Marco A. Sanchez

Nucleoside and Nucleobase Transporters in Parasitic Protozoa

One 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

Cloning of a novel inosine-guanosine transporter gene from Leishmania donovani by functional rescue of a transport-deficient mutant.

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 donovanideficient 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 LdNT2re-established 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 μm) and guanosine (K m = 1.7 μm) and does not recognize other naturally occurring nucleosides. Expression ofLdNT2 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.

Arsenic transport by zebrafish aquaglyceroporins

BackgroundArsenic is one of the most ubiquitous toxins and endangers the health of tens of millions of humans worldwide. It is a mainly a water-borne contaminant. Inorganic trivalent arsenic (AsIII) is one of the major species that exists environmentally. The transport of AsIII has been studied in microbes, plants and mammals. Members of the aquaglyceroporin family have been shown to actively conduct AsIII and its organic metabolite, monomethylarsenite (MAsIII). However, the transport of AsIII and MAsIII in in any fish species has not been characterized.ResultsIn this study, five members of the aquaglyceroporin family from zebrafish (Danio rerio) were cloned, and their ability to transport water, glycerol, and trivalent arsenicals (AsIII and MAsIII) and antimonite (SbIII) was investigated. Genes for at least seven aquaglyceroporins have been annotated in the zebrafish genome project. Here, five genes which are close homologues to human AQP3, AQP9 and AQP10 were cloned from a zebrafish cDNA preparation. These genes were named aqp3, aqp3l, aqp9a, aqp9b and aqp10 according to their similarities to the corresponding human AQPs. Expression of aqp9a, aqp9b, aqp3, aqp3l and aqp10 in multiple zebrafish organs were examined by RT-PCR. Our results demonstrated that these aquaglyceroporins exhibited different tissue expression. They are all detected in more than one tissue. The ability of these five aquaglyceroporins to transport water, glycerol and the metalloids arsenic and antimony was examined following expression in oocytes from Xenopus leavis. Each of these channels showed substantial glycerol transport at equivalent rates. These aquaglyceroporins also facilitate uptake of inorganic AsIII, MAsIII and SbIII. Arsenic accumulation in fish larvae and in different tissues from adult zebrafish was studied following short-term arsenic exposure. The results showed that liver is the major organ of arsenic accumulation; other tissues such as gill, eye, heart, intestine muscle and skin also exhibited significant ability to accumulate arsenic. The zebrafish larvae also accumulate considerable amounts of arsenic.ConclusionThis is the first molecular identification of fish arsenite transport systems and we propose that the extensive expression of the fish aquaglyceroporins and their ability to transport metalloids suggests that aquaglyceroporins are the major pathways for arsenic accumulation in a variety of zebrafish tissues. Uptake is one important step of arsenic metabolism. Our results will contribute to a new understanding of aquatic arsenic metabolism and will support the use of zebrafish as a new model system to study arsenic associated human diseases.

Cloning and Functional Expression of a Gene Encoding a P1 Type Nucleoside Transporter from Trypanosoma brucei

Nucleoside transporters are likely to play a central role in the biochemistry of the parasite Trypanosoma brucei, since these protozoa are unable to synthesize purinesde novo and must salvage them from their hosts. Furthermore, nucleoside transporters have been implicated in the uptake of antiparasitic and experimental drugs in these and other parasites. We have cloned the gene for a T. brucei nucleoside transporter, TbNT2, and shown that this permease is related in sequence to mammalian equilibrative nucleoside transporters. Expression of theTbNT2 gene in Xenopus oocytes reveals that the permease transports adenosine, inosine, and guanosine and hence has the substrate specificity of the P1 type nucleoside transporters that have been previously characterized by uptake assays in intact parasites.TbNT2 mRNA is expressed in bloodstream form (mammalian host stage) parasites but not in procyclic form (insect stage) parasites, indicating that the gene is developmentally regulated during the parasite life cycle. Genomic Southern blots suggest that there are multiple genes related in sequence to TbNT2, implying the existence of a family of nucleoside transporter genes in these parasites.

Functional expression and characterization of a purine nucleobase transporter gene from Leishmania major

Leishmania major, like all the other kinetoplastid protozoa, are unable to synthesize purines and rely on purine nucleobase and nucleoside acquisition across the parasite plasma membrane by specific permeases. Although, several genes have been cloned that encode nucleoside transporters in Leishmania and Trypanosoma brucei, much less progress has been made on nucleobase transporters, especially at the molecular level. The studies reported here have cloned and expressed the first gene for a L. major nucleobase transporter, designated LmaNT3. The LmaNT3 permease shows 33% identity to L. donovani nucleoside transporter 1.1 (LdNT1.1) and is, thus, a member of the equilibrative nucleoside transporter (ENT) family. ENT family members identified to date are nucleoside transporters, some of which also transport one or several nucleobases. Functional expression studies in Xenopus laevis oocytes revealed that LmaNT3 mediates high levels of uptake of hypoxanthine, xanthine, adenine and guanine. Moreover, LmaNT3 is an high affinity transporter with Km values for hypoxanthine, xanthine, adenine and guanine of 16.5±1.5, 8.5±0.6, 8.5±1.1 and 8.8±4.0 μM, respectively. LmaNT3 is, thus, the first member of the ENT family identified in any organism that functions as a nucleobase rather than nucleoside or nucleoside/nucleobase transporter.

Molecular genetic analysis of purine nucleobase transport in Leishmania major.

