PATHOGENESIS OF NEONATAL ESCHERICHIA COLI K1 INFECTIONS

新生儿大肠杆菌 K1 感染的发病机制

• 批准号: 2067475
• 负责人: Jane L. Burns
• 金额: $ 15.39万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2067475
• 关键词: Escherichia coli; Escherichia coli infections; disease /disorder model; gastrointestinal epithelium; gene deletion mutation; genetic library; laboratory rat; molecular cloning; newborn animals; nucleic acid sequence; pathologic process; plasmids; site directed mutagenesis; structural genes; tissue /cell culture; transposon /insertion element; virulence

Escherichia coli is the most common cause of neonatal Gram-negative sepsis and meningitis, occurring in 1 in 1000 live births. Despite the fact that over 100 capsular types have been identified in E. coli, K1 strains predominate, causing 40% of the bacteremia infections and 80% of the meningitis in neonates. Colonization with K1 occurs primarily at parturition with maternal GI tract colonization resulting in newborn colonization. For infants to become bacteremia, the organism must penetrate the GI mucosal barrier. The focus of this proposed research is the pathogenesis of neonatal infections, specifically the mechanisms by which E. coli K1 penetrates the intestinal mucosa. This project will examine the hypothesis that virulent E. coli K1 strains are able to enter the bloodstream of young infants because they have the ability to invade GI epithelial cells. Using a tissue culture model to examine invasion and an infant rat model of bacteremia to confirm virulence, this research will undertake the molecular analysis of the bacterial gene(so involved in the invasion of GI epithelium by E. coli K1. Transposon mutagenesis with Tnpho A has been used to derive mutants of a well-characterized, invasive 018:K1:H7 strain, RS218, which are unable to invade both in the tissue culture and infant rat models. A non- invasive mutant with a single transposon insertion, RS218-B3, has been selected for further characterization: it does not have a mutation within an operon encoding known virulence factors including K1 capsule, LPS or Type 1 pili or S-fimbriae. This and other non-invasive mutants will be used to clone invasion-related determinants: the DNA sequences flanking the TnphoA insertions will be cloned and sequenced and used to identify corresponding sequences from a cosmid library of DNA from the invasive parent strain, RS21. In parallel experiments, direct cloning of the invasion determinant(s) into a non-invasion E. coli strain is underway: a library has been constructed and is being screened for invasion. Complementation of the mutants will be attempted and nucleotide sequence and gene product analysis of putative invasion gene(s) will be performed. These studies will form the basis for the identification of a likely invasion-related structural gene. A deletion mutant of this gene will be constructed and its phenotype will be examined in both the tissue culture and infant rat models of invasion. These studies should help us understand the pathogenesis of E. coli K1 infections in newborn infants. In the future, this understanding may lead to improved strategies for the prevention and treatment of these life-threatening neonatal infections.

大肠杆菌是新生儿革兰氏阴性菌的最常见原因 败血症和脑膜炎，发生率为千分之一的活产儿。 尽管 事实上，在大肠杆菌 K1 中已鉴定出 100 多种荚膜类型 菌株占主导地位，导致 40% 的菌血症感染和 80% 的菌血症感染 新生儿脑膜炎。 K1 的定殖主要发生在 分娩时母体胃肠道定植导致新生儿 殖民化。 要使婴儿出现菌血症，机体必须 穿透胃肠道粘膜屏障。 这项研究的重点是新生儿的发病机制 感染，特别是大肠杆菌 K1 渗透的机制 肠粘膜。 该项目将检验以下假设： 剧毒大肠杆菌 K1 菌株能够进入年轻人的血液 婴儿，因为它们具有侵入胃肠道上皮细胞的能力。 使用组织培养模型检查入侵和幼鼠模型 菌血症以确定毒力，本研究将进行 细菌基因的分子分析（因此参与入侵 大肠杆菌 K1 的胃肠道上皮。 Tnpho A 的转座子诱变已被用于衍生突变体 一种特征明确的侵入性 018:K1:H7 菌株 RS218，无法 侵入组织培养物和幼年大鼠模型。 一个非 具有单个转座子插入的侵入突变体 RS218-B3 已被 选择用于进一步表征：它内部没有突变 编码已知毒力因子的操纵子，包括 K1 胶囊、LPS 或 1 型菌毛或 S 菌毛。 该突变体和其他非侵入性突变体将 用于克隆入侵相关决定簇：侧翼 DNA 序列 TnphoA 插入将被克隆和测序并用于识别 来自侵入性DNA粘粒文库的相应序列 亲本菌株，RS21。 在平行实验中，直接克隆 正在将入侵决定因素转化为非入侵大肠杆菌菌株： 图书馆已经建成，正在筛查入侵情况。 将尝试突变体的互补并确定核苷酸序列 并对假定的入侵基因进行基因产物分析。 这些研究将构成确定可能的 侵袭相关结构基因。 该基因的缺失突变体将 构建并在组织中检查其表型 培养和幼年大鼠入侵模型。 这些研究应该有助于我们了解大肠杆菌 K1 的发病机制 新生儿感染。 未来，这种理解可能会 从而改进预防和治疗这些疾病的策略 危及生命的新生儿感染。