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

项目总结 食源性致病菌沙门氏菌是研究遗传调控的重要模式生物 以及细菌致病机制。沙门氏菌致病的必要条件是直接注射效应剂 沙门氏菌致病性三型分泌系统(T3SS)编码的蛋白质进入宿主细胞 岛1(SPI1)。这种关键的毒力因子受到控制，以应对过多的环境和 调节信号，指示系统在宿主中的适当时间和位置表达。我们的长- 学期目标是了解实现这一精确监管的整体信号集成。SPI1监管 电路由三个类似AraC的调节器控制，Hild、HilC和RTSA在复杂的前馈中起作用 调控环控制Hila的表达，编码SPI1结构基因的直接调节因子。 大部分调控输入是在HILD水平上整合的，包括在HILD mRNA翻译或稳定性方面。 HILD基因有一个不寻常的300核苷酸3‘非翻译区(UTR)，作为一个独立的 模块来赋予信使核糖核酸的不稳定性。一个主要的假设是，希尔德3‘UTR是一个关键的 用于整合调节信号的节点。初步数据显示，mrna的稳定性受一种 Rho介导的3‘非编码区转录终止相互作用的新机制 依赖核糖核酸酶E的降解。此外，这些活动由sRNA独立控制。 这项建议的第一个目的是确定通过Hild 3‘UTR调控的sRNA和顺式作用位点。 相互作用的sRNA将使用无偏见的分子技术进行鉴定，碱基配对得到确认 通过诱变。缺失分析将确定Rho在3‘非编码区中的作用部位。由此产生的希尔德 具有sRNA结合位点或Rho利用位点突变的mRNAs为进一步的机制提供了工具 分析。第二个目标是描述通过hILD的转录后调控机制。 3‘非编码区。Rho、RNaseE和小RNA在3‘端的产生和/或处理中的作用 在Hild中，将使用标记和深层序列分析来监测3‘非编码区。体外转录将 更准确地定义Rho在创建终止的Hild记录中的作用。它们之间的相互作用 因素将揭示这一新规定的机械性细节。SP1 T3SS调节电路用于 作为理解整合宿主环境信号以控制复合体的范例 毒力表型。对这个系统的分析对于我们理解这种重要的病原体是至关重要的。

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.