amount of a JNK analogue increased, and production of reactive oxygen was identified by DAB staining. A similar necrotic reaction along stem and petioles was observed in LeHT1-silenced R plants infected with the DNA virus Bean dwarf mosaic virus and the RNA viruses Cucumber mosaic virus and Tobacco mosaic virus. These results constitute the first evidence for a necrotic response backing natural resistance to TYLCV in tomato, confirming that plant defense is organized in multiple layers. They demonstrate that the hexose transporter LeHT1 is essential for the expression of natural resistance against TYLCV and its expression correlates with inhibition of virus replication and movement.

A Sugar-Inducible Protein Kinase, VvSK1, Regulates Hexose Transport and Sugar Accumulation in Grapevine Cells

In grapevine (Vitis vinifera), as in many crops, soluble sugar content is a major component of yield and economical value. This paper identifies and characterizes a Glycogen Synthase Kinase3 protein kinase, cloned from a cDNA library of grape Cabernet Sauvignon berries harvested at the ripening stage. This gene, called VvSK1, was mainly expressed in flowers, berries, and roots. In the berries, it was strongly expressed at postvéraison, when the berries accumulate glucose, fructose, and abscisic acid. In grapevine cell suspensions, VvSK1 transcript abundance is increased by sugars and abscisic acid. In transgenic grapevine cells overexpressing VvSK1, the expression of four monosaccharide transporters (VvHT3, VvHT4, VvHT5, and VvHT6) was up-regulated, the rate of glucose uptake was increased 3- to 5-fold, and the amount of glucose and sucrose accumulation was more than doubled, while the starch amount was not affected. This work provides, to our knowledge, the first example of the control of sugar uptake and accumulation by a sugar-inducible protein kinase.

Detection and Localization of GLUTs 1, 2, 3 and 5 in Donkey Spermatozoa

GLUTs are a family of proteins that facilitate the transport of glucose and other hexoses through the plasma membrane of the cells. GLUTs are present in mammalian spermatozoon's membrane in different isoforms and they supply metabolic substrates for all the cell's activities such as motility, homoeostasis and fertilization. As studies about donkey spermatozoa and their metabolism are lacking, this study was aimed at detecting GLUTs 1, 2, 3 and 5 presence by western blotting technique and at determining their localization on the plasma membrane by indirect immunofluorescence. Each protein showed a typical localization on the sperm cells' plasma membrane, differencing the one to the other on the basis of the hexose they transport. We also highlighted some differences between GLUTs distribution and molecular weight in donkey spermatozoa and its nearest relative, the horse.

Transcriptomic profiling of the Saccharomyces cerevisiae response to quinine reveals a glucose limitation response attributable to drug-induced inhibition of glucose uptake.

Quinine has been employed in the treatment of malaria for centuries and is still used against severe Plasmodium falciparum malaria. However, its interactions with the parasite remain poorly understood and subject to debate. In this study, we used the Saccharomyces cerevisiae eukaryotic model to better understand quinine's mode of action and the mechanisms underlying the cell response to the drug. We obtained a transcriptomic profile of the yeast's early response to quinine, evidencing a marked activation of genes involved in the low-glucose response (e.g., CAT8, ADR1, MAL33, MTH1, and SNF3). We used a low inhibitory quinine concentration with no detectable effect on plasma membrane function, consistent with the absence of a general nutrient starvation response and suggesting that quinine-induced glucose limitation is a specific response. We have further shown that transport of [(14)C]glucose is inhibited by quinine, with kinetic data indicating competitive inhibition. Also, tested mutant strains deleted for genes encoding high- and low-affinity hexose transporters (HXT1 to HXT5, HXT8, and HXT10) exhibit resistance phenotypes, correlating with reduced levels of quinine accumulation in the mutants examined. These results suggest that the hexose transporters are facilitators of quinine uptake in S. cerevisiae, possibly through a competitive inhibition mechanism. Interestingly, P. falciparum is highly dependent on glucose uptake, which is mediated by the single-copy transporter PfHT1, a protein with high homology to yeast's hexose transporters. We propose that PfHT1 is an interesting candidate quinine target possibly involved in quinine import in P. falciparum, an uptake mechanism postulated in recent studies to occur through a still-unidentified importer(s).

