Detailed analysis of Cryptosporidium non-coding gene expression

摘要 隐孢子虫是一种人畜共患病的尖端复合体原生寄生虫，最近被确认为第二流行的寄生虫。 全球婴儿腹泻病原体(1-6)。它通过口腔-粪便途径传播，它可以在 免疫功能受损，因为没有被批准在这一人群中使用的疗法。分子 隐孢子虫的寄生虫学一直很缺乏，但随着体外培养技术的出现，这种情况正在改变。 培养系统、更好的寄生虫浓缩方案和新的遗传系统(8-11)。本项目建议书 重点分析了隐孢子虫的转录和宿主与病原菌的相互作用。具体来说，我们 建议生成候选分子，用于对最近的假设进行实验验证，即隐孢子虫 通过输出长的非编码RNA分子(LncRNA)改变宿主细胞的反式基因表达 宿主细胞基因的表达，从而影响宿主细胞的反应和发病机制。我们小组最近的工作和 其他人(7，9，12)揭示了微小隐孢子虫的转录组是复杂的，并承载了各种非编码 RNA，包括长的和短的。陈先明博士最近的工作是在宿主细胞中检测到微小弧菌的转录本 并证明了几种微小隐孢子虫的lncRNAs在引入时会影响宿主细胞的基因表达(13- 18)。可悲的是，由于与微小隐孢子虫合作的历史限制，转录数据严重缺乏 这种重要的病原体。建立在我们已证明的生成和共享微小隐孢子虫转录的记录基础上 数据，本项目建议系统地表征微小隐孢子虫编码和非编码RNA，包括 用PacBio从寄生虫的几个发育阶段、感染前和感染后(体外)提取小RNA ISO-Seq(19)确定研究小RNA的完整转录本和Illumina技术作为目标1。 重点研究宿主与病原菌的相互作用。生物信息学将用于识别选定的lncRNA 可能需要在陈博士的实验室进行宿主细胞效应或基因敲除的实验测试的分子 通过鲍里斯·斯特里潘博士实验室的CRISPR。此外，能够产生数十亿次读取的NovaSeq(20) 将用于体外严重感染(0-48小时)和未感染的宿主细胞的深层RNA测序 描述感染后三个月宿主和病原体的基因转录变化 时间点。必须确定在寄主病原体中可能起作用的转录反应的特征。 互动。它们将揭示寄生虫生存机制、发病机制发展的新见解 并揭示了未来治疗干预措施亟需的新目标(21-24)。

ABSTRACT Cryptosporidium is a zoonotic apicomplexan protist parasite recently identified as the second most prevalent diarrheal pathogen of infants globally (1-6). It is spread via an oral-fecal route and it can be lethal in the immunocompromised since there are no therapeutics approved for use in this population. The molecular parasitology of Cryptosporidium has been lacking but this situation is changing with the recent advent of in vitro culture systems, better parasite enrichment protocols and a nascent genetic system (8-11). This project proposal focuses on an analysis of Cryptosporidium transcription and the host-pathogen interaction. Specifically, we propose to generate candidate molecules for experimental testing of the recent hypothesis that Cryptosporidium alters host-cell gene expression in trans, via the export of long non-coding RNA molecules (lncRNA) that alter host cell gene expression thereby affecting host cell response and pathogenesis. Recent work by our group and others (7, 9, 12) has revealed that the C. parvum transcriptome is complex and laden with a variety of non-coding RNAs, both long and short. Recent work by Dr. Xian-Ming Chen has detected C. parvum transcripts in host-cell nuclei and demonstrated that several C. parvum lncRNAs, when introduced affect host-cell gene expression (13- 18). Sadly, due to the historical limitations of working with C. parvum, transcriptional data are sorely lacking for this important pathogen. Building on our proven track record of generating and sharing C. parvum transcriptional data, this project proposes to systematically characterize C. parvum coding and non-coding RNAs including small RNAs from several developmental stages of the parasite, pre- and post-infection (in vitro) using PacBio Iso-Seq (19) to identify complete transcripts and Illumina technologies to study small RNAs as Aim 1. Aim 2 focuses on examining the host-pathogen interaction. Bioinformatics will be used to identify select lncRNA molecules that may warrant experimental testing in the laboratory of Dr. Chen for host cell effects or knockout via CRISPR in the laboratory of Dr. Boris Striepen. Additionally, NovaSeq, which generates billions of reads (20) will be used for deep RNA sequencing of in vitro heavily-infected (0-48 hr) and uninfected host cells to characterize the gene transcriptional changes occurring in both the host and pathogen at three post-infection time-points. It is imperative to characterize transcriptional responses that may play a role in the host-pathogen interaction. They will expose new insights into parasite survival mechanisms, the development of pathogenesis and reveal much needed new targets for future therapeutic interventions (21-24).