E. coli Growth Parameters in the Intestine

描述（由申请人提供）：控制大肠杆菌生长和非生长状态之间转换的细菌严格反应系统。大肠杆菌在动物定殖中的作用尚未得到评价。对营养限制的严格反应涉及独特核苷酸鸟苷3 '，5'-双焦磷酸（ppGpp）的快速积累，其在生长过渡期间引起基因表达的广泛重编程。ppGpp的合成和降解是动态的，允许细胞快速适应压力，然后在条件允许时快速恢复生长。拟议的研究计划将确定肠道定植是否取决于肠道中细菌的最大生长速率或细菌对营养限制的反应和适应肠道环境的能力。具体而言，我们将解决四个问题，关于营养的可用性在小鼠肠道和适应E。大肠杆菌进入肠道环境。(1)E.大肠杆菌在小鼠肠道中的存在是一种盛宴和饥饿？我们已经确定了ppGpp依赖基因，作为细胞生长状态的分子信标，这将使我们能够问E。大肠杆菌在肠道中经历不连续或连续的营养利用率。荧光蛋白报告基因的融合将被用来可视化与共聚焦显微镜的感染小鼠肠道的粘液层内的单个细菌细胞的生长状态。或者，我们将使用原位杂交来测量这些生长调节基因在单个细菌细胞中的表达。这些实验将揭示肠道环境的性质，因为它是由殖民细菌感知。(2)维持殖民化所需的最低增长率是多少？在实验中，我们将通过用一系列经过遗传操作的菌株感染小鼠来解决这个问题，这些菌株可以独立于环境信号固定其ppGpp水平，因此具有固定的生长速率。我们将确定这些菌株定植的相对适应性，并测量它们的体内生长速率。(3)生长较快的菌株是否胜过生长较慢的菌株？具有固定生长速率的菌株将相互竞争。(4)是调整ppGpp水平的能力（即，增长率）比维持最大对数增长率更重要？固定的ppGpp菌株将与野生型竞争，其可以正常调节其生长速率。这些实验将在模式生物E. coliK-12，应该会导致对殖民细菌生理学的新见解。因为定殖是感染和疾病的第一步，这项工作将扩展到E。大肠杆菌O 157：H7的研究，以寻找大肠杆菌病及其致病机制的共同主题。这些问题的答案将导致更好地了解胃肠道健康和潜在的新战略，以打击胃肠道感染。 公共卫生相关性：这项建议是基于假设，即E。大肠杆菌必须适当地控制其生长速率以在肠道中定殖并实现高群体。在检验这一假设时，我们将确定动物定植是否取决于肠道中细菌的最大生长速率或细菌对营养限制的反应能力，并通过调整其生长速率来适应。这些问题的答案将提供有关宿主肠道细菌生理学的新信息，并更好地了解胃肠道健康，这将导致对抗胃肠道感染的新策略。

DESCRIPTION (provided by applicant): The bacterial stringent response system that controls transitions between growth and non-growth states in E. coli has not been evaluated with respect to its role in animal colonization. The stringent response to nutrient limitation involves the rapid accumulation of a unique nucleotide, guanosine 3', 5'-bispyrophosphate (ppGpp), which causes extensive reprogramming of gene expression during growth transitions. Synthesis and degradation of ppGpp is dynamic, allowing the cell to rapidly adapt to stress and then rapidly resume growth when conditions allow. The proposed research plan will determine whether intestinal colonization is dependent on maximal growth rate of the bacteria in the intestine or the ability of the bacteria to respond to nutrient limitation and adapt to the intestinal environment. Specifically, we will address four questions with regard to nutrient availability in the mouse intestine and the adaptation of E. coli to the intestinal environment. (1) Does E. coli lead a feast and famine existence in the mouse intestine? We have identified ppGpp-dependent genes to use as molecular beacons of the growth status of the cell, which will allow us to ask whether or not E. coli experiences discontinuous or continuous nutrient availability in the intestine. Gene fusions to fluorescent protein reporters will be used to visualize with confocal microscopy the growth status of individual bacterial cells within the mucus layer of infected mouse intestines. Alternatively we will use in situ hybridization to measure expression of these growth regulated genes in single bacterial cells. These experiments will reveal the nature of the intestinal environment as it is perceived by colonized bacteria. (2) What is the minimum growth rate required to sustain colonization? Experimentally, we will address this question by infecting mice with a series of strains that have been genetically manipulated to fix their ppGpp levels independently of environmental signals and hence have fixed growth rates. We will determine the relative fitness of these strains for colonization and measure their in vivo growth rates. (3) Do faster growing strains out-compete strains with slower growth? The strains with fixed growth rates will be competed against each other. (4) Is the ability to adjust ppGpp levels (i.e., growth rate) more important than sustaining the maximal logarithmic growth rate? The fixed ppGpp strains will be competed with the wild type, which can adjust its growth rate normally. These experiments, to be conducted in the model organism E. coli K-12, should lead to novel insights into the physiology of colonized bacteria. Because colonization is the first step in infection and disease, this work will be extended to E. coli O157:H7 in the future to search for common themes in commensalism and pathogenesis. The answers to these questions will lead to a better understanding of gastrointestinal health and potentially to novel strategies for combating gastrointestinal infections. PUBLIC HEALTH RELEVANCE: This proposal is based on the hypothesis that E. coli must appropriately control its growth rate to colonize and achieve high populations in the intestine. In testing this hypothesis, we will determine whether animal colonization is dependent on maximal growth rate of the bacteria in the intestine or the ability of the bacteria to respond to nutrient limitation and adapt by adjusting their growth rate. The answers to these questions will provide new information about the physiology of intestinal bacteria within their host and a better understanding of gastrointestinal health, which will lead to novel strategies for combating gastrointestinal infections.