Both insect and mammalian life cycle stages of Leishmania mexicana take up glucose and express all three isoforms encoded by the LmGT glucose transporter gene family. To evaluate glucose transporter function in intact parasites, a null mutant line has been created by targeted disruption of the LmGT locus that encompasses the LmGT1, LmGT2, and LmGT3 genes. This Δlmgt null mutant exhibited no detectable glucose transport activity. The growth rate of the Δlmgt knockout in the promastigote stage was reduced to a rate comparable with that of WT cells grown in the absence of glucose. Δlmgt cells also exhibited dramatically reduced infectivity to macrophages, demonstrating that expression of LmGT isoforms is essential for viability of amastigotes. Furthermore, WT L. mexicana were not able to grow as axenic culture form amastigotes if glucose was withdrawn from the medium, implying that glucose is an essential nutrient in this life cycle stage. Expression of either LmGT2 or LmGT3, but not of LmGT1, in Δlmgt null mutants significantly restored growth as promastigotes, but only LmGT3 expression substantially rescued amastigote growth in macrophages. Subcellular localization of the three isoforms was investigated in Δlmgt cells expressing individual LmGT isoforms. Using anti-LmGT antiserum and GFP-tagged LmGT fusion proteins, LmGT2 and LmGT3 were localized to the cell body, whereas LmGT1 was localized specifically to the flagellum. These results establish that each glucose transporter isoform has distinct biological functions in the parasite
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 ⌬tgst2, the modest impairment in ⌬tggt1 and ⌬tggt1/⌬tgst2 mutants is because of a minor delay in their intracellular replication, which is a direct consequence of the abolished import of glucose. The ⌬tggt1 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 ⌬tggt1 culture fails to accentuate its intrinsic growth defect and prompts it to procure glutamine to sustain its metabolism. Unexpectedly, in vivo virulence of ⌬tggt1 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.glucose transport ͉ glutamine metabolism ͉ genetic manipulation
All parasitic protozoa studied to date are incapable of purine biosynthesis and must therefore salvage purine nucleobases or nucleosides from their hosts. This salvage process is initiated by purine transporters on the parasite cell surface. We have used a mutant line (TUBA5) of Leishmania donovani that is deficient in adenosine͞pyrimi-dine nucleoside transport activity (LdNT1) to clone genes encoding these nucleoside transporters by functional rescue. Parasitic protozoa of the genus Leishmania are the etiological agents of leishmaniasis, a disease that affects an estimated 12 million people worldwide (1) and ranges from the disfiguring cutaneous form to fatal visceral leishmaniasis (2). Because current empirically identified drugs suffer from many deficiencies, including toxicity and resistance, it is important to identify unique biochemical targets that could be exploited for rational development of improved therapies. Perhaps the most striking metabolic discrepancy between parasites and their hosts is the purine pathway. Whereas most mammalian cells synthesize purines de novo, all parasitic protozoa studied to date are unable to synthesize purines (3) and consequently must rely on purine acquisition from their hosts for survival and growth. The first step in this salvage pathway involves the transport of these substrates across the parasite plasma membrane. Moreover, these purine transporters initiate the uptake of certain pyrazolopyrimidine analogs of hypoxanthine and inosine that are toxic to both Leishmania and Trypanosoma (4). These pyrazolopyrimidines, such as allopurinol, allopurinol riboside, and formycin B, are subsequently metabolized to the nucleotide level by the parasite metabolic machinery and incorporated into RNA, metabolic transformations that do not occur in mammalian cells (4). Both the essential nutritional function of these transporters and their roles in mediating the toxicities of well-characterized antiparasitic agents provide compelling rationale to study these membrane permeases at the molecular level.Biochemical and genetic studies have established that Leishmania donovani parasites express two distinct nucleoside transporters with nonoverlapping substrate specificities (5). One transporter mediates the uptake of adenosine and pyrimidine nucleosides and also transports tubercidin, a cytotoxic analog of adenosine, whereas the other transporter allows membrane permeation of guanosine, inosine, and formycin B (5). Parasites deficient in either or both transport activities have been isolated by mutagenesis with N-methyl-NЈ-nitro-N-nitrosoguanidine followed by selection in tubercidin or formycin B (6). The availability of these null mutants provided a functional strategy for cloning genes encoding each of these nucleoside permeases.In the present study, we have transfected the adenosine͞pyrimidine nucleoside transport-deficient TUBA5 cell line with a cosmid library containing inserts of L. donovani genomic DNA (7) and screened individual transfectants for restoration of tubercidin...
