A New Quorum-Sensing Autoinducer Acts with the RhIR Receptor to Control Virulence and Biofilms in Pseudomonas Aeruginosa

项目总结 医院获得性继发感染是一个日益严重的全球性问题。的确，多种药物 耐药铜绿假单胞菌是美国医院获得性感染的主要原因。 铜绿假单胞菌现在是疾控中心ESKAPE病原体名单上的优先病原体。铜绿假单胞菌感染是一种特殊的 囊性纤维化、微生物角膜炎、三度烧伤单位以及癌症患者和艾滋病毒患者的问题。 铜绿假单胞菌的毒力和生物膜的形成依赖于细菌的细胞间通讯过程。 这被称为群体感应。已知铜绿假单胞菌群体感应电路具有两种典型的Luxi/R型 信号通路：LASI/R和RhLI/R，它们加在一起，估计控制着基因组中10%的基因。 已知电路的功能如下：LASI产生并且LASR响应自动诱导剂30C12-高丝氨酸 内酯。LasR：3OC12-高丝氨酸内酯复合体激活许多基因的转录，包括rhLR， 编码第二个群体感应受体。RhlR与自身诱导剂C4-高丝氨酸内酯结合，产物 莱利的。RhlR：C4-高丝氨酸内酯也控制着一大批基因，包括那些编码毒力的基因 如绿青素、弹力酶和鼠李糖脂。通常情况下，群体感应鲁西型和 LuxR型基因(即LASI-LasR和RHLI-RHLR)具有相同的表型，因为这对基因的每个组成部分 需要另一个来发挥作用。然而，使用生物膜分析，转录报告分析，RNA-SEQ研究， 和动物感染分析，我发现RhlR既指导RhlI依赖的调节子，也指导RhlI非依赖的调节子。 我发现另一种由PqsE硫酯酶合成的自身诱导剂驱动RhlI- 独立的RhlR调节子。我证明了，虽然规范的RhlR-RhlI系统是可有可无的，但RhlR- PqsE系统是两种动物形成生物膜和毒力所必需的关键的群体感应系统 感染的模型。最重要的是，这些研究表明，与激活生物膜形成的激光受体不同， RhlR作为生物膜发育的抑制因子发挥作用。在这里，我建议确定(1)RHL群体感知 与CBR营养传感系统融合以抑制生物膜的形成和(2)哪些因素允许生物膜 当LAS、RHL和CBR系统的感官线索缺失时形成。拟议的研究将有助于 群体感应控制生物膜形成的机理及揭示群体感应的机理 与其他感觉信号系统相交，这对理解基础铜绿假单胞菌生物学至关重要 以及成功开发反法定人数感知策略。

PROJECT SUMMARY Hospital-acquired secondary infections are an escalating problem of global significance. Indeed, multi-drug resistant Pseudomonas aeruginosa is the leading cause of hospital-acquired infections in the USA and P. aeruginosa is now a priority pathogen on the CDC ESKAPE pathogen list. P. aeruginosa infection is a particular problem in cystic fibrosis, microbial keratitis, in third-degree burn units, and in cancer sufferers and HIV patients. P. aeruginosa virulence and biofilm development depend on the bacterial cell-to-cell communication process called quorum sensing. The known P. aeruginosa quorum-sensing circuit possesses two canonical LuxI/R type signaling pathways: LasI/R and RhlI/R, that, together, control an estimated 10% of the genes in the genome. The known circuit functions as follows: LasI produces and LasR responds to the autoinducer 3OC12-homoserine lactone. The LasR:3OC12-homoserine lactone complex activates transcription of many genes including rhlR, encoding a second quorum-sensing receptor. RhlR binds to the autoinducer C4-homoserine lactone, the product of RhlI. RhlR:C4-homoserine lactone also directs a large regulon of genes including those encoding virulence factors such as pyocyanin, elastases, and rhamnolipids. Typically, mutations in quorum-sensing luxI-type and luxR-type genes (i.e., lasI-lasR and rhlI-rhlR) confer identical phenotypes because each component of the pair needs the other to function. However, using biofilm analyses, transcriptional reporter assays, RNA-seq studies, and animal infection assays, I discovered that RhlR directs both RhlI-dependent and RhlI-independent regulons. I discovered that an alternative autoinducer, synthesized by the PqsE thioesterase enzyme, drives the RhlI- independent RhlR regulon. I demonstrated that while the canonical RhlR-RhlI system is dispensable, the RhlR- PqsE system is the crucial quorum-sensing system required for biofilm formation and for virulence in two animal models of infection. Most importantly, these studies revealed that unlike LasR that activates biofilm formation, RhlR functions as a repressor of biofilm development. Here, I propose to determine (1) how Rhl quorum sensing converges with Cbr nutrient sensing system to repress biofilm formation and (2) what factors allow biofilm formation when the sensory cues for Las, Rhl and Cbr systems are absent. The proposed research will contribute a mechanistic understanding of quorum-sensing control of biofilm formation and uncover how quorum sensing intersects with other sensory signaling systems, which is crucial for understanding basic P. aeruginosa biology and for successful development of anti-quorum-sensing strategies.