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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

Generation of cell lines over expressing ASS.(A) Diagram of recombinant LdASS constructs used in this study. The black boxes represent the V5 epitope and grey striped boxes represent hexahistidine tags. The solid gray box indicates the enzyme active site and G or S indicates the residue at position 128. SSL indicates the 3 residues at the C terminus that have been described as a PTS1 glycosomal targeting signal. V5His-LdASSWT and LdASSWT-V5His: ASS native sequence with epitope tags at the amino terminus and carboxy terminal respectively. V5His-LdASSG128S and LdASSG128S-V5His: ASS native sequence with a serine substitution at glycine 128 and epitope tags at the amino terminus and carboxy terminal respectively. (B) Western blot analysis: Equal amounts of parasite lysates (106 cell equivalents/lane) of untransfected Ld1S2D or cells stably transfected with empty vector or constructs shown above in each lane, taken at 2 different stages (Pro-promastigotes and Ama- amastigotes) were separated by SDS page and transferred to nitrocellulose membranes. The Western blots were probed with anti-LdASS, anti-V5, anti-Tubulin antibodies or pre immune sera (left panel: short exposure and right panel: long exposure). The arrow indicates the endogenous LdASS.
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pntd-0001849-g001: Generation of cell lines over expressing ASS.(A) Diagram of recombinant LdASS constructs used in this study. The black boxes represent the V5 epitope and grey striped boxes represent hexahistidine tags. The solid gray box indicates the enzyme active site and G or S indicates the residue at position 128. SSL indicates the 3 residues at the C terminus that have been described as a PTS1 glycosomal targeting signal. V5His-LdASSWT and LdASSWT-V5His: ASS native sequence with epitope tags at the amino terminus and carboxy terminal respectively. V5His-LdASSG128S and LdASSG128S-V5His: ASS native sequence with a serine substitution at glycine 128 and epitope tags at the amino terminus and carboxy terminal respectively. (B) Western blot analysis: Equal amounts of parasite lysates (106 cell equivalents/lane) of untransfected Ld1S2D or cells stably transfected with empty vector or constructs shown above in each lane, taken at 2 different stages (Pro-promastigotes and Ama- amastigotes) were separated by SDS page and transferred to nitrocellulose membranes. The Western blots were probed with anti-LdASS, anti-V5, anti-Tubulin antibodies or pre immune sera (left panel: short exposure and right panel: long exposure). The arrow indicates the endogenous LdASS.

Mentions: Functional characterization of genes in Leishmania often involves creation of targeted gene deleted parasites and analysis of the effect of gene loss on parasite survival and infectivity. Such a strategy was not successful in our study since LdASS is present on chromosome 23 that has at least 3 copies in the L. donovani 1S2D genome (Personal communication, Peter Myler). Future studies will focus on the deletion of all the copies of the L. donovani ASS gene to address this question. Alternatively, to characterize LdASS and its function, we employed a dominant negative approach. Four cell lines were created expressing the wild type or mutant form of LdASS (G128S) with V5epitopes and hexasitidine tags at either the N-terminal (V5His-LdASSWT, V5His-LdASSG128S) or C-terminal (LdASSWT-V5His, LdASSG128S-V5His) ends (Fig. 1A). All four exhibited similar growth kinetics in vitro either in promastigote or axenic amastigote forms (data not shown). Immunoblot analysis of total cell lysates from these transfectants cultured as the promastigote or axenic amastigote forms indicated that the exogenous protein reacted with both the anti-LdASS and anti-V5 antibodies, was present in excess of the native protein in both life cycle stages, the recombinant form of LdASS was detected predominantly as a doublet that migrated at 47 kDa and 49 kDa, and the expression resulted in extra bands (Fig. 1B; V5His-LdASSWT (lanes 1, 5); V5His-LdASSG128S (lanes 2, 6); LdASSWT-V5His (lanes 3, 7); LdASSG128S-V5His (lanes 4, 8)). Longer exposure time was required to reveal native ASS protein (Fig. 1B; lanes 9–12). The endogenous LdASS both in non-transfected and vector alone transfected parasites migrated at 48 kDa, reacted with the anti-LdASS antibody but not with the anti-V5 and was exclusively expressed in the amastigote form (Fig. 1.B; lanes 11 and 12). The anti-tubulin blot showed that the same quantity of lysates was loaded in each lane on the gel (Fig. 1B). Parasite lysates probed with normal rabbit serum (NRS) did not show any reactivity supporting the specificity of the reactive anti-sera.


