Molecular genetic analysis of purine nucleobase transport in <i>Leishmania major</i>

Ortiz, Diana; Sánchez, Marco A.; Pierce, Steven; Herrmann, Timo; Kimblin, Nicola; Bouwer, H. G. A.; Landfear, Scott M.

doi:10.1111/j.1365-2958.2007.05730.x

Cited by 44 publications

(58 citation statements)

References 44 publications

(49 reference statements)

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“…Generation of the ⌬lmant-null mutant and complementation with the LmaNT3 and LmaNT4 genes have been described previously (7).…”

Section: Methodsmentioning

confidence: 99%

“…Salvage of these essential nutrients is initiated by uptake of nucleobases and nucleosides across the parasite plasma membrane by permeases of the ENT (equilibrative nucleoside transporter) family (SLC29 family). The Leishmania major genome (3) encodes five members of this family, NT1.1, NT1.2, and NT2, which are nucleoside transporters (4,5), and NT3 and NT4, which are purine nucleobase permeases (6,7). Previous functional characterization of LmaNT3 (Lma indicates that the transporter is from L. major) revealed that it mediates the uptake of hypoxanthine, adenine, guanine, and xanthine with K m values in the low micromolar range (6).…”

mentioning

confidence: 99%

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An Acid-activated Nucleobase Transporter from Leishmania major

Ortiz¹,

Sánchez²,

Koch

et al. 2009

Journal of Biological Chemistry

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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 K m 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.Leishmania species are parasitic protozoa that infect humans and animals and cause a spectrum of diseases in tropical and subtropical regions of the world (1). The life cycle consists of three principal stages: flagellated promastigotes that colonize the midgut of the sand fly vector, metacyclic promastigotes that live in the mouth parts of the sand fly and are infectious to mammalian hosts, and non-flagellated amastigotes that reside within acidified phagolysosomal vesicles of vertebrate host macrophages.Like all other parasitic protozoa examined, Leishmania species are unable to synthesize the purine ring de novo and must import purines from both the insect and mammalian hosts (2). Salvage of these essential nutrients is initiated by uptake of nucleobases and nucleosides across the parasite plasma membrane by permeases of the ENT (equilibrative nucleoside transporter) family (SLC29 family). The Leishmania major genome (3) encodes five members of this family, NT1.1, NT1.2, and NT2, which are nucleoside transporters (4, 5), and NT3 and NT4, which are purine nucleobase permeases (6, 7). Previous functional characterization of LmaNT3 (Lma indicates that the transporter is from L. major) revealed that it mediates the uptake of hypoxanthine, adenine, guanine, and xanthine with K m values in the low micromolar range (6). In contrast, at neutral pH, LmaNT4 promotes the uptake of only adenine. Nonetheless, studies with the ⌬lmant4-null mutant (7) revealed that LmaNT4 was essential for optimal viability of L. major amastigotes in primary murine macrophages. In contrast, deletion of the LmaNT3 genes in the ⌬lmant3-null mutant was without effect on amastigote viabili...

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“…Generation of the ⌬lmant-null mutant and complementation with the LmaNT3 and LmaNT4 genes have been described previously (7).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

An Acid-activated Nucleobase Transporter from Leishmania major

Ortiz¹,

Sánchez²,

Koch

et al. 2009

Journal of Biological Chemistry

Self Cite

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“…However, while a double null mutant of the nucleoside transporters (⌬nt1 ⌬nt2) was viable (58), it was impossible to generate a dual null mutant (⌬nt3 ⌬nt4) of the nucleobase transporters (74). This result implies that purine nucleobases play a particularly important role in purine salvage.…”

Section: Purine Transportersmentioning

confidence: 82%

“…Gene knockout studies have revealed that none of these 4 purine permeases is essential for viability of either promastigotes (insect stage) or amastigotes (intramacrophage vertebrate stage) (58,74). However, while a double null mutant of the nucleoside transporters (⌬nt1 ⌬nt2) was viable (58), it was impossible to generate a dual null mutant (⌬nt3 ⌬nt4) of the nucleobase transporters (74).…”

