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）以及通过利用小区信号（例如，自诱导物-2，吲哚）。微流控装置将用于二次筛选，并为应用构建设计师设计的多物种生物膜。范式转变是控制生物膜以实现工程和医学目的（例如，生物腐蚀、生物催化、根修复、食物中毒），而以前生物膜主要作为消除它们的手段进行研究。此外，我们的目标是通过操纵信号调节剂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，而不是发现抗菌剂）。我们最近发现E.大肠杆菌和假单胞菌对它们不合成的信号作出反应（高丝氨酸内酯影响大肠杆菌。大肠杆菌生物膜而吲哚影响假单胞菌的生物膜），对信号的竞争激烈到信号被改变的程度（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性基因座（例如，吲哚、尿嘧啶），且生物膜可以通过全局调节剂分散。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沿着假单胞菌和硫还原菌），使我们能够研究生物膜的形成和毒力在一个现实的环境（即，多物种生物膜）。所提出的方法的新奇来自（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第一批研究之一），（ii）研究细胞信号的浓度依赖性相互作用，其中许多是我们最近才鉴定的，关于生物膜形成，（iii）构建设计师多物种生物膜，（iv）以及利用微流体装置来仔细控制多物种生物膜中各种信号的混合物的浓度和梯度。以这种方式，该建议包括复杂的生物系统（致病性/非致病性E。coli、大肠杆菌E. coli/硫还原菌、E.大肠杆菌/假单胞菌）、基因组学（DNA微阵列）、细胞信号传导、微图案化和微流体、蛋白质工程（DNA改组）沿着细胞筛选（FACS）以调节生物膜形成和细胞定殖。 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3%的GNP），为燃料电池形成氢，农业中的根修复和生物控制，以及微流体装置中的图案化。如果毒力基因也可以在生物膜中得到控制，那么可以设想新的治疗方法，用于80%的细菌感染，这些细菌感染发生在抗生素通常无效的生物膜中。

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.