TRANSCRIPTOME ANALYSIS OF LEISHMANIA PANAMENSIS DEVELOPMENTAL STAGES

描述(申请人提供)：本申请集中在利什曼原虫属的锥虫，利什曼原虫是利什曼病的病原体。20多种利什曼原虫能够在人类身上感染和致病，它们的流行病学特征涉及相当大范围的宿主和昆虫媒介，这表明它们具有特定的适应能力。利什曼病是仅次于疟疾的第二大寄生虫杀手，对社会和经济条件的恶化具有重大影响。目前还没有可用的疫苗，目前的治疗方案有严重的局限性。利什曼原虫(Viannia)亚属代表了利什曼原虫的一个独特的系统发育和进化群。尽管这些寄生虫在中南美洲流行，并与皮肤和粘膜皮肤病有关，但它们的毒力机制和发病机制尚未得到广泛研究。利什曼原虫的生活史在形态上有两种截然不同的形式：通过受感染的雌性沙蝇媒介的叮咬，鞭毛化的准环前鞭毛虫开始向脊椎动物宿主传播。在哺乳动物宿主中，利什曼原虫是专性的细胞内寄生虫，在巨噬细胞的晚期内切/溶酶体内复制，并可持续数月或数年。这一建议建立在对L.(V.)巴拿马虫慢性感染，以及前鞭毛体和感染性无鞭毛体的体外培养。我们建议在分子水平上通过使用深度测序技术(RNA-Seq)监测转录情况并通过“核糖体图谱”检查mRNA的翻译特征来研究差异。传统上，DNA微阵列被用来调查基因表达的全球模式和变化。最近的开创性实验引入了RNA-Seq，这是一种采用下一代测序的方法，即大规模的cDNA并行测序。我们还将通过实施新建立的“核糖体图谱”方法来探索潜在的翻译调控机制(S)，该方法将核糖体在信使核糖核酸上的占有率与相对信使核糖核酸的翻译效率联系起来。建议的研究将进一步加深我们对L.(V.)泛美利什曼原虫的生物学，增强了我们对万寿菊亚属基因组编码能力的了解，有助于确定翻译调控在利什曼原虫中可能产生的影响，并有可能识别巨噬细胞感染的重要基因。识别的基因可能是治疗和疾病控制的潜在免疫治疗和药理学靶点。

DESCRIPTION (provided by applicant): This application focuses on trypanosomatid protozoa of the genus Leishmania, the causative agent of leishmaniasis. More than 20 species of Leishmania parasites are able to infect and cause disease in humans and their epidemiological features involve a considerable range of reservoir hosts and insect vectors, suggesting specific adaptations. Leishmaniasis is the second biggest parasitic killer following malaria, with a significant impact on deterioration of social and economic conditions. There is no vaccine available and current therapeutic regimens have serious limitations. The Leishmania (Viannia) subgenus represents a distinct phylogenetic and evolutionary group of Leishmania. Despite the prevalence of these parasites in Central and South America and their association with cutaneous and mucocutaneous disease, their mechanisms of virulence and pathogenesis have not been extensively studied. The Leishmania life cycle entails two morphologically distinct forms: transmission to the vertebrate host is initiated by flagellated metacyclic promastigotes via the bite of an infected female sandfly vector. In the mammalian host, Leishmania are obligate intracellular parasites that replicate within late endosomal/lysosomal compartments of macrophages and may be persistent for months or years. This proposal builds on the availability of a robust murine model for L. (V.) panamensis chronic infection, as well as promastigote and infectious axenic amastigote in vitro cultures. We propose to study differentiation at the molecular level by monitoring the transcriptional landscape using deep-sequencing technologies (RNA-Seq) and examining translation features of mRNA by "ribosome profiling". Traditionally, DNA microarrays were used to survey global patterns and changes in gene expression. Recent pioneering experiments have introduced RNA-Seq, a methodology employing next generation sequencing, i.e. massive parallel sequencing of cDNA. We will also explore potential translation regulatory mechanism(s) by implementing the newly-established "ribosome profiling" methodology, which correlates ribosome occupancy on mRNA with relative mRNA translation efficiencies. The proposed studies will further our understanding of L. (V.) panamensis biology, enhance our understanding of the genome coding capacity of the subgenus Viannia, contribute to determine the possible impact of translational control in Leishmania and have the potential to identify genes important for macrophage infection. The identified genes could be of interest as potential immunotherapeutic and pharmacological targets for treatment and disease control.