SdiA regulation of EHEC virulence

性状（由申请方提供）：肠出血性大肠杆菌。大肠杆菌（EHEC）是一种致命的食源性病原体，在世界范围内引起出血性结肠炎和溶血性尿毒综合征（HUS）。肠出血性大肠杆菌的主要宿主是牛群。肠出血性大肠杆菌定植于牛的直肠肛门连接处（RAJ），利用肠上皮细胞消失（LEE）基因位点形成附着和消失（AE）病变。此外，肠出血性大肠杆菌还需要谷氨酸抗性（gad）系统在这些动物的酸性胃中生存并成功地在RAJ定植。LEE和gad基因的协调表达通过SdiA转录因子实现。SdiA是LuxR的同源物，LuxR是参与细菌群体感应（QS）的转录因子，作为细菌自诱导物酰基高丝氨酸内酯（阿勒）的受体发挥作用。虽然E.大肠杆菌（包括EHEC）不产生AHLs，它们有能力通过SdiA对这些信号做出反应。PI报告说，SdiA感应反刍动物微生物群产生的AHLs，以抑制LEE基因的表达，这是胃肠道（GI）隔室中不必要的能量消耗，并激活gad基因的表达，以在EHEC到达酸性胃之前引发其耐酸性。这种协调的SdiA介导的基因调控对于EHEC在牛体内的有效定殖是必要的。大多数LuxR型转录因子需要阿勒信号作为折叠开关。虽然最初提出SdiA也是这种情况，但越来越多的证据表明，SdiA可以在没有AHLs的情况下调节几个基因的转录，包括gad基因。我们解决了SdiA结合到两个AHL，氧代-C6-高丝氨酸内酯（HSL），和氧代-C8-HSL，以及在阿勒的情况下的结构。两种AHL结合形式的SdiA的晶体结构彼此完全重叠。然而，与没有阿勒相比，SdiA AHL结合蛋白的整体结构中存在谨慎的结构变化，这在该蛋白的螺旋-转角-螺旋（HTH）DNA结合结构域中更明显。这种结构变化赋予这种蛋白质的阿勒结合形式更高的DNA结合亲和力。这些结构研究还表明，EHEC产生一种SdiA内源性配体，鉴定为1-辛酰基-rac-甘油，一种构成三酰基甘油结构单元之一的单酰基甘油。因此，本申请的具体目的是：具体目的1.鉴定SdiA内源性配体的合成途径。具体目标2。定义SdiA配体结合口袋内不同信号识别和功能的要求。具体目标3。确定SdiA依赖性gad调节的分子机制。

DESCRIPTION (provided by applicant): Enterohemorrhagic E. coli (EHEC) is a deadly food borne pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome (HUS) worldwide. The main reservoir for EHEC is cattle herds. EHEC colonizes the recto anal junction (RAJ) of cattle, forming attaching and effacing (AE) lesions employing the locus of enterocyte effacement (LEE) genes. Additionally, EHEC also requires the glutamate acid resistance (gad) system to survive the acidic stomachs of these animals and successfully colonize the RAJ. Coordinated expression of the LEE and gad genes is achieved through the SdiA transcription factor. SdiA is a homolog of LuxR, which is a transcription factor involved in bacterial quorum sensing (QS), functioning as a receptor for the bacterial autoinducer acyl-homoserine lactone (AHL). Although E. coli (including EHEC) does not produce AHLs, they have the ability to respond to these signals through SdiA. The PI reported that SdiA senses AHLs produced by the rumminal microbiota to repress expression of the LEE genes, which is an unnecessary expense of energy in this gastrointestinal (GI) compartment, and activate expression of the gad genes to prime EHEC's acid resistance before it reaches the acidic stomachs. This coordinated SdiA-mediated gene regulation is necessary for EHEC's efficient colonization of cattle. Most LuxR-type transcription factors require the AHL signal as a folding switch. Although it was initially proposed that this scenario was also the case for SdiA, there is mounting evidence that SdiA can regulate transcription of several genes, including the gad genes, in the absence of AHLs. We solved the structures of SdiA bound to two AHLs, oxo-C6-homoserine lactone (HSL), and oxo-C8-HSL, as well as in the absence of AHL. The crystal structures of both AHL-bound forms of SdiA overlay perfectly with each other. However, there is a discreet structural change in the overall structure of the SdiA AHL-bound protein compared to no AHL, which is more pronounced in the helix-turn-helix (HTH) DNA binding domain of this protein. This structural change confers higher DNA binding affinity to the AHL bound form of this protein. These structural studies also revealed that EHEC produces an SdiA endogenous ligand identified as 1-Octanoyl-rac-glycerol, a monoacylglycerol that constitutes one of the building blocks of triacylglycerols. Accordingly the Specific Aims of this application are: Specific Aim 1. Identify te synthetic pathway of the SdiA endogenous ligand. Specific Aim 2. Define the requirements for different signal recognition and function within the SdiA ligand-binding pocket. Specific Aim 3. Determine the molecular mechanism of SdiA- dependent gad regulation.