Transcriptome analysis of Entamoeba development using RNA-sequencing

描述(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。溶组织埃希氏菌的感染循环始于对包囊的摄取，包囊是一种不分裂的形式，由于有保护性的细胞壁，它能够在环境中生存。在摄取后，包囊进入肠道，在那里经历排出，产生增殖性滋养体形式，这可能会导致疾病。一些滋养体会被包囊，使它们被排泄出来，并继续感染新的宿主。不幸的是，尽管发育级联对于病原体的传播和疾病的发生是必不可少的，但由于缺乏良好的体外系统来研究溶组埃希氏菌的阶段转换，对这一过程的调控的研究一直受到阻碍。我们将利用已建立的方法在入侵埃希氏菌中进行诱导囊化和激化，这是一种与组织溶解埃希氏菌高度相关的爬行动物寄生虫，在体外可以实现这两个过程的高水平。我们建议使用RNA转录本的深度测序(RNA-seq)在整个生命周期(包囊化和激发化)中建立入侵E.inadens的转录组。这种方法利用了下一代测序技术，以前曾被用于在许多系统中开发转录本。对于滋养体，在包囊形成期间(8、12、24和48小时)、成熟包囊(72小时)以及排出寄生虫的早期(30分钟、2小时、8小时)和晚期(48小时)将进行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，以识别发育调节基因。关于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。数据将存放在EuPathDB中，供社区随时使用。这项工作将对内阿米巴的激活和包囊化的调控提供洞察力，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.