Environmental modulation of the virulence program of enterhemorrhagic E. coli

肠出血性大肠杆菌(EHEC)是一种严重的食物和水传播病原体，是出血性结肠炎和溶血性尿毒症综合征的主要病因。虽然在EHEC致病机制方面已经取得了重大进展，但我们仍然不完全了解环境应激对EHEC毒力的影响。我们的研究表明，暴露在环境应激下会引起一系列特异的、高度调控的反应，这些反应不仅可以保护EHEC免受应激的影响，还可以调节毒力潜力和抗菌素敏感性。我们研究了暴露在包括酸、胆盐和短链脂肪酸(SCFA)在内的压力下的影响，这些压力是在宿主内外遇到的制剂，并发现了显著的证据表明，这些压力非但没有损害EHEC的生存，反而增强了它的适应性和引起感染的能力。我们这项研究计划的长期目标是系统地了解EHEC如何应对这些常见的环境压力，并特别强调病原体的生存和毒力潜力。*我们的第一个目标是确定EHEC对选定的压力做出反应的分子机制。具体地说，我们计划确定胆汁和胆盐如何增强EHEC抵抗人类防御素的能力，人类防御素是由肠道产生的对抗细菌病原体的分子。我们的最新数据表明，EHEC可能正在利用接触胆汁/胆盐来武装自己对抗防御素。我们的目标是确定这种抗性的分子基础。其次，我们有证据表明，病原体在食品加工行业清洗屠体和通过肠道转运过程中遇到单链脂肪酸时，会调节其运动能力。运动性为病原体在肠道转运过程中提供了深刻的优势，帮助它避免了有害的场所，并找到了有利的生态位。我们计划研究SCFAs如何在胃肠道的不同区域和外部环境中遇到，调节病原体的运动性，以及这种调节是如何调节的。*我们的第二个目标是研究使用选定的抗菌剂来增强胃酸对EHEC的自然杀灭作用的方法。从我们最近的研究中，我们知道了EHEC如何对酸做出反应，特别是通过增加修复其受损DNA的机制。我们计划使用靶向抗菌剂，防止病原体在酸应激后修复其DNA，并在体外和体内实验中比较它们的作用。使用EHEC和感染小鼠的EHEC相关病原体，我们将比较抗菌剂在小鼠台面和胃中暴露在酸中后损害这两种病原体生存的能力。通过这样做，我们应该能够利用胃酸应激的自然抗菌作用，在不破坏有益的共生菌群的情况下，以杀死新摄入的EHEC为目标。这一策略可以用来开发一种抗菌剂，用于在摄入之前应用于可能受到污染的食物。由于EHEC在酸性食物中存活，这也可能危及它在那里的生存。*这项研究提案将使我们能够在我们重大进展的基础上，深入调查常见环境因素对特定关键毒力特性和途径的影响，确定途径的分子基础和调控。这项研究的结果将加深我们对EHEC和相关肠道病原体对选定的环境应激反应的生物学途径的理解，并可能提供新的分子靶点，可以在食品加工过程中利用来调节这些病原体的毒力潜力。

Enterohemorrhagic E.coli (EHEC) is a serious food and water-borne pathogen and a leading cause of hemorrhagic colitis and hemolytic uremic syndrome. While major advances have been made in the field of EHEC pathogenesis, we still do not fully understand the impact of environmental stress on EHEC virulence. Our research has revealed that exposure to environmental stress elicits a set of specific, highly regulated responses designed not only to protect EHEC from the stress but also to modulate virulence potential and antimicrobial susceptibility. We have examined the impact of exposure to stresses including acid, bile salts and short chain fatty acids (SCFA), agents encountered both inside and outside the host and have found striking evidence that these stresses, rather than compromising EHEC survival, enhance its fitness and ability to cause infection. Our long term goal for this research proposal is to develop a systematic understanding of how EHEC responds to these commonly encountered environmental stresses with specific emphasis on pathogen survival and virulence potential. *Our first objective is to define the molecular mechanisms underlying EHEC's response to the selected stresses. Specifically, we plan to determine how bile and bile salts enhance EHEC's ability to resist human defensins, molecules produced by the intestines against bacterial pathogens. Our latest data indicate that EHEC may be using exposure to bile/bile salts to arm itself against defensins. Our aim is to define the molecular basis of this resistance. Secondly, we have evidence that the pathogen modulates its motility in response to SCFAs that it encounters during washing of meat carcasses in the food processing industry and during transit through the intestines. Motility provides the pathogen with a profound advantage during intestinal transit, helping it to avoid detrimental locales and to find favorable niches. We plan to examine how SCFAs encountered in different regions of the GI tract and in the external environment, modulate the pathogen's motility and how this modulation is regulated. *Our second objective is to investigate ways of enhancing the natural killing action of gastric acid on EHEC using selected antimicrobials. We know from our recent research how EHEC responds to acid, in particular by increasing mechanisms to repair its damaged DNA. We plan to employ targeted antimicrobials that prevent the pathogen from repairing its DNA after acid stress and to compare their action in both in vitro and in vivo experiments. Using EHEC and an EHEC-related pathogen that infects mice, we will compare the ability of the antimicrobials to compromise survival of both pathogens after exposure to acid on the bench top and in the stomach of mice. By doing so, we should be able to exploit the natural antimicrobial action of gastric acid stress in a way that targets the killing of newly ingested EHEC without damaging the beneficial commensal flora. This strategy could be used to develop an antimicrobial agent for application to potentially contaminated foods prior to ingestion. Since EHEC survives in acidic foods, this could also compromise its survival there.*This research proposal will permit us to build on our significant progress with an in-depth investigation of the effect of commonly encountered environmental agents on specific critical virulence properties and pathways, identifying the molecular basis and regulation of pathways. Results of this research will enhance our understanding of the biological pathways engaged during the response of EHEC and related enteric pathogens to selected environmental stresses and may provide new molecular targets that can be exploited during food processing to modulate the virulence potential of these pathogens.