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：
10058036
负责人：
Hema Prasad Narra
金额：
$ 23.7万
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 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10058036
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.

项目概要恙虫病东方体 (Ot) 是一种高毒力、恙螨传播的严格细胞内革兰氏阴性细菌已知会引起人类丛林斑疹伤寒，这是一种“恙虫三角区”特有的疾病，导致超过每年有 10 亿人面临风险，并造成超过 100 万起病例。抗原和遗传变异 Ot 菌株之间的差异是一种既定现象，但我们对 Ot 基因组调控的认识宿主与病原体的相互作用仍处于起步阶段。细菌小调节 RNA (sRNA) 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 sRNA 的作用通过与目标 mRNA 结合；顺式作用 sRNA 转录为与其靶 RNA 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、目标 mRNA 的稳定/降解来确定细菌毒力，以及翻译监管。尽管它们作为潜在的治疗靶点很重要，但其特性和功能 Ot sRNA 的数量仍然是个谜。阻碍探索此类监管的主要瓶颈 Ot 中的网络属于“粘性”基因组，其间存在着由广泛的同源重组驱动的转座子、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 基因组作为闭合环状染色体的测序和注释来克服，包括原型和高致病性卡普菌株（OtK）。长期目标是定义 sRNA- Ot 中的 mRNA 相互作用，我们利用该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 基因组不编码经典细菌 sRNA 4.5S 和 6S，表明我们假设发挥独特的 sRNA 库在微血管内皮转录后基因表达中发挥重要作用，作为首选、主要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 OtK 的非编码转录组与相互作用过程中相应的蛋白质组相关宿主内皮。在目标 1 中，我们将对所有表达的 sRNA 和编码转录本进行编目，并确定通过差异RNA OtK感染人/小鼠内皮细胞期间它们的转录起始位点测序。然后，目标 2 将识别并验证所有新型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技术相结合方法和我们之前记录的 sRNA 鉴定和表征经验致病性立克次体种。结果将通过初步的机制理解对该领域产生积极影响 Ot中核调节回路对宿主-病原体相互作用和灌木丛病发病机制的贡献斑疹伤寒，使得能够发现用于设计/开发新的和改进的疗法的新靶标。