Leishmania major and all other parasitic protozoa are unable to synthesize purines de novo and are therefore reliant upon uptake of preformed purines from their hosts via nucleobase and nucleoside transporters. L. major expresses two nucleobase permeases, NT3 that is a high affinity transporter for purine nucleobases and NT4 that is a low affinity transporter for adenine. nt3(–/–) null mutant promastigotes were unable to replicate in medium containing 10 μM hypoxanthine, guanine, or xanthine and replicated slowly in 10 μM adenine due to residual low affinity uptake of that purine. The NT3 transporter mediated the uptake of the anti‐leishmanial drug allopurinol, and the nt3(–/–) mutants were resistant to killing by this drug. Expression of the NT3 permease was profoundly downregulated at the protein but not the mRNA level in stationary phase compared with logarithmic phase promastigotes. The nt4(–/–) null mutant was quantitatively impaired in survival within murine bone marrow‐derived macrophages. Extensive efforts to generate an nt3(–/–)/nt4(–/–) dual null mutant were not successful, suggesting that one of the two nucleobase permeases must be retained for robust growth of the parasite. The phenotypes of these null mutants underscore the importance of purine nucleobase transporters in the Leishmania life cycle and pharmacology.

Molecular and functional characterization of the first nucleobase transporter gene from African trypanosomes.

African trypanosomes are unable to synthesize purines and depend upon purine nucleoside and nucleobase transporters to salvage these compounds from their hosts. To understand the crucial role of purine salvage in the survival of these parasites, a central objective is to identify and characterize all of the purine permeases that mediate uptake of these essential nutrients. We have cloned and functionally expressed in a purine nucleobase transport deficient strain of Saccharomyces cerevisiae a novel nucleobase transporter gene, TbNT8.1, from Trypanosoma brucei. The permease encoded by this gene mediates the uptake of hypoxanthine, adenine, guanine, and xanthine with Kms in the low micromolar range. The TbNT8.1 protein is a member of the equilibrative nucleoside transporter (ENT) family of permeases that occur in organisms as diverse as protozoa and mammals. TbNT8.1 is distinct from other ENT permeases that have been identified in trypanosomes in utilizing multiple purine nucleobases, rather than purine nucleosides, as substrates and is hence the first bona fide nucleobase permease identified in these parasites. Furthermore, unlike the mRNAs for other purine transporters, TbNT8.1 mRNA is significantly more abundant in insect stage procyclic forms than in mammalian stage bloodstream forms, and the TbNT8.1 permease thus may represent a major route for purine nucleobase uptake in procyclic trypanosomes.

A constitutive pan-hexose permease for the Plasmodium life cycle and transgenic models for screening of antimalarial sugar analogs

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 (Äm˜87 µM), mannose (Äi˜93 µM), fructose (Äi˜0.54 mM), and galactose (Ki˜5 mM) in Leishmania mexicana mutant and Xenopus laevis; and, therefore, is functionally equivalent to HT1 of P. falciparum (Glc, Äm˜175 µM;Man, iζ˜276 µM;Fru, iζ˜1.25 mM;Gal, iζ˜5.86mM). Notably, a glucose analog, C3361, attenuated hepatic (IC50˜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.—Blume, M., Hliscs, M., Rodriguez‐Contreras, D., Sanchez, M., Landfear, S., Lucius, R., Matuschewski, K., Gupta, N. A constitutive pan‐hexose permease for the Plasmodium life cycle and transgenic models for screening of antimalarial sugar analogs. FASEB J. 25, 1218–1229 (2011). www.fasebj.org

Two Novel Nucleobase/Pentamidine Transporters from Trypanosoma brucei

African trypanosomes are unable to synthesize purines de novo and must salvage preformed purine nucleosides and nucleobases from their hosts. The Trypanosoma brucei genome project has identified 12 members of the equilibrative nucleoside transporter family, most of which have been characterized previously as nucleoside and/or nucleobase transporters. Here the 11th member of this family, TbNT11.1, has been functionally expressed in null mutants of Leishmania that are deficient in purine nucleoside or nucleobase uptake and identified as a high-affinity purine nucleobase transporter. Expression of TbNT11.1 in Xenopus oocytes revealed that it is also a transporter for the diamidine drug pentamidine that is the principal drug employed to treat early stage human African trypanosomiasis and may thus contribute to the uptake of this therapeutically important compound. In addition, characterization of the 12th member of the family, TbNT12.1, reveals that it is an adenine/pentamidine transporter.

An Acid-activated Nucleobase Transporter from Leishmania major

Parasitic protozoa are unable to synthesize purines de novo and must import preformed purine nucleobases or nucleosides from their hosts. Leishmania major expresses two purine nucleobase transporters, LmaNT3 and LmaNT4. Previous studies revealed that at neutral pH, LmaNT3 is a broad specificity, high affinity nucleobase transporter, whereas LmaNT4 mediates the uptake of only adenine. Because LmaNT4 is required for optimal viability of the amastigote stage of the parasite that lives within acidified phagolysomal vesicles of mammalian macrophages, the function of this permease was examined under acidic pH conditions. At acidic pH, LmaNT4 acquires the ability to transport adenine, hypoxanthine, guanine, and xanthine with Km values in the micromolar range, indicating that this transporter is activated at low pH. Thus, LmaNT4 is an acid-activated purine nucleobase transporter that functions optimally under the physiological conditions the parasite is exposed to in the macrophage phagolysosome. In contrast, LmaNT3 functions optimally at neutral pH. Two-electrode voltage clamp experiments performed on LmaNT3 and LmaNT4 expressed in Xenopus oocytes revealed substrate-induced inward directed currents at acidic pH, and application of substrates induced acidification of the oocyte cytosol. These observations imply that LmaNT3 and LmaNT4 are nucleobase/proton symporters.