Apoptotic proteins in Leishmania donovani: in silico screening, modeling, and validation by knock-out and gene
Ketan Kumar1, Lucien Crobu2, Rokhaya Thiam2
1Department of Biochemistry, School of Life Sciences, Central University of Rajasthan, Ajmer 305817, India - University of Montpellier, CNRS, IRD, University Hospital Center (CHU) of Montpellier, MiVEGEC, Department of Parasitology-Mycology, 34295 Montpellier cedex 5, France.
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.
Insights
Researchers identified novel apoptotic proteins in Leishmania donovani, crucial for parasite survival. These findings offer potential new therapeutic targets for visceral leishmaniasis, a neglected tropical disease.
Area of Science:
- Parasitology
- Molecular Biology
- Tropical Neglected Diseases
Background:
- Visceral leishmaniasis (VL) is a severe, vector-borne disease disproportionately affecting vulnerable populations.
- Understanding parasite cell death mechanisms in Leishmania donovani is critical for developing new treatments.
- No apoptotic partner proteins had been previously identified in L. donovani.
Purpose of the Study:
- To identify and characterize apoptotic proteins in Leishmania donovani.
- To investigate the role of these proteins in parasite cell death and survival.
- To explore potential therapeutic targets for visceral leishmaniasis.
Main Methods:
- Bioinformatic search for mammalian Bcl-2 family protein orthologs in L. donovani.
- Molecular docking and dynamics simulations to analyze protein-protein interactions.
- CRISPR-Cas9 gene editing to create knockout and overexpression mutants.
- Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and quantitative expression profiling.
Main Results:
- Identified one anti-apoptotic and two pro-apoptotic orthologs in L. donovani.
- Demonstrated stable complex formation between pro-apoptotic and anti-apoptotic proteins via molecular simulations.
- CRISPR-Cas9 generated mutants showed altered responses to apoptotic stimuli, implicating these proteins in parasite apoptosis.
Conclusions:
- The identified apoptotic proteins are involved in Leishmania donovani cell death pathways.
- These proteins play a significant role in parasite survival.
- The characterized apoptotic machinery represents a promising target for novel anti-leishmanial therapeutics.
