MOLECULAR MECHANISMS TO ATTENUATE LEISHMANIA PARASITE

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菌的新方法，我们开始了了解寄生虫从无毒(前鞭毛体)向强(无鞭毛体)分化的机制的研究。这种转变是由寄生虫中发生的基因变化控制的。我们开发了一种模拟利什曼原虫从前鞭毛体向无鞭毛体分化的培养系统。我们合理地认为遗传变化应该在形态变化之前进行，因此我们试图鉴定在寄生虫分化开始后立即表达的基因，并将其描述为即刻早期基因。为了鉴定早期基因，在诱导分化前和分化后(1、2、4、8、16、24和48小时)收集RNA样本。这些RNA的特征首先是与三个先前鉴定的基因杂交，这些基因的表达在前无鞭毛体和无鞭毛体之间存在差异调节。结果表明，在分化过程中被关闭的基因反应迅速，而上调的基因上调的动力学较慢。这些基因表达的差异动力学表明，这些RNA样本可以用来产生探针，以筛选在分化开始后立即表达的新基因的文库。这些新基因可能会提供靶标来阻断寄生虫的分化，并可用于减毒寄生虫疫苗。

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 parasite differentiation from avirulent(promastigote)to virulent(amastigote)form. This transformation is controlled by the genetic changes which take place in the parasite. We have developed a culture system which mimics the differentiation of Leishmania parasite from promastigotes to amastigotes. We rationalized that the genetic changes should proceed the morphological changes, therefore we made attempted to identify genes that are expressed immediately after the parasite differentiation is initiated, and are described as immediate early genes. To identify immediate early genes, RNA samples were collected before and at time intervals (1, 2, 4, 8, 16, 24, and 48 hrs) after induction of differentiation from promastigotes and amastigotes. These RNAs were first characterized by hybridization with three previuosly identified genes whose expression is differentially regulated between the pro-and the amastigotes. The results suggested that genes which were turned off over the course of differentiation did so rapidly while the genes that are up-regulated, the kinetics of up-regulation is slower. The differential kinetics of expression of these genes indicates that these RNA samples can be used to generate probes to screen libraries for novel genes which are expressed immediately after the initiation of differentiation. These novel genes may provide targests to block parasite differentiation and be useful for attenuated parasite vaccines.