Re-Wiring Cellular Metabolism to Control Biofilm Formation and Virulence BY...

重新连接细胞代谢以控制生物膜形成和毒力...

• 批准号: 8128599
• 负责人: Thomas K Wood
• 金额: --
• 依托单位: TEXAS ENGINEERING EXPERIMENT STATION
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-15 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8128599
• 关键词: Agriculture; Antibiotics; Bacteria; Bacterial Infections; Biocontrols; Biological Models; Cells; Corrosion; DNA Microarray Chip; DNA Shuffling; Engineering; Environment; Enzymes; Escherichia coli; Food Poisoning; Gene Expression; Genes; Genomics; Hydrogen; Indoles; Investigation; Medical; Metabolic; Metabolism; Microbial Biofilms; Microfluidic Microchips; Microfluidics; Pathogenicity; Pattern; Protein Engineering; Screening procedure; Signal Transduction; Sulfur-Reducing Bacteria; Uracil; VAI-2; Virulence; antimicrobial; complex biological systems; genetic regulatory protein; homoserine lactone; novel; receptor

DESCRIPTION (provided by applicant): This project will utilize metabolic engineering to control the supramolecular assembly known as a bacterial biofilm as well as control virulence by evolving signal receptor proteins (e.g., SdiA, Hha, YmgB, and MqsR) and by utilizing cell signals (e.g., autoinducer-2, indole). Microfluidic devices will be used for secondary screening and to build designer, engineered, multi-species biofilms for applications. The paradigm shift is in controlling biofilms to achieve engineering and medical aims (e.g., biocorrosion, biocatalysis, rhizoremediation, food poisoning) whereas previously biofilms have been studied primarily as a means toward eradicating them. In addition, we aim to control biofilm formation and virulence genes via manipulation of signal regulators rather than try to eliminate the bacterium (i.e., control gene expression via cell signaling rather than discover antimicrobials). We have recently discovered that E. coli and pseudomonads respond to signals they do not synthesize (homoserine lactones influence E. coli biofilms while indole influences those of pseudomonads), that competition for signals is intense to the extent that signals are altered (e.g., indole is hydroxylated by bacteria that do not synthesize it and then regulates a different set of genes), that biofilm signals control pathogenicity loci (e.g., indole, uracil), and that biofilms may be dispersed via global regulators. Here, we will use a simple model system (pathogenic and non-pathogenic Escherichia coli along with pseudomonads and sulfur-reducing bacteria) that allows us to investigate biofilm formation and virulence in a realistic environment (i.e., multi-species biofilms). The novelty of the proposed approach arises from (i) protein engineering of regulatory proteins to control biofilms including formation, dispersal, and virulence (this is one of the first studies to evolve regulators rather than enzymes), (ii) investigation of the concentration-dependent interaction of cell signals, many that we have only recently identified, on biofilm formation, (iii) building designer multi-species biofilms, (iv) and utilizing microfluidic devices to carefully control concentrations and gradients of mixtures of the various signals in multi-species biofilms. In this way, this proposal includes complex biological system (pathogenic/non-pathogenic E. coli, E. coli/sulfur-reducing bacteria, E. coli/ pseudomonads), genomics (DNA microarrays), cell signaling, micro-patterning and microfluidics, and protein engineering (DNA shuffling) along with cell screening (FACS) to tune biofilm formation and cell colonization. If biofilms can be controlled, then they may be used for many diverse applications including reducing corrosion ($276 billion/yr problem in the U.S. or 3% GNP), forming hydrogen for fuel cells, rhizoremediation and biocontrol in agriculture, and patterning in microfluidic devices. If virulence genes can also be controlled in biofilms, then novel treatments can be envisioned for the 80% of bacterial infections that occur in biofilms where antibiotics are often ineffective.

描述（由申请人提供）： 该项目将利用代谢工程来控制被称为细菌生物膜的超分子组装，并通过进化信号受体蛋白（例如 SdiA、Hha、YmgB 和 MqsR）和利用细胞信号（例如 autoinducer-2、吲哚）。微流体装置将用于二次筛选，并为应用构建设计、工程、多物种生物膜。范式转变是控制生物膜以实现工程和医学目标（例如生物腐蚀、生物催化、根部修复、食物中毒），而以前生物膜的研究主要是作为根除生物膜的一种手段。此外，我们的目标是通过操纵信号调节器来控制生物膜形成和毒力基因，而不是试图消除细菌（即通过细胞信号传导控制基因表达而不是发现抗菌药物）。我们最近发现大肠杆菌和假单胞菌会对它们不合成的信号做出反应（高丝氨酸内酯影响大肠杆菌生物膜，而吲哚影响假单胞菌的生物膜），信号的竞争非常激烈，以至于信号被改变（例如吲哚）被不合成它的细菌羟基化，然后调节一组不同的基因），生物膜信号控制致病位点（例如吲哚，尿嘧啶），生物膜可能通过全球监管机构分散。在这里，我们将使用一个简单的模型系统（致病性和非致病性大肠杆菌以及假单胞菌和硫还原细菌），使我们能够研究现实环境中的生物膜形成和毒力（即多物种生物膜）。所提出方法的新颖性源于（i）调节蛋白的蛋白质工程，以控制生物膜，包括形成、分散和毒力（这是进化调节剂而不是酶的首批研究之一），（ii）对浓度的研究我们最近才发现细胞信号之间的相互作用对生物膜形成的影响，(iii) 构建设计师多物种生物膜，(iv) 并利用微流体装置仔细控制各种信号混合物的浓度和梯度在多物种生物膜中。这样，该提案包括复杂的生物系统（致病性/非致病性大肠杆菌、大肠杆菌/硫还原菌、大肠杆菌/假单胞菌）、基因组学（DNA微阵列）、细胞信号传导、微图案和微流体、蛋白质工程（DNA 改组）以及细胞筛选 (FACS)，以调整生物膜形成和细胞定植。 如果生物膜可以被控制，那么它们可以用于许多不同的应用，包括减少腐蚀（美国每年 2760 亿美元的问题或国民生产总值的 3%）、为燃料电池形成氢气、农业中的根部修复和生物控制以及微流体装置中的图案化。如果生物膜中的毒力基因也可以被控制，那么就可以设想针对抗生素通常无效的生物膜中发生的 80% 细菌感染的新治疗方法。