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Leishmania donovani argininosuccinate synthase is an active enzyme associated with parasite pathogenesis.

Lakhal-Naouar I, Jardim A, Strasser R, Luo S, Kozakai Y, Nakhasi HL, Duncan RC - PLoS Negl Trop Dis (2012)

Bottom Line: Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid).The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA.Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Bethesda, MD, USA.

ABSTRACT

Background: Gene expression analysis in Leishmania donovani (Ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS), that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo.

Methodology/principal findings: Two parasite cell lines either over expressing wild type LdASS or a mutant form (G128S) associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid). However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver.

Conclusion/significance: Our study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a potential drug target.

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Related in: MedlinePlus

Subcellular organelle fractionation.(A) Cleared lysates (lane 1) from untransfected Ld1S2D parasites (upper panel) or V5-HisLdASSWT over expressing amastigotes (lower panel) were fractionated into cytosol (lane 2) and crude organelles (lane 3) and used for immuno-blot with anti-LdASS antibody. (B) V5-HisLdASSWT over expressing amastigotes were lysed then cleared (lane 1) and fractionated into cytosol (lane 2) and crude organelles (lanes 3–6). Crude organelles were treated with protease K in absence (lane 4) or presence of triton (lane 6). Proteins were separated by SDS page, transferred to nitrocellulose membranes, and then bound with anti-LdASS, anti-HGPRT and anti-TatD antibodies. (C) The crude organelle fraction of V5His-LdASSWT over expressing amastigotes was further fractionated on a linear 20–70% sucrose gradient. Sucrose fractions 1–21 were assayed for the presence of LdASS by Western blot analysis using anti-V5 antibody. Here only fractions 1–14 are shown on the membrane, L: crude organelle fraction layered on the gradient, +: cleared lysate as positive control.
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pntd-0001849-g006: Subcellular organelle fractionation.(A) Cleared lysates (lane 1) from untransfected Ld1S2D parasites (upper panel) or V5-HisLdASSWT over expressing amastigotes (lower panel) were fractionated into cytosol (lane 2) and crude organelles (lane 3) and used for immuno-blot with anti-LdASS antibody. (B) V5-HisLdASSWT over expressing amastigotes were lysed then cleared (lane 1) and fractionated into cytosol (lane 2) and crude organelles (lanes 3–6). Crude organelles were treated with protease K in absence (lane 4) or presence of triton (lane 6). Proteins were separated by SDS page, transferred to nitrocellulose membranes, and then bound with anti-LdASS, anti-HGPRT and anti-TatD antibodies. (C) The crude organelle fraction of V5His-LdASSWT over expressing amastigotes was further fractionated on a linear 20–70% sucrose gradient. Sucrose fractions 1–21 were assayed for the presence of LdASS by Western blot analysis using anti-V5 antibody. Here only fractions 1–14 are shown on the membrane, L: crude organelle fraction layered on the gradient, +: cleared lysate as positive control.

Mentions: IFA experiments suggested that LdASS was found in a subcellular structure that was distinct from the microbody organelles containing the proteins HGPRT, LdIMPDH and LdPEX14 which define glycosomes [35]–[37]. In order to define the LdASS localization, we performed a subcellular fractionation of whole cell lysates of axenic Leishmania amastigotes from untransfected parasites or from parasites expressing V5His-LdASSWT. Both parasite strains showed a similar distribution of LdASS with a dual compartmentalization between cytosol (Fig. 6A, lane 2) and crude organelles (Fig. 6A, lane 3). For the later experiments, we used only the LdASS over expressing parasites since the distribution of over expressing LdASS mirrors the distribution of endogenous LdASS and the higher expression of recombinant ASS permits detection in further fractionation.


Leishmania donovani argininosuccinate synthase is an active enzyme associated with parasite pathogenesis.

Lakhal-Naouar I, Jardim A, Strasser R, Luo S, Kozakai Y, Nakhasi HL, Duncan RC - PLoS Negl Trop Dis (2012)

Subcellular organelle fractionation.(A) Cleared lysates (lane 1) from untransfected Ld1S2D parasites (upper panel) or V5-HisLdASSWT over expressing amastigotes (lower panel) were fractionated into cytosol (lane 2) and crude organelles (lane 3) and used for immuno-blot with anti-LdASS antibody. (B) V5-HisLdASSWT over expressing amastigotes were lysed then cleared (lane 1) and fractionated into cytosol (lane 2) and crude organelles (lanes 3–6). Crude organelles were treated with protease K in absence (lane 4) or presence of triton (lane 6). Proteins were separated by SDS page, transferred to nitrocellulose membranes, and then bound with anti-LdASS, anti-HGPRT and anti-TatD antibodies. (C) The crude organelle fraction of V5His-LdASSWT over expressing amastigotes was further fractionated on a linear 20–70% sucrose gradient. Sucrose fractions 1–21 were assayed for the presence of LdASS by Western blot analysis using anti-V5 antibody. Here only fractions 1–14 are shown on the membrane, L: crude organelle fraction layered on the gradient, +: cleared lysate as positive control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475689&req=5

pntd-0001849-g006: Subcellular organelle fractionation.(A) Cleared lysates (lane 1) from untransfected Ld1S2D parasites (upper panel) or V5-HisLdASSWT over expressing amastigotes (lower panel) were fractionated into cytosol (lane 2) and crude organelles (lane 3) and used for immuno-blot with anti-LdASS antibody. (B) V5-HisLdASSWT over expressing amastigotes were lysed then cleared (lane 1) and fractionated into cytosol (lane 2) and crude organelles (lanes 3–6). Crude organelles were treated with protease K in absence (lane 4) or presence of triton (lane 6). Proteins were separated by SDS page, transferred to nitrocellulose membranes, and then bound with anti-LdASS, anti-HGPRT and anti-TatD antibodies. (C) The crude organelle fraction of V5His-LdASSWT over expressing amastigotes was further fractionated on a linear 20–70% sucrose gradient. Sucrose fractions 1–21 were assayed for the presence of LdASS by Western blot analysis using anti-V5 antibody. Here only fractions 1–14 are shown on the membrane, L: crude organelle fraction layered on the gradient, +: cleared lysate as positive control.
Mentions: IFA experiments suggested that LdASS was found in a subcellular structure that was distinct from the microbody organelles containing the proteins HGPRT, LdIMPDH and LdPEX14 which define glycosomes [35]–[37]. In order to define the LdASS localization, we performed a subcellular fractionation of whole cell lysates of axenic Leishmania amastigotes from untransfected parasites or from parasites expressing V5His-LdASSWT. Both parasite strains showed a similar distribution of LdASS with a dual compartmentalization between cytosol (Fig. 6A, lane 2) and crude organelles (Fig. 6A, lane 3). For the later experiments, we used only the LdASS over expressing parasites since the distribution of over expressing LdASS mirrors the distribution of endogenous LdASS and the higher expression of recombinant ASS permits detection in further fractionation.

Bottom Line: Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid).The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA.Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Bethesda, MD, USA.

ABSTRACT

Background: Gene expression analysis in Leishmania donovani (Ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS), that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo.

Methodology/principal findings: Two parasite cell lines either over expressing wild type LdASS or a mutant form (G128S) associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid). However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver.

Conclusion/significance: Our study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a potential drug target.

Show MeSH
Related in: MedlinePlus