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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

项目概要食源性病原体沙门氏菌是了解遗传调控的重要模式生物沙门氏菌引起疾病的必要条件是直接注射效应物。通过沙门氏菌致病性编码的三型分泌系统（T3SS）将蛋白质进入宿主细胞岛 1 (SPI1) 这一关键毒力因子是根据大量的环境和因素而受到控制的。决定系统在宿主体内适当的时间和地点表达的调节信号。术语目标是了解实现这种精确调节的整体信号集成。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控制 hilA 表达的调节环，编码 SPI1 结构基因的直接调节因子。许多调控输入在 HilD 水平上整合，包括 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)，充当独立的赋予 mRNA 稳定性的模块一个主要假设是 hilD 3' UTR 是一个关键因素。初步数据表明 mRNA 稳定性受调节信号调节。涉及 Rho 介导的 3’UTR 转录终止之间相互作用的新机制此外，这些活性均由 sRNA 独立控制。该提案的第一个目标是确定通过 hilD 3' UTR 进行调节的 sRNA 和顺式作用位点。将使用公正的分子技术鉴定相互作用的 sRNA，并确认碱基配对通过突变分析将确定 3’UTR 中 Rho 作用的位点。 sRNA 结合位点或 Rho 利用位点发生突变的 mRNA 为进一步的机制提供了工具第二个目的是通过 hilD 表征转录后调控机制。 3' UTR。Rho、RNase E 和小 RNA 在 3' 末端的创建和/或加工中的作用将使用标记和深度序列分析来监测 hilD 3' UTR。更准确地定义 Rho 在创建终止的 hilD 转录本中的作用。这些因素将揭示这种新颖调节的机制细节。作为理解宿主环境信号的整合以控制复杂的范例该系统的毒力表型分析对于我们了解这种重要的病原体至关重要。