Brain Endothelial Cell Receptor for Escherichia coli

描述（由申请人提供）：大肠杆菌K1是新生儿脑膜炎的最常见原因。在过去的几十年里，抗生素治疗的无效和抗生素耐药大肠杆菌菌株的出现意味着对新的治疗和预防方法的需求仍未得到满足。对发病机制每一步所涉及的机制的不完全理解归因于这一不良结果。例如，大肠杆菌K1进入构成血脑屏障的人脑微血管内皮细胞（HBMEC）并破坏紧密连接（TJs）的机制尚不清楚。我们已经确定大肠杆菌外膜蛋白A （OmpA）与内皮细胞gp96 （Ecgp）相互作用；一种在HBMEC上特异性表达的受体，侵入并破坏tj。我们的研究结果进一步支持了OmpA-Ecgp相互作用的重要性：1)在新生小鼠或大鼠模型中，缺乏OmpA或表达无功能OmpA的大肠杆菌菌株不会诱发脑膜炎；2)Ecgp表达被抑制的小鼠对大肠杆菌感染具有抗性。有趣的是，OmpA与Ecgp的相互作用触发了一氧化氮（NO）的产生，这是由于iNOS激活，从而增强了受体的表达，使细菌更有效地入侵。与此一致的是，iNOS-/-小鼠对大肠杆菌感染具有耐药性，并且在高级别菌血症期间给予iNOS特异性抑制剂氨基胍（AG）可以预防脑膜炎的发生。利用新的计算机建模方法来研究OmpA和Ecgp的相互作用，并确定阻止大肠杆菌入侵HBMEC的小分子抑制剂。在体内和体外，三种小分子对大肠杆菌在HBMEC中的侵袭均有80%以上的抑制作用。我们的研究还表明，在大肠杆菌感染后，Ecgp与HBMEC膜上的Robo4的相互作用增加。此外，结合肌动蛋白和b-连环蛋白的GTPase激活蛋白IQGAP1似乎在入侵过程中发挥作用。IQGAP1是Stat3的客户蛋白，Stat3与Ecgp相关，表明IQGAP1可能通过传递Ecgp介导的信号诱导大肠杆菌侵袭。因此，我们的假设是OmpA和Ecgp的相互作用是启动诱导大肠杆菌入侵和血脑屏障通透性增加的信号事件的基础。在目标1中，我们建议定义协调Ecgp/Robo4与OmpA相互作用的Ecgp结合域。接下来，为了了解Ecgp与Robo4的相互作用是否有助于诱导NO产生的信号事件，从而调节IQGAP1与b-CAT的关联，将其从TJs中移除，将在Aim 2中进行测试。然后，在Aim 3中，我们将修饰拮抗剂以获得更高的抑制效率，并将其偶联到纳米颗粒上，纳米颗粒将携带大量iNOS抑制剂递送到大脑，以预防大肠杆菌诱导的新生大鼠脑膜炎。转化医学是这一应用的结果，其中基础生物学研究和开发新的预防策略的应用技术将得到整合。

DESCRIPTION (provided by applicant): Escherichia coli K1 is the most common cause of meningitis in neonates. Ineffectiveness of antibiotic therapy over the last few decades and the emergence of antibiotic resistant E. coli strains imply that there is a great unmet need for new methods of treatment and prevention. Incomplete understanding of the mechanisms involved at every step of pathogenesis is attributed to this poor outcome. For example, the mechanisms by which E. coli K1 enters the human brain microvascular endothelial cells (HBMEC) that constitute the BBB and disrupts tight junctions (TJs) are poorly understood. We have established that outer membrane protein A (OmpA) of E. coli interacts with endothelial cell gp96 (Ecgp); a receptor specifically expressed on HBMEC, to invade and disrupt the TJs. The importance of OmpA-Ecgp interaction is further supported by our findings that 1) E. coli strains that either lack OmpA or express non-functional OmpA do not induce meningitis in a newborn mouse or rat model and 2) Mice in which Ecgp expression was suppressed were resistant to E. coli infection. Intriguingly, OmpA interaction with Ecgp triggers the production of nitric oxide (NO) due to iNOS activation and thereby enhances the expression of the receptor to allow the bacteria to invade more efficiently. In agreement, iNOS-/- mice are resistant to E. coli infection and also administration of an iNOS specific inhibitor, aminoguanidine (AG), during high-grade bacteremia prevented the occurrence of meningitis. Novel computer modeling methods were utilized to study the interaction of OmpA and Ecgp and to identify small molecule inhibitors that prevent the E. coli invasion of HBMEC. Three small molecules exhibited more than 80% inhibition of E. coli invasion in HBMEC both in vitro and in vivo. Our studies have also revealed that Ecgp interaction with Robo4 at the HBMEC membrane increases upon infection with E. coli. Further, a GTPase activating protein, IQGAP1, which binds both actin and b-catenin, appears to play a role in the invasion process. IQGAP1 is a client protein for Stat3, which was shown to be associated with Ecgp, indicating that IQGAP1 might be relaying Ecgp mediated signals to induce E. coli invasion. Thus, our hypothesis is that the interaction of OmpA and Ecgp is fundamental to initiate signaling events that induce E. coli invasion and increased permeability of the BBB. In Aim 1, we propose to define the binding domains of Ecgp that orchestrate the interaction of Ecgp/Robo4 with OmpA. Next, to understand whether Ecgp interaction with Robo4 contributes to signaling events to induce NO production and thereby modulating IQGAP1 association with b-CAT to dislodge it from TJs will be tested in Aim 2. Then, in Aim 3 we will modify the antagonists for higher inhibition efficiency and couple them to nanoparticles, which will carry a load of iNOS inhibitors to deliver to brain to prevent E. coli induced meningitis in newborn rats. Translational medicine is the outcome of this application in which studies of basic biology and applied technology to develop new strategies of prevention will be integrated.