Genetic Manipulation of Leishmania donovani to Explore the Involvement of Argininosuccinate Synthase in Oxidative Stress Management

Abstract

Reactive oxygen and nitrogen species (ROS and RNS) produced by the phagocytic cells are the most common arsenals used to kill the intracellular pathogens. However, Leishmania, an intracellular pathogen, has evolved mechanisms to survive by counterbalancing the toxic oxygen metabolites produced during infection. Polyamines, the major contributor in this anti-oxidant machinery, are largely dependent on the availability of L-arginine in the intracellular milieu. Argininosuccinate synthase (ASS) plays an important role as the rate-limiting step required for converting L-citrulline to argininosuccinate to provide arginine for an assortment of metabolic processes. Leishmania produce an active ASS enzyme, yet it has an incomplete urea cycle as it lacks an argininosuccinate lyase (ASL). There is no evidence for endogenous synthesis of L-arginine in Leishmania, which suggests that these parasites salvage L-arginine from extracellular milieu and makes the biological function of ASS and the production of argininosuccinate in Leishmania unclear. Our previous quantitative proteomic analysis of Leishmania promastigotes treated with sub-lethal doses of ROS, RNS, or a combination of both, led to the identification of several differentially expressed proteins which included ASS. To assess the involvement of ASS in stress management, a mutant cell line with greatly reduced ASS activity was created by a double-targeted gene replacement strategy in L. donovani promastigote. Interestingly, LdASS is encoded by three copies of allele, but Western blot analysis showed the third allele did not appear to express ASS. The free thiol levels in the mutant LdASS-/-/+ cell line were decreased. Furthermore, the cell viability in L-arginine depleted medium was greatly attenuated on exposure to different stress environments and was adversely impacted in its ability to infect mice. These findings suggest that ASS is important for Leishmania donovani to counterbalance the stressed environments encountered during infection and can be targeted for chemotherapeutic purpose to treat visceral leishmaniasis.

Genomic insights into virulence mechanisms of Leishmania donovani: evidence from an atypical strain

“…Terbinafine resistance locus protein has previously been identified to mediate efflux of terbinafine in L. major [ 32 ] and argininosuccinate synthase gene has been identified to be associated with antimony resistance in L. infantum [ 33 ]. Also an up regulation of argininosuccinate synthase gene was seen in Leishmania parasites as a response to oxidative stress and arginine starvation and the viability of the parasites were seen to notably reduce when the gene was down regulated, indicating an important role of the argininosuccinate synthase gene in infectivity and survival of the parasites in the mammalian host [ 34 , 35 ].…”

Integrated genomic and metabolomic profiling of ISC1, an emerging Leishmania donovani population in the Indian subcontinent

“…In ISC1 versus CG we detected a higher concentration of citrulline and a lower concentration of argininosuccinate. In organisms with a complete urea cycle, argininosuccinate is then further converted to arginine and fumarate by argininosuccinate lyase (ASL), however, in Leishmania this enzyme is missing ( Sardar et al, 2016 ). This difference is of particular interest since both argininosuccinate and arginine deprivation have been linked with reduced thiol content ( Sardar et al, 2016 ; Mandal et al, 2016 ) and ASS mutants have shown a lower virulence than WT parasites ( Lakhal-Naouar et al, 2012 ).…”

Arginine and Polyamines Fate in Leishmania Infection

“…Yet, ASS activity converting citrulline to arginosuccinate has been demonstrated. It seems that ASS activity is not related with L -arginine synthesis, but with important biological functions of arginosuccinate in the establishment of the infection ( Lakhal-Naouar et al, 2012 ; Sardar et al, 2016 ).…”