Limits...
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.

Show MeSH

Related in: MedlinePlus

An inactive LdASS enzyme induces a loss of virulence in vivo.(A) Data presented are the average number of parasites per organ from 9 animals in liver (black bars) or spleen (gray bars). Error bars indicate the standard error. Means that are significantly different by One way ANOVA (Tukey HSD) are indicated. The experiment was repeated three times with similar results. (B) Western blot with anti-LdASS and anti-V5 antibodies using an equal number of lysed promastigotes (V5His-LdASSWT, V5His-LdASSG128S and vector control) cultured for three weeks after recovery from 5 week infection of mice.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3475689&req=5

pntd-0001849-g007: An inactive LdASS enzyme induces a loss of virulence in vivo.(A) Data presented are the average number of parasites per organ from 9 animals in liver (black bars) or spleen (gray bars). Error bars indicate the standard error. Means that are significantly different by One way ANOVA (Tukey HSD) are indicated. The experiment was repeated three times with similar results. (B) Western blot with anti-LdASS and anti-V5 antibodies using an equal number of lysed promastigotes (V5His-LdASSWT, V5His-LdASSG128S and vector control) cultured for three weeks after recovery from 5 week infection of mice.

Mentions: The higher expression of the LdASS enzyme in the amastigote stage suggested that ASS may be important for parasite survival and pathogenesis. To determine the role of LdASS in Leishmania pathogenesis, we infected susceptible BALB/c mice with either wild type parasites transfected with empty vector or parasites over expressing wild type or mutant LdASS gene. Five weeks after infection, the parasite load was determined from both liver and spleen by the serial dilution method. Vector control and V5His-LdASSWT parasites survived well at 5 weeks post-infection in both liver and spleen (Fig. 7A). However, V5His-LdASSG128S parasites had reduced survival in the animals showing significant reductions (p<0.05) of parasite burdens in both liver and spleen compared to the vector control parasite and LdASSWT over expressing parasites. The altered virulence of the point mutant over expressing parasite can be attributed to the expression of the recombinant mutant LdASSG1218S protein, which was confirmed by Western blot analysis in the absence of drug selection in vitro after recovery of parasites from mice (Fig. 7B). The mouse infection experiment was repeated twice with similar findings. We demonstrate here that dominant negative effect of over expression of the non-functional LdASSG128S leads to a reduction of pathogenicity in mice (400 and 8 fold less, in liver and spleen respectively compared to LdASSWT transfected; 413 and 23 times less, in liver and spleen respectively compared to the vector control transfected). These data suggest that LdASS is essential for parasite survival and growth in the mammalian host.


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)

An inactive LdASS enzyme induces a loss of virulence in vivo.(A) Data presented are the average number of parasites per organ from 9 animals in liver (black bars) or spleen (gray bars). Error bars indicate the standard error. Means that are significantly different by One way ANOVA (Tukey HSD) are indicated. The experiment was repeated three times with similar results. (B) Western blot with anti-LdASS and anti-V5 antibodies using an equal number of lysed promastigotes (V5His-LdASSWT, V5His-LdASSG128S and vector control) cultured for three weeks after recovery from 5 week infection of mice.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0001849-g007: An inactive LdASS enzyme induces a loss of virulence in vivo.(A) Data presented are the average number of parasites per organ from 9 animals in liver (black bars) or spleen (gray bars). Error bars indicate the standard error. Means that are significantly different by One way ANOVA (Tukey HSD) are indicated. The experiment was repeated three times with similar results. (B) Western blot with anti-LdASS and anti-V5 antibodies using an equal number of lysed promastigotes (V5His-LdASSWT, V5His-LdASSG128S and vector control) cultured for three weeks after recovery from 5 week infection of mice.
Mentions: The higher expression of the LdASS enzyme in the amastigote stage suggested that ASS may be important for parasite survival and pathogenesis. To determine the role of LdASS in Leishmania pathogenesis, we infected susceptible BALB/c mice with either wild type parasites transfected with empty vector or parasites over expressing wild type or mutant LdASS gene. Five weeks after infection, the parasite load was determined from both liver and spleen by the serial dilution method. Vector control and V5His-LdASSWT parasites survived well at 5 weeks post-infection in both liver and spleen (Fig. 7A). However, V5His-LdASSG128S parasites had reduced survival in the animals showing significant reductions (p<0.05) of parasite burdens in both liver and spleen compared to the vector control parasite and LdASSWT over expressing parasites. The altered virulence of the point mutant over expressing parasite can be attributed to the expression of the recombinant mutant LdASSG1218S protein, which was confirmed by Western blot analysis in the absence of drug selection in vitro after recovery of parasites from mice (Fig. 7B). The mouse infection experiment was repeated twice with similar findings. We demonstrate here that dominant negative effect of over expression of the non-functional LdASSG128S leads to a reduction of pathogenicity in mice (400 and 8 fold less, in liver and spleen respectively compared to LdASSWT transfected; 413 and 23 times less, in liver and spleen respectively compared to the vector control transfected). These data suggest that LdASS is essential for parasite survival and growth in the mammalian host.

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