Asymmetric Signal Transduction through Paralogs That Comprise a Genetic Switch for Sugar Sensing in Saccharomyces cerevisiae

Efficient uptake of glucose is especially critical to Saccharomyces cerevisiae because its preference to ferment this carbon source demands high flux through glycolysis. Glucose induces expression of HXT genes encoding hexose transporters through a signal generated by the Snf3 and Rgt2 glucose sensors that leads to depletion of the transcriptional regulators Mth1 and Std1. These paralogous proteins bind to Rgt1 and enable it to repress expression of HXT genes. Here we show that Mth1 and Std1 can substitute for one another and provide nearly normal regulation of their targets. However, their roles in the glucose signal transduction cascade have diverged significantly. Mth1 is the prominent effector of Rgt1 function because it is the more abundant of the two paralogs under conditions in which both are active (in the absence of glucose). Moreover, the cellular level of Mth1 is quite sensitive to the amount of available glucose. The abundance of Std1 protein, on the other hand, remains essentially constant over a similar range of glucose concentrations. The signal generated by low levels of glucose is amplified by rapid depletion of Mth1; the velocity of this depletion is dependent on both its rate of degradation and swift repression of MTH1 transcription by the Snf1-Mig1 glucose repression pathway. Quantitation of the contributions of Mth1 and Std1 to regulation of HXT expression reveals the unique roles played by each paralog in integrating nutrient availability with metabolic capacity: Mth1 is the primary regulator; Std1 serves to buffer the response to glucose.

Solute carrier family 2, member 9 and uric acid homeostasis

The goal of this article is to review the possible physiological roles of the recently identified urate transporter, solute carrier family 2 (facilitated glucose transporter), member 9 (SLC2A9), in the renal handling of urate. Glucose transporter 9 is a high affinity hexose transporter encoded by the SLC2A9 gene found on human chromosome 4. The two splice variants SLC2A9b and SLC2A9a are expressed in the apical and basolateral membranes, respectively, of the proximal convoluted tubule. Recent reports have found significant correlations between two different sets of single nucleotide polymorphisms in SLC2A9. In one case, they are associated with increases in plasma urate levels and/or the incidence of hypertension or gout. The second set of single nucleotide polymorphisms correlate with hypouricaemia in Japanese patients. Expression of SLC2A9a and b in Xenopus laevis oocytes shows that these proteins mediate rapid urate fluxes and can exchange glucose for urate. Indirect evidence also suggests that the transporter is electrogenic. This review proposes that SLC2A9 contributes significantly in two ways to the fluxes of urate across the proximal convoluted tubule. Firstly, the apical expression of SLC2A9b secretes urate back into the urine in exchange for lumenal glucose. Secondly, the basolateral membrane SLC2A9a could be the primary route for urate movement out of the epithelium into the peritubular space.

Dynamic carbon transfer during pathogenesis of sunflower by the necrotrophic fungus Botrytis cinerea: from plant hexoses to mannitol

The main steps for carbon acquisition and conversion by Botrytis cinerea during pathogenesis of sunflower cotyledon were investigated here. A sequential view of soluble carbon metabolites detected by NMR spectroscopy during infection is presented. Disappearance of plant hexoses and their conversion to fungal metabolites were investigated by expression analysis of an extended gene family of hexose transporters (Bchxts) and of the mannitol pathway, using quantitative PCR. In order to analyse the main fungal metabolic routes used by B. cinerea in real time, we performed, for the first time, in vivo NMR analyses during plant infection. During infection, B. cinerea converts plant hexoses into mannitol. Expression analysis of the sugar porter gene family suggested predominance for transcription induced upon low glucose conditions and regulated according to the developmental phase. Allocation of plant hexoses by the pathogen revealed a conversion to mannitol, trehalose and glycogen for glucose and a preponderant transformation of fructose to mannitol by a more efficient metabolic pathway. Uptake of plant hexoses by B. cinerea is based on a multigenic flexible hexose uptake system. Their conversion into mannitol, enabled by two simultaneously expressed pathways, generates a dynamic intracellular carbon pool.

