Mechanisms for bacterial dissemination in corneal infection

角膜感染中细菌传播的机制

• 批准号: 9918910
• 负责人: Vincent Nieto
• 金额: $ 4.79万
• 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 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9918910
• 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; Pseudomonas aeruginosa infection; 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; human pathogen; in vivo; in vivo Model; mouse model; mutant; novel strategies; prevent; protein expression; severe injury; 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.

项目概要 铜绿假单胞菌是导致致盲性角膜疾病的最常见原因之一，而 也是威胁生命的医院感染（例如肺炎、菌血症、 尿路感染 (UTI) 和囊性纤维化 (CF)，针对免疫功能低下和 重伤患者。 Fleiszig 实验室的出版物表明，抽搐运动是一种 与表面相关的运动类型，有助于铜绿假单胞菌渗透的能力 体外人角膜上皮细胞多层，对铜绿假单胞菌的发病机制至关重要 体内小鼠模型的角膜感染。角膜中铜绿假单胞菌发病机制的关键是 细菌侵入角膜上皮细胞的能力。而铜绿假单胞菌突变体 缺乏抽搐运动可以侵入上皮细胞，并在其内部复制，就像 野生型细菌，它们退出所进入的细胞的能力降低。在我的 在 Fleiszig 实验室的博士后研究期间，我使用成像和各种其他方法来研究 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 这种退出并不一定伴随着细胞死亡，这表明主动/有意的机制 贡献。我的数据进一步表明，当抽搐突变体侵入角膜并在角膜中复制时 上皮细胞，它们与野生型铜绿假单胞菌的不同之处在于无法将自身分布在 细胞质并以聚集体形式积累。我还筛选了一个突变体库 出口能力，并发现两种磷脂酶 PlcB 或 PA2155 中的突变体 退出有缺陷。与抽搐突变体相反，磷脂酶突变体正常传播 遍及宿主细胞的细胞质。因此，我的数据机械地将退出过程分为 两个阶段，一个阶段依赖于抽搐，另一个阶段依赖于磷脂酶。我的 退出的理论模型是铜绿假单胞菌利用抽搐运动来避免形成生物膜 聚集在细胞内并进入宿主细胞质膜，在那里它们使用 磷脂酶活性（例如 PlcB 和 PA2155）改变质膜以提供出口 路线。因此，在目标 1 中，我将鉴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，在目标 2 中，我将确定磷脂酶是否 通过其酶活性促进退出。同时有助于我们对 P 的理解。 铜绿假单胞菌发病机制，该项目最终可能有助于制定铜绿假单胞菌发病机制 通过阻止细菌渗透到我们的身体来预防和治疗感染 保护性表面上皮。