ESCHERICHIA COLI INVASION OF BRAIN ENDOTHELIAL CELLS

大肠杆菌入侵脑内皮细胞

基本信息

批准号：
6373584
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
项目类别：
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/6373584
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

项目摘要

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）：细菌性脑膜炎尽管仍然与高发病率和死亡率相关化疗和支持治疗方面的进展。大肠杆菌是最常见的引起新生儿脑膜炎的革兰氏阴性菌。大多数病例发展由于血行传播，但尚不清楚大肠杆菌是如何循环的。大肠杆菌穿过大脑微血管内皮，该内皮构成了血脑屏障。 PI已证明大脑的入侵大肠杆菌 K1 介导的微血管内皮细胞 (BMEC) 因素。两个独立的基因位点，由 PI 鉴定为致病性岛屿（paiA 和 paiB）显然与大肠杆菌脑膜炎有关。派阿含有被确定为主要入侵基因的 20 kb 大肠杆菌位点 K1 大肠杆菌菌株 RS218 中的簇。大肠杆菌的 TnphoA 插入突变体 RS218在组织培养中无法侵入BMEC并导致血行新生大鼠模型中的脑膜炎。 paiA上编码为ibe10，它编码显示完整膜特征的 8.2 kDa 蛋白质具有四个跨膜结构域的蛋白质。重组 Ibe10 蛋白是能够阻断大肠杆菌K1对BMEC的侵袭。 30% 的人中检测到 Ibe10 引起脑膜炎的 K1 大肠杆菌临床分离株。 Ibe10 的六个克隆已从 lambda Gem-12 文库中分离出来。重组质粒携带 15 kb 大肠杆菌片段能够补充非侵入性 TnphoA 突变体 10A-23。并且，在附录中，PI 报告说他已经使用 18 成功地赋予大肠杆菌 K12 菌株侵袭性 paiA 的 kb 片段。这项工作的长期目标是开发新颖的预防和治疗大肠杆菌脑膜炎的策略（例如疫苗）基于对分子机制的充分理解大肠杆菌侵入血脑屏障的原因。 PI 假设大肠杆菌侵入血脑的表型屏障由致病性大肠杆菌 K1 特异性基因编码决定因素，pai。该提案包含两个目标：目标 1：进一步描述该角色的特征入侵基因簇 paiA 和/或 paiB 在大肠杆菌发病机制中的作用使用体外（BMEC 侵入）和体内（幼鼠）模型来治疗脑膜炎以及分子和遗传学方法。目标 2：确定函数和通过BMEC鉴定入侵蛋白ibe10的功能域 ibe10 受体、体外诱变和表位作图。