Chowdhury Sayan, Mukherjee Tulika, Chowdhury Somenath Roy, Sengupta Souvik, Mukhopadhyay Sibabrata, Jaisankar Parasuraman, Majumder Hemanta K
Molecular Parasitology Laboratory, Indian Institute of Chemical Biology, Kolkata, India.
Antimicrob Agents Chemother. 2014;58(4):2186-201. doi: 10.1128/AAC.02193-13. Epub 2014 Jan 27.
The unicellular organism Leishmania undergoes 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 in Leishmania parasites. 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 translocated Leishmania donovani endonuclease G (LdEndoG) from mitochondria to the nucleus, which was responsible for the DNA degradation process. Conditional antisense knockdown of L. donovani metacaspase (LdMC), as well as EndoG, -subverted death of the parasite and rescued cell cycle arrest in G1 phase. 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.
单细胞生物利什曼原虫会因外部应激或接触抗利什曼原虫药物而经历类似凋亡的细胞死亡。在此，我们表明3 - O，28 - O - 二琥珀酰桦木醇（DiSB），一种有效的拓扑异构酶IB型抑制剂，通过产生氧化应激诱导寄生虫细胞死亡。死亡过程的特征类似于在高等真核生物中看到的程序性细胞死亡（PCD）。在当前研究中，活性氧（ROS）的产生，随后线粒体膜电位（ΔΨm）的去极化，导致利什曼原虫寄生虫的ATP生成减少。这进一步产生正反馈以产生大量ROS，进而导致氧化性DNA损伤和基因组DNA片段化。用DiSB处理前鞭毛体诱导了metacaspase蛋白的高表达水平，导致这种单细胞生物的细胞死亡。PCD对苄氧羰基 - 缬氨酸 - 丙氨酸 - 天冬氨酸（OMe） - 氟甲基酮（zVAD - fmk）不敏感，表明死亡过程与半胱天冬酶的激活无关。DiSB处理使杜氏利什曼原虫内切核酸酶G（LdEndoG）从线粒体转移到细胞核，这负责DNA降解过程。有条件地反义敲低杜氏利什曼原虫metacaspase（LdMC）以及EndoG，可颠覆寄生虫的死亡并挽救G1期的细胞周期停滞。目前关于与寄生虫PCD途径相关的效应分子的研究应有助于阐明PCD的机制，并且也可能用于抗利什曼原虫化疗。
