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转换的细菌严格反应系统尚未就其在动物定植中的作用进行评估。对营养限制的严格反应涉及一种独特核苷酸鸟苷3',5'-二焦磷酸（ppGpp）的快速积累，这在生长过渡期间导致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，然后在条件允许的情况下迅速恢复生长。拟议的研究计划将确定肠道定植是否取决于细菌在肠道内的最大生长速度，还是细菌对营养限制的反应能力和对肠道环境的适应能力。具体来说，我们将解决四个问题，关于营养物质在小鼠肠道和大肠杆菌对肠道环境的适应。(1)大肠杆菌是否在小鼠肠道中导致了盛宴和饥荒的存在？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，这将使我们能够询问大肠杆菌在肠道中是否经历间断或连续的营养可用性。荧光蛋白报告基因融合将用于共聚焦显微镜观察感染小鼠肠道黏液层内单个细菌细胞的生长状况。另外，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本质，因为它是由定植细菌感知。(2)维持定殖所需的最低生长速率是多少？在实验上，我们将通过用一系列菌株感染小鼠来解决这个问题，这些菌株经过基因操纵，使它们的ppGpp水平不受环境信号的影响而固定，从而具有固定的生长速度。我们将确定这些菌株的相对适合定植和测量他们的体内生长速度。(3)生长快的菌株是否胜过生长慢的菌株？具有固定生长速率的菌株将相互竞争。(4)调整ppGpp水平（即增长率）的能力是否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，可以正常调节其生长速度。这些实验将在模式生物大肠杆菌K-12中进行，应该会对定植细菌的生理学产生新的见解。由于定植是感染和疾病的第一步，这项工作将在未来扩展到大肠杆菌O157:H7，以寻找共生和发病机制的共同主题。这些问题的答案将导致对胃肠道健康的更好理解，并有可能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.