ROVA REGULON AND VIRULENCE OF YERSINIA ENTEROCOLITICA

描述（由申请方提供）：小肠结肠炎耶尔森菌是一种革兰氏阴性病原体，可引起一系列临床综合征，但主要与胃肠道疾病相关。从Y. enterocolitica和Y.假结核此外，这些肠道病原体已作为细菌入侵的重要模型，这一过程主要由inv编码。由于这些细菌在实验室中易于操作，并且存在用于解剖宿主-病原体相互作用的优良小鼠感染模型，因此可以在分子水平上表征毒力特性，例如侵袭。在进一步了解inv及其毒力作用的研究中，我们鉴定了一个基因rovA，它在实验室和感染过程中调节inv的表达。随后的研究表明，RovA作为一种DNA结合蛋白，通过置换inv启动子上的阻遏物复合物来促进inv表达。rovA突变体的毒性低于野生型菌株或inv突变体。RovA毒力缺陷的特征是减少全身传播和增加口服感染后的LD 50。使用腹膜内途径的感染消除了RovA缺陷，表明RovA是发生在肠/结肠中/来自肠/结肠或派伊尔集合淋巴结中的事件所必需的。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调节额外的毒力决定因子。然而，RovA似乎没有调节先前确定的毒力决定因素的表达。使用全基因组微阵列分析，我们确定了64个基因可能受到RovA的调控，这表明调节子可能相当大。长期目标是（i）了解RovA如何调控这些基因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。具体来说，我们提出以下几点：（目的1）什么是RovA调节子在感染过程中的作用？最近，我们对肠道病原体如何与肠粘膜相互作用并全身传播的理解发生了显着变化，但问题仍然存在。此外，关于这些细菌如何从宿主传播到宿主的问题更容易处理。鉴于已知的毒力缺陷的rovA突变体，我们假设，RovA调节子将发挥作用，在这些主机-病原体的相互作用，并认为一个更详细的了解野生型和rovA突变体的这些方面的感染是必要的。(Aim 2）哪些rrg对毒力很重要，它们如何促成个体RovA相关表型？(Aim 3）所有的rrg都是以与inv相同的方式监管的吗？通过了解这些基因产物表达的时间和位置，结合这些基因突变表型的信息，我们可能能够更好地了解它们的功能。 公共卫生相关性：胃肠道疾病是婴儿发病和死亡以及成人发病的重要原因。我们的长期目标是在分子水平上充分了解小肠结肠炎耶尔森氏菌的毒力因子及其对疾病过程的作用。这将扩大我们的理解和能力，不仅与耶尔森氏菌，但与许多其他胃肠道病原体以及治疗干预。

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.