# Apoptotic proteins in Leishmania donovani: in silico screening, modeling, and validation by knock-out and gene expression analysis

## TL;DR

This study identifies and validates apoptotic proteins in Leishmania donovani, offering new insights into parasite cell death and potential therapeutic targets.

## Contribution

The study reports the first apoptotic partner proteins in Leishmania donovani and validates their role in apoptosis using CRISPR-Cas9 and gene expression analysis.

## Key findings

- Molecular docking and dynamics simulations showed stable interactions between pro-apoptotic and anti-apoptotic proteins in L. donovani.
- CRISPR-Cas9 and TUNEL assays confirmed the involvement of identified proteins in parasite apoptosis.
- Gene expression profiling supported the role of these proteins in parasite survival.

## Abstract

Visceral leishmaniasis, a life-threatening vector-borne illness that disproportionately affects children and elderly immunocompromised people, is a primary tropical neglected disease. No apoptotic partner proteins have yet been reported in Leishmania donovani, while their identification could contribute to knowledge on parasite cell death and the establishment of alternative therapeutics. We searched for mammalian Bcl-2 family protein orthologs and found one anti-apoptotic and two pro-apoptotic orthologs in L. donovani. A pro-death aquaporin protein, due to its characteristic BH3 domain known to interact with pro-apoptotic proteins in mammalian Bcl-2 family proteins, was also included in this study. Molecular docking and molecular dynamics simulations were conducted to assess protein-protein interactions between the identified apoptotic proteins and mimic mammalian intrinsic apoptotic pathways. The results showed that both pro-apoptotic proteins interacted with the hydrophobic pocket of the anti-apoptotic ortholog, forming a stable complex. This interaction may represent a critical event in an apoptotic pathway in L. donovani. To further characterise it, we used CRISPR-Cas9 approaches to target the identified proteins. Pure knocked population mutants, and episomal over-expressing mutant cells were exposed to apoptotic stimuli. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and quantitative expression profiling suggested that these proteins are involved in the parasite’s apoptosis and could play a role in its survival.