Molecular Mechanism of Leishmaniasis

摘要：利什曼原虫引起人类疾病（利什曼病），其临床症状从自愈的皮肤病变到致命的内脏感染。由于缺乏对利什曼原虫致病机制的了解，在世界范围内，特别是对美国军人及其家属和在流行地区居住或旅行的游客构成了严重的公共卫生风险。作为了解利什曼原虫发病分子机制的第一步，我们已经开始分析寄生虫生命周期中从无毒形式转化为有毒形式的过程，并针对这些过程控制寄生虫的生长。细胞器的独特结构和功能特征，如细胞骨架和鞭毛器，在利什曼原虫的传染性中所起的作用仍然不清楚。为了确定控制生长的基因，我们首次从L. donovani中分离到一个编码中心蛋白的基因。中心体蛋白是钙结合的细胞骨架蛋白，是中心体复制或分离所必需的。中心蛋白mRNA和蛋白水平在培养过程中呈指数增长，在固定期下降到较低水平。n端缺失中心蛋白的表达显著降低了寄生虫的生长速度，并且发现在G2/M期比对照细胞明显更多的细胞被阻滞。这些研究表明，中心蛋白可能在利什曼原虫的生长中起功能作用。程序性细胞死亡（PCD）是细胞生物学的重要组成部分，被认为不仅仅是为了调节多细胞生物的生长和发育。然而，最近的研究表明PCD在单细胞生物中存在，并假设PCD在单细胞生物的生物学中具有功能作用。据推测，为了促进和维持种群内的克隆性，锥虫必须发展出一种利他机制来控制生长。我们想探究PCD在利什曼原虫中是否存在，如果存在，抗利什曼原虫药物是否能诱导PCD。这将为开发未来的抗利什曼病药物提供机会。在目前的研究中，我们已经证明了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程序性细胞死亡（PCD）的一些特征。我们报道了PCD在固定期培养中启动，在抗利什曼药物（Pentostam和两性霉素B）治疗后，在无性系无性系和promastigotes的积极生长培养中启动。然而，这两种细胞类型对抗利什曼药物的反应不同。因此，这些研究为了解利什曼原虫的发病机制以及如何利用利什曼原虫来控制寄生虫的生长提供了基础。

Summary: Leishmania parasite causes human disease (Leishmaniasis) with clinical symptoms ranging from self healing cutaneous lesions to fatal visceral infection. The lack of understanding of the mechanism by which Leishmania parasite causes disease poses a serious public health risk worldwide and in particular for U.S. military personnel their families and tourists either living or travelling in endemic areas. As a first step towards understanding the molecular mechanism of Leishmania pathogenesis, we have began to analyze the processes that are involved in parasite life cycle in transformation from an avirulent to virulent form and to target such processes to control growth of the parasite. The role of unique structural and functional features of organelles, like the cytoskeleton and the flagellar apparatus, in infectivity of Leishmania, is still obscure. In order to identify genes that control growth, we have isolated for the first time a gene encoding for centrin from L. donovani. Centrin is calcium binding cytoskeletal protein essential for centrosome duplication or segregation. The levels of centrin mRNA and protein were high during the exponential growth of the parasite in culture and declined to a low level in the stationary phase. Expression of N-terminal deleted centrin in the parasite significantly reduces its growth rate and it was found that significantly more cells are arrested in the G2/M stage than in control cells. These studies indicate that centrin may have a functional role in Leishmania growth. Programmed cell death (PCD) is an essential part of cell biology and is thought to have evolved not only to regulate growth and development in multicellular organisms. However, recent studies, which showed the existence of PCD in unicellular organisms, have postulated a functional role of PCD in the biology of unicellular organisms. It has been postulated that in order to promote and maintain clonality within the population, the Trypanosomatids must have developed an altruistic mechanism to control growth. We wanted to explore whether PCD exists in Leishmania and if so can antiLeishmanial drugs induce it. This will provide an opportunity to develop future antiLeishmanial drugs. In the present study we have demonstrated some features characterizing programmed cell death (PCD) in the unicellular protozoan parasite Leishmania donovani, the causative agent of visceral Leishmaniasis. We report that PCD is initiated in stationary phase cultures of promastigotes and both in actively growing cultures of axenic amastigotes and promastigotes upon treatment with anti Leishmanial drugs (Pentostam and amphotericin B). However, the two cell types respond to antiLeishmanial drugs differently. Therefore, these studies provide the basis for understanding the mechanism of Leishmania pathogenesis and how it can be exploited in order to control parasite growth.