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

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(例如自动诱导剂-2、吲哚)来控制毒力。微流控设备将被用于二次筛选，并为应用构建设计的、工程的、多物种生物膜。范式的转变是控制生物膜以实现工程和医学目的(例如，生物腐蚀、生物催化、根际修复、食物中毒)，而以前的研究主要是将生物膜作为根除它们的手段。此外，我们的目标是通过操纵信号调节器来控制生物膜的形成和毒力基因，而不是试图消除细菌(即通过细胞信号控制基因表达，而不是发现抗菌剂)。我们最近发现，大肠杆菌和假单胞菌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，吲哚影响假单胞菌的信号)，对信号的竞争激烈到信号改变的程度(例如，吲哚被不合成吲哚的细菌羟化，然后调节一组不同的基因)，生物膜信号控制致病基因(例如吲哚、尿嘧啶)，生物膜可能通过全球调节因子分散。在这里，我们将使用一个简单的模型系统(致病和非致病的大肠杆菌以及假单胞菌和硫还原细菌)，使我们能够在现实环境(即多物种生物膜)中研究生物膜的形成和毒力。该方法的新颖之处在于：(I)控制生物膜的调节蛋白的蛋白质工程，包括形成、扩散和毒力(这是进化调节剂而不是酶的首批研究之一)，(Ii)研究细胞信号与浓度的相互作用，我们最近才发现许多细胞信号在生物膜形成上的依赖于浓度的相互作用，(Iii)构建设计的多物种生物膜，(Iv)并利用微流控设备仔细控制多物种生物膜中各种信号的混合浓度和梯度。通过这种方式，这一建议包括复杂的生物系统(致病/非致病的大肠杆菌，大肠杆菌/硫还原细菌，大肠杆菌/假单胞菌)，基因组学(DNA微阵列)，细胞信号，微图案化和微流体，以及蛋白质工程(DNA改组)和细胞筛选(FAC)，以调节生物膜的形成和细胞的定植。 如果生物膜可以被控制，那么它们可能被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3%的GNP)，形成燃料电池的氢气，农业中的根际修复和生物控制，以及微流控设备中的图案化。如果毒力基因也可以在生物膜中控制，那么可以设想新的治疗方法来治疗发生在抗生素往往无效的生物膜中的80%的细菌感染。

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.