Development of recombinase-based tools to study established infections

摘要 利用动物模型研究突变细菌的毒力是细菌致病的主要方法。 研究。然而，在大多数情况下，我们正在研究突变株建立感染的能力。如果 突变是有缺陷的，它被清除了，从而阻止了我们稍后评估该毒力因子的作用 在感染中。这一点很重要，因为患者是持续感染的，而不是在第一次接触时 与病原体有关。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。 该系统在体外几天内稳定关闭，但可以被诱导有效地去除所选择的毒力基因。 在这里，我们建议在铜绿假单胞菌角膜感染的小鼠模型上测试该系统。这个 我们将检查的候选毒力因素是III型分泌系统(T3SS)，一种分子注射器，它是 对铜绿假单胞菌首先确定感染的能力来说是绝对关键的。 在目标1中，我们将检查重组酶在体内处于关闭状态的稳定性，以及 后期去除T3SS对铜绿假单胞菌维持感染能力的影响。在目标2中，我们将 检测去除T3SS对免疫细胞募集和细胞因子产生的影响。我们还将 开发一种基于重组酶的融合系统，旨在产生与效应器的b-内酰胺酶融合 蛋白质。这个系统将允许我们检查T3SS在已确定的感染中针对哪些细胞。 综上所述，我们提议开发一套工具，首次探索 铜绿假单胞菌在感染生根后毒力基因的变化。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可以应用于任何数量的系统，如生物膜的形成 或混合微生物群落的建立，通过初始建立阶段进行到 成熟的结构。

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.