Development of recombinase-based tools to study established infections

摘要 使用动物模型研究突变细菌的毒力是细菌致病的主要手段 research.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 突变体是有缺陷的，它被清除，从而阻止我们评估的作用，毒力因子以后 在感染中。这一点很重要，因为患者表现为持续感染，而不是在第一次接触时 感染了病原体我们已经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地去除选择的毒力基因。 在这里，我们建议在绿脓杆菌角膜感染的小鼠模型中测试该系统。的 我们将研究的候选毒力因子是III型分泌系统（T3 SS），这是一种分子注射器， 这对铜绿假单胞菌首先建立感染的能力绝对至关重要。 在目的1中，我们将检查重组酶“关闭”状态在体内的稳定性，以及重组酶的作用。 在后期去除T3 SS对铜绿假单胞菌维持感染的能力的影响。在目标2中，我们 检查去除T3 SS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β-内酰胺酶融合到效应子 proteins.该系统将使我们能够检查在已建立的感染中T3 SS靶向哪些细胞。 总而言之，我们建议开发一套工具来首次探索这种贡献 铜绿假单胞菌的毒力基因后，感染已经扎根。除了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应用于任何数量的系统，如生物膜形成 或混合微生物群落的建立，其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.