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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.

项目总结恙虫病东方体(Ot)是一种由恙螨携带的高度毒力的细胞内严格的革兰氏阴性细菌已知会在人类中引起丛林斑疹伤寒，这种疾病是一种地方性疾病，由一种每年有10亿人面临风险，并对100多万起病例负有责任。抗原性和遗传变异性在OT菌株之间是一个既定的现象，但我们对OT基因组在宿主与病原体的相互作用仍处于初级阶段。细菌小分子调控RNA(SRNAs)直到最近才成为基因表达的关键转录后调节因子。其中，反式作用的sRNA发挥作用通过与靶基因结合(S)，顺式作用的sRNA被转录成与其靶基因反义；以及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调控通过启动/终止转录、稳定/降解靶基因mRNAs而产生的细菌毒力对翻译的规范。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 OTsRNAs的发现仍然是个谜。阻碍探索这种监管的一个主要瓶颈 OT中的网络是指由广泛的同源重组所驱动的“粘性”基因组。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为闭合的、环形的染色体而被克服，包括典型的和高致病性的卡普病毒株(OTK)。有一个定义sna的长期目标- 在OT中的mRNA相互作用，我们已经利用这个资源进行生物信息学预测和后续验证识别OTK中的sRNA。我们有趣的初步发现表明，OTK基因组并不编码经典细菌sRNAs 4.5S和6S，表明一个独特的sRNA谱系，我们假设它扮演一个微血管内皮细胞转录后基因表达的重要作用哺乳动物宿主中的靶细胞生态位。因此，本申请的目的是确定编码和OTK的非编码转录本与相应的蛋白质组在与宿主内皮细胞。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，并确定差异RNA检测其在人/鼠内皮细胞感染OTK过程中的转录起始位置测序。然后，目标2将识别和验证所有新的反式和顺式编码的同源mRNA靶标 SRNA。我们将应用尖端的RNA测序平台与先进的基于组学的方法和我们以前记录的识别和鉴定sRNA的经验致病立克次体种类。这些结果将通过第一次机械理解对该领域产生积极影响 OT中的核转录调节回路在宿主-病原体相互作用和CLUB发病机制中的作用斑疹伤寒，使发现设计/开发新的和改进的疗法的新靶点成为可能。