CONTROL OF LEISHMANIA BY PROGRAMMED CELL DEATH (APOPTOSI

利什曼原虫会引起人类疾病，临床症状从自我愈合的皮肤损害到致命的内脏感染。此外，在流行地区，感染艾滋病毒的人特别容易感染利什曼原虫。对这种寄生虫的细胞生物学和致病机制缺乏了解，使得控制这一严重的世界性健康风险的任务变得困难。在国内，这尤其令美国军事人员、他们的家人和其他前往或居住在疫情地区的旅行者感到担忧。为了找到控制这种病原体的新方法，我们开始了了解利什曼原虫细胞程序性死亡(细胞凋亡)机制的研究。程序性细胞死亡(PCD)是一种以前仅与多细胞生物体有关的现象，用于调节生长和发育。有人认为，像锥虫这样的单细胞生物，为了促进和维持种群内的遗传稳定，应该通过类似PCD的途径去除不适合的细胞。在本研究中，我们证明了PCD存在于单细胞原生动物寄生虫杜氏利什曼原虫中，杜氏利什曼原虫是内脏利什曼病的病原体。利什曼原虫的生活史涉及两种主要的发育形式(前鞭毛体和无鞭毛体)。我们报道了PCD在前鞭毛体和无菌无鞭毛体中都是在它们达到培养的静止阶段时启动的。此外，五价锑和两性霉素B等药物可以在这些细胞中诱导PCD，这两种药物是治疗人类内脏利什曼病的标准疗法。然而，这两种药物的处理在诱导前鞭毛体和无菌无鞭毛体的PCD方面显示出差异。静止期和药物处理细胞中的PCD与核固缩、DNA梯带形成和TUNEL阳性反应有关。使用含有四肽DEVD的底物，我们证明了在PCD诱导时诱导了caspase3样酶的活性。出乎我们意料的是，在某些高等真核分裂细胞中，已知的细胞死亡诱导剂放线菌亚胺抑制了前鞭毛体细胞死亡的诱导，而不是无菌无鞭毛体细胞死亡的诱导，这是因为缺乏caspase样活性和DNA梯带的缺失。另一方面，常用于诱导哺乳动物细胞死亡的抗生素G418可引起无菌无鞭毛体的PCD，但不能显著诱导前无鞭毛体的PCD。因此，这表明利什曼原虫的两种细胞类型对细胞死亡信号的反应不同，可能对治疗这种疾病构成挑战。在单细胞生物体中，如利什曼原虫，正在研究PCD的诱导机制和参与PCD途径的因素。

The Leishmania parasite causes human disease with clinical symptoms ranging from-self healing cutaneous lesions to a fatal visceral infection. Additionally, in endemic areas, people infected with HIV are especially prone to Leishmania infection. The lack of understanding of cell biology and pathogenic mechanisms of this parasite makes the task of controlling this grave, worldwide health risk difficult. Closer to home it is particularly of concern to U.S. military personnel, their families and other travellers visiting or living in the endemic areas. To find novel methods for control of this pathogen,we have initiated study to understand the mechanism of programmed cell death (apoptosis) in Leishmania. Programmed Cell Death (PCD) is a phenomenon previously associated exclusively with multicellular organisms to regulate growth and development. It has been argued that unicellular organisms such as trypanosomatids, in order to promote and maintain genetic stability within the population, should get rid of unfit cells via a pathway similar to PCD. In the present study we have demonstrated that PCD exists in a unicellular protozoan parasite Leishmania donovani, a causative agent of visceral leishmaniasis. Two major developmental forms (promastigote and amastigote) are involved in the Leishmania life cycle. We report that PCD is initiated in both promastigotes and axenic amastigotes when they reach stationary phase in culture. In addition, PCD can be induced in these cells by drugs, pentavalent antimony [Pentostam] and amphotericin B, which are standard treatments for human visceral leishmaniasis. However, the treatment with these two drugs revealed differences in the induction of PCD in promastigotes and axenic amastigotes. The PCD in stationary phase and drug-treated cells is associated with nuclear condensation, DNA ladder formation and positive TUNEL assay. Using a substrate containing the tetra-peptide DEVD, the cleavage site for caspase 3-like proteases, we demonstrated that a caspase-like activity is induced at the time of PCD induction. To our surprise cycloheximide, a known inducer of cell death in certain higher eukaroytic cells, suppressed the induction of cell death in promastigotes and not in axenic amastigotes as judged by the lack of caspase-like activity and absence of DNA ladder. On the other hand antibiotic drug, geneticin (G418) commonly used to induce cell death in mammalian cells induced PCD in axenic amastigotes and was not able to significantly induce PCD in promastigotes. Thereby, suggesting that the two cell types of Leishmania respond differently to cell death signals and could present a challenge to treat this disease. Studies are on going to understand the mechanism of PCD induction and to characterize factors that are involved in the PCD pathway in unicellular organism such as Leishmania.