Abstract
BackgroundGene 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 FindingsTwo 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/SignificanceOur 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.
Highlights
Leishmaniasis represents a group of parasitic diseases caused by infection with a parasite of the genus Leishmania that is transmitted to the host by the phlebotomine sandfly bite
In an attempt to identify new potential drug targets, we focused our research on Leishmania donovani argininosuccinate synthase (LdASS), which is more highly expressed in the virulent form of the parasite
In previous studies aimed at identifying biomarkers associated with the attenuation of the centrin gene-deleted cell line, we found that the argininosuccinate synthase (ASS) was down regulated in the amastigote stage when cell division is disrupted in this cell line [2,4]
Summary
Leishmaniasis represents a group of parasitic diseases caused by infection with a parasite of the genus Leishmania that is transmitted to the host by the phlebotomine sandfly bite. Additional uncharacterized proteins have been identified in studies of L. donovani gene expression. Argininosuccinate synthase (ASS) was identified as a biomarker of attenuation in the L. donovani centrin deleted cell line [4]. ASS expression was lost when parasites differentiate into the amastigote stage and the attenuated phenotype is manifest. It was shown that the virulent strain expresses a higher level of ASS in the amastigote life cycle stage responsible for the disease [4]. ASS characterization in Leishmania could reveal whether it is a virulence factor for drug therapy targeting or for manipulation to create a genetically defined attenuated parasite vaccine candidate. 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
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