A Synthetic Approach for Bacterial-Mammalian Cell Binding

项目摘要 合成生物学和分子生物学的进展已经改变了基因工程领域， 特别是在为细胞和生物体添加新功能的背景下。对细菌的基因操作 为解决旧的和新出现的生物医学问题提供了新的机会。为了充分利用 这些基因工程细菌在生物医学领域，新的工具，以检测和跟踪他们在体内， 必需的.分子成像比传统的诊断工具更有优势，因为它可以识别 并实时跟踪宿主环境中的这些细菌。然而，目前的成像造影剂 是非特异性的、非选择性的，并且通常识别“死”细菌。在本提案中，我们建议使用 细菌铁载体，由细菌分泌的金属结合分子，其结合细菌表面的不同受体， 膜，作为核成像的造影剂。细菌的铁载体已经进化到可以作为金属 螯合剂用于多种金属，其中大多数对铁显示出高结合亲和力。铁载体是 被认为是进入细菌细胞的“门户”，经常被用作“特洛伊木马”策略， 运送药物对抗抗药性细菌。建议的战略，以提供放射性核素，如64铜 使用这种途径将能够对不同的野生型和工程化细菌进行分子成像， 铁载体特异性方式。在目标1中，我们将确定以下的体内稳定性和靶向能力： 金属载体/64 Cu复合物定位静态野生型细菌。在目标2中，我们将改造大肠杆菌Nissle， 表达GFP结合表面纳米抗体以结合表达表面GFP的转移性癌细胞， 评价金属载体/64 Cu复合物对这些工程菌的靶向能力。我们将评估 示踪剂是否可以在体内独特的小生境中追踪工程细菌，并评估其 功能稳定性与此同时我们将优化和开发一种定量核成像方法， 可以检测到“活”细菌和病变时，他们是显着小于目前检测到的 现有的诊断测试和成像方法。与传统的制造技术相比， 探针，这种策略旨在通过结合金属附着的功能大大简化过程 和细胞识别整合成一个分子如果成功的话，它将能够创造新的知识， 生命系统中的金属载体-金属、金属载体-细菌、金属载体-宿主和细菌-宿主相互作用 这将有助于创造先进的工具和战略。

Project Summary Synthetic biology and advances in molecular biology have transformed the field of genetic engineering, particularly in the context of adding new function to cells and organisms. Genetic manipulation of bacteria has opened new opportunities to address old and emerging biomedical problems. To harness the full potential of these genetically engineered bacteria in the biomedical domain, new tools to detect and track them in vivo are required. Molecular imaging is advantageous over traditional diagnostic tools because it enables identification and tracking of these bacteria in the host environment in real-time. However, current imaging contrast agents are non-specific, non-selective and typically identify “dead” bacteria. In this proposal we propose to use bacterial siderophores, metal binding molecules secreted by bacteria that bind to distinct receptors on bacterial membrane, as contrast agents for nuclear imaging. Bacterial siderophores have evolved to serve as metal chelators for a wide variety of metals with the majority showing a high binding affinity for iron. Siderophores are considered “gateways” into the bacterial cell and have often been exploited as a ‘Trojan horse’ strategy to deliver drugs against antibiotic-resistant bacteria. The proposed strategy to deliver radionuclides such as 64Cu using this pathway will enable molecular imaging of a diverse array of wildtype and engineered bacteria in a siderophore-specific manner. In Aim 1 we will determine the in vivo stability and targeting ability of metallophore/64Cu complexes to locate static wildtype bacteria . In Aim 2 we will engineer E.coli Nissle to express GFP binding surface nanobodies to bind to metastatic cancer cells expressing surface GFP and evaluate the targeting ability of metallophore/64Cu complexes to these engineered bacteria. We will evaluate whether the tracers can track down the engineered bacteria at unique niches in the body and assess its functional stability at the same time. We will optimize and develop a quantitative nuclear imaging method that can detect “live” bacteria and the lesions when they are significantly smaller than those currently detected with existing diagnostic tests and imaging methods. Compared to traditional techniques used to manufacture probes, this strategy seeks to simplify the process considerably by combining the function of metal attachment and cell recognition into a single molecule. If successful, it will enable creation of new knowledge about metallophore-metal, metallophore-bacteria, metallophore-host and bacteria-host interactions in living systems that would help create advanced tools and strategies.