MOLECULAR MECHANISMS TO ATTENUATE LEISHMANIA PARASITE

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，潜伏感染可在感染艾滋病毒后重新激活。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疫情地区的其他旅行者尤其感到担忧。为了寻找新的方法来控制这种病原体，我们已经开始研究寄生虫从无毒（promastigote）到有毒（amastigote）的分化机制。这种转变是由发生在寄生虫体内的基因变化控制的。我们开发了一个模拟利什曼原虫从原鞭毛体到无尾鞭毛体分化的培养系统。利用这种培养系统，我们已经鉴定和克隆了差异表达基因。对这两个基因的基因组克隆进行了分离和全序列分析，发现一个具有482个氨基酸的开放阅读框（ORF），主要表达于promastigotes，另一个具有902个氨基酸的开放阅读框（ORF），主要表达于amastigotes。数据库搜索表明，没有从其他生物中克隆出这些蛋白的同源物。调控分化的遗传变化应该先于形态变化，因此我们试图研究在寄生虫分化开始后立即表达的差异基因的表达，并将其描述为即时早期基因。分别在诱导分化前和诱导分化后的时间间隔（1、2、4、8、16、24和48小时）采集RNA样本。这些rna与四个差异调控基因杂交，包括上面提到的两个。结果表明，在分化过程中被关闭的基因在短短1小时内就关闭了；而上调的基因则在16小时后逐渐增加到峰值。因此，直接早期基因可以作为阻断利什曼原虫分化过程的靶点。此外，这些基因表达的差异动力学表明，这些RNA样本可以用来生成探针，以筛选分化开始后立即表达的其他新基因文库。我们已经开始合作筛选L. major微阵列的直接早期基因，并已开始开发L. donovani微阵列。这些新基因可能为阻断寄生虫分化提供药物靶点，或用于制造寄生虫减毒疫苗。

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 and latent infections can reactivate upon acquisition of HIV. 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 the 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. Using this culture system we have identified and cloned differentially expressed genes. The genomic clones for two such genes were isolated and complete sequence analysis revealed one with an open reading frame (ORF) of 482 amino acids predominantly expressed in promastigotes and a second with a 902 amino acid ORF predominantly expressed in amastigotes. Database searches indicated that no homologues of these proteins have been cloned from other organisms. The genetic changes that regulate differentiation should preceed the morphological changes, therefore we are attempting to study the expression of differentially expressed genes immediately after the parasite differentiation is initiated, and thus described as 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 hybridized with four differentially regulated genes including the two that were mentioned above. The results suggested that genes which were turned off over the course of differentiation did so in as little as 1 hour; while the genes that are up-regulated, gradually increased to their peak by 16 hours. Thus the immediate early genes could be used as targets for blocking the process of differentation in Leishmania. Further, the differential kinetics of expression of these genes indicates that these RNA samples can be used to generate probes to screen libraries for other novel genes which are expressed immediately after the initiation of differentiation. We have initiated collaboration to screen a L. major microarray for immediate early genes and have begun development of a L. donovani microarray. These novel genes may provide drug targests to block parasite differentiation or be useful to create attenuated parasite vaccines.