SdiA regulation of EHEC virulence

描述（由申请人提供）：肠出血性大肠杆菌（EHEC）血清型O157:H7是世界各地血性腹泻和溶血性尿毒症综合征暴发的原因。肠出血性大肠杆菌的主要宿主是牛群。牛肠出血性大肠杆菌的定植需要肠细胞消退（LEE）区域的功能位点，该区域参与肠粘连，并在肠细胞上形成称为附着和消退（AE）病变，以及功能性SdiA转录因子。SdiA是一种群体感应转录因子，需要其自诱导剂酰基-高丝氨酸内酯（AHL）才能正常折叠和发挥功能。肠出血性大肠杆菌含有SdiA，但不产生AHL自诱导剂，因此必须感知环境中其他细菌产生的AHL。这些观察结果，结合报告显示AHL在牛瘤胃中很突出，表明SdiA- AHL介导的肠出血性大肠杆菌基因调节对牛胃肠道（GI）定植所必需的重要作用。我们证明，无论是合成ahl，还是从牛瘤胃中提取的ahl，都能抑制LEE基因的表达。然而，饮食在调节瘤胃内AHL水平中的作用及其与肠出血性大肠杆菌脱落的相关性仍然知之甚少。此外，SdiA调节转录的机制仍然未知。最后，sdiA突变体在牛肠道内的分布及其衰减的确切原因仍有待确定。因此，本研究的具体目的是：具体目的1：评估牛瘤胃样本中ahl的存在，以及它们与饮食和肠出血性大肠杆菌脱落的关系。专项目标2：确定sdia依赖性基因调控的分子机制。具体目标3：表征牛肠内sdiA突变体的分布，并更好地定义该突变体在牛体内定植能力下降的基础。公共卫生相关性：肠出血性大肠杆菌（EHEC）血清型O157:H7是导致世界各地出血性腹泻暴发的病原体。肠出血性大肠杆菌的主要宿主是牛群。因此，减少牛肠出血性大肠杆菌的定植和脱落对人类健康非常重要。在这项拨款申请中，我们的目标是了解细菌细胞之间的信号机制如何通过牛瘤胃中存在的一种称为酰基-高丝氨酸内酯的小分子来控制肠出血性大肠杆菌基因的表达，这种基因对牛肠道的毒力和定植是必要的。

DESCRIPTION (provided by applicant): Enterohemorrhagic E. coli (EHEC) serotype O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome throughout the world. The main reservoir for EHEC is cattle herds. EHEC colonization of cattle has been shown to require a functional locus of enterocyte effacement (LEE) region, involved in intestinal adhesion, and formation of lesions on enterocytes named attaching and effacing (AE) lesions, as well as a functional SdiA transcription factor. SdiA is a quorum sensing transcription factor that requires its autoinducer, an acyl-homoserine lactone (AHL) for proper folding and function. EHEC harbors SdiA, but does not produce an AHL autoinducer, consequently having to sense AHLs produced by other species of bacteria in the environment. These observations, combined with reports showing that AHLs are prominent within cattle rumen, suggest an important role for SdiA- AHL mediated regulation of EHEC genes necessary for colonization of the bovine gastrointestinal (GI) tract. We demonstrated that both synthetic AHLs, as well as AHLs extracted from cattle rumen repress expression of the LEE genes. However, the role of diet in modulating AHL levels within the rumen, and their correlation with EHEC shedding remain poorly understood. Furthermore, the mechanisms by which SdiA regulates transcription are still unknown. Finally, the distribution of an sdiA mutant within the intestinal tract of cattle, as well as a definitive reason underlying its attenuation remains to be established. Accordingly the Specific Aims of this study are: Specific Aim 1: To assess the presence of AHLs in rumen samples from cattle, as well as their correlation with diet and EHEC shedding. Specific Aim 2: Determine the molecular mechanisms of SdiA-dependent gene regulation. Specific Aim 3: Characterize the distribution of an sdiA mutant within the bovine intestine, and better define the basis for this mutant's diminished ability to colonize cattle. PUBLIC HEALTH RELEVANCE: Enterohemorrhagic E.coli (EHEC) serotype O157:H7 is the agent responsible for outbreaks of bloody diarrhea throughout the world. The main reservoir for EHEC is cattle herds. Consequently, diminishing cattle colonization and shedding of EHEC is of great interest to human health. In this grant proposal, we aim to understand how a signaling mechanism between bacterial cells through a small molecule called acyl-homoserine lactone, which is present in the rumen of cattle, controls expression of EHEC genes necessary for virulence and colonization of the bovine intestine.