Development of recombinase-based tools to study established infections

开发基于重组酶的工具来研究已确定的感染

基本信息

批准号：
10201472
负责人：
Arne Rietsch
金额：
$ 20.13万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-24 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10201472
关键词：
Alleles Animal Model Bacteria Bacterial Eye Infections Biological Assay Blindness Cells Complex Cornea Development Disease Enterobacteria phage P1 Cre recombinase Excision Eye Eye Infections Factor Analysis Fluorescence Resonance Energy Transfer Generations Genes Genetic Recombination Goals Immune In Vitro Infection Keratitis Methods Microbial Biofilms Modeling Molecular Monitor Mus Mutate Neutrophilic Infiltrate Noise Pathogenesis Patients Phase Phenotype Plant Roots Production Proteins Pseudomonas aeruginosa Pseudomonas aeruginosa infection Research Role Structure Surface Syringes System Testing Time Type III Secretion System Pathway Virulence Virulence Factors Work base beta-Lactamase cell type cytokine design experimental study genetic analysis in vivo insight microbial community mouse model mutant novel pathogen prevent programs public health relevance recombinase recruit stability testing tissue culture tool

项目摘要

Abstract The use of animal models to study the virulence of mutant bacteria is a staple of bacterial pathogenesis research. However, in most instances we are studying the ability of a mutant to establish an infection. If the mutant is defective, it is cleared, thereby preventing us from assessing the role of that virulence factor later on in the infection. This is important, since patients present with an ongoing infection, not at the time of first contact with the pathogen. We have developed a recombinase based system to remove virulence factors after infection. The system is stably off for days in vitro, but can be induced to efficiently remove the virulence gene of choice. Here we propose to test this system in a mouse model of Pseudomonas aeruginosa corneal infection. The candidate virulence factor we will examine is the type III secretion system (T3SS), a molecular syringe that is absolutely critical for the ability of P. aeruginosa to establish an infection in the first place. In Aim 1, we will examine the stability of the recombinase “off” state in vivo, as well as the effect of removing the T3SS at later stages on the ability of P. aeruginosa to maintain the infection. In Aim 2, we will examine the effect of removing the T3SS on immune cell recruitment and cytokine production. We will also develop a recombinase-based fusion system, that is designed to generate b-lactamase fusions to effector proteins. This system will allow us to examine which cells are targeted by the T3SS in an established infection. Taken together, we are proposing to develop a set of tools to explore, for the first time, the contribution of P. aeruginosa virulence genes after the infection has taken root. Beyond the novel insights into P. aeruginosa pathogenesis, the tools we will develop will can be applied to any number of systems, such as biofilm formation or establishment of mixed microbial communities, that proceed through an initial establishment phase to the mature structure.

抽象的使用动物模型研究突变细菌的毒力是细菌发病机制的主要内容然而，在大多数情况下，我们正在研究突变体建立感染的能力。突变体有缺陷，它被清除，从而阻止我们稍后评估该毒力因子的作用这很重要，因为患者处于持续感染状态，而不是在第一次接触时。我们开发了一种基于重组酶的系统来消除感染后的毒力因子。该系统在体外稳定关闭数天，但可以被诱导以有效去除所选的毒力基因。在这里，我们建议在铜绿假单胞菌角膜感染的小鼠模型中测试该系统。我们将检查的候选毒力因子是 III 型分泌系统 (T3SS)，这是一种分子注射器对于铜绿假单胞菌首先建立感染的能力绝对至关重要。在目标 1 中，我们将检查体内重组酶“关闭”状态的稳定性，以及在目标 2 中，我们将在后期去除 T3SS 来影响铜绿假单胞菌维持感染的能力。我们还将检查去除 T3SS 对免疫细胞招募和细胞因子产生的影响。开发基于重组酶的融合系统，旨在生成与效应子融合的β-内酰胺酶该系统将使我们能够检查在已确定的感染中哪些细胞是 T3SS 的目标。综上所述，我们建议开发一套工具来首次探索贡献感染扎根后铜绿假单胞菌毒力基因的变化超越对铜绿假单胞菌的新见解。发病机制，我们将开发的工具将应用于许多系统，例如生物膜形成或建立混合微生物群落，通过初始建立阶段进行结构成熟。