CONTROL OF LEISHMANIA BY PROGRAMMED CELL DEATH (APOPTOSI

利什曼原虫引起人类疾病，其临床症状从自愈皮肤病变到致命的内脏感染不等。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。由于缺乏对这种寄生虫的细胞生物学和致病机制的了解，控制这种严重的全球健康风险的任务变得困难。在美国本土，美国军事人员、他们的家人以及访问或居住在流行地区的其他旅行者尤其值得关注。为了找到控制这种病原体的新方法，我们已经开始研究利什曼原虫程序性细胞死亡（凋亡）的机制。程序性细胞死亡（PCD）是一种以前只与多细胞生物有关的调节生长发育的现象。有人认为，单细胞生物，如锥虫，为了促进和维持群体内的遗传稳定性，应该通过类似于PCD的途径去除不适合的细胞。在目前的研究中，我们已经证明PCD存在于内脏利什曼病的病原体——多诺瓦利什曼原虫的单细胞原生动物寄生虫中。利什曼原虫的生命周期涉及两种主要的发育形式（promastigote和amastigote）。我们报道了PCD在原无性系和无性系无性系中都是在培养达到固定阶段时启动的。此外，药物、五价锑（Pentostam）和两性霉素B （amphotericin B）可以在这些细胞中诱导PCD，这是人类内脏利什曼病的标准治疗方法。然而，用这两种药物治疗显示在诱导PCD在promastigotes和axenic amastigotes的差异。固定期和药物处理细胞的PCD与核凝聚、DNA阶梯形成和TUNEL检测阳性有关。使用含有四肽DEVD （caspase 3样蛋白酶的裂解位点）的底物，我们证明了在PCD诱导时可以诱导caspase 3样活性。令我们惊讶的是，环己亚胺，一种已知的在某些高级真核细胞中细胞死亡的诱导剂，抑制了原无性系细胞死亡的诱导，而不是在无性系细胞中，这是由缺乏caspase样活性和缺乏DNA阶梯来判断的。另一方面，在哺乳动物细胞中常用的诱导细胞死亡的抗生素药物遗传素（G418）在无性系无性系中诱导PCD，而在promastigotes中不能显著诱导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提出挑战。研究将继续了解PCD诱导的机制，并表征单细胞生物（如利什曼原虫）中PCD途径所涉及的因素。

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.