Functional analysis of an AMP forming acetyl CoA synthetase from Leishmania donovani by gene overexpression and targeted gene disruption approaches

Abstract

Leishmaniasis, a neglected tropical disease is endemic in 98 countries and >350 million people are at risk of getting the infection. The existing chemotherapy of Leishmaniasis is limited due to adverse effects, resistance to existing drugs and increasing cases of HIV-Leishmaniasis co-infection. Hence, there is a need to identify novel metabolic pathways for design of new chemical entities. Acetyl-CoA synthetase (AceCS) is an enzyme of acetate metabolic pathway whose functions are unknown in Leishmania parasite. AceCS from Leishmania donovani (LdAceCS) is significantly different from human host to be explored as a potential drug candidate to develop parasite specific inhibitors. To dissect the functions of LdAceCS in Leishmania promastigotes, two approaches were followed. LdAceCS overexpressing parasites were generated by episomal expression of LdAceCS in promastigotes and single knockout (SKO) cell lines of LdAceCS were generated by targeted gene disruption. An insight into the phenotypic changes undergone by the overexpressors revealed an increase in LdAceCS activity, total lipid content, infectivity and ergosterol levels by ~2.2, 2.2, 1.65 and 3 fold respectively with respect to wild type. Similarly SKO transgenic parasites exhibited ~2.5, 3, 1.5 and 3 fold decrease in activity, total lipid content, infectivity and ergosterol respectively. Repeated attempts to generate null mutants failed thus indicating that LdAceCS is essential for the parasite and can be selectively targeted to combat Leishmania infection. The present study demonstrates that LdAceCS is important for in vitro macrophage infection and is also essential for biosynthesis of total lipids and ergosterol.