Mechanisms of Dysbiosis by Attaching and Effacing Pathogens

描述(申请人提供)：致病性肠出血性大肠杆菌(EPEC)是发展中国家幼儿胃肠道疾病的常见原因。这些细菌和相关的小鼠病原体轮状柠檬酸杆菌被归类为附着和消除(A/E)细菌，因为它们被摄入后，密切地附着在宿主肠道细胞上并导致其消失。A/E细菌会导致胃肠道中通常自然存在的细菌群发生变化，导致生物失调和疾病。虽然A/E细菌诱导的病理的分子基础已经被很好地研究了，但使这些病原体能够成功地竞争资源并驱动生物失调的机制还不是很清楚。此外，通过形成基座样结构与宿主细胞亲密黏附的健康益处目前尚不清楚，这是A/E病原体特有的特征。这项研究的长期目标是确定导致病原体诱导的肠道生物失调的机制。具体地说，我们的目标是1)说明呼吸在感染期间对A/E病原体生长的作用，以及2)定义 管控轮齿藻与宿主细胞的亲密附着所提供的健康益处的机制。我们的假设是，与上皮细胞的紧密黏附为A/E病原体提供了呼吸所需的氧气，从而允许比发酵的共生微生物更有效地产生能量。为了解决我们的假设，将构建具有关键呼吸酶(BD氧化酶和甲酸脱氢酶)和附着基因(底座诱导效应蛋白EspH)的轮状芽孢杆菌菌株，并结合生长试验、组织培养试验和共聚焦显微镜对其进行体外鉴定。然后给小鼠灌胃单一或混合接种，以进行竞争性感染，以测试体内呼吸和附着的益处。然后，无菌小鼠感染将使我们能够隔离病原体和微生物区系特定成员之间的相互作用。对寄主和病原体基因的定量聚合酶链式反应将提供作为寄主反应一部分的细胞因子表达水平的信息，以及判断不同实验条件下轮状芽孢杆菌代谢状态的指标。这项研究的完成将为病原体诱导的肠道生物失调的机制提供有价值的信息，并为制定未来预防A/E病原体传播和疾病的战略奠定坚实的基础。

DESCRIPTION (provided by applicant): Enteropathogenic E. coli (EPEC) are common causes of gastrointestinal illness in very young children in developing nations. These and the related murine pathogen, Citrobacter rodentium, are classified as attaching and effacing (A/E) bacteria because upon ingestion, they intimately attach to and cause effacement of host intestinal cells. A/E bacteria induce shifts in the bacteria groups typically present naturally in the gastrointestinl tract, resulting in dysbiosis and disease. While the molecular basis of A/E bacteria-induced pathology is well studied, the mechanisms that enable these pathogens to successfully compete for resources and drive dysbiosis are not well understood. Additionally, the fitness benefit of intimate adherence to host cells through formation of pedestal-like structures, a feature unique to A/E pathogens, is currently unknown. The long-term objectives of this study are to define the mechanisms that result in pathogen-induced intestinal dysbiosis. Specifically, we aim to 1) illustrate the role of respiration to outgrowth of A/E pathogens during infection and 2) define the mechanisms that govern the fitness benefit provided by C. rodentium intimate attachment to host cells. Our hypothesis states that close adherence to epithelial cells provides A/E pathogens with access to oxygen for respiration, therefore allowing for more efficient energy generation over fermenting commensal microbes. To address our hypothesis, C. rodentium strains will be constructed with disruptions in key respiratory enzymes (bd oxidase and formate dehydrogenase) and attachment genes (pedestal-inducing effector protein EspH) and characterized in vitro with a combination of growth assays, tissue culture assays, and confocal microscopy. Mice will then be orally gavaged with single or mixed inocula for competitive infections to test the benefit derived from respiration and attachment in vivo. Germ-free mouse infections will then allow us to isolate interactions between pathogen and specific members of the microbiota. Quantitative PCR on host and pathogen genes will provide information on the levels of cytokine expression as part of the host response as well as a gauge to judge the metabolic state of C. rodentium under different experimental conditions. Completion of this study will provide valuable information on the mechanisms that result in pathogen-induced gut dysbiosis and a strong foundation on which to develop future strategies to prevent A/E pathogen transmission and disease.