SdiA regulation of EHEC virulence

描述(申请人提供)：肠出血性大肠杆菌(EHEC)O157：H7血清型导致世界各地爆发血性腹泻和溶血性尿毒症综合征。EHEC的主要蓄水池是牛群。EHEC对牛的定植需要一个功能性的肠细胞消失区(Lee)区域，参与肠粘连，并在肠上皮细胞上形成称为附着和消失性(AE)病变的病变，以及一个功能性的SdiA转录因子。SdiA是一种群体感应转录因子，需要其自身诱导剂，即酰基高丝氨酸内酯(AHL)才能正确折叠和发挥功能。EHEC含有SdiA，但不产生AHL自身诱导子，因此必须感应环境中其他种类的细菌产生的AHL。这些观察，结合报道显示AHL在牛瘤胃内显著存在，表明SdiA-AHL介导的EHEC基因调控对牛胃肠道定植所必需的基因具有重要作用。我们证明了人工合成的AHL和从牛瘤胃中提取的AHL都能抑制Lee基因的表达。然而，日粮在调节瘤胃内AHL水平中的作用及其与EHEC脱落的相关性仍然知之甚少。此外，SdiA调控转录的机制仍不清楚。最后，sdiA突变在牛肠道中的分布，以及其衰减的确切原因仍有待确定。因此，这项研究的具体目标是：具体目标1：评估牛瘤胃样品中AHLS的存在及其与日粮和EHEC脱落的相关性。具体目的2：确定SdiA依赖的基因调控的分子机制。具体目标3：确定一个sdiA突变体在牛肠中的分布，并更好地确定该突变体在牛的定居能力减弱的基础。公共卫生相关性：肠出血性大肠杆菌(EHEC)O157：H7血清型是导致全世界爆发血性腹泻的病原体。EHEC的主要蓄水池是牛群。因此，减少EHEC对牛的定植和脱落对人类健康具有重大意义。在这项拨款提案中，我们的目标是了解细菌细胞之间的信号机制是如何通过一种名为酰基高丝氨酸内酯的小分子来控制EHEC基因的表达，这些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.