TRANSCRIPTOME ANALYSIS OF LEISHMANIA PANAMENSIS DEVELOPMENTAL STAGES

描述（由申请人提供）：本申请关注利什曼原虫属的锥虫原虫，利什曼病的病原体。超过20种利什曼原虫能够感染人类并引起疾病，其流行病学特征涉及相当大范围的储库宿主和昆虫载体，表明特定的适应性。利什曼病是继疟疾之后的第二大寄生虫杀手，对社会和经济状况的恶化产生重大影响。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）亚属代表利什曼原虫的一个独特的系统发育和进化组。尽管这些寄生虫在中美洲和南美洲的流行及其与皮肤和粘膜皮肤疾病的关系，其毒力和发病机制尚未得到广泛研究。利什曼原虫的生命周期需要两种形态上不同的形式：通过感染雌性白蛉媒介的叮咬，由有鞭毛的后环前鞭毛体启动向脊椎动物宿主的传播。在哺乳动物宿主中，利什曼原虫是专性细胞内寄生虫，在巨噬细胞的晚期内体/溶酶体隔室内复制，并且可能持续数月或数年。这一建议建立在一个强大的鼠模型的可用性L。（五）Panamensis慢性感染，以及前鞭毛体和感染性无鞭毛体体外培养。我们建议通过使用深度测序技术（RNA-Seq）监测转录景观并通过“核糖体分析”检查mRNA的翻译特征来研究分子水平上的分化。传统上，DNA微阵列用于调查基因表达的全球模式和变化。最近的开创性实验已经引入了RNA-Seq，这是一种采用下一代测序的方法，即cDNA的大规模平行测序。我们还将通过实施新建立的“核糖体分析”方法来探索潜在的翻译调控机制，该方法将核糖体在mRNA上的占据与相对mRNA翻译效率相关联。 这些研究将进一步加深我们对L.（五）Panamensis生物学，增强我们对Viannia亚属基因组编码能力的理解，有助于确定利什曼原虫翻译控制的可能影响，并有可能鉴定对巨噬细胞感染重要的基因。所鉴定的基因可能作为潜在的免疫学和药理学靶点用于治疗和疾病控制。 公共卫生相关性：寄生原生动物是全球传染病的主要原因，因此是对公共卫生的最严重威胁之一。其中包括非洲锥虫，它是非洲锥虫病或人类昏睡病的病原体，也是牛、家猪和其他农场动物的一种消耗性和致命性疾病（Nagana），对非洲大陆大部分地区的经济造成深远影响。除非得到治疗，非洲昏睡病总是致命的;没有疫苗被批准，而且药物库非常有限，通常存在严重缺陷，如高毒性和新出现的耐药性

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. PUBLIC HEALTH RELEVANCE: Parasitic protozoa are a major cause of global infectious diseases and thus, represent one of the most serious threats to public health. Among these are African trypanosomes, the causative agents of African trypanosomiasis or sleeping sickness in humans and a wasting and fatal disease (Nagana) in cattle, domestic pigs and other farm animals causing a profound effect on the economy of much of the continent. Unless treated, African sleeping sickness is always fatal; no vaccine has been approved and there is a very limited arsenal of drugs with generally severe shortcomings, such as high toxicity and emerging resistance