TY - JOUR
T1 - Leishmania major lacking arginase (ARG) are auxotrophic for polyamines but retain infectivity to susceptible BALB/c mice
AU - Reguera, Rosa M.
AU - Balaña-Fouce, Rafael
AU - Showalter, Melissa
AU - Hickerson, Suzanne
AU - Beverley, Stephen M.
N1 - Funding Information:
We thank A. Fairlamb, S. Roberts and B. Ullman for discussions and/or sharing preliminary data. This work was supported by NIH Grant 21903 (SMB) and in part by Instituto de Salud Carlos III (grant PI06302 and RICET) from Ministerio de Salud y Consumo from the Spanish Kingdom, and by Junta de Castilla y León grant LE002A/08 to RMR.
PY - 2009/5
Y1 - 2009/5
N2 - Polyamines are essential metabolites in eukaryotes participating in a variety of proliferative processes, and in trypanosomatid protozoa play an additional role in the synthesis of the critical thiol trypanothione. Whereas the polyamine biosynthesis arising from l-ornithine has been well studied in protozoa, the metabolic origin(s) of l-ornithine have received less attention. Arginase (EC 3.5.3.1) catalyzes the enzymatic hydrolysis of l-arginine to l-ornithine and urea, and we tested the role of arginase in polyamine synthesis by the generation of an arg- knockout in Leishmania major by double targeted gene replacement. This mutant lacked arginase activity and required the nutritional provision of polyamines or l-ornithine for growth. A complemented line (arg-/+ARG) expressing arginase from a multi-copy expression vector showed 30-fold elevation of arginase activity, similar polyamine and ornithine levels as the wild-type, and resistance to the inhibitors α-difluoromethylornithine (DFMO) and Nω-hydroxy-l-arginine (NOHA). This established that arginase is the major route of polyamine synthesis in promastigotes cultured in vitro. The arg- parasites retained the ability to differentiate normally to the infective metacyclic stage, and were able to induce progressive disease following inoculation into susceptible BALB/c mice, albeit less efficiently than WT parasites. These data suggest that the infective amastigote form of Leishmania, which normally resides within an acidified parasitophorous vacuole, can survive in vivo through salvage of host polyamines and/or other molecules, aided by the tendency of acidic compartments to concentrate basic metabolites. This may thus contribute to the relative resistance of Leishmania to ornithine decarboxylase (ODC) inhibitors. The availability of infective, viable, arginase-deficient parasites should prove useful in dissecting the role of l-arginine metabolism in both pro- and anti-parasitic responses involving host nitric oxide synthase, which requires l-arginine to generate NO.
AB - Polyamines are essential metabolites in eukaryotes participating in a variety of proliferative processes, and in trypanosomatid protozoa play an additional role in the synthesis of the critical thiol trypanothione. Whereas the polyamine biosynthesis arising from l-ornithine has been well studied in protozoa, the metabolic origin(s) of l-ornithine have received less attention. Arginase (EC 3.5.3.1) catalyzes the enzymatic hydrolysis of l-arginine to l-ornithine and urea, and we tested the role of arginase in polyamine synthesis by the generation of an arg- knockout in Leishmania major by double targeted gene replacement. This mutant lacked arginase activity and required the nutritional provision of polyamines or l-ornithine for growth. A complemented line (arg-/+ARG) expressing arginase from a multi-copy expression vector showed 30-fold elevation of arginase activity, similar polyamine and ornithine levels as the wild-type, and resistance to the inhibitors α-difluoromethylornithine (DFMO) and Nω-hydroxy-l-arginine (NOHA). This established that arginase is the major route of polyamine synthesis in promastigotes cultured in vitro. The arg- parasites retained the ability to differentiate normally to the infective metacyclic stage, and were able to induce progressive disease following inoculation into susceptible BALB/c mice, albeit less efficiently than WT parasites. These data suggest that the infective amastigote form of Leishmania, which normally resides within an acidified parasitophorous vacuole, can survive in vivo through salvage of host polyamines and/or other molecules, aided by the tendency of acidic compartments to concentrate basic metabolites. This may thus contribute to the relative resistance of Leishmania to ornithine decarboxylase (ODC) inhibitors. The availability of infective, viable, arginase-deficient parasites should prove useful in dissecting the role of l-arginine metabolism in both pro- and anti-parasitic responses involving host nitric oxide synthase, which requires l-arginine to generate NO.
KW - Infections
KW - Nitric oxide
KW - Null mutant
KW - Polyamines
KW - Trypanosomatid protozoan
KW - l-arginine
KW - l-ornithine
UR - http://www.scopus.com/inward/record.url?scp=60649087303&partnerID=8YFLogxK
U2 - 10.1016/j.molbiopara.2009.01.001
DO - 10.1016/j.molbiopara.2009.01.001
M3 - Article
C2 - 19393161
AN - SCOPUS:60649087303
SN - 0166-6851
VL - 165
SP - 48
EP - 56
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 1
ER -