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)

Generation of cell lines over expressing ASS.(A) Diagram of recombinant LdASS constructs used in this study. The black boxes represent the V5 epitope and grey striped boxes represent hexahistidine tags. The solid gray box indicates the enzyme active site and G or S indicates the residue at position 128. SSL indicates the 3 residues at the C terminus that have been described as a PTS1 glycosomal targeting signal. V5His-LdASSWT and LdASSWT-V5His: ASS native sequence with epitope tags at the amino terminus and carboxy terminal respectively. V5His-LdASSG128S and LdASSG128S-V5His: ASS native sequence with a serine substitution at glycine 128 and epitope tags at the amino terminus and carboxy terminal respectively. (B) Western blot analysis: Equal amounts of parasite lysates (106 cell equivalents/lane) of untransfected Ld1S2D or cells stably transfected with empty vector or constructs shown above in each lane, taken at 2 different stages (Pro-promastigotes and Ama- amastigotes) were separated by SDS page and transferred to nitrocellulose membranes. The Western blots were probed with anti-LdASS, anti-V5, anti-Tubulin antibodies or pre immune sera (left panel: short exposure and right panel: long exposure). The arrow indicates the endogenous LdASS.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0001849-g001: Generation of cell lines over expressing ASS.(A) Diagram of recombinant LdASS constructs used in this study. The black boxes represent the V5 epitope and grey striped boxes represent hexahistidine tags. The solid gray box indicates the enzyme active site and G or S indicates the residue at position 128. SSL indicates the 3 residues at the C terminus that have been described as a PTS1 glycosomal targeting signal. V5His-LdASSWT and LdASSWT-V5His: ASS native sequence with epitope tags at the amino terminus and carboxy terminal respectively. V5His-LdASSG128S and LdASSG128S-V5His: ASS native sequence with a serine substitution at glycine 128 and epitope tags at the amino terminus and carboxy terminal respectively. (B) Western blot analysis: Equal amounts of parasite lysates (106 cell equivalents/lane) of untransfected Ld1S2D or cells stably transfected with empty vector or constructs shown above in each lane, taken at 2 different stages (Pro-promastigotes and Ama- amastigotes) were separated by SDS page and transferred to nitrocellulose membranes. The Western blots were probed with anti-LdASS, anti-V5, anti-Tubulin antibodies or pre immune sera (left panel: short exposure and right panel: long exposure). The arrow indicates the endogenous LdASS.
Mentions: Functional characterization of genes in Leishmania often involves creation of targeted gene deleted parasites and analysis of the effect of gene loss on parasite survival and infectivity. Such a strategy was not successful in our study since LdASS is present on chromosome 23 that has at least 3 copies in the L. donovani 1S2D genome (Personal communication, Peter Myler). Future studies will focus on the deletion of all the copies of the L. donovani ASS gene to address this question. Alternatively, to characterize LdASS and its function, we employed a dominant negative approach. Four cell lines were created expressing the wild type or mutant form of LdASS (G128S) with V5epitopes and hexasitidine tags at either the N-terminal (V5His-LdASSWT, V5His-LdASSG128S) or C-terminal (LdASSWT-V5His, LdASSG128S-V5His) ends (Fig. 1A). All four exhibited similar growth kinetics in vitro either in promastigote or axenic amastigote forms (data not shown). Immunoblot analysis of total cell lysates from these transfectants cultured as the promastigote or axenic amastigote forms indicated that the exogenous protein reacted with both the anti-LdASS and anti-V5 antibodies, was present in excess of the native protein in both life cycle stages, the recombinant form of LdASS was detected predominantly as a doublet that migrated at 47 kDa and 49 kDa, and the expression resulted in extra bands (Fig. 1B; V5His-LdASSWT (lanes 1, 5); V5His-LdASSG128S (lanes 2, 6); LdASSWT-V5His (lanes 3, 7); LdASSG128S-V5His (lanes 4, 8)). Longer exposure time was required to reveal native ASS protein (Fig. 1B; lanes 9–12). The endogenous LdASS both in non-transfected and vector alone transfected parasites migrated at 48 kDa, reacted with the anti-LdASS antibody but not with the anti-V5 and was exclusively expressed in the amastigote form (Fig. 1.B; lanes 11 and 12). The anti-tubulin blot showed that the same quantity of lysates was loaded in each lane on the gel (Fig. 1B). Parasite lysates probed with normal rabbit serum (NRS) did not show any reactivity supporting the specificity of the reactive anti-sera.

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