Mechanisms for bacterial dissemination in corneal infection

角膜感染中细菌传播的机制

• 批准号: 9541945
• 负责人: Vincent Nieto
• 金额: $ 6.3万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9541945
• 关键词: Actins; Active Sites; Affect; Alleles; Bacteremia; Bacteria; Bioinformatics; Candidate Disease Gene; Cell Death; Cell membrane; Cells; Cellular biology; Cornea; Corneal Diseases; Cytoplasm; Data; Development; Disease; Doctor of Philosophy; Electron Microscopy; Electrons; Engineering; Epithelial; Epithelial Cells; Epithelium; Event; Eye; Fellowship; Fibrosis; Genes; Human; Image; Immunocompromised Host; Immunofluorescence Microscopy; In Vitro; Individual; Infection; Injury; Invaded; Knock-out; Lead; Libraries; Life; Mediating; Methods; Microbial Biofilms; Microtubules; Modeling; Motor; Movement; Mus; Mutation; Nosocomial Infections; Outcome; Pathogenesis; Patients; Penetration; Phospholipase; Pilum; Plasma Cells; Pneumonia; Polymerase Chain Reaction; Postdoctoral Fellow; Process; Pseudomonas aeruginosa; Publications; Publishing; Regulation; Role; Route; Second Messenger Systems; Solid; Suggestion; Surface; Testing; Theoretical model; Time; Training; Transcript; Urinary tract infection; Vacuole; Variant; Virulence; Western Blotting; appendage; bacterial genetics; cell motility; corneal epithelium; depolymerization; differential expression; experimental study; follow-up; host-microbe interactions; in vivo; in vivo Model; injured; mouse model; mutant; novel strategies; pathogen; prevent; protein expression; tool; trafficking; transcriptome sequencing

Project Summary Pseudomonas aeruginosa is among the most common causes of blinding corneal disease, while also being a major cause of life threating nosocomial infections such as pneumonia, bacteremia, urinary tract infections (UTIs), and cytstic fibrosis (CF), targeting immunocompromised and critically injured patients. Publications from the Fleiszig lab have shown that twitching motility, a type of surface associated movement, contributes to the ability of P. aeruginosa to penetrate human corneal epithelial cell multilayers in vitro and is critical to pathogenesis of P. aeruginosa corneal infection in a mouse model in vivo. Key to P. aeruginosa pathogenesis in the cornea is the capacity of the bacteria to invade corneal epithelial cells. While P. aeruginosa mutants that lack twitching motility can invade epithelial cells, and replicate inside them just as efficiently as wildtype bacteria, they have reduced capacity for exiting cells they have entered. During my postdoctoral fellowship in the Fleiszig lab, I used imaging and various other methods to study the mechanisms by which P. aeruginosa exits epithelial cells. Importantly, my preliminary data show that exit does not necessarily follow cell death, suggesting active/deliberate mechanisms contribute. My data further show that when twitching mutants invade and replicate in corneal epithelial cells, they differ from wildtype P. aeruginosa in being unable to distribute themselves in the cytoplasm and instead accumulate in aggregates. I have also screened a mutant library for exit capacity, and have found that mutants in either of two phospholipases, PlcB or PA2155, are exit defective. In contrast to twitching mutants, the phospholipase mutants spread normally throughout the host cell cytoplasm. Thus, my data mechanistically separate the exit process into two stages one dependent on twitching and the other dependent on phospholipases. My theoretical model for exit is that P. aeruginosa uses twitching motility to avoid forming a biofilm aggregate inside the cell and to access the host cell plasma membrane, where they use phospholipase activity (e.g. of PlcB and PA2155) to alter the plasma membrane to provide an exit route. Thus, in aim 1 I will the identify the genes transcripts that impact twitching mutant aggregation and exit compared to wildtype, and in aim 2 I will determine if phospholipases facilitate exit through their enzymatic activity. While contributing to our understanding of P. aeruginosa pathogenesis, this project could ultimately contribute to development of strategies for preventing and treating infections that act by preventing bacterial penetration through our protective surface epithelia.

项目摘要 铜绿假单胞菌是盲目角膜疾病的最常见原因之一，而 也是威胁性医院感染的主要生命原因，例如肺炎，菌血症， 尿路感染（UTI）和细胞纤维化（CF），靶向免疫功能低下和 严重受伤的患者。 Fleiszig Lab的出版物表明，Twitching Motility，一个 与表面相关运动的类型，有助于铜绿假单胞菌穿透的能力 人角膜上皮细胞多层体外，对于铜绿假单胞菌的发病机理至关重要 小鼠模型中的角膜感染。角膜中铜绿假单胞菌发病机理的关键是 细菌侵入角膜上皮细胞的能力。而铜绿假单胞菌突变体 缺乏抽搐的运动性会侵入上皮细胞，并在其中复制，就像 野生型细菌，它们减少了退出已进入细胞的能力。在我期间 弗莱西格实验室的博士后奖学金，我使用成像和其他各种方法来研究 铜绿假单胞菌退出上皮细胞的机制。重要的是，我的初步数据显示 该出口不一定遵循细胞死亡，表明有主动/故意的机制 贡献。我的数据进一步表明，抽搐突变体入侵并在角膜中复制 上皮细胞，它们与野生型铜绿假单胞菌不同，无法分配自己 细胞质并积聚在聚集体中。我还筛选了一个突变库的 出口容量，发现两种磷脂酶中的两个PLCB或PA2155中的突变体是 退出有缺陷。与抽搐突变体相反，磷脂酶突变体正常扩散 在整个宿主细胞细胞质中。因此，我的数据将出口过程机械地分开 两个阶段一个依赖于抽搐，另一个取决于磷脂酶。我的 出口的理论模型是，铜绿假单胞菌使用抽搐运动避免形成生物膜 在细胞内部聚集并访问宿主细胞质膜，它们在其中使用 磷脂酶活性（例如PLCB和PA2155的）以改变质膜以提供出口 路线。因此，在目标1中，我将确定影响抽搐突变体的基因转录本 与野生型相比，聚集和退出，在AIM 2中，我将确定磷脂酶是否是否 通过其酶活性促进退出。同时为我们的理解做出了贡献。 铜绿发病机理，该项目最终可能有助于制定策略 防止和治疗通过防止细菌穿透我们的感染 保护性表面上皮。