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Decoding the coding and non-coding transcriptomes of Orientia tsutsugamushi in target host cells

解码靶宿主细胞中恙虫病东方体的编码和非编码转录组

基本信息

批准号：
10206039
负责人：
Hema Prasad Narra
金额：
$ 19.75万
依托单位：
UNIVERSITY OF TEXAS MED BR GALVESTON
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：
2020-07-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10206039
关键词：
Acute Adult Respiratory Distress Syndrome Amino Acyl Transfer RNA Anti-Bacterial Agents Antibiotics Antigenic Variation Asia Australia Bacteria Binding Bioinformatics Biology Blood Coagulation Disorders Catalogs Cells Chiggers China Chromosomes Climacteric Code DNA Transposable Elements Data Development Disease Documentation Doxycycline Elements Endemic Diseases Endothelial Cells Endothelium Environment Evaluation Event Evolution Experimental Animal Model Family Far East Fever Gastrointestinal Hemorrhage Gene Duplication Gene Expression Gene Expression Regulation Genetic Genetic Transcription Genome Goals Gram-Negative Bacteria Growth Human Incidence India Indonesia Infection Intercistronic Region Investigation Japan Kidney Failure Knowledge Korea Life Cycle Stages Ligands Mammalian Cell Meningoencephalitis Messenger RNA Mus Orientia tsutsugamushi Outcome Outcome Study Papua New Guinea Pathogenesis Pathogenicity Patients Plasmids Play Predisposition Prevalence Process Proteome RNA Regulation Regulator Genes Repetitive Sequence Reporting Ribosomal RNA Ribosomes Rickettsia Rickettsiaceae Risk Role Scrub Typhus Sequence Analysis Small RNA Soldier South Africa South America Southeastern Asia Taiwan Therapeutic Transcript Transcription Initiation Site Translations Untranslated RNA Vaccines Validation Vietnam Virulence Virulent War World Health Organization World War II antimicrobial arthropod-borne base bioinformatics resource contig design experience follow-up homologous recombination human disease improved in silico infancy insight mortality novel novel diagnostics novel therapeutics pathogen pathogenic bacteria prevent sequencing platform therapeutic target transcription termination transcriptome transcriptome sequencing treatment choice

项目摘要

PROJECT SUMMARY Orientia tsutsugamushi (Ot) is a highly virulent, chigger-borne, strictly intracellular Gram-negative bacterium known to cause scrub typhus in humans, a disease endemic to `Tsutsugamushi triangle' placing more than a billion people at risk and responsible for more than a million cases every year. Antigenic and genetic variability among Ot strains is an established phenomenon, yet our appreciation of the regulation of Ot genomes during host-pathogen interactions remains in its infancy. Bacterial small regulatory RNAs (sRNAs) have only recently emerged as critical post-transcriptional regulators of gene expression. Among these, trans-acting sRNAs act by binding to target mRNA(s); cis-acting sRNAs are transcribed antisense to their target RNA; and riboswitches alter gene expression by interacting with either small ligands or metabolites. Small RNAs regulate bacterial virulence by initiation/termination of transcription, stabilization/degradation of target mRNAs, and regulation of translation. Despite their importance as potential therapeutic targets, the identities and functions of Ot sRNAs have remained a mystery. A major bottleneck precluding the exploration of such regulatory networks in Ot pertains to the `sticky' genomes punctuated by extensive homologous recombination driven by transposons, conjugative elements, repetitive sequences, and gene duplication events, but this hurdle can now be overcome based on the sequencing and annotation of six Ot genomes as closed, circular chromosomes, including that of prototypical and highly pathogenic Karp strain (OtK). With a long-term goal of defining sRNA- mRNA interactions in Ot, we have employed this resource for bioinformatic predictions and follow-up validation to identify sRNAs in OtK. Our intriguing preliminary findings suggest that OtK genome does not encode for classical bacterial sRNAs 4.5S and 6S, indicating a unique sRNA repertoire which we hypothesize to play an important role in post-transcriptional gene expression in microvascular endothelium as the preferred, primary target cell niche in mammalian hosts. Accordingly, the objective of this application is to determine the coding and non-coding transcriptomes of OtK in correlation with the corresponding proteome during interactions with the host endothelium. In Aim 1, we will catalogue all expressed sRNAs and coding transcripts and determine their transcriptional start sites during OtK infection of human/mouse endothelial cells by differential RNA sequencing. Aim 2 will then identify and validate cognate mRNA targets for all novel trans- and cis-encoded sRNAs. We will apply a cutting-edge RNA-sequencing platform in conjunction with advanced omics-based approaches and our previously documented experience with the identification and characterization of sRNAs in pathogenic Rickettsia species. The outcomes will positively impact the field via first mechanistic understanding of the contributions of riboregulatory circuitry in Ot to the host-pathogen interactions and pathogenesis of scrub typhus, enabling the discovery of novel targets for the design/development of new and improved therapeutics.

项目摘要恙虫病东方体（Orientia tutsugamushi，Ot）是一种由恙螨传播的革兰氏阴性菌已知会引起人类恙虫病，这是一种“恙虫病三角区”特有的疾病， 10亿人处于危险之中，每年造成100多万起病例。抗原和遗传变异性在Ot菌株中，这是一个既定的现象，但我们对Ot基因组在宿主-病原体相互作用仍处于初期阶段。细菌小调控RNA（sRNAs）最近才被发现，成为基因表达的关键转录后调节因子。其中，反式作用的sRNAs 通过与靶mRNA结合;顺式作用sRNA被转录为其靶RNA的反义;和核糖开关通过与小配体或代谢物相互作用来改变基因表达。小RNA调节通过转录的起始/终止、靶mRNA的稳定化/降解的细菌毒力，以及翻译规范。尽管它们作为潜在的治疗靶点很重要，但它们的身份和功能 Ot sRNAs的基因组结构仍然是个谜。一个主要的瓶颈阻碍了这种监管的探索， Ot中的网络属于“粘性”基因组，其被广泛的同源重组打断，转座子，接合元件，重复序列和基因复制事件，但这个障碍现在可以基于六个Ot基因组的测序和注释作为封闭的环状染色体，包括典型和高致病性Karp株（OtK）。长期目标是定义sRNA mRNA相互作用的Ot，我们已经采用了生物信息学预测和后续验证这一资源来鉴定OtK中的sRNA。我们有趣的初步研究结果表明，OtK基因组不编码经典的细菌sRNAs 4.5S和6S，表明一个独特的sRNA库，我们假设发挥作用，在微血管内皮细胞转录后基因表达中的重要作用，哺乳动物宿主中的靶细胞小生境。因此，本申请的目的是确定编码和非编码转录组与相应的蛋白质组的相互作用过程中，宿主内皮细胞在目标1中，我们将对所有表达的sRNA和编码转录本进行分类，并确定通过差异RNA检测OtK感染人/小鼠内皮细胞过程中的转录起始位点测序目标2将识别和验证所有新的反式和顺式编码的同源mRNA靶点， sRNAs。我们将应用尖端的RNA测序平台，结合先进的基于组学的方法和我们以前记录的sRNA鉴定和表征的经验，致病性立克次氏体这些成果将通过第一次机械理解对该领域产生积极影响 Ot中的核糖核酸调控回路对宿主-病原体相互作用和灌木丛的发病机制的贡献斑疹伤寒，能够发现新的目标，设计/开发新的和改进的治疗。