Section: Purine Transportersmentioning

confidence: 99%

“…Subsequently, two additional members of this gene family were identified in the then-emerging genome sequence of Leishmania major. These genes were designated NT3 (74) and NT4 (73) and were shown by functional expression to encode purine nucleobase transporters (the substrates hypoxanthine, xanthine, adenine, and guanine). While hENT1 is a nonconcentrative permease, all 4 of the Leishmania purine transporters are active proton-coupled symporters that harness energy from the electrochemical gradient across the plasma membrane to concentrate their substrates within the parasite (73,93).…”

Section: Purine Transportersmentioning

confidence: 99%

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Nutrient Transport and Pathogenesis in Selected Parasitic Protozoa

Landfear

2011

Eukaryot Cell

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Parasitic protozoa, such as malaria parasites, trypanosomes, and Leishmania, acquire a plethora of nutrients from their hosts, employing transport proteins located in the plasma membrane of the parasite. Application of molecular genetic approaches and the completion of genome projects have allowed the identification and functional characterization of a cohort of transporters and their genes in these parasites. This review focuses on a subset of these permeases that have been studied in some detail, that import critical nutrients, and that provide examples of approaches being undertaken broadly with these and other parasite transporters. Permeases reviewed include those for hexoses, purines, iron, polyamines, carboxylates, and amino acids. Topics of special emphasis include structure-function approaches, critical roles for transporters in parasite viability and physiology, regulation of transporter expression, and subcellular targeting. Investigations of parasite transporters impact a broad spectrum of basic biological problems in these protozoa.Parasitic protozoa are responsible for a host of devastating diseases worldwide, including malaria, African trypanosomiasis, Chagas' disease, leishmaniases, toxoplasmosis, and many others. While all organisms must acquire nutrients from their environment, the parasitic mode of life has a number of consequences regarding nutrient uptake. First, the parasite must compete with its insect and vertebrate hosts for acquisition of many essential compounds and thus must evolve efficient uptake mechanisms. Second, the typical parasitic life cycle entails at least two hosts, one of which is often an invertebrate vector and the other a vertebrate host. Thus, the microbe cycles between multiple physiologically distinct milieus that may present pronounced differences in available nutrients, pH, temperature, ionic composition, etc. The parasite must express nutrient uptake systems that accommodate these profound alterations in environment and may employ regulatory mechanisms to alter the level of uptake according to nutrient availability and/or life cycle stage. The capacity of a parasite to import critical nutrients is central to its ability to be transmitted, to infect a host, and to cause disease and hence is an important component of pathogenesis. Figure 1 illustrates the interaction with the mammalian host of 3 parasites discussed in this review, Plasmodium, Trypanosoma brucei, and Leishmania, and reveals how the unique niche in the host dictates the mode of nutrient acquisition. African trypanosomes are extracellular parasites that live in the blood and interstitial spaces of the host and thus salvage nutrients directly from these extracellular fluids. In contrast, both Plasmodium and Leishmania live within parasitophorous vacuoles inside the red blood cell and macrophage, respectively. Intracellular parasitism provides constraints and opportunities regarding nutrient acquisition, specifically the need to import nutrients across 3 membrane systems and potentially the opportunity ...

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Purine and pyrimidine transporters of pathogenic protozoa – conduits for therapeutic agents

Campagnaro

Koning

2020

Medicinal Research Reviews

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Purines and pyrimidines are essential nutrients for any cell. Most organisms are able to synthesize their own purines and pyrimidines, but this ability was lost in protozoans that adapted to parasitism, leading to a great diversification in transporter activities in these organisms, especially for the acquisition of amino acids and nucleosides from their hosts throughout their life cycles. Many of these transporters have been shown to have sufficiently different substrate affinities from mammalian transporters, making them good carriers for therapeutic agents. In this review, we summarize the knowledge obtained on purine and pyrimidine activities identified in protozoan parasites to date and discuss their importance for the survival of these parasites and as drug carriers, as well as the perspectives of developments in the field.

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Molecular genetic analysis of purine nucleobase transport in Leishmania major

Cited by 44 publications

References 44 publications

An Acid-activated Nucleobase Transporter from Leishmania major

An Acid-activated Nucleobase Transporter from Leishmania major

Nutrient Transport and Pathogenesis in Selected Parasitic Protozoa

Purine and pyrimidine transporters of pathogenic protozoa – conduits for therapeutic agents

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