MOLECULAR MECHANISMS TO ATTENUATE LEISHMANIA PARASITE

利什曼原虫引起人类疾病，临床症状从自愈性皮肤损伤到致命的内脏感染。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，使得控制这种严重的全球健康风险的任务变得困难。在离家更近的地方，美国军事人员、他们的家人和访问或居住在流行地区的其他旅行者尤其关注这一问题。为了找到控制这种病原体的新方法，我们已经开始研究寄生虫从无毒力（前鞭毛体）到有毒力（无鞭毛体）形式的分化机制。这种转变是由寄生虫体内发生的遗传变化控制的。我们已经开发了一个培养系统，模仿利什曼原虫的分化，从前鞭毛体无鞭毛体。我们合理化的遗传变化应该进行形态学的变化，因此，我们试图确定基因表达后立即寄生虫分化开始，并被描述为立即早期基因。为了鉴定立即早期基因，在诱导前鞭毛体和无鞭毛体分化之前和之后的时间间隔（1、2、4、8、16、24和48小时）收集RNA样品。这些RNA首先通过与三个先前鉴定的基因杂交来表征，所述基因的表达在原无鞭毛体和无鞭毛体之间差异调节。结果表明，在分化过程中关闭的基因如此迅速，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样品可用于产生探针以筛选文库中分化开始后立即表达的新基因。这些新的基因可以提供阻断寄生虫分化的靶点，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.