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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

Immunofluorescence analysis of LdASS.L. donovani amastigote cells were stained with affinity purified antibody against LdASS as primary, and Alexa488-conjugated anti-rabbit IgG (green) as secondary antibodies. Panel A: upper row: untransfected parasites, middle row: V5His-LdASSWT transfected parasites, lower row: LdASSWT-V5His transfected parasites. Panel B: untransfected parasites labeled with biotinylated anti-LdASS (green) and anti-HGPRT antibodies (red). The nuclei and kinetoplast are stained with DAPI (blue). The merge of the images is shown in the column labeled “Merge” in the figure. The right panel shows the bright field images. The white bar represents the scale: 10 µm.
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pntd-0001849-g003: Immunofluorescence analysis of LdASS.L. donovani amastigote cells were stained with affinity purified antibody against LdASS as primary, and Alexa488-conjugated anti-rabbit IgG (green) as secondary antibodies. Panel A: upper row: untransfected parasites, middle row: V5His-LdASSWT transfected parasites, lower row: LdASSWT-V5His transfected parasites. Panel B: untransfected parasites labeled with biotinylated anti-LdASS (green) and anti-HGPRT antibodies (red). The nuclei and kinetoplast are stained with DAPI (blue). The merge of the images is shown in the column labeled “Merge” in the figure. The right panel shows the bright field images. The white bar represents the scale: 10 µm.

Mentions: Having shown that LdASS has enzymatic activity, to gain a better understanding of its physiological role we investigated the subcellular localization of this enzyme as an indication of the pathways in which ASS might be involved. Analysis of the deduced amino acid sequence revealed that LdASS has a C-terminal Ser-Ser-Leu amino acid sequence (PTS1 signal), a targeting signal that directs proteins for glycosomal import [22] (Fig. S1). To determine whether LdASS has glycosomal localization, the antibodies raised against L. donovani ASS were used for immunofluorescence studies. IFA analysis showed both untransfected Ld1S2D or Ld1S2D parasites over expressing LdASS (V5His-LdASSWT and LdASSWT-V5His cell lines) had a punctate pattern (Fig. 3A) suggesting that LdASS may be localized in an intracellular organelle. No cell-associated fluorescence was seen with the cells stained with the secondary antibody only (data not shown).


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)

Immunofluorescence analysis of LdASS.L. donovani amastigote cells were stained with affinity purified antibody against LdASS as primary, and Alexa488-conjugated anti-rabbit IgG (green) as secondary antibodies. Panel A: upper row: untransfected parasites, middle row: V5His-LdASSWT transfected parasites, lower row: LdASSWT-V5His transfected parasites. Panel B: untransfected parasites labeled with biotinylated anti-LdASS (green) and anti-HGPRT antibodies (red). The nuclei and kinetoplast are stained with DAPI (blue). The merge of the images is shown in the column labeled “Merge” in the figure. The right panel shows the bright field images. The white bar represents the scale: 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0001849-g003: Immunofluorescence analysis of LdASS.L. donovani amastigote cells were stained with affinity purified antibody against LdASS as primary, and Alexa488-conjugated anti-rabbit IgG (green) as secondary antibodies. Panel A: upper row: untransfected parasites, middle row: V5His-LdASSWT transfected parasites, lower row: LdASSWT-V5His transfected parasites. Panel B: untransfected parasites labeled with biotinylated anti-LdASS (green) and anti-HGPRT antibodies (red). The nuclei and kinetoplast are stained with DAPI (blue). The merge of the images is shown in the column labeled “Merge” in the figure. The right panel shows the bright field images. The white bar represents the scale: 10 µm.
Mentions: Having shown that LdASS has enzymatic activity, to gain a better understanding of its physiological role we investigated the subcellular localization of this enzyme as an indication of the pathways in which ASS might be involved. Analysis of the deduced amino acid sequence revealed that LdASS has a C-terminal Ser-Ser-Leu amino acid sequence (PTS1 signal), a targeting signal that directs proteins for glycosomal import [22] (Fig. S1). To determine whether LdASS has glycosomal localization, the antibodies raised against L. donovani ASS were used for immunofluorescence studies. IFA analysis showed both untransfected Ld1S2D or Ld1S2D parasites over expressing LdASS (V5His-LdASSWT and LdASSWT-V5His cell lines) had a punctate pattern (Fig. 3A) suggesting that LdASS may be localized in an intracellular organelle. No cell-associated fluorescence was seen with the cells stained with the secondary antibody only (data not shown).

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