Molecular Basis for Pseudomonas Recognition of Epithelia

假单胞菌识别上皮细胞的分子基础

• 批准号: 6986114
• 负责人: BARBARA I KAZMIERCZAK
• 金额: $ 31.62万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6986114
• 关键词: Pseudomonas aeruginosa; apoptosis; bacteria infection mechanism; bacterial proteins; biological signal transduction; cell cell interaction; cytotoxicity; enteric bacteria; enzyme linked immunosorbent assay; epithelium; host organism interaction; laboratory mouse; microarray technology; molecular genetics; polymerase chain reaction; southern blotting; virulence

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is an opportunistic gram-negative pathogen that causes acute, hospital-acquired infections as well as chronic disease in patients with cystic fibrosis. Though the organism is ubiquitous in the environment and possesses many virulence factors found in other gram-negative bacteria, it rarely causes disease in healthy hosts. Acute infection by P. aeruginosa usually occurs in the setting of pre-existing epithelial tissue damage; this is mirrored by increased bacterial adherence, invasion and cytotoxicity toward damaged epithelial monolayers in vitro. This grant proposal describes experiments to elucidate the molecular basis for P. aeruginosa interactions with epithelial cells. Its primary objective is to understand how pathogen-host cell interactions result in the activation of contact-dependent virulence factors such as the Type III secretion system. We have defined a novel set of P. aeruginosa mutants that can no longer interact with epithelial cells to promote internalization. The ability of these mutants to cause in vitro cytotoxicity will be directly assayed in Specific Aim #1, allowing us to identify a subset of mutants deficient for interactions required for Type III mediated virulence. These mutants will be tested in a murine model of acute pneumonia to confirm that these mutations also result in attenuated virulence toward the intact host. We also present preliminary data about two mutants, ide-2 and ide-9, which demonstrates that we have already identified two putative P. aeruginosa proteins required for epithelial cell interactions leading to Type III dependent virulence. Specific Aims #2 and #3 discuss in detail the characterization of these two mutants. The characterization of bacterial molecules and signaling pathways underlying host cell recognition and contact-dependent virulence in P. aeruginosa will provide novel targets for treating and preventing Pseudomonas colonization and disease. Such insights are likely to prove applicable to other gram-negative pathogens of animals and plants, which similarly activate Type III secretion of bacterial effectors required for virulence in response to poorly understood signals that follow host cell contact.

描述（由申请人提供）：铜绿假单胞菌是一种机会主义的革兰氏阴性病原体，可导致囊性纤维化患者的急性，医院获得性感染以及慢性疾病。尽管该生物在环境中无处不在，并且在其他革兰氏阴性细菌中发现了许多毒力因子，但它很少引起健康宿主的疾病。铜绿假单胞菌的急性感染通常发生在预先存在的上皮组织损伤的情况下。在体外，对受损上皮单层受损的细菌粘附，侵袭和细胞毒性的增加反映了这一点。该赠款提案描述了阐明铜绿假单胞菌相互作用与上皮细胞的分子基础的实验。它的主要目标是了解病原体宿主细胞相互作用如何导致接触依赖性毒力因子（例如III型分泌系统）的激活。 我们已经定义了一组新型的铜绿假单胞菌突变体，这些突变体无法再与上皮细胞相互作用以促进内在化。这些突变体引起体外细胞毒性的能力将直接在特定的目标＃1中进行测定，从而使我们能够确定缺乏III型介导的毒力所需相互作用的突变体的子集。这些突变体将在急性肺炎的鼠模型中进行测试，以确认这些突变还会导致对完整宿主的毒力减弱。我们还提供了有关两个突变体IDE-2和IDE-9的初步数据，这些数据表明我们已经确定了上皮细胞相互作用所需的两个假定的铜绿假单胞菌蛋白，导致III型依赖性毒力。具体目的＃2和＃3详细讨论了这两个突变体的表征。 铜绿假单胞菌中宿主细胞识别和接触依赖性的毒力的细菌分子的表征和信号通路将为治疗和预防假单胞菌定殖和疾病提供新的目标。这种见解可能证明适用于动物和植物的其他革兰氏阴性病原体，这些病原体类似地激活了毒力所需的III型细菌效应子的分泌，以响应于伴随着宿主细胞接触的信号较低的信号。