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。溶组织芽胞杆菌的感染周期从摄入囊肿开始，囊肿是一种不分裂的形式，由于有保护性细胞壁，它能够在环境中生存。摄入后，囊肿进入肠道，在那里它经历了排泄，产生增生的滋养体形式，这是能够引起疾病的。一些滋养体会形成囊体，排出体外，继续感染新的寄主。不幸的是，尽管发育级联对病原体传播和疾病病因至关重要，但由于缺乏良好的体外系统来研究溶组织芽胞杆菌阶段转化，对这一过程的调控研究受到了阻碍。我们将利用已建立的方法，在E. invadens（一种与E. histolytica高度相关的爬行类寄生虫）中诱导胞内和胞外，在体外可以实现高水平的这两个过程。我们建议利用RNA转录本的深度测序（RNA-seq）来开发入侵芽孢杆菌在整个生命周期（胞内和胞外）的转录组。该方法利用了下一代测序技术，并已在许多系统中用于开发转录组。将对滋养体、囊肿形成（8、12、24和48小时）、成熟囊肿（72小时）以及早期（30分钟、2小时、8小时）和晚期（48小时）排出的寄生虫进行rna测序。序列数据将被映射到基因组中，并通过生物信息学方法进行分析，以确定发育调节基因。基因注释和选择性剪接的信息也可以从RNA-seq方法中收集到。数据将存储在EuPathDB中，以便社区随时可用。这项工作将为内阿米巴原虫胞缩和胞缩的调控提供深入的见解，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.