Detailed analysis of Cryptosporidium non-coding gene expression

隐孢子虫非编码基因表达的详细分析

基本信息

批准号：
10092931
负责人：
Jessica C Kissinger
金额：
$ 18.88万
依托单位：
UNIVERSITY OF GEORGIA
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10092931
关键词：
Affect Back Bioinformatics Biological Birds Caliber Cell Nucleus Cell Volumes Cells Chlorine Clustered Regularly Interspaced Short Palindromic Repeats Code Collaborations Complex Cryptosporidiosis Cryptosporidium Cryptosporidium parvum DNA Data Data Analyses Development Diarrhea Distant Expressed Sequence Tags Fibroblasts Future Genbank Gene Expression Generations Genes Genetic Genetic Transcription Genome Hela Cells Human Immune response Immunocompromised Host In Vitro Infant Infection Ingestion Initiator Codon Institutes Joints Knock-out Laboratories Length Libraries Life Life Cycle Stages Location Molecular Nature Oocysts Oral Parasites Parasitology Pathogenesis Pattern Phenotype Play Population Process Protein Isoforms Proteins Protocols documentation Publications RNA RNA Splicing Reporting Research Research Personnel Role Route Side Small RNA Sporozoites Surveys System Technology Terminator Codon Testing Therapeutic Therapeutic Intervention Thinness Time Transcript Trust Untranslated RNA Untranslated Regions Validation Water Work Zoonoses base c new design insight pathogen professor response telomere transcriptome transcriptome sequencing

项目摘要

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).

抽象的隐孢子虫是一种人畜共患的顶端复门原生生物寄生虫，最近被确定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，在人体中可能是致命的免疫功能低下，因为没有批准用于该人群的治疗方法。分子隐孢子虫的寄生虫学一直缺乏，但随着最近体外试验的出现，这种情况正在改变培养系统、更好的寄生虫富集方案和新生的遗传系统 (8-11)。本项目建议书重点分析隐孢子虫转录和宿主-病原体相互作用。具体来说，我们提议生成候选分子，用于实验测试最近的假设，即隐孢子虫通过输出长非编码 RNA 分子 (lncRNA) 反式改变宿主细胞基因表达宿主细胞基因表达从而影响宿主细胞反应和发病机制。我们小组最近的工作和其他人 (7, 9, 12) 揭示了 C. parvum 转录组是复杂的并且充满了各种非编码 RNA，有长的和短的。陈贤明博士最近的工作在宿主细胞中检测到了 C. parvum 转录本细胞核，并证明一些 C. parvum lncRNA 在引入后会影响宿主细胞基因表达 (13- 18）。遗憾的是，由于使用 C. parvum 的历史限制，转录数据非常缺乏这种重要的病原体。以我们在生成和共享 C. parvum 转录方面的良好记录为基础数据，该项目建议系统地表征 C. parvum 编码和非编码 RNA，包括使用 PacBio 检测来自寄生虫多个发育阶段、感染前和感染后（体外）的小 RNA Iso-Seq (19) 用于识别完整转录本，并使用 Illumina 技术来研究小 RNA，如目标 1 和目标 2 所示重点检查宿主与病原体的相互作用。生物信息学将用于识别选定的lncRNA 可能需要在陈博士实验室进行宿主细胞效应或敲除实验测试的分子通过 Boris Striepen 博士实验室的 CRISPR 技术。此外，NovaSeq 可生成数十亿次读取 (20) 将用于体外重度感染（0-48小时）和未感染宿主细胞的深度RNA测序描述感染后三个时期宿主和病原体中发生的基因转录变化时间点。必须表征可能在宿主病原体中发挥作用的转录反应相互作用。他们将揭示对寄生虫生存机制、发病机制发展的新见解并揭示未来治疗干预急需的新目标 (21-24)。