ROVA REGULON AND VIRULENCE OF YERSINIA ENTEROCOLITICA

描述（由申请人提供）：小肠结肠炎耶尔森菌是一种革兰氏阴性病原体，可导致一系列临床综合征，但主要与胃肠道疾病相关。从小肠结肠炎耶氏菌和假结核耶氏菌的研究中出现了一些重要的发病机制范式。此外，这些肠致病菌已成为细菌入侵的重要模型，这一过程主要由侵入体编码。由于在实验室中易于操作这些细菌，并且存在一种用于解剖宿主-病原体相互作用的优秀小鼠感染模型，因此可以在分子水平上表征入侵等毒力特性。在进一步了解inv及其在毒力中的作用的研究中，我们发现了一个基因rovA，它在实验室和感染过程中都能调节inv的表达。随后的研究表明，RovA作为一种DNA结合蛋白，通过取代反转录启动子上的抑制复合物来促进反转录表达。rovA突变体的毒力低于野生型菌株和inv突变体。rovA毒力缺陷的特点是全身传播减少，口腔感染后LD50增加。使用ip途径的感染消除了rovA缺陷，这表明在肠道/结肠或Peyer's patch中发生的事件需要rovA。由于rovA突变体的毒力缺陷比单独的inv突变体更显著，这表明rovA调节了其他毒力决定因素。然而，RovA似乎没有调节先前确定的毒力决定因素的表达。通过全基因组微阵列分析，我们鉴定出64个可能受RovA调控的基因，表明该调控子可能相当大。长期目标是(i)了解这些基因如何受RovA调控，以及如何与其他毒力因子的表达协调，以及（ii）确定哪些RovA调控基因（rrg）有助于毒力。具体而言，我们提出以下建议：（Aim 1）在感染过程中RovA调控的作用是什么？近年来，我们对肠道病原体如何与肠黏膜相互作用和系统传播的理解发生了重大变化，但问题仍然存在。此外，关于这些细菌如何在宿主之间传播的问题更容易处理。鉴于已知的rovA突变体的毒力缺陷，我们假设rovA调控子将在这些宿主-病原体相互作用中发挥作用，并认为在感染的这些方面对野生型和rovA突变体有更详细的了解是必要的。（目标2）哪些rrgs对毒力很重要，它们如何促成个体rova相关表型？（目标3）所有rrgs的监管方式是否与invv相同？通过了解这些基因产物在何时何地表达，并结合这些基因突变的表型信息，我们可能能够更好地了解它们的功能。

DESCRIPTION (provided by applicant): Yersinia enterocolitica is a Gram-negative pathogen responsible for a range of clinical syndromes but is primarily associated with gastrointestinal disorders. A number of important paradigms of pathogenesis have emerged from the studies of Y. enterocolitica and Y. pseudotuberculosis. In addition these enteropathogens have served as important models of bacterial invasion, a process primarily encoded by inv. Virulence properties such as invasion can be characterized at the molecular level due to the ease of manipulation of these bacteria in the laboratory and the existence of an excellent murine model of infection for dissecting the host-pathogen interaction. In studies to further our knowledge of inv and its role in virulence we identified a gene, rovA, that regulates expression of inv both in the laboratory and during infection. Subsequent studies demonstrated that RovA acts as a DNA binding protein and promotes inv expression by displacing a repressor complex from the inv promoter. The rovA mutant was less virulent than either the wild type strain or the inv mutant. The rovA virulence defect is characterized by reduced systemic dissemination and an increased LD50 after oral infection. Infection using the i.p. route abrogates the rovA defect, suggesting RovA is required for events occurring either in/from the intestine/colon or in the Peyer's patch. Because the rovA mutant virulence defect was more significant than that of an inv mutant alone, this suggested RovA regulates additional virulence determinants. However, RovA did not appear to regulate the expression of previously identified virulence determinants. Using whole genome microarray analysis we identified 64 genes potentially regulated by RovA, suggesting the regulon may be quite large. The long-term goals are (i) to understand how these genes are regulated by RovA and how that is coordinated with expression of other virulence factors, and (ii) to determine which RovA regulated genes (rrg) contribute to virulence. Specifically we propose the following: (Aim 1) What is the role of the RovA regulon during infection? Recently our understanding of how enteric pathogens interact with the intestinal mucosa and spread systemically has changed significantly but questions still remain. In addition, questions regarding how these bacteria spread from host-to-host are more tractable. Given the known virulence defects of the rovA mutant, we hypothesize that the RovA regulon will play a role in these host-pathogen interactions and feel that a more detailed understanding of both wild type and the rovA mutant with respect to these aspects of infection is warranted. (Aim 2) Which rrgs are important for virulence and how do they contribute to individual RovA-associated phenotypes? (Aim 3) Are all rrgs regulated in the same way as inv? By knowing when and where these gene products are expressed combined with information regarding the phenotype of mutations in these genes we may be able to gain a better understanding of their function. PUBLIC HEALTH RELEVANCE: Gastrointestinal disease is a significant cause of morbidity and mortality in infants and of morbidity in adults. Our long-range goal is to gain a full understanding of the Yersinia enterocolitica virulence factors and their contribution to the disease process at the molecular level. This will expand our understanding and ability to intervene therapeutically not only with Yersinia but with many other gastrointestinal pathogens as well.