Transcriptome analysis of Entamoeba development using RNA-sequencing

描述（由申请人提供）：溶组织内阿米巴每年感染超过5亿人，是人类寄生虫死亡的主要原因。E.溶组织菌开始于包囊的摄入，包囊是一种非分裂形式，由于保护性细胞壁而能够在环境中存活。摄入后，囊肿进入肠道，在那里它经历脱囊以产生增殖的滋养体形式，这能够引起疾病。有些滋养体会包囊，使它们排出体外，继续感染新的宿主。不幸的是，虽然发育级联是必不可少的病原体的传播和疾病的原因，研究到这一过程的调控一直受到阻碍，缺乏一个良好的体外系统研究大肠杆菌。溶组织分期转换。我们将利用已建立的方法在E. invadens是一种与E.其中两种过程的高水平可以在体外实现。我们建议建立一个E.使用RNA转录物的深度测序（RNA-seq）在整个生命周期期间（包囊形成和脱囊形成）对入侵体进行测序。该方法利用了下一代测序技术，并且先前已用于在许多系统中开发转录组。将对滋养体、包囊形成期间（8、12、24和48 h）、成熟包囊（72 h）以及早期（30 min、2 h、8 h）和晚期（48 h）脱囊寄生虫进行RNA-seq。序列数据将被映射到基因组，并通过生物信息学方法进行分析，以确定发育调控基因。关于基因注释和可变剪接的信息也可以从RNA-seq方法中收集。数据将存放在EuPathDB中，社区可以随时使用。这项工作将提供深入了解内阿米巴的脱囊和成囊的调节，并为未来研究这些重要过程的机制提供丰富的途径。

DESCRIPTION (provided by applicant): Entamoeba histolytica infects over 500 million people annually and is a leading cause of parasitic death in humans. The infectious cycle of E. histolytica begins with the ingestion of the cyst, a non-dividing form that is able to survive in the environment due to a protective cell wall. After ingestion, the cyst travels to the intestine, where it undergoes excystation to produce the proliferative trophozoite form, which is capable of causing disease. Some trophozoites will encyst, allowing them to be excreted and to go on to infect new hosts. Unfortunately, although the developmental cascade is essential to pathogen transmission and disease causation, research into the regulation of this process has been hampered by the lack of a good in vitro system for studying E. histolytica stage conversion. We will take advantage of established methods for inducible encystation and excystation in E. invadens, a reptilian parasite highly related to E. histolytica in which high levels of both processes can be achieved in vitro. We propose to develop a transcriptome of E. invadens during the entire life cycle (encystation and excystation) using deep sequencing of RNA transcripts (RNA-seq). This method takes advantage of next generation sequencing technologies and has previously been used to develop transcriptomes in numerous systems. RNA-seq will be performed for trophozoites, during cyst formation (8, 12, 24 and 48 h), mature cysts (72h), and early (30min, 2h, 8h) and late (48h) excysting parasites. Sequence data will be mapped to the genome and analyzed by bioinformatics methods to identify developmentally regulated genes. Information on gene annotation and alternative splicing can also be gleaned from the RNA-seq approach. Data will be deposited in EuPathDB where it will readily be available to the community. This work will provide insight into the regulation of excystation and encystation in Entamoeba and generate a wealth of avenues for future studies on the mechanisms of these vital processes.