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

• 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
• 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

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 研究。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。如果 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 在感染中。这很重要，因为患者正在持续感染，而不是在第一次接触时 与病原体。我们开发了一种基于重组酶的系统来去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。这 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 在后期去除 T3SS 会影响铜绿假单胞菌维持感染的能力。在目标 2 中，我们将 检查去除 T3SS 对免疫细胞招募和细胞因子产生的影响。我们还将 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 蛋白质。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。 总而言之，我们建议开发一套工具来首次探索贡献 感染生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，我们将开发的工具可以应用于任何数量的系统，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。