Disuccinyl Betulin Triggers Metacaspase-Dependent Endonuclease G-Mediated Cell Death in Unicellular Protozoan Parasite Leishmania donovani

Abstract

ABSTRACTThe unicellular organismLeishmaniaundergoes apoptosis-like cell death in response to external stress or exposure to antileishmanial agents. Here, we showed that 3-O,28-O-disuccinyl betulin (DiSB), a potent topoisomerase type IB inhibitor, induced parasitic cell death by generating oxidative stress. The characteristic feature of the death process resembled the programmed cell death (PCD) seen in higher eukaryotes. In the current study, the generation of reactive oxygen species (ROS), followed by the depolarization of mitochondrial membrane potential (ΔΨm), caused a loss in ATP production inLeishmaniaparasites. This further gave positive feedback to produce a large amount of ROS, which in turn caused oxidative DNA lesions and genomic DNA fragmentation. The treatment of promastigotes with DiSB induced high expression levels of metacaspase protein that led to cell death in this unicellular organism. The PCD was insensitive to benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk), suggesting that the death process was not associated with the activation of caspases. DiSB treatment translocatedLeishmania donovaniendonuclease G (LdEndoG) from mitochondria to the nucleus, which was responsible for the DNA degradation process. Conditional antisense knockdown ofL. donovanimetacaspase (LdMC), as well as EndoG, -subverted death of the parasite and rescued cell cycle arrest in G1phase. The present study on the effector molecules associated with the PCD pathway of the parasite should help to manifest the mechanisms of PCD and also might be exploited in antileishmanial chemotherapy.