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E. coli Growth Parameters in the Intestine

肠道内大肠杆菌生长参数

基本信息

批准号：
7860306
负责人：
TYRRELL CONWAY
金额：
$ 18.75万
依托单位：
UNIVERSITY OF OKLAHOMA
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-06-05 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7860306
关键词：
Address Animal Model Animals Applications Grants Bacteria Cells Confocal Microscopy Disease Environment Escherichia coli Escherichia coli O157 Famines Foundations Future Gene Expression Gene Fusion Genes Genetic Programming Growth Guanosine Health Heart In Situ Hybridization In Vitro Individual Infection Intestines Knowledge Lead Life Style Measures Metabolism Molecular Monitor Mucous body substance Mus Nature Nucleotides Nutrient Pathogenesis Physiology Population Proteins Relative (related person)Reporter Reporter Genes Research Role Series Signal Transduction Stress Symbiosis System Testing United States National Institutes of Health Work base combat commensal microbes enteric pathogen experience fitness gastrointestinal gastrointestinal infection in vivo insight mutant novel novel strategies pathogenic Escherichia coli programs public health relevance research study response

项目摘要

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.

描述(申请人提供)：在大肠杆菌中控制生长和非生长状态之间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的核苷酸--鸟苷3‘，5’-二焦磷酸(PpGpp)的快速积累，这会导致生长过渡期间基因表达的广泛重新编程。PpGpp的合成和降解是动态的，使细胞能够快速适应压力，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取决于肠道细菌的最大生长速度，还是取决于细菌对营养限制的反应和适应肠道环境的能力。具体地说，我们将解决四个问题，涉及小鼠肠道内营养的可获得性和大肠杆菌对肠道环境的适应。(1)在小鼠的肠道中，大肠杆菌会导致暴食和饥荒吗？我们已经确定了ppGpp依赖的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了不连续或连续的营养供应。将基因融合到荧光蛋白报告程序将用共聚焦显微镜显示受感染小鼠肠道粘液层中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，正如被定植的细菌所感知的那样。(2)维持殖民所需的最低增长率是多少？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作，可以独立于环境信号固定它们的ppGpp水平，从而具有固定的生长率。我们将确定这些菌株的相对定植适合性，并测量它们在体内的生长速度。(3)生长较快的品系是否胜过生长较慢的品系？具有固定生长速度的菌株将相互竞争。(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，野生型能够正常调节其生长速度。这些实验将在模式生物E.ColiK-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，以寻找共生和致病的共同主题。对这些问题的回答将有助于更好地了解胃肠道健康，并可能产生抗击胃肠道感染的新战略。与公共卫生相关：这项建议是基于这样一个假设，即大肠杆菌必须适当控制其生长速度才能在肠道内定居并实现高种群。在检验这一假设时，我们将确定动物的定植是否取决于肠道细菌的最大生长速度，或者细菌通过调整生长速度对营养限制做出反应和适应的能力。这些问题的答案将提供有关肠道细菌在宿主体内的生理学的新信息，并更好地了解胃肠道健康，这将导致抗击胃肠道感染的新策略。