Single Cell Cloning of Leishmania Parasites in Purine-Defined Medium: Isolation of Drug-Resistant Variants

Adenylosuccinate Synthetase and Adenylosuccinate Lyase Deficiencies Trigger Growth and Infectivity Deficits in Leishmania donovani

“…LdBob promastigotes were cultured at ambient temperature in modified Dulbecco's modified Eagle mediumLeishmania (35) as described (16) and supplemented with 5% FBS, whereas axenic amastigotes were propagated at 37°C as detailed (33,34). Plating methods and single-cell cloning protocols for L. donovani promastigotes are outlined elsewhere (35,36). Wild type and ⌬aah parasites were propagated in 100 M xanthine as the purine nutrient, the ⌬aah/⌬adss, ⌬adss [pADSS], and ⌬asl[pASL] cells were grown in 100 M adenine, and the ⌬adss and ⌬asl knock-outs were grown in modified Dulbecco's modified Eagle medium-Leishmania supplemented with 100 M adenine and 20 M dCF.…”

Purine salvage in Leishmania: complex or simple by design?

“…are digenetic parasites, existing as highly motile, flagellated promastigotes that reside freely within the midgut of the sandfly vector and as amotile amastigotes that replicate within the phagolysosome of the mammalian macrophage. Most of the initial investigations on the purine pathway were undertaken with an attenuated L. donovani clone [59] derived from the 1S-2D strain that was originally adapted to axenic culture by Dennis Dwyer [60–62]. Consequently, studies on parasite infectivity and virulence were precluded.…”

Adenine Aminohydrolase from Leishmania donovani

“…Creation of Null Mutants-The ⌬aah knock-out was generated from wild type parasites by double targeted gene replacement using the transfection parameters and plating techniques reported previously (44,55). The linearized drug resistance cassettes enclosing the AAH flanks were excised from pX63-PAC-⌬aah and pXG-BSD-⌬aah with HindIIIBglII and SgrAI-BamHI, respectively, and used to sequentially create AAH/⌬aah::PAC (AAH/aah) heterozygotes and ⌬aah::PAC/⌬aah::BSD (⌬aah) null mutants.…”