PURINE TRANSPORT AND SALVAGE IN PARASITIC PROTOZOAOne distinctive feature of the biochemistry of parasitic protozoa is their absolute reliance upon the salvage of preformed purines from their vertebrate and invertebrate hosts. While many mammalian cells possess the innate ability to synthesize purines de novo, all protozoa so far examined that exhibit a parasitic life style lack these biosynthetic pathways and have therefore elaborated a variety of salvage pathways ( Fig. 1) that enable them to acquire preformed purines from their hosts (14). These nutrients are imported from the host as either nucleosides or nucleobases, either of which can serve as a purine source for the parasite. The first step in these salvage pathways entails the uptake of the purine nucleosides or nucleobases from the host milieu and is mediated by various nucleoside or nucleobase transporters, located in the plasma membrane of the parasite, that provide substrate-specific permeation routes. While the enzymes of purine salvage have been studied in considerable molecular detail (7), the identification of individual purine transporters at the molecular level is a more recent development. However, over the past several years a plethora of genes encoding nucleoside or nucleobase permeases from parasitic protozoa have been identified and functionally expressed. The increasing understanding of these carriers at the molecular level provides an appropriate setting for a timely review of these important transporters and a comparison of their properties to related and distinct purine permeases of other organisms. This article will concentrate primarily on purine transporters from the Kinetoplastid parasites Leishmania and Trypanosoma brucei, since these are the protozoa with which the majority of the studies have been accomplished. However, we will also briefly discuss related work on transporters from the Apicomplexa Plasmodium falciparum and Toxoplasma gondii. This review does not attempt to be exhaustive but focuses rather upon recent results utilizing molecular genetic approaches.A principal reason for the interest in purine transport in these protozoa is the essential nature of purine salvage for this large family of parasites. Thus, while some permeases may provide nutrients that are nonessential, albeit advantageous, to the fitness of the organism in its natural environment, the purine transporters as a group are likely to be required for parasite viability in all life cycle stages. A second compelling reason to study these transporters is that they also mediate the uptake of a variety of cytotoxic drugs, many, but not all, of which are purine homologs (11,13,49). Consequently, the import of subversive substrates that are metabolized, often uniquely, by parasite purine salvage enzymes is absolutely dependent upon the purine permeases. Thus, this family of permeases plays an important role in the delivery of drugs or experimental therapeutic compounds, and mutations in these carriers can cause drug resistance (5, 13, 36). STUDIES OF PURINE TR...
Purine transport is an indispensable nutritional function for protozoan parasites, since they are incapable of purine biosynthesis and must, therefore, acquire purines from the host milieu. Exploiting a mutant cell line (FBD5) of Leishmania donovani deficient in inosine and guanosine transport activity, the gene encoding this transporter (LdNT2) has been cloned by functional rescue of the mutant phenotype. LdNT2 encodes a polypeptide of 499 amino acids that shows substantial homology to other members of the equilibrative nucleoside transporter family. Molecular analysis revealed that LdNT2 is present as a single gene copy within the leishmanial genome and encodes a single transcript of 3 kilobase pairs. Transfection of FBD5 parasites with LdNT2 reestablished their ability to take up inosine and guanosine with a concurrent restoration of sensitivity to the inosine analog formycin B. Kinetic analyses reveal that LdNT2 is highly specific for inosine (K m ؍ 0.3 M) and guanosine (K m ؍ 1.7 M) and does not recognize other naturally occurring nucleosides. Expression of LdNT2 cRNA in Xenopus oocytes significantly augmented their ability to take up inosine and guanosine, establishing that LdNT2 by itself suffices to mediate nucleoside transport. These results authenticate genetically and biochemically that LdNT2 is a novel nucleoside transporter with an unusual and strict specificity for inosine and guanosine.Leishmania donovani is a protozoan parasite and the etiologic agent of visceral leishmaniasis, a devastating and invariably fatal disease if untreated. The parasite exhibits an intricate life cycle in which the extracellular, flagellated promastigote exists in the phlebotomine sandfly vector, and the intracellular amastigote resides in the phagolysosome of macrophages and other reticuloendothelial cells of the mammalian host. Drugs are the only defense against visceral leishmaniasis, but the efficacy of these empirically derived agents is compromised both by drug toxicity and resistance (1). Thus, it is increasingly imperative to identify new and unique biochemical targets in the parasite for potential therapeutic exploitation.Among the most conspicuous metabolic differences between parasites and their mammalian hosts is the purine pathway. Whereas animal cells synthesize purine nucleotides de novo, all protozoan parasites are incapable of synthesizing purines and depend upon purine acquisition from their hosts to survive and proliferate (2). Hence, each genus of parasite has evolved a unique complement of purine salvage enzymes in order to scavenge purines from the host milieu (2). The first step in this salvage process involves the translocation of purines across the parasite plasma membrane, a process mediated by membrane permeases. These permeases also initiate the uptake of pyrazolopyrimidine nucleobase and nucleoside analogs of hypoxanthine and inosine that are selectively toxic to Leishmania spp. (3, 4). Thus, purine transporters play vital roles in both purine nutrition and antiparasitic drug targeting i...
Arsenic exposure is associated with hypertension, diabetes and cancer. Some mammals methylate arsenic. Saccharomyces cerevisiae hexose permeases catalyze As(OH) 3 uptake. Here we report that mammalian glucose transporter GLUT1 catalyzes As(OH) 3 and CH 3 As(OH) 2 uptake in yeast or in Xenopus laevis öocytes. Expression of GLUT1 in a yeast lacking other glucose transporters allows for growth on glucose. Yeast expressing yeast HXT1 or rat GLUT1 transport As(OH) 3 and CH 3 As (OH) 2 . The K m of GLUT1 is to 1.2 mM for CH 3 As(OH) 2 , compared to a K m of 3 mM for glucose. Inhibition between glucose and CH 3 As(OH) 2 is noncompetitive, suggesting differences between the translocation pathways of hexoses and arsenicals. Both human and rat GLUT1 catalyze uptake of both As(OH) 3 and CH 3 As(OH) 2 in öocytes. Thus GLUT1 may be a major pathway uptake of both inorganic and methylated arsenicals in erythrocytes or the epithelial cells of the blood-brain barrier, contributing to arsenic-related cardiovascular problems and neurotoxicity.Arsenic ranks first on the United States Government's Comprehensive Environmental Response, Compensation, and Liability (Superfund) Act Priority List of Hazardous Substances
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.