Host-derived glucose and its transporter in the obligate intracellular pathogen Toxoplasma gondii are dispensable by glutaminolysis

Toxoplasma gondii, as an obligate intracellular and promiscuous pathogen of mammalian cells, utilizes host sugars for energy and to generate glycoconjugates that are important to its survival and virulence. Here, we report that T. gondii glucose transporter (TgGT1) is proficient in transporting mannose, galactose, and fructose besides glucose, and serves as a major hexose transporter at its plasma membrane. Toxoplasma harbors 3 additional putative sugar transporters (TgST1-3), of which TgST2 is expressed at its surface, whereas TgST1 and TgST3 are intracellular. Surprisingly, TgGT1 and TgST2 are nonessential to the parasite as their ablations inflict only a 30% or no defect in its intracellular growth, respectively. Indeed, Toxoplasma can also tolerate the deletion of both genes while incurring no further growth phenotype. Unlike Deltatgst2, the modest impairment in Deltatggt1 and Deltatggt1/Deltatgst2 mutants is because of a minor delay in their intracellular replication, which is a direct consequence of the abolished import of glucose. The Deltatggt1 displays an attenuated motility in defined minimal media that is rescued by glutamine. TgGT1-complemented parasites show an entirely restored growth, motility, and sugar import. The lack of exogenous glucose in Deltatggt1 culture fails to accentuate its intrinsic growth defect and prompts it to procure glutamine to sustain its metabolism. Unexpectedly, in vivo virulence of Deltatggt1 in mice remains unaffected. Taken together, our data demonstrate that glucose is nonessential for T. gondii tachyzoites, underscore glutamine is a complement substrate, and provide a basis for understanding the adaptation of T. gondii to diverse host cells.

Comparison of effects of green tea catechins on apicomplexan hexose transporters and mammalian orthologues

Here we have investigated the inhibitory properties of green tea catechins on the Plasmodium falciparum hexose transporter (PfHT), the Babesia bovis hexose transporter 1 (BboHT1) and the mammalian facilitative glucose transporters, GLUT1 and GLUT5, expressed in Xenopus laevis oocytes. (−)-Epicatechin-gallate (ECG) and (−)-epigallocatechin-gallate (EGCG) inhibited d-glucose transport by GLUT1 and PfHT, and d-fructose transport by GLUT5, with apparent Ki values between 45 and 117μM. BboHT1 was more potently inhibited by the ungallated catechins (−)-epicatechin (EC) and (−)-epigallocatechin (EGC), with apparent Ki values of 108 and 168μM, respectively. Site-directed mutagenesis experiments provided little further support for previously reported models of catechin binding to hexose transporters. Furthermore, P. falciparum growth inhibition by catechins was not affected by the external d-glucose concentration. Our results provide new data on the inhibitory action of catechins against sugar transporters but were unable to elucidate the antimalarial mechanism of action of these agents.

Increased hexose transport in the roots of tomato plants submitted to prolonged hypoxia

We investigated the effects of prolonged hypoxia on the sugar uptake in tomato (Solanum lycopersicum L. var. MP-1) roots. Hydroponic cultures of whole tomato plants were submitted to hypoxic treatment for 1 week, and the roots were analyzed for sugar concentrations, hexose uptake and hexose transporter expression level. Contrary to what has been observed after anoxic shock or short-term hypoxic treatment, we show that sugar concentrations increase and hexose uptake is up-regulated in the roots after 1 week of hypoxic treatment. Increased hexose transport is concomitant with the induction of the hexose transporter gene LeHT2. These responses may be due either to a direct effect of low O(2) supply, or to a secondary effect associated with the increase in sugar concentrations, which, typically, develops in most hypoxic plants.