A New Quorum-Sensing Autoinducer Acts with the RhlR 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 生物膜形成所需的因子，如绿青素、弹性酶、鼠李糖脂和基因。通常情况下，突变 在群体感应中，Luxi类型和LuxR类型的基因(即LASI-LasR和rhLI-RHLR)赋予相同的表型 因为这两个组件中的每个组件都需要另一个组件才能发挥作用。然而，使用生物膜分析，转录 记者分析、RNA-Seq研究和动物感染分析，我发现RhlR引导两种RhlI- 依赖的和RhlI非依赖的调节因子。重要的是，我证明了Δ基因突变的无细胞培养液，即， 缺乏C4-高丝氨酸内酯，含有刺激RhlR依赖的基因表达的活性。我 假设RhlR对传统的C4-高丝氨酸内酯以外的另一种配体有反应 自动诱导器。支持这一观点，我证明了PqsE酶是合成替代配体所必需的。 最后，我论证了虽然RhlR-RhlI系统是可有可无的，但RhlR-PqsE系统是关键 在两种动物感染模型中，生物膜的形成和毒力所需的群体感应系统。这里, 我建议1)确定替代配体的化学结构，并确定它如何与RhlR相互作用； 2)表征PqsE活性部位，发现PqsE小分子抑制剂，鉴定和表征 参与替代配体合成的其他因素；3)定位替代配体和/或C4-高丝氨酸 内酯依赖型RhlR调节子(S)在单细胞水平和生物膜发育水平上的作用 社区。这项拟议的研究将有助于对一个人使用的化学词典有重要的见解 在生物膜和疾病中具有重要临床意义的细菌。此外，拟议的研究将提供一个 从机制上理解群体感应如何调节毒力和生物膜的形成，这对 了解铜绿假单胞菌的基本生物学和成功开发反群体感应策略。

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, rhamnolipids and genes required for biofilm formation. 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. Importantly, I showed that ΔrhlI mutant cell-free culture fluids, i.e., that lack C4-homoserine lactone, contain an activity that stimulates RhlR-dependent gene expression. I hypothesize that RhlR responds to an alternative ligand, in addition to the traditional C4-homoserine lactone autoinducer. Supporting this notion, I showed that the enzyme PqsE is required for alternative ligand synthesis. Finally, I demonstrated that while the 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. Here, I propose to 1) determine the chemical structure of the alternative ligand and define how it interacts with RhlR; 2) characterize the PqsE active site, discover small molecule inhibitors of PqsE, and identify and characterize additional factors involved in alternative ligand synthesis; 3) map the alternative ligand and/or C4-homoserine lactone-dependent RhlR regulon(s) required for biofilm development at the single cell level and at the level of community. The proposed research will contribute significant insights about the chemical lexicon used by a clinically important bacterium in biofilms and in disease. Moreover, the proposed research will provide a mechanistic understanding of how quorum sensing regulates virulence and biofilm formation, which is crucial for understanding basic P. aeruginosa biology and for successful development of anti-quorum-sensing strategies.