Mechanism of Infectivity Acquisition in African Trypanosomes

描述(申请人提供)：解开人类病原体的生物学对于理解致病机制和识别在宿主中生存所必需的基因是基本的。这项应用的重点是原生动物寄生虫布鲁氏锥虫，这种寄生虫会在撒哈拉以南非洲的人和动物中造成毁灭性的疾病。没有疫苗，治疗药物副作用严重，疗效下降。布氏锥虫在哺乳动物宿主和食血采采蝇载体之间经历了一个复杂的生活史，其中包括细胞形态、代谢、信号通路和基因表达的变化。因此，这些寄生虫进化出适应能力，以便在采采蝇的肠道和唾液腺以及哺乳动物宿主的血液中生存。采采蝇在摄食受感染的宿主后，呈细长、中间和粗壮的血液形态。在苍蝇体内，中肠粗壮的形式分化为非感染性的原环状形式。感染性的重新获得是通过一个复杂的发育程序实现的，最终在采采期的唾液腺中产生具有感染性的后循环。尽管苍蝇体内锥虫发育的复杂本质已经被认识到一个多世纪，但分子机制仍然神秘，部分原因是采采蝇带来的实验挑战。我们发现，布鲁氏毛滴虫RNA结合蛋白RBP6在培养的非感染性原环状结构中的过表达启动了采采蝇的发育阶段，并最终产生了表达变异表面糖蛋白(VSG)外壳的感染性顺环体。我们的第一个目标将是描述RBP6的作用机制。由于RBP6似乎是触发一系列事件的“主调控因子”，因此确定RBP6的主要信使核糖核酸靶标至关重要。这将提供有关分化早期阶段所涉及的基因产物的关键信息，并为形成一个可检验的假说，即从原环状向上鞭毛体的转变过程中发生的细胞适应。我们的第二个目标将是提供上胚体和后周期的转录和蛋白质组学图谱，并破译后周期发生的生物学。最后，除了VSG外，新基因的发现将是破译感染力获得过程的重大突破，这些基因是元环分化所必需的。综上所述，我们的研究计划提供了独特的机会来阐明从原环到半环的分化程序，并揭示一种重要的RNA结合蛋白的作用方式。

DESCRIPTION (provided by applicant): Unraveling the biology of human pathogens is fundamental toward understanding mechanisms of pathogenesis and identifying genes essential for survival in the host. This application focuses on the protozoan parasite Trypanosoma brucei, which causes devastating diseases in humans and animals in sub-Saharan Africa. There are no vaccines, and therapeutic drugs have serious side effects and decreasing efficacy. T. brucei undergoes a complex life cycle between the mammalian host and the blood-feeding tsetse fly vector (Diptera: Glossinidae), which among others involves changes in cell morphology, metabolism, signaling pathways and gene expression. Consequently, these parasites have evolved adaptations to allow for their survival in both the gut and salivary glands of the tsetse fly, as well as in the bloodstream of their mammalian host. Upon feeding on an infected host, the tsetse fly takes up slender, intermediate and stumpy bloodstream forms. In the fly midgut stumpy forms differentiate into non-infectious procyclic forms. Reacquisition of infectivity is achieved through a complex developmental program that culminates in the tsetse salivary glands with the generation of infectious metacyclics. Although the intricate nature of trypanosome development in the fly has been recognized for more than a century, the molecular mechanisms are still mysterious, due in part to the experimental challenges posed by the tsetse fly. We found that overexpression of the T. brucei RNA-binding protein RBP6 in cultured non-infectious procyclic forms initiates differentiation into the developmental stages found in tsetse flies and culminates with the generation of infective metacyclics expressing the variant surface glycoprotein (VSG) coat. Our first goal will be to delineate the mechanism of action of RBP6. As RBP6 appears to be a "master regulator" triggering a cascade of events, it will be critical to identify the primary mRNA targets of RBP6. This will provide crucial information about gene products involved in the early stages of differentiation and for formulating a testable hypothesis about the cellular adaptations occurring in the transition from procyclic to epimastigotes forms. Our second goal will be to provide a transcriptomic and proteomic map of epimastigotes and metacyclics and to decode the biology of metacyclogenesis. Finally, the discovery of novel genes, besides VSG, required for metacyclic differentiation will be a major breakthrough toward deciphering how the process of acquisition of infectivity is brought about. Taken together our research plan provides unique opportunities to illuminate the differentiation program from procyclic to metacyclic and reveal the mode of action of an important RNA binding protein.