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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

Comparison of LdASS localization with LdPEX14 and LdIMPDH.(A) L. donovani amastigotes were stained with antibodies against LdPEX14 and biotinylated anti-LdASS antibodies as primary, then with Alexa568 (red)-conjugated anti-rabbit IgG and Streptavidivin FITC as secondary. The nucleus and kinetoplast were stained with DAPI. (B) L. donovani amastigotes were stained with antibodies against affinity purified LdASS and guinea pig anti-LdIMPDH antibodies as primary, then with Alexa488 (red)-conjugated anti-rabbit IgG and Alexa564 (red)-conjugated anti-Guinea-pig IgG as secondary. The nucleus and kinetoplast were stained with DAPI. (C) L. donovani amastigotes were stained with rabbit anti-LdPEX14 and Guinea pig anti- LdIMPDH as primary, and Alexa488 (green)-conjugated anti-rabbit IgG and Alexa568 (red)-conjugated anti-guinea pig IgG as secondary antibodies. The nucleus and kinetoplast were stained with DAPI. The merge of the images is shown in the column labeled “Merge”. The right panel shows the bright field images. The white bar represents the scale that corresponds to 10 µm.
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pntd-0001849-g005: Comparison of LdASS localization with LdPEX14 and LdIMPDH.(A) L. donovani amastigotes were stained with antibodies against LdPEX14 and biotinylated anti-LdASS antibodies as primary, then with Alexa568 (red)-conjugated anti-rabbit IgG and Streptavidivin FITC as secondary. The nucleus and kinetoplast were stained with DAPI. (B) L. donovani amastigotes were stained with antibodies against affinity purified LdASS and guinea pig anti-LdIMPDH antibodies as primary, then with Alexa488 (red)-conjugated anti-rabbit IgG and Alexa564 (red)-conjugated anti-Guinea-pig IgG as secondary. The nucleus and kinetoplast were stained with DAPI. (C) L. donovani amastigotes were stained with rabbit anti-LdPEX14 and Guinea pig anti- LdIMPDH as primary, and Alexa488 (green)-conjugated anti-rabbit IgG and Alexa568 (red)-conjugated anti-guinea pig IgG as secondary antibodies. The nucleus and kinetoplast were stained with DAPI. The merge of the images is shown in the column labeled “Merge”. The right panel shows the bright field images. The white bar represents the scale that corresponds to 10 µm.

Mentions: IFA performed using anti-LdASS and anti-LdPEX14 antibodies with non-transfected L. donovani showed that LdASS localized to small cellular structures that were distributed throughout the cell body. These structures however, did not co-localize with glycosomal membrane associated protein LdPEX14 [36] (Fig. 5A). As another example of a PTS1 containing glycosomal protein, IMPDH localization was compared to LdASS. Affinity purified anti-LdASS did not co-localize with guinea pig anti-LdIMPDH (Fig. 5B). However, similar co-localization experiments performed using anti-LdIMPDH and anti-LdPEX14 antibodies confirmed that both of these proteins were present in the glycosome (Fig. 5C). These results suggest that although LdASS interacts with some of the glycosomal import machinery, it is localized in a subcellular structure that is different from the glycosomes.


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)

Comparison of LdASS localization with LdPEX14 and LdIMPDH.(A) L. donovani amastigotes were stained with antibodies against LdPEX14 and biotinylated anti-LdASS antibodies as primary, then with Alexa568 (red)-conjugated anti-rabbit IgG and Streptavidivin FITC as secondary. The nucleus and kinetoplast were stained with DAPI. (B) L. donovani amastigotes were stained with antibodies against affinity purified LdASS and guinea pig anti-LdIMPDH antibodies as primary, then with Alexa488 (red)-conjugated anti-rabbit IgG and Alexa564 (red)-conjugated anti-Guinea-pig IgG as secondary. The nucleus and kinetoplast were stained with DAPI. (C) L. donovani amastigotes were stained with rabbit anti-LdPEX14 and Guinea pig anti- LdIMPDH as primary, and Alexa488 (green)-conjugated anti-rabbit IgG and Alexa568 (red)-conjugated anti-guinea pig IgG as secondary antibodies. The nucleus and kinetoplast were stained with DAPI. The merge of the images is shown in the column labeled “Merge”. The right panel shows the bright field images. The white bar represents the scale that corresponds to 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0001849-g005: Comparison of LdASS localization with LdPEX14 and LdIMPDH.(A) L. donovani amastigotes were stained with antibodies against LdPEX14 and biotinylated anti-LdASS antibodies as primary, then with Alexa568 (red)-conjugated anti-rabbit IgG and Streptavidivin FITC as secondary. The nucleus and kinetoplast were stained with DAPI. (B) L. donovani amastigotes were stained with antibodies against affinity purified LdASS and guinea pig anti-LdIMPDH antibodies as primary, then with Alexa488 (red)-conjugated anti-rabbit IgG and Alexa564 (red)-conjugated anti-Guinea-pig IgG as secondary. The nucleus and kinetoplast were stained with DAPI. (C) L. donovani amastigotes were stained with rabbit anti-LdPEX14 and Guinea pig anti- LdIMPDH as primary, and Alexa488 (green)-conjugated anti-rabbit IgG and Alexa568 (red)-conjugated anti-guinea pig IgG as secondary antibodies. The nucleus and kinetoplast were stained with DAPI. The merge of the images is shown in the column labeled “Merge”. The right panel shows the bright field images. The white bar represents the scale that corresponds to 10 µm.
Mentions: IFA performed using anti-LdASS and anti-LdPEX14 antibodies with non-transfected L. donovani showed that LdASS localized to small cellular structures that were distributed throughout the cell body. These structures however, did not co-localize with glycosomal membrane associated protein LdPEX14 [36] (Fig. 5A). As another example of a PTS1 containing glycosomal protein, IMPDH localization was compared to LdASS. Affinity purified anti-LdASS did not co-localize with guinea pig anti-LdIMPDH (Fig. 5B). However, similar co-localization experiments performed using anti-LdIMPDH and anti-LdPEX14 antibodies confirmed that both of these proteins were present in the glycosome (Fig. 5C). These results suggest that although LdASS interacts with some of the glycosomal import machinery, it is localized in a subcellular structure that is different from the glycosomes.

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