Regulation of the Salmonella Pathogenicity Island 1 Type III Secretion System via the hilD 3' untranslated region

通过 hilD 3 非翻译区调节沙门氏菌致病性岛 1 III 型分泌系统

• 批准号: 10527931
• 负责人: JAMES M. SLAUCH
• 金额: $ 22.65万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-20 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10527931
• 关键词: 3' Untranslated Regions; Affect; Animal Model; Bacteria; Bacterial Infections; Base Pairing; Binding Sites; Bioinformatics; Biological Assay; Cells; Complex; Data; Development; Diarrhea; Disease; Epithelial Cells; Genetic; Genetic Transcription; Genetic Translation; Goals; Half-Life; Human; In Vitro; Inflammatory; Injections; Intestines; Invaded; Knowledge; Laboratories; Maps; Mediating; Messenger RNA; Methods; Microbiology; Modeling; Molecular; Monitor; Mutagenesis; Mutate; Mutation; Nucleotides; Pathogenesis; Pathogenicity Island; Phenotype; Post-Transcriptional Regulation; Prevention; Process; Proteins; Regulation; Role; SP1 gene; Salmonella; Sequence Analysis; Signal Transduction; Site; Small RNA; Spottings; Structural Genes; System; Systems Analysis; Techniques; Time; Transcript; Translations; Type III Secretion System Pathway; Untranslated Regions; Variant; Virulence; Virulence Factors; Work; deletion analysis; experimental study; foodborne pathogen; improved; in vivo; mRNA Stability; mRNA Transcript Degradation; mutant; novel; pathogen; response; rho; ribonuclease E; tool; trait; transcription termination

PROJECT SUMMARY The foodborne pathogen Salmonella is an important model organism for understanding genetic regulation and bacterial pathogenesis. A requisite for Salmonella to cause disease is the direct injection of effector proteins into host cells via a Type Three Secretion System (T3SS) encoded on Salmonella Pathogenicity Island 1 (SPI1). This critical virulence factor is controlled in response to a plethora of environmental and regulatory signals that dictate expression of the system at the proper time and place in the host. Our long- term goal is to understand overall signal integration that allows this precise regulation. The SPI1 regulatory circuit is controlled by three AraC-like regulators, HilD, HilC, and RtsA, which act in a complex feed-forward regulatory loop to control expression of hilA, encoding the direct regulator of the SPI1 structural genes. Much of the regulatory input is integrated at the level of HilD, including at hilD mRNA translation or stability. The hilD mRNA has an unusual 300 nucleotide 3’ untranslated region (UTR) that acts as an independent module to confer instability to the mRNA. A primary hypothesis is that the hilD 3’ UTR serves as a critical node for integration of regulatory signals. Preliminary data show that mRNA stability is regulated by a novel mechanism involving interaction between Rho-mediated transcriptional termination at the 3’ UTR and RNase E-dependent degradation. Moreover, these activities are independently controlled by sRNAs. The first aim of this proposal is to identify sRNAs and cis-acting sites that regulate via the hilD 3’ UTR. Interacting sRNAs will be identified using an unbiased molecular technique, with base pairing confirmed by mutagenesis. Deletion analysis will identify the site of Rho action in the 3’ UTR. The resulting hilD mRNAs with mutations in sRNA binding sites or Rho-utilization site provide tools for further mechanistic analyses. The second aim is to characterize the mechanism of post-transcriptional regulation via the hilD 3' UTR. The roles of Rho, RNase E, and the small RNAs in the creation and/or processing of the 3’ ends in the hilD 3’ UTR will be monitored using tagging and deep sequence analysis. In vitro transcription will more precisely define the action of Rho in creating terminated hilD transcripts. The interactions of these factors will reveal the mechanistic details of this novel regulation. The SP1 T3SS regulatory circuit serves as a paradigm for understanding the integration of host environmental signals to control a complex virulence phenotype. Analysis of this system is critical to our understanding of this important pathogen.

项目摘要 食源性致病菌沙门氏菌是了解遗传调控的重要模式生物 和细菌致病性。沙门氏菌致病的一个必要条件是直接注射效应子 通过沙门氏菌致病性编码的三型分泌系统（T3 SS）将蛋白质导入宿主细胞 岛1（SPI 1）。这种关键的毒力因子是在响应过多的环境和 调控信号，指示系统在宿主中的适当时间和位置表达。我们长久以来- 术语目标是理解允许这种精确调节的总体信号集成。SPI 1监管 电路由三个类似AraC的调节器HilD、HilC和RtsA控制，它们以复杂的前馈方式起作用 调节环控制hilA的表达，编码SPI 1结构基因的直接调节子。 许多调控输入在HilD水平整合，包括在hilD mRNA翻译或稳定性。 hilD mRNA有一个不寻常的300个核苷酸的3'非翻译区（UTR），作为一个独立的非翻译区。 模块赋予mRNA不稳定性。一个主要的假设是hilD 3' UTR作为一个关键的 用于整合调节信号的节点。初步数据表明，mRNA的稳定性是由一个 涉及Rho介导的3' UTR转录终止之间相互作用的新机制 和RNase E依赖性降解。此外，这些活性由sRNA独立控制。 该建议的第一个目的是鉴定通过hilD 3' UTR调节的sRNA和顺式作用位点。 将使用无偏分子技术鉴定相互作用的sRNA，并确认碱基配对 通过诱变。缺失分析将鉴定3' UTR中Rho作用的位点。结果hilD 在sRNA结合位点或Rho利用位点具有突变的mRNA为进一步的机制研究提供了工具。 分析。第二个目的是通过hilD来描述转录后调节的机制。 3'UTR。Rho、RNase E和小RNA在3'末端的产生和/或加工中的作用 在hilD中，将使用标记和深度序列分析来监测3' UTR。体外转录将 更精确地定义Rho在产生终止的hilD转录物中的作用。它们之间的相互作用 这些因素将揭示这一新规则的机制细节。SP1 T3 SS调节电路用于 作为理解宿主环境信号的整合以控制复合体的范例， 毒力表型分析这一系统对于我们了解这一重要病原体至关重要。