Microfabricated 3D Environments for Characterizing Bacterial Group Behaviors

描述（由申请人提供）：这项工作的目标是为研究受控环境条件下的细菌群体行为提供一个平台，允许研究有关与毒力和增强的抗生素和免疫抗性相应的状态的发生和维持的基本问题。在这些研究中，一种基于多光子光刻（MPL）的新方法将被开发出来，用于使用生物构建块（蛋白质）来微加工定义的任意地形，这将被研究作为一种产生能够支持细胞聚集、生长和群体行为的微腔的手段，重点是群体感应和生物膜沉积由机会性环境细菌铜绿假单胞菌作为模型系统。这种细菌与许多人类疾病有关，包括囊性纤维化个体的持续感染。为了实现这些目标，本研究将推进MPL技术的发展，重点研究材料和微腔特性的表征，并将这项最先进的技术应用于评估铜绿假单胞菌在蛋白质微腔中的行为。该项目代表了德克萨斯大学奥斯汀分校Jason Shear教授的研究小组（化学/生物化学系）和Marvin Whiteley教授（微生物系）的合作研究。预计所产生的数据将为未来的研究提供重要信息，旨在确定针对细菌通信系统的新疗法。虽然我们建议铜绿假单胞菌作为这些研究的测试细菌，但它们很容易扩展到其他细菌物种，甚至更复杂的多物种群落。具体目标是：(1)优化制造具有可调质量传输到外部环境的三维细菌微腔的柔性平台；(2)评估基于蛋白质的三维微腔在研究细菌群体行为方面的效用。公共卫生相关性：拟议研究的目标是开发一个平台，用于研究受控环境条件下细菌群体的行为，从而研究与毒力和增强的抗生素和免疫耐药性相应的状态的发生和维持相关的基本问题。在这些研究中，将研究一种利用生物构建块进行微加工的新方法，这种方法可以制造出能够支持细胞聚集、生长和群体行为的微腔。预计所产生的数据将为旨在确定针对细菌通信系统的新疗法的研究提供重要信息。

DESCRIPTION (provided by applicant): The goal of this work is to produce a platform for studying bacterial group behavior under controlled environmental conditions, allowing fundamental questions to be investigated regarding the onset and maintenance of states corresponding to virulence and enhanced antibiotic and immune resistance. In these studies, a novel approach based on multiphoton lithography (MPL) will be developed for microfabricating defined, arbitrary topographies using biological building blocks (proteins) which will be investigated as a means to produce microcavities capable of supporting cell aggregration, growth, and group behavior, focusing on quorum sensing and biofilm deposition by the opportunistic environmental bacterium, Pseudomonas aeruginosa, as a model system. This bacterium has relevance to a number of human diseases, including persistant infections in cystic fibrosis individuals. Toward these ends, the proposed studies will advance the underlying MPL technology, focusing on characterization of material and microcavity properties, and will apply this state-of-the-art technology to an evaluation of P. aeruginosa behavior in protein-based microcavities. This project represents collaborative studies between Prof. Jason Shear's research group (Chemistry/Biochemistry Department), and that of Prof. Marvin Whiteley (Microbiology Department) at UT Austin. It is anticipated that the data generated will provide important information for future studies aimed at identifying novel therapeutics targeting bacterial communication systems. Although we are proposing P. aeruginosa as the test bacterium for these studies, they could easily be extended to other bacterial species and even to more complex multi-species communities. The Specific Aims are: (1) To Optimize a flexible platform for fabricating 3D bacterial microcavities having tunable mass-transport to the outside environment; and (2) To assess the utility of 3D protein-based microcavities for studying bacterial group behavior. PUBLIC HEALTH RELEVANCE: The goal of the proposed research is to develop a platform for studying bacterial group behavior under controlled environmental conditions, allowing fundamental questions to be investigated regarding the onset and maintenance of states corresponding to virulence and enhanced antibiotic and immune resistance. In these studies, a novel approach for microfabricating defined, arbitrary topographies using biological building blocks will be investigated as a means to produce microcavities capable of supporting cell aggregration, growth, and group behavior. It is anticipated that the data generated will provide important information for studies aimed at identifying novel therapeutics targeting bacterial communication systems.