Brain Endothelial Cell Receptor for Escherichia coli

描述（由申请方提供）：大肠埃希菌K1是新生儿脑膜炎的最常见病因。过去几十年来抗生素治疗的无效和耐药大肠杆菌的出现。大肠杆菌菌株的感染意味着对新的治疗和预防方法存在巨大的未满足的需求。对发病机制的每一步的不完全理解是导致这种不良结局的原因。例如，E. coli K1进入人脑微血管内皮细胞（HBMEC）并破坏紧密连接（TJ）的机制尚不清楚。我们已经确定了E.大肠杆菌与内皮细胞gp 96（Ecgp）相互作用，侵入并破坏TJ; OmpA-Ecgp相互作用的重要性进一步得到以下结果的支持：1）E.缺乏OmpA或表达非功能性OmpA的大肠杆菌菌株在新生小鼠或大鼠模型中不诱导脑膜炎;大肠杆菌感染。有趣的是，OmpA与Ecgp的相互作用由于iNOS活化而触发一氧化氮（NO）的产生，从而增强受体的表达，使细菌更有效地侵入。与此一致，iNOS-/-小鼠对E.大肠杆菌感染，也管理的iNOS特异性抑制剂，氨基胍（AG），在高级别菌血症预防脑膜炎的发生。利用计算机模拟方法研究OmpA和Ecgp的相互作用，并筛选出抑制E.大肠杆菌侵袭HBMEC。三种小分子对大肠杆菌的抑制率均在80%以上。大肠杆菌在HBMEC中的侵袭能力。我们的研究还表明，当感染E.杆菌此外，GT3活化蛋白IQGAP 1结合肌动蛋白和β-连环蛋白，似乎在侵袭过程中发挥作用。IQGAP 1是Stat 3的客户蛋白，Stat 3被证明与Ecgp相关，表明IQGAP 1可能中继Ecgp介导的信号以诱导E.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E.大肠杆菌侵袭和血脑屏障通透性增加。在目的1中，我们提出定义的结合域的Ecgp编排的相互作用的Ecgp/Robo 4与OmpA。接下来，为了理解Ecgp与Robo 4的相互作用是否有助于诱导NO产生的信号传导事件，从而调节IQGAP 1与b-CAT的结合以将其从TJ中驱逐，将在目标2中进行测试。然后，在目标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iNOS抑制剂负载到大脑中以防止E。coli诱导的新生大鼠脑膜炎。转化医学是这种应用的结果，其中基础生物学和应用技术的研究，以开发新的预防策略将被整合。

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.