IbeA受体波形蛋白(VIM)介导的新生儿细菌性脑膜炎三联特征的分子机制

Despite the availability of bactericidal antibiotics over the last sixty years, bacterial meningitis has remained one of the top ten causes of infection-related death worldwide (mortality rates: 25-35%), and about 40% of the survivors sustain neurological sequelae. This high morbidity and mortality is due to the lack of integrated studies on the three interrelated hallmark features of bacterial meningitis: (a) NFkB activation in CNS, (b) pathogen invasion and (c) neutrophil transmigration (PMNT) across the blood-brain barrier (BBB), which consists mainly of brain microvascular endothelial cells (BMEC). Our previous studies showed that the E. coli IbeA receptor vimentin (Vim) plays an important role in the pathogenesis of neonatal sepsis and meningitis (NSM), which remains a major cause of death in newborns, especially in low-birth weight infants. The ibeA locus is able to modulate expression of several virulence factors (e.g., fim, ibeB, ompA) and predominantly contribute to E. coli K1-caused early-onset human NSM by modulating the three interrelated hallmark features of this disease. Vim has emerged as an organizer of a number of critical proteins involved in cell adhesion/migration and cell signaling. Vim-mediated signaling is required for IbeA-induced pathogenicities. Lipid rafts (LRs) serve as a signaling platform for Vim, PSF (IbeA co-receptor), α7 nAChR (regulator of inflammation), and other signaling molecules. Our recent studies have shown that Vim could regulate IbeA-induced NFkB signaling. Vim forms a complex with IkB, NFkB and tubulins in the resting cells through its head domain. Vim-mediated modulation of the ubiquitin proteasome system (UPS) is essential for NFkB activation. IbeA is able to induce the IkB kinase activation, dissociation of the Vim/IkB-NFkB complex, and expression of Vim, α7 nAChR and proinflammatory factors (PIFs). Nuclear translocation of NFkB is correlated with increased PSF in the nucleus. Circulating BMECs (cBMEC) in the blood could be used as cell-based biomarkers for single cell profiling of the BBB injury using infectomic approaches. Most importantly, we have shown that IbeA-induced NFkB activation, bacterial invasion, and PMNT are significantly reduced in Vim KO (-/-) mice, which are consistent with the in vitro findings. Based on these results, we hypothesize that Vim contributes to IbeA-induced cytoplasmic activation and nuclear translocation of NFkB. Subsequent upregulation of Vim, α7 nAChR and PIFs leads to CNS inflammation/injury, which can be detected by cBMEC, and blocked by Vim inhibitors. Our hypothesis will be examined with the following specific aims: 1). Examine how Vim modulates the three interrelated hallmark features of bacterial meningitis using the cell culture systems, animal models (Vim +/+ vs Vim -/-), and genetic approaches. 2). Investigate effects of Vim-targeted inhibitors on E. coli K1 infection in vitro (BMEC), ex vivo (organotypic hippocampal culture), and in vivo (mouse model).

抗生素用于临床虽已60年之久, 细菌性脑膜炎仍然是致死性的中枢神经系统(CNS)感染性疾病。其病死率仍高达25～35%，而幸存者中神经、精神后遗症高达40%。该病难治的重要原因是对细菌性脑膜炎三联炎症特征[即NF-κB激活, 细菌入侵血脑屏障(BBB)与白细胞进入CNS]的病理机制尚无统一认识。我们的前期研究证明新生儿E. coli脑膜炎三联炎症特性是由IbeA受体波形蛋白（VIM）所介导的。IbeA是引起新生儿早期E. coli感染的重要毒力因子，并参与多个毒力因子的调控。IbeA如何通过VIM调控细菌性脑膜炎三联炎症特性有待进一步探讨。因此，本项目将从细胞（体外BBB模型），组织器官（海马回切片）及整体水平（VIM基因敲除小鼠动物实验）等不同层面，采用最新单细胞感染组学技术监测BBB功能，揭示E. coli脑膜炎时VIM是如何介导及调节细菌性脑膜炎三联炎症特征，为其防治提供重要依据。

项目背景: 细菌性脑膜炎(BM)仍然是严重的危及生命的中枢神经系统(CNS)感染性疾病。其死亡率仍然可高达25～35%，而幸存者高达40%留有CNS后遗症。该病难治的重要原因是对BM三联炎症特征[即NF-κB激活, 细菌入侵血脑屏障(BBB)与白细胞(PMN)进入CNS)]的病理机制尚无统一认识。我们的前期研究证明新生儿E. coli BM三联炎症特性是由IbeA受体波形蛋白（VIM）所介导的。IbeA是引起新生儿早期E. coli感染并参与多个毒力基因调控的重要毒力因子。本项目进一步探讨了IbeA如何通过VIM调控BM三联炎症特性。主要研究内容:本项目从细胞、组织器官及整体水平（体内BBB模型、VIM基因敲除小鼠）等不同层面，探讨并揭示E. coli脑膜炎时VIM是如何介导及调节NF-κB通路, 细菌入侵BBB与白细胞进入CNS等三个相互关联的BM特征，并为其防治提供重要理论依据。重要结果: 1、VIM抑制剂(WFA)能有效抑制三联炎症特征,这一过程呈量效关系。2、无论是环境因素，如尼古丁(NT)，还是E. coli或毒力因子，引起的BBB的破坏及功能异常，都会导致血中细胞水平的生物标志物cBMEC显著升高,提示cBMEC可作为潜在的BBB损伤的诊断指标。3、实验证明无论敲除小鼠VIM基因或致病菌毒力因子IbeA均会导致三联炎症特征显著降低。4、NF-κB抑制剂CAPE的阻断作用与VIM的存在显著相关。5.α7nAChR抑制剂(MEM)能有效抑制三联炎症特征,这一过程呈量效关系。6.无论是药物敏感菌株，如E44，还是多重耐药菌株(如IHE2015) 所引发的菌血症与脑膜炎均可被MEM治愈。MEM能明显保护病原菌引起的BBB损伤。7. 我们首次证明MEM能显著升高PMN的胞外杀菌网络(NET)活性。关键数据及其科学意义: 通过VIM基因敲除小鼠E. coli BM三联炎症特征的影响的研究验证了VIM如何在致病过程中发挥作用。同时，模拟了E. coli BM自然感染全过程，其在体内通过肠道-血循环-脑的致病过程，从整体进一步明了病原体-宿主之间如何协同作用而致病的。我们系统阐述了BM早期CNS炎症和BBB损伤之间的内在致病机理。进一步完善了病原菌入侵，炎症信号途径的活化与反馈，以及白细胞经穿BBB迁徙的BM三联症致病机制的理论模型。

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