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

一种新型群体感应自诱导剂与 RhlR 受体共同作用来控制铜绿假单胞菌的毒力和生物膜

• 批准号: 10202818
• 负责人: Sampriti Mukherjee
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10202818
• 关键词: Active Sites; Anabolism; Animal Model; Animals; Antibiotics; Attenuated; Bacteria; Bacterial Physiology; Behavior; Binding; Biochemical; Biological Assay; Biology; Biophysics; Burn Units; Cell Communication; Cell Signaling Process; Cells; Centers for Disease Control and Prevention (U.S.); Chemical Structure; Chemicals; Chemistry; Clinical; Communication; Communities; Complex; Control Groups; Cystic Fibrosis; Detection; Development; Disease; ESKAPE pathogens; Elastases; Engineering; Enzymes; Foundations; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Screening; Genetic Transcription; Genome; HIV; Hospitals; Image; Individual; Industrialization; Infection; Keratitis; Knowledge; Laboratories; Life Style; Ligands; Liquid substance; Malignant Neoplasms; Maps; Medical; Microbial Biofilms; Microscopy; Modeling; Mutagenesis; Mutation; Nosocomial Infections; Pathway interactions; Patients; Pattern; Phenotype; Play; Population; Population Density; Process; Production; Proteins; Pseudomonas aeruginosa; Pseudomonas aeruginosa infection; Pyocyanine; Regulation; Regulon; Reporter; Research; Resistance; Resolution; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Structure; Surface; System; Time; Virulence; Virulence Factors; Work; antimicrobial; biophysical model; clinically relevant; combat; design; extracellular; flasks; homoserine lactone; human pathogen; insight; microbial; multidrug-resistant Pseudomonas aeruginosa; mutant; novel; priority pathogen; programs; quorum sensing; receptor; response; rhamnolipid; secondary infection; small molecule inhibitor; spatiotemporal; stem; theories; transcriptome sequencing; transcriptomics

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.

项目概要 事实上，医院获得性继发感染是一个日益严重的全球性问题。 耐药铜绿假单胞菌是美国和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 病原体清单上的优先病原体。铜绿假单胞菌感染是一种特殊的病原体。 囊性纤维化、微生物性角膜炎、三度烧伤病房以及癌症患者和艾滋病毒患者的问题。 铜绿假单胞菌毒力和生物膜发育取决于细菌细胞间的通讯过程 已知的铜绿假单胞菌群体感应电路具有两种典型的 LuxI/R 类型。 信号传导：LasI/R 和 RhlI/R 通路共同控制着基因组中大约 10% 的基因。 已知的电路功能如下：LasI 产生自诱导剂 3OC12-高丝氨酸，LasR 响应自诱导剂 3OC12-高丝氨酸 LasR:3OC12-高丝氨酸内酯复合物激活许多基因的转录，包括 rhlR、 编码第二个群体感应受体 RhlR 与自诱导剂 C4-高丝氨酸内酯（产物）结合。 RhlI 的 RhlR:C4-高丝氨酸内酯还指导大量基因调节，包括编码毒力的基因。 诸如绿脓素、弹性蛋白酶、鼠李糖脂和生物膜形成所需的基因等因子通常是突变。 群体感应 luxI 型和 luxR 型基因（即 lasI-lasR 和 rhlI-rhlR）赋予相同的表型 因为这对组件中的每个组件都需要另一个组件才能发挥作用。 通过记者分析、RNA-seq 研究和动物感染分析，我发现 RhlR 指导 RhlI- 重要的是，我证明了 ΔrhlI 突变体无细胞培养液，即 缺乏 C4-高丝氨酸内酯，含有刺激 RhlR 依赖性基因表达的活性。 除了传统的 C4-高丝氨酸内酯外，RhlR 还响应替代配体 为了支持这一观点，我证明了酶 PqsE 是替代配体合成所必需的。 最后，我证明了虽然RhlR-RhlI系统是可有可无的，但RhlR-PqsE系统是至关重要的 两种动物感染模型中生物膜形成和毒力所需的群体感应系统。 我建议 1) 确定替代配体的化学结构并定义它如何与 RhlR 相互作用； 2）表征PqsE活性位点，发现PqsE的小分子抑制剂，并鉴定和表征 参与替代配体合成的其他因素；3) 绘制替代配体和/或 C4-高丝氨酸图谱； 单细胞水平和细胞水平生物膜发育所需的内酯依赖性 RhlR 调节子 拟议的研究将为人们所使用的化学词典提供重要的见解。 此外，拟议的研究将提供生物膜和疾病中临床上重要的细菌。 对群体感应如何调节毒力和生物膜形成的机制理解，这对于 了解铜绿假单胞菌的基本生物学并成功开发抗群体感应策略。