TY - JOUR
T1 - Genetic analysis of spermidine synthase from Leishmania donovani
AU - Roberts, Sigrid C.
AU - Jiang, Yuqui
AU - Jardim, Armando
AU - Carter, Nicola S.
AU - Heby, Olle
AU - Ullman, Buddy
N1 - Funding Information:
This work was supported by grants AI10096 (S.R.) and AI41622 (B.U.) from the National Institute of Allergy and Infectious Disease and by the Swedish Natural Science Research Council (O.H.). Buddy Ullman is a Burroughs Wellcome Fund Scholar in Molecular Parasitology, and this work was supported in part by a grant from The Burroughs Wellcome Fund. We would like to thank Francy Pesek-Jardim and Birgitta Grahn for their excellent help with the cell culture and HPLC analyses, respectively.
PY - 2001/7
Y1 - 2001/7
N2 - The polyamine biosynthetic pathway of protozoan parasites has been validated as a target in antiparasitic chemotherapy. To investigate this pathway at the biochemical and genetic level in a model parasite, the gene encoding spermidine synthase (SPDSYN), a key polyamine biosynthetic enzyme, has been cloned and sequenced from Leishmania donovani. The L. donovani SPDSYN gene encodes a polypeptide of 300 amino acids that exhibits 56% amino acid identity with the human counterpart. SPDSYN is present as a single copy gene in the leishmanial genome and encodes a 1.6 kb transcript. Employing SPDSYN flanking sequences to construct drug resistance cassettes, a Δspdsyn knockout strain of L. donovani was created by double targeted gene replacement. This Δspdsyn line could not convert putrescine to spermidine and was auxotrophic for polyamines. The polyamine auxotrophy could be circumvented by exogenous spermidine but not by putrescine (1,4-diaminobutane), cadaverine (1,5-diaminopentane), 1,3-diaminopropane, or spermine. Incubation of the null mutant in polyamine-deficient medium resulted in a rapid depletion in the intracellular spermidine level with a concomitant elevation of the putrescine pool. In addition, the level of trypanothione, a spermidine-containing thiol, was reduced, whereas the glutathione pool increased 3-4-fold. These data establish that SPDSYN is an essential enzyme in L. donovani promastigotes. The molecular and cellular reagents created in this investigation provide a foundation for subsequent structure-function and inhibitor design studies on this key polyamine biosynthetic enzyme.
AB - The polyamine biosynthetic pathway of protozoan parasites has been validated as a target in antiparasitic chemotherapy. To investigate this pathway at the biochemical and genetic level in a model parasite, the gene encoding spermidine synthase (SPDSYN), a key polyamine biosynthetic enzyme, has been cloned and sequenced from Leishmania donovani. The L. donovani SPDSYN gene encodes a polypeptide of 300 amino acids that exhibits 56% amino acid identity with the human counterpart. SPDSYN is present as a single copy gene in the leishmanial genome and encodes a 1.6 kb transcript. Employing SPDSYN flanking sequences to construct drug resistance cassettes, a Δspdsyn knockout strain of L. donovani was created by double targeted gene replacement. This Δspdsyn line could not convert putrescine to spermidine and was auxotrophic for polyamines. The polyamine auxotrophy could be circumvented by exogenous spermidine but not by putrescine (1,4-diaminobutane), cadaverine (1,5-diaminopentane), 1,3-diaminopropane, or spermine. Incubation of the null mutant in polyamine-deficient medium resulted in a rapid depletion in the intracellular spermidine level with a concomitant elevation of the putrescine pool. In addition, the level of trypanothione, a spermidine-containing thiol, was reduced, whereas the glutathione pool increased 3-4-fold. These data establish that SPDSYN is an essential enzyme in L. donovani promastigotes. The molecular and cellular reagents created in this investigation provide a foundation for subsequent structure-function and inhibitor design studies on this key polyamine biosynthetic enzyme.
KW - Gene knockouts
KW - Leishmania donovani
KW - Polyamines
KW - Spermidine synthase
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U2 - 10.1016/S0166-6851(01)00293-6
DO - 10.1016/S0166-6851(01)00293-6
M3 - Article
C2 - 11420108
AN - SCOPUS:0035400392
SN - 0166-6851
VL - 115
SP - 217
EP - 226
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 2
ER -