Leishmania donovani Argininosuccinate Synthase Is an Active Enzyme Associated with Parasite Pathogenesis

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.

In silicoidentification of metabolic enzyme drug targets inBurkholderia pseudomallei

“…The rest of the chokepoint enzymes in Table 3 were components of various biosynthesis pathways. These enzymes included argininosuccinate synthase (argG), which catalyzes the second to last step in L-arginine biosynthesis, and is associated with pathogenesis in the parasite Leishmania donovani (150), Streptococcus pneumoniae (151), and B. cenocepacia (152). The chokepoint enzyme 3-isopropylmalate dehydrogenase (leuB) is the third enzyme specific to leucine biosynthesis in microorganisms (153), and has been investigated as an antibacterial target in M. tuberculosis (154).…”

Extracellular vesicles from human liver stem cells restore argininosuccinate synthase deficiency

“…Eluted proteins were then evaluated for ASS1 activity using a modified version of the assay described by Lakhal-Naouar et al [ 22 ]. Briefly, eluted protein samples (12.5 μl per reaction) were resuspended in reaction buffer (20 mM Tris-HCl, pH 7.8, 4 mM ATP, 4 mM citrulline, 4 mM aspartate, 6 mM MgCl2, 20 mM KCl, and 0.2 units of pyrophosphatase) to a final volume of 20 μl.…”

“…Due to the spontaneous release of Pi in the absence of substrates, the final concentration of Pi from affinity-purified proteins and/or rat liver extract (RLE; served as positive control for the reaction) were determined by subtracting the mean concentration of Pi obtained in the absence of substrates from the mean concentration of Pi obtained in the presence of substrates. The specific ASS1 activity was determined using the following formula: nanomoles of phosphates released/mg protein/30 min [ 22 ]. In selected experiments, ASS1-siRNA transfected ASS1-HLSC were used.…”

Standardized methods to generate mock (spiked) clinical specimens by spiking blood or plasma with cultured pathogens

“…, , ; Lakhal‐Naouar et al . ). Frozen aliquots of live organisms of this strain are available from the authors or any of the laboratories that are referenced above (Debrabant et al .…”

“…This strain of the protozoan parasite has been characterized by sequencing multiple gene segments and aligning to database genomic sequence Selvapandiyan et al 2001;Debrabant et al 2002;Goyal et al 2006;Gannavaram et al 2011Gannavaram et al , 2012Gannavaram et al , 2014Lakhal-Naouar et al 2012). Frozen aliquots of live organisms of this strain are available from the authors or any of the laboratories that are referenced above (Debrabant et al 2004).…”