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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).

摘要隐孢子虫是一种人畜共患的顶复门原生寄生虫，最近被确定为第二大流行全球范围内的婴儿肺炎病原体（1-6）。它是通过口腔-粪便途径传播的，免疫受损，因为没有批准用于该人群的治疗剂。分子隐孢子虫的寄生虫学一直缺乏，但这种情况正在改变，最近出现的体外培养系统，更好的寄生虫富集方案和新生遗传系统（8-11）。该项目提案重点分析隐孢子虫的转录和宿主-病原体相互作用。我们特别建议产生候选分子，用于实验测试最近的假设，即隐孢子虫通过输出改变宿主细胞基因表达的长链非编码RNA分子（lncRNA），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， Others（7，9，12）已经揭示了C.小孢子转录组是复杂的，并载有各种非编码 RNA，长的和短的。陈先明博士最近的工作检测到了C。宿主细胞中的微小转录物核，并证明了几个C.当引入时，小lncRNA影响宿主细胞基因表达（13- 18）。 18）。可悲的是，由于与C。小，转录数据非常缺乏，这个重要的病原体。基于我们在生成和共享C.小转录数据，本项目提出系统地表征C.小RNA编码和非编码RNA，包括来自寄生虫几个发育阶段的小RNA，感染前和感染后（体外），使用PacBio Iso-Seq（19）鉴定完整转录物，Illumina技术研究小RNA作为目标1。目的2 重点是研究宿主-病原体相互作用。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的实验测试的分子在鲍里斯·斯特里彭（Boris Striepen）博士的实验室里，此外，NovaSeq，它产生了数十亿的读取（20）将用于体外严重感染（0-48小时）和未感染宿主细胞的深度RNA测序，描述了在感染后三个时间点宿主和病原体中发生的基因转录变化，时间点。这是必要的，以确定转录反应，可能发挥作用，在宿主-病原体互动他们将揭示寄生虫生存机制的新见解，并揭示了未来治疗干预急需的新靶点（21-24）。