Identification and characterization of bax inhibitor-1 of leishmania donovani and developing new strategies for anti leishmanial therapeutics

Abstract

Leishmaniasis is a vector-borne disease that causes significant morbidity and mortality. Leishmania belongs to the trypanosomatidae family from the order Kinetoplastida. There are at least 20 different species of Leishmania that can cause human infection. Human leishmaniasis is of three types - cutaneous leishmaniasis, mucocutaneous leishmaniasis, and visceral leishmaniasis. Visceral leishmaniasis is the most dangerous form of leishmaniasis. It is spread from the bite of a female sandfly of the genus Phlebotomus and Lutzomyia, and affects the internal organs of body like spleen and liver. It also causes anemia and severe fever. Leishmania has a digenic life cycle – it exists as promastigotes in the sandfly which transforms into amastigotes in the mammalian host. In India, cutaneous and visceral leishmaniasis are more predominant. The traditional treatments for leishmaniasis are Pentavalent antimony, Miltefosine, and Amphotericin B. All these drugs cause significant toxicity to the patient’s body. Above all, the report of drug resistance has been reported in different places around the globe in the past two decades. All this causes difficulties in handling this global threat. Thus, there is an urgent need to discover new drugs and drug targets to combat this protozoan parasitic disease. The genome of Leishmania is approximately 30kb in size and most of the genes and cellular pathways are yet unexplored. The knowledge about the cell death pathway is not adequate in Leishmania. The existence of cell death effectors in protozoan parasites is yet a matter of debate. In Leishmania, different stimuli induce the cell death phenotype which is similar to that of mammals. Despite the presence of the phenotypic cell death markers, the key cell death regulators like caspases and Bax are absent in Leishmania. Since apoptosis regulators are not present in Leishmania, the cell death process has been referred to as apoptosis-like in these protozoan parasites. The knowledge about Leishmania cell death is fragmented, and it is still not fully understood. This gap in understanding largely stems from the difficulty in identifying key proteins linked to mammalian apoptosis in Leishmania, even with the use of bioinformatics methods. Research has demonstrated that the pathway of programmed cell death serves a crucial role in multicellular organisms, facilitating the removal of cellular debris, pathogens, and infected cells, while also contributing to their normal growth and development. Given this understanding, it is pertinent to explore whether similar predetermined mechanisms exist within protozoan parasites. In most of these organisms, the evidence of apoptosis-like death is mitochondrial transmembrane potential disruption (mainly due to mitochondrion permeabilization) and nuclear DNA degradation. In Leishmania, investigators primarily focus on two principal indicators of cell death: the degradation of DNA and the production of reactive oxygen species (ROS) within the parasite. While many regulators of cell death are lacking in Leishmania, several other proteins that have been demonstrated to serve as potential regulators of cell death have been discovered. One of the well-identified cell death regulators is endonuclease G (LdBPK_100600.1). Under normal conditions, this enzyme is localized in the mitochondrion of promastigotes as well as of axenic amastigotes where it appears to be essential for parasite survival. Different apoptotic stimuli, such as H2O2 or miltefosine, induce EndoG translocation from the mitochondrion to the nucleus where it forms a complex with the Flap endonuclease-1 (FEN-1) and the nuclease TatD. This results in DNA degradation through endonuclease and exonuclease activities, notably on single stranded DNA. Metacaspases are cysteine peptidases that have been identified in fungi, plants, and protozoa. They possess a caspase-like domain homologous to the p20 subunit of caspases, with the catalytic diad histidine/cysteine, and a C-terminal proline-rich domain homologous to the p10 subunit of caspases.In L. donovani, two metacaspases have been described: LdMC1 (DQ367530) and LdMC2 (DQ367531). LdMCA has no caspase-like activity but it possesses trypsine-like activity towards arginine at position P1 that depends on the catalytic diad H147/C202.Since metacaspase is not directly involved in the regulation of apoptosis, it is not suitable to use it as an apoptotic marker. Further research is necessary in this area to clarify its role. In the context of cell death research of Leishmania, it is important to identify cell death regulators that may act as pro-apoptotic or anti-apoptotic factors. Also, it is important to understand the different types of cell death pathways that are functional under different kinds of cell death stimuli. Our research identified a novel regulator of cell death in L. donovani, a homolog of the Bax Inhibitor-1 gene, termed LdBI-1. Although LdBI-1 shows limited sequence similarity to its human counterpart, it retains conserved transmembrane domains. Using CRISPR/CAS9, we created a LdBI-1 disrupted cell line. Our results show that LdBI-1 is not essential for parasite survival, but its disruption increases susceptibility to cell death stimuli, indicating a protective role. Overexpression of LdBI-1 was found to negatively regulate autophagy which is crucial for L. donovani cellular fitness. The disruption also impaired mitochondrial bioenergetics and calcium homeostasis, reducing the infectivity of promastigotes. We observed a negative correlation between LdBI-1 expression and the autophagy-related gene ATG8, highlighting LdBI-1's role in autophagy modulation. LdBI-1 disrupted cells failed to transition to the amastigote form under stress, underscoring its importance in the parasite's life cycle. These findings suggest that LdBI-1 is vital for regulating autophagy, cell death, and mitochondrial function, making it a potential target for antileishmanial therapies.