High Efficiency Plating Method for Leishmania Promastigotes in Semidefined or Completely-Defined Medium

Iovannisci, David M.; Ullman, Buddy

doi:10.2307/3281131

Cited by 144 publications

(66 citation statements)

References 10 publications

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“…To determine the role of the LmGT transporters in parasite growth, we examined the growth of WT and ⌬lmgt promastigotes in DME-L medium (17) with and without 25 mM glucose and with and without 5 mM proline, an amino acid that can be used as an alternative energy source by Leishmania promastigotes (26). In medium containing both glucose and proline, ⌬lmgt parasites grew less rapidly and to a lower cell density than WT cells, doubling from 1 ϫ 10 7 to 2 ϫ 10 7 cells per ml in Ϸ60 h compared with Ϸ20 h for WT cells and attaining a density of Ϸ2.5 ϫ 10 7 cells per ml compared with Ϸ5 ϫ 10 7 cells per ml for WT cells [compare WT(ϩG, ϩP) to ⌬lmgt(ϩG, ϩP), Fig.…”

Section: Resultsmentioning

confidence: 99%

Genetic characterization of glucose transporter function inLeishmania mexicana

Burchmore

Rodriguez‐Contreras

McBride

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

127

165

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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 Furthermore, WT L. mexicana were not able to grow as axenic culture form amastigotes if glucose was withdrawn from the medium, implying that glucose is an essential nutrient in this life cycle stage.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

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Section: Resultsmentioning

confidence: 99%

Genetic characterization of glucose transporter function inLeishmania mexicana

Burchmore

Rodriguez‐Contreras

McBride

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

127

165

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“…Parasites were cultivated in DME-L, a completely defined Dulbecco's modified Eagle-based medium that was specifically developed for growing Leishmania promastigotes (33). Transfected parasites were maintained in a modified DME-L medium, DME-L-CS, in which the bovine serum albumin component of DME-L was replaced with 10% chicken serum to avert polyamine oxidasemediated polyamine toxicity (8).…”

Section: Methodsmentioning

confidence: 99%

Arginase Plays a Pivotal Role in Polyamine Precursor Metabolism in Leishmania

Roberts

Tancer

Polinsky

et al. 2004

Journal of Biological Chemistry

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158

193

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The polyamine pathway of protozoan parasites has been successfully targeted in anti-parasitic therapies and is significantly different from that of the mammalian host. To gain knowledge into the metabolic routes by which parasites synthesize polyamines and their precursors, the arginase gene was cloned from Leishmania mexicana, and ⌬arg null mutants were created by double targeted gene replacement and characterized. The ARG sequence exhibited significant homology to ARG proteins from other organisms and predicted a peroxisomal targeting signal (PTS-1) that steers proteins to the glycosome, an organelle unique to Leishmania and related parasites. ARG was subsequently demonstrated to be present in the glycosome, whereas the polyamine biosynthetic enzymes, in contrast, were shown to be cytosolic. The ⌬arg knockouts expressed no ARG activity, lacked an intracellular ornithine pool, and were auxotrophic for ornithine or polyamines. The ability of the ⌬arg null mutants to proliferate could be restored by pharmacological supplementation, either with low putrescine or high ornithine or spermidine concentrations, or by complementation with an arginase episome. Transfection of an arg construct lacking the PTS-1 directed the synthesis of an arg that mislocalized to the cytosol and notably also complemented the genetic lesion and restored polyamine prototrophy to the ⌬arg parasites. This molecular, biochemical, and genetic dissection of ARG function in L. mexicana promastigotes establishes: (i) that the enzyme is essential for parasite viability; (ii) that Leishmania, unlike mammalian cells, expresses only one ARG activity; (iii) that the sole vital function of ARG is to provide polyamine precursors for the parasite; and (iv) that ARG is present in the glycosome, but this subcellular milieu is not essential for its role in polyamine biosynthesis.

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“…All Escherichia coli transformations were performed in the DH5␣ strain (Invitrogen) by using usual methodologies (14). L. donovani promastigotes were cultured at 26°C in DME-L medium (Invitrogen) as described (15). The construction and phenotypic characterization of the ⌬ldnt1/⌬ldnt2 L. donovani null mutant in which both copies of LdNT1 and LdNT2 were eliminated by targeted gene replacement followed by loss-of-heterozygosity will be described elsewhere.…”

Section: Methodsmentioning

confidence: 99%

Second-site Suppression of a Nonfunctional Mutation within the Leishmania donovani Inosine-Guanosine Transporter

Arastu‐Kapur¹,

Arendt²,

Purnat

et al. 2005

Journal of Biological Chemistry

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LdNT2 is a member of the equilibrative nucleoside transporter family, which possesses several conserved residues located mainly within transmembrane domains. One of these residues, Asp 389 within LdNT2, was shown previously to be critical for transporter function without affecting ligand affinity or plasma membrane targeting. To further delineate the role of Asp 389 in LdNT2 function, second-site suppressors of the ldnt2-D389N null mutation were selected in yeast deficient in purine nucleoside transport and incapable of purine biosynthesis. A library of random mutants within the ldnt2-D389N background was screened in yeast for restoration of growth on inosine. Twelve different clones were obtained, each containing secondary mutations enabling inosine transport. One mutation, N175I, occurred in four clones and conferred augmented inosine transport capability compared with LdNT2 in yeast. N175I was subsequently introduced into an ldnt2-D389N construct tagged with green fluorescent protein and transfected into a ⌬ldnt1/⌬ldnt2 Leishmania donovani knockout. GFP-N175I/D389N significantly suppressed the D389N phenotype and targeted properly to the plasma membrane and flagellum. Most interestingly, N175I increased the inosine K m by 10-fold within the D389N background relative to wild type GFP-LdNT2. Additional substitutions introduced at Asn 175 established that only large, nonpolar amino acids suppressed the D389N phenotype, indicating that suppression by Asn 175 has a specific size and charge requirement. Because multiple suppressor mutations alleviate the constraint imparted by the D389N mutation, these data suggest that Asp 389 is a conformationally sensitive residue. To impart spatial information to the clustering of second-site mutations, a three-dimensional model was constructed based upon members of the major facilitator superfamily using threading analysis. The model indicates that Asn 175 and Asp 389 lie in close proximity and that the second-site suppressor mutations cluster to one region of the transporter.

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High Efficiency Plating Method for Leishmania Promastigotes in Semidefined or Completely-Defined Medium

Cited by 144 publications

References 10 publications

Genetic characterization of glucose transporter function inLeishmania mexicana

Genetic characterization of glucose transporter function inLeishmania mexicana

Arginase Plays a Pivotal Role in Polyamine Precursor Metabolism in Leishmania

Second-site Suppression of a Nonfunctional Mutation within the Leishmania donovani Inosine-Guanosine Transporter

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