Afreen Nawaz

Conference 2024 Live Talk

Talk Title

Serine o-acetyltransferase protein in Leishmania donovani: Orchestrating thiol-based drug resistance and oxidative stress defense

Authors and Affiliations

Afreen1 and Vahab Ali1
1. Department of Biochemistry, ICMR-Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna-800007, India

Abstract

Background

Visceral Leishmaniasis (VL), a neglected tropical disease caused by the parasite Leishmania donovani, remains a global threat that requires effective chemotherapy since there is still no vaccine available for human use. The current therapeutics are not sufficient to keep up with the emerging cases of drug resistance, Post-KalaAzar Dermal Leishmaniasis and HIV-VL coinfection, highlighting the need for investigating new drug targets. Leishmania parasites have a unique trypanothione-based thiol metabolism for maintaining redox homeostasis and antioxidative stress defense. The synthesis of trypanothione, however, depends on the availability of cysteine. Leishmania can synthesize cysteine by de novo cysteine biosynthetic pathway involving two key proteins- serine o-acetyltransferase (SAT) and cysteine synthase (CS).

Methods

In order to investigate the role of SAT protein, LdSAT gene was cloned into pXG-GFP+ vector for episomal expression of SAT in Amp B sensitive L. donovani (LdSAT-OE). SAT enzymatic assay, GFP fluorescence, immunoblotting and PCR confirmed the SAT overexpression. Cell viability and antioxidant enzyme assays were performed. J774A.1 macrophage infectivity of LdSAT-OE was assessed to decipher the role of SAT in coping with the hostile conditions inside the macrophage. The expression pattern of thiol pathway proteins under drug pressure and oxidative stress was analyzed by immunoblotting.

Results

LdSAT-OE parasites showed higher tolerance to drug pressure and heavy metal stress. In LdSAT-OE, a reduction in intracellular reactive oxygen species (ROS) was seen along with an increase in thiol content. Additionally, the ROS-mediated increase in the expression of LdSAT, LdCS, LdTryS, and cTXNPx demonstrates an important cross talk between SAT and thiol pathway proteins in fighting oxidative stress and maintaining redox balance. LdSAT overexpression was also found to augment antioxidant enzyme activities. In vitro macrophage infectivity revealed that SAT protein overexpression enhances parasite infectivity.

Conclusions

Our results provide the first insight into the essentiality of SAT protein in parasite infectivity and survival under drug pressure and oxidative stress. The crucial role of SAT protein in modulating thiol based antioxidative defense mechanism and its absence in human host, makes it an attractive target for anti-leishmanial drug development.