SdiA regulation of EHEC virulence

描述(申请人提供)：肠出血性大肠杆菌(EHEC)是一种致命的食源性病原体，可在全球范围内引起出血性结肠炎和溶血性尿毒症综合征(HUS)。EHEC的主要蓄水池是牛群。EHEC在牛的直肠肛门交界处(Raj)定植，利用肠细胞消失(Lee)基因形成附着和消失(AE)病变。此外，EHEC还需要谷氨酸抗性(GAD)系统才能在这些动物的酸性胃中生存并成功地在Raj上定居。Lee和Gad基因的协同表达是通过SdiA转录因子实现的。SdiA是LuxR的同源物，LuxR是一种参与细菌群体感应(QS)的转录因子，它是细菌自身诱导剂酰基高丝氨酸内酯(AHL)的受体。虽然大肠杆菌(包括EHEC)不产生AHL，但它们有能力通过SdiA对这些信号做出反应。PI报道，SdiA感知瘤胃微生物区系产生的AHL，以抑制Lee基因的表达，这是胃肠道(GI)区不必要的能量消耗，并在EHEC到达酸性胃之前激活Gad基因的表达，以启动EHEC的耐酸性。这种协调的SdiA介导的基因调控对于EHEC有效地在牛身上定植是必要的。大多数LuxR型转录因子需要AHL信号作为折叠开关。虽然最初提出SdiA也是如此，但越来越多的证据表明，在缺乏AHL的情况下，SdiA可以调节包括Gad基因在内的几个基因的转录。我们解决了SdiA与两个AHL结合的结构，即oxo-C6-高丝氨酸内酯(HSL)和oxo-C8-HSL，以及在没有AHL的情况下。两种AHL结合形式的SdiA的晶体结构完美地重叠在一起。然而，与没有AHL相比，SdiA AHL结合蛋白的整体结构有一个谨慎的结构变化，这在该蛋白的螺旋-转角-螺旋(HTH)DNA结合域中更加明显。这种结构变化使该蛋白的AHL结合形式具有更高的DNA结合亲和力。这些结构研究还显示，EHEC产生一种内源性SdiA配体，被鉴定为1-辛酰基-RAC-甘油，这是一种构成三酰甘油的单酰基甘油。因此，本应用的具体目的是：1.确定SdiA内源配体的合成途径。具体目标2.确定SdiA配体结合口袋内不同信号识别和功能的要求。具体目的3.确定SdiA依赖的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.