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

项目摘要 医院获得性继发感染是一个日益严重的全球性问题。事实上，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CDC ESKAPE病原体列表上的优先病原体。铜绿假单胞菌感染是一种特殊的 在囊性纤维化、微生物性角膜炎、三度烧伤、癌症患者和HIV患者中， P.铜绿假单胞菌的毒力和生物膜的形成依赖于细菌细胞间的通讯过程 叫做群体感应已知的铜绿假单胞菌群体感应电路具有两个典型的LuxI/R类型 信号通路：LasI/R和RhII/R，它们共同控制基因组中估计10%的基因。 已知的回路功能如下：LasI产生和LasR响应自诱导物3 OC 12-高丝氨酸 内酯。LasR：3 OC 12-高丝氨酸内酯复合物激活许多基因的转录， 编码第二群体感应受体。RhlR与自身诱导物C4-高丝氨酸内酯结合， 的RhlI。RhlR：C4-高丝氨酸内酯还指导包括编码毒力的那些基因的大的调节子 因子如绿脓菌素、弹性蛋白酶和鼠李糖脂。通常，群体感应luxI型和 luxR型基因（即，lasI-lasR和rhlI-rhlR）赋予相同的表型，因为该对的每个组分 需要另一个来运作然而，使用生物膜分析，转录报告基因测定，RNA-seq研究， 和动物感染试验，我发现RhlR指导RhlI依赖性和RhlI非依赖性调节子。 我发现了另一种自诱导物，由PqsE硫酯酶合成，驱动RhII- 独立RhlR调节子。我证明了，虽然典型的RhlR-RhlI系统是稳定的，但RhlR-RhlI系统是稳定的。 PqsE系统是两种动物生物膜形成和毒力产生的关键群体感应系统 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LasR不同， RhlR作为生物膜发育的阻遏物起作用。在这里，我建议确定（1）Rhl群体感应 与CBR营养传感系统收敛以抑制生物膜形成和（2）什么因素允许生物膜 当Las、Rhl和Cbr系统的感觉线索不存在时，这项研究将有助于 对生物膜形成的群体感应控制的机械理解，并揭示群体感应如何 与其他感觉信号系统交叉，这对于理解铜绿假单胞菌的基本生物学至关重要 以及反聚量感应策略的成功发展。

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.