Enterohemorrhagic Escherichia coli virulence regulation by non-coding RNAs

描述（由申请人提供）：小的非编码RNA（sRNA）转录后调节原核生物中的无数细胞过程。然而，有相对缺乏的知识，sRNA调节细菌的毒力，尽管有迹象表明，这发生在几种病原体。本申请的主要目标是鉴定和表征控制肠出血性大肠杆菌（EHEC）毒力的sRNA，EHEC是一组新兴的食源性非侵入性肠道病原体，其引起从非血性腹泻到潜在致命的溶血性尿毒综合征（HUS）的疾病。为了实现这一目标，我提出了以下三个目标：1）研究sRNA在调控已知EHEC毒力因子中的作用。强有力的初步证据表明，sRNA可以调节编码Tir的mRNA，Tir是EHEC定植能力的关键蛋白之一。在确认了初步数据后，我将进行遗传筛选，以确定感兴趣的sRNA。2)鉴定可能在毒力调节中发挥作用的EHEC sRNA。生物信息学和“深度测序”方法将用于对EHEC sRNA进行全面筛查。3)描述目标1和2中确定的选定EHEC sRNA在毒力、靶点和作用机制中的作用。我将在编码感兴趣的sRNA的基因中创建框内缺失，并在体外和体内检查这些突变体的毒力相关表型。这些突变体也将用于微阵列分析，以鉴定由这些sRNA调控的靶标。然后，我将确定sRNAs控制毒力基因表达的机制。这种方法似乎是合理的，因为我已经证实了几个预测的sRNA可能参与毒力。公共卫生相关性：这项工作将产生重要病原体中小转录组的基本新知识，并为控制细菌毒力的过程提供新的见解。此外，这些研究将有助于小RNA调控机制的知识不断增长，这是原核和真核领域的一个非常感兴趣的领域。最后，更好地了解肠出血性大肠杆菌毒力的调节可能会导致更好地了解治疗方案，并为这一重要的人类病原体开发新的治疗方法。

DESCRIPTION (provided by applicant): Small non-coding RNAs (sRNAs) post-transcriptionally regulate a myriad of cellular processes in prokaryotes. However, there is relatively scant knowledge of sRNAs that regulate bacterial virulence, despite indications that this occurs in several pathogens. The principal goal of this application is to identify and characterize sRNAs that control the virulence of Enterohemorrhagic Escherichia coli (EHEC), an emerging group of food-borne non-invasive intestinal pathogens that cause illnesses ranging from non-bloody diarrhea to the potentially fatal hemolytic uremic syndrome (HUS). In order to address this goal, I propose the following three aims: 1) Examine the role of sRNAs in the regulation of known EHEC virulence factors. Strong preliminary evidence indicates that a sRNA may regulate the mRNA coding for Tir, one of the crucial proteins in the colonization abilities of EHEC. After confirming the preliminary data I will carry out a genetic screen to identify the sRNA of interest. 2) Identify EHEC sRNAs likely to play a role in virulence regulation. Both bioinformatic and 'deep-sequencing' approaches will be used to conduct a comprehensive screen for EHEC sRNAs. 3) Characterize the role in virulence, the target(s), and the mechanisms of action of selected EHEC sRNAs identified in Aims 1 and 2. I will create in-frame deletions in the genes encoding the sRNAs of interest and examine those mutants for virulence-related phenotypes both in vitro and in vivo. These mutants will also be used in microarray analyses to identify the targets regulated by these sRNAs. I will then determine the mechanisms by which sRNAs govern virulence gene expression. This approach appears to be sound as I have already confirmed several predicted sRNAs which may be involved in virulence. PUBLIC HEALTH RELEVANCE: This work will yield fundamental new knowledge of the small transcriptome in an important pathogen as well as provide new insights into the processes that govern bacterial virulence. In addition, these studies will contribute to the growing body of knowledge of the mechanisms of small RNA regulation, an area of great interest in both prokaryotic and eukaryotic fields. Finally, a greater understanding of the regulation of EHEC virulence may lead to better understanding of treatment options and to the development of novel therapeutics for this important human pathogen.