SdiA regulation of EHEC virulence

性状（由申请方提供）：肠出血性大肠杆菌。O 157：H7型肠出血性大肠杆菌（EHEC）引起了世界范围内的出血性腹泻和溶血性尿毒综合征的爆发。肠出血性大肠杆菌的主要宿主是牛群。牛的EHEC定植已被证明需要肠细胞消失（LEE）区域的功能位点，该区域参与肠粘连和肠细胞损伤（称为附着和消失（AE）损伤）的形成，以及功能性SdiA转录因子。SdiA是一种群体感应转录因子，需要其自身诱导物，酰基高丝氨酸内酯（阿勒）的正确折叠和功能。EHEC携带SdiA，但不产生阿勒自身诱导物，因此必须感测环境中其他细菌物种产生的AHL。这些观察结果与显示AHL在牛瘤胃内突出的报道相结合，表明阿勒介导的EHEC基因的调节对于牛胃肠道（GI）道的定殖是必要的，具有重要作用。我们证明，无论是合成的AHLs，以及从牛瘤胃提取的AHLs抑制LEE基因的表达。然而，在调节瘤胃内阿勒水平的饮食的作用，以及它们与EHEC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以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：评估牛瘤胃样品中AHLs的存在，以及它们与饮食和EHEC排出的相关性。具体目标2：确定SdiA依赖性基因调控的分子机制。具体目标3：表征sdiA突变体在牛肠道内的分布，并更好地定义该突变体在牛体内定植能力降低的基础。公共卫生相关性：肠出血性大肠杆菌（EHEC）血清型O 157：H7是引起全世界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，减少牛的肠出血性大肠杆菌定植和脱落对人类健康具有重大意义。在这项资助提案中，我们的目标是了解细菌细胞之间的信号传导机制如何通过一种称为酰基高丝氨酸内酯的小分子（存在于牛的瘤胃中）控制EHEC基因的表达，这些基因是牛肠道毒力和定植所必需的。

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.