Apoptotic proteins inLeishmania donovani:In silicoscreening, modelling, and validation by knock-out and gene expression analysis

Abstract

AbstractVisceral leishmaniasis (VL), a life-threatening vector-borne illness that disproportionately affects children and elderly immunocompromised people, is a primary tropical neglected disease. No apoptotic partner proteins inL. donovanihave been reported yet, which might contribute to the knowledge of parasite cell death and the establishment of alternative therapeutics. We used the Orthologues algorithm to search for the mammalian Bcl-2 family proteins orthologs, one anti-apoptotic and two pro-apoptotic, inL. donovani. We also included a pro-death aquaporin (AQP) protein due to its characteristic BH3 domain, which is known to interact with pro-apoptotic proteins in mammals. Molecular docking and molecular dynamics simulation studies were conducted to assess the protein-protein interaction between the identified apoptotic proteins and mimic mammalian intrinsic apoptotic pathways. The results showed that the pro-apoptotic protein interacted with the hydrophobic pocket of the anti-apoptotic ortholog, forming a stable complex, which may represent a critical event in the apoptotic pathways of leishmaniasis. To further establish an apoptotic pathway inL. donovani, we used several CRISPR-Cas9 approaches to target the identified proteins. The pure knocked population mutants, and episomal over-expressing mutant cells were exposed to apoptotic stimuli. TUNEL assay and quantitative expression profiling suggested that these proteins are needed during the parasite’s apoptosis and could play a role in the parasite’s survival.Author SummaryVisceral leishmaniasis, a fatal systemic infection affecting internal organs, is one of three types of leishmaniasis in mammals alongside cutaneous and mucocutaneous leishmaniasis. It predominantly occurs in tropical and subtropical climatic zones,Leishmania donovanipredominant in the Indian subcontinent andLeishmania infantumin the Mediterranean basin, the Middle East, Central Asia, South America, and Central America. This disease primarily affects children, immunocompromised adults, and the elderly.L donovani,transmitted by the infected sandflies complete its life cycle in humans, serving as reservoir. During its life cycle, at a particular stage, the parasite undergoes apoptotic-like events, yet underlying proteins or key factors remain unidentified. Using computational methods, we screened theL. donovanigenome for potential candidate genes of the Bcl-2 family apoptotic proteins. We biologically/experimentally validated ourin-silicofindings using molecular editing tools like CRISPR-Cas9, advancing our understanding of the parasite’s apoptotic pathway. Targeting this pathway could lead to more effective therapeutics against visceral leishmaniasis.