ESCHERICHIA COLI INVASION OF BRAIN ENDOTHELIAL CELLS

大肠杆菌入侵脑内皮细胞

• 批准号: 6170086
• 负责人: SHENG-HE HUANG
• 金额: $ 10.57万
• 依托单位: CHILDREN'S HOSPITAL OF LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6170086
• 关键词: Escherichia coli; Escherichia coli infections; bacterial cytopathogenic effect; bacterial genetics; bacterial meningitis; brain cell; gene complementation; host organism interaction; laboratory rat; membrane proteins; phenotype; site directed mutagenesis; transposon /insertion element; vascular endothelium

DESCRIPTION (Adapted from the applicant's abstract): Bacterial meningitis continues to be associated with high morbidity and mortality despite advances in chemotherapy and supportive care. E. coli is the most common gram-negative organism that causes neonatal meningitis. Most cases develop as a result of hematogenous spread, but it is not clear how circulating E. coli traverse the brain microvascular endothelium, which constitutes the blood brain barrier. The PI has demonstrated that the invasion of brain microvascular endothelial cells (BMEC) by E. coli K1 is mediated by multiple factors. Two separate genetic loci, identified by the PI as pathogenicity islands (paiA and paiB) apparently contribute to E. coli meningitis. PaiA contains the 20 kb E. coli locus identified as the major invasion gene cluster in K1 E. coli strain RS218. A TnphoA insertion mutant of E. coli RS218 was unable to invade BMEC in tissue culture and to cause hematogenous meningitis in a newborn rat model. Encoded on paiA is ibe10, which encodes an 8.2 kDa protein displaying the characteristics of an integral membrane protein with four transmembrane domains. A recombinant Ibe10 protein was able to block invasion of BMEC by E. coli K1. Ibe10 was detected in 30% of clinical isolates of K1 E. coli causing meningitis. Six clones for Ibe10 have been isolated from a lambda Gem-12 library. A recombinant plasmid carrying a 15 kb E. coli fragment was able to complement the non-invasive TnphoA mutant 10A-23. And, in an appendix, the PI reports that he has succeeded in conferring invasiveness on a strain of E. coli K12 using an 18 kb fragment of paiA. The long term goal of this work is to develop novel strategies for the prevention and treatment of E. coli meningitis (such as vaccines) based on a full understanding of the molecular mechanisms responsible for E. coli invasion of the blood-brain barrier. The PI hypothesizes that the phenotype of E. coli invasion of the blood-brain barrier is encoded by the pathogenic E. coli K1-specific genetic determinants, the pai's. The proposal comprises two aims: Aim 1: To further characterize the role of the invasion gene cluster paiA and/or paiB in the pathogenesis of E. coli meningitis by using in vitro (BMEC invasion) and in vivo (infant rat) models and molecular and genetic approaches. Aim 2: To determine the function and functional domains of invasion protein ibe10 by identification of the BMEC receptor for ibe10, in vitro mutagenesis and epitope mapping.

描述（改编自申请人的摘要）：细菌性脑膜炎 尽管 化学疗法和支持性护理的进步。 大肠杆菌是最常见的 引起新生儿脑膜炎的革兰氏阴性生物。 大多数情况都会发展 由于血源扩散的结果，尚不清楚如何循环E。 大肠杆菌穿越脑微血管内皮，构成 血脑屏障。 PI证明了大脑的侵袭 大肠杆菌K1的微血管内皮细胞（BMEC）由多个介导 因素。 两个单独的遗传基因座，由PI鉴定为致病性 岛屿（PAIA和PAIB）显然有助于大肠杆菌脑膜炎。 Paia 包含20 kb的大肠杆菌基因座，被鉴定为主要侵袭基因 簇在K1大肠杆菌菌株RS218中。 大肠杆菌的tnphoa插入突变体 RS218无法入侵组织培养物中的BMEC，并引起血源性 新生大鼠模型中的脑膜炎。 在PAIA上编码是IBE10，该编码编码 8.2 kDa蛋白显示整体膜的特性 具有四个跨膜结构域的蛋白质。 重组IBE10蛋白是 能够阻止大肠杆菌K1对BMEC的入侵。 在30％的30％中检测到IBE10 K1大肠杆菌的临床分离株，导致脑膜炎。 ibe10的六个克隆 已从lambda Gem-12库中隔离。 重组质粒 携带15 kb的大肠杆菌片段能够补充非侵入性 tnphoa突变体10a-23。 而且，在附录中，Pi报告说他有 成功地使用18 Paia的Kb碎片。 这项工作的长期目标是开发小说 预防和治疗大肠杆菌脑膜炎的策略（例如 疫苗）基于对分子机制的完全理解 负责大肠杆菌入侵血脑屏障。 pi 假设大肠杆菌侵入血脑的表型 障碍由致病性大肠杆菌K1特异性遗传编码 决定因素，pai's。 该提案包括两个目的：目标1：进一步表征角色 大肠杆菌发病机理中的入侵基因簇PAIA和/或PAIB 脑膜炎通过体外（BMEC侵袭）和体内（婴儿大鼠）模型 以及分子和遗传方法。 目标2：确定功能和 通过识别BMEC的侵袭蛋白IBE10的功能域 IBE10的受体，体外诱变和表位图。