ROVA REGULON AND VIRULENCE OF YERSINIA ENTEROCOLITICA

描述(由申请人提供)：小肠结肠炎耶尔森氏菌是一种革兰氏阴性病原体，可引起一系列临床症状，但主要与胃肠道疾病有关。从小肠结肠炎耶尔森菌和假结核耶尔森菌的研究中，出现了许多重要的致病模式。此外，这些肠道病原体已经成为细菌入侵的重要模式，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，并且存在一个良好的小鼠感染模型来解剖宿主与病原体的相互作用，因此可以在分子水平上表征这些毒力特性，如侵袭性。在进一步了解inv及其在毒力中的作用的研究中，我们发现了一种名为rova的基因，它在实验室和感染期间调节inv的表达。后来的研究表明，RoVa作为DNA结合蛋白，通过取代inv启动子上的阻遏复合体来促进inv的表达。RoVa突变体的致病力低于野生型菌株或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感染。Route消除了RoVa缺陷，表明RoVa对于发生在/来自肠/结肠或Peyer‘s区的事件是必需的。由于RoVa突变体的毒力缺陷比inv突变体更显著，这表明RoVa调节着额外的毒力决定因素。然而，RoVa似乎并不调节以前发现的毒力决定因素的表达。通过全基因组芯片分析，我们发现了可能受RoVa调控的基因，提示该基因的调控子可能相当大。长期目标是(I)了解这些基因是如何被RoVa调控的，以及它是如何与其他毒力因子的表达相协调的，以及(Ii)确定哪些RoVa调控基因(RRG)对毒力起作用。具体地说，我们提出如下建议：(目标1)RoVa调节子在感染过程中起什么作用？最近，我们对肠道病原体如何与肠粘膜相互作用并系统传播的理解发生了显着变化，但问题仍然存在。此外，关于这些细菌如何在宿主之间传播的问题更容易解决。鉴于已知的RoVa突变体的毒力缺陷，我们假设RoVa调节子将在这些宿主-病原体相互作用中发挥作用，并认为有必要更详细地了解野生型和RoVa突变体在这些感染方面的作用。(目标2)哪些RRG对毒力是重要的，它们如何影响与RoVa相关的单个表型？(目标3)所有RRG的监管方式是否与INV相同？通过了解这些基因产物在何时何地表达，结合关于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率和死亡率以及成人发病率的重要原因。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菌的毒力因子及其在疾病过程中的作用。这将扩大我们的理解和能力，不仅对耶尔西氏菌，而且对许多其他胃肠道病原体进行治疗干预。

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.