Designing real-time bacterial reporting of enzymes secreted by mammalian cells

哺乳动物细胞的体外培养一直是帮助研究人员了解从疾病进展到宿主-微生物相互作用的广泛细胞和多细胞过程的重要工具。然而，它的潜力受到已经开发的用于哺乳动物细胞分析的分析工具的严重限制，几乎没有工具适合在其培养环境中和长时间尺度上对哺乳动物细胞进行实时分析。随着该领域向包含动态细胞外基质(ECM)的更复杂的培养环境发展，这种需求变得更加强烈，在这种环境中，时空数据更重要、更丰富，但更难访问。新的报告或传感器模式的设计将对哺乳动物细胞的研究产生革命性的影响，这些新的报告或传感器模式可能会在原位发挥作用，靶向特定的蛋白质，放大信号，持续数天，并再生。仅举一个说明性的例子，这些工具可以帮助解开蛋白质的各个角色，这些蛋白质共同协调与疾病相关的过程，如细胞外基质重塑。我们建议设计一种工程菌，它可以报告上皮细胞分泌的酶的丰富性，同时基于一种正交的和必要的营养物质表现出严格控制的增殖。这一策略的特点是将大肠杆菌细胞直接引入哺乳动物细胞培养中，并且直到最近才有可能，因为在内在生物遏制方面的进展。文献一般表明，上皮细胞暴露在非致病性、非粘附性和非侵袭性细菌中并不会显著改变关键信号分子的表达。此外，虽然细菌可能具有一些固有的能力来感知某些哺乳动物蛋白质，但这些系统知之甚少，范围有限。然而，蛋白质工程和基因电路设计的最新进展使人们能够对几个小分子输入信号做出反应。在这项研究计划中，我们利用我们过去在合成营养缺陷性生物遏制方面的工作，专注于为我们独特的菌株配备创新的上皮细胞分泌酶的传感模式。我们的第一个目标是探索将这些细菌菌株直接引入哺乳动物细胞培养，重点放在建立长期稳定机制的可行性上。除了努力在每天传代后获得相对可预测的细菌和哺乳动物细胞比例外，我们还将确定不会导致我们选择研究的分泌蛋白的表达水平发生重大变化的细菌制度。我们的第二个目标是开发新的检测细胞外空间分泌酶的报告程序。我们的第三个目标是研究如何将有关分泌蛋白丰度的信息传递回细菌细胞，以及细菌的靶向反应的设计。这项研究的预期结果是开发了一个技术平台，用于阐明与生物医学相关的更广泛的基本现象。

In vitro culturing of mammalian cells has been a crucial tool in helping researchers understand a broad range of cellular and multicellular processes, from disease progression to host-microbe interactions. However, its potential has been severely limited by the analytical tools that have been developed for mammalian cell analysis, with few tools suitable for real-time analysis of mammalian cells within their culturing environment and over long timescales. This need is heightened as the field moves toward more complex culturing environments that contain dynamic extracellular matrices (ECMs), where spatiotemporal data is more consequential and richer but harder to access. The design of new reporter or sensor modalities that could potentially function in situ, target specific proteins, amplify signal, endure over multiple days, and regenerate would be transformative for the study of mammalian cells. As just one illustrative example, such tools could help disentangle the individual roles of proteins that collectively orchestrate disease-related processes such as ECM remodeling. We propose the design of engineered bacteria that can report the abundance of enzymes secreted by epithelial cells while exhibiting tightly controlled proliferation based on an orthogonal and essential nutrient. This strategy features the direct introduction of Escherichia coli cells to mammalian cell culture and is only recently possible given advances in intrinsic biological containment. Literature generally indicates that exposure of epithelial cells to bacteria that are non-pathogenic, non-adherent, and non-invasive does not dramatically alter expression of key signaling molecules. Additionally, while bacteria may possess some inherent ability to sense certain mammalian proteins, these systems are poorly understood and limited in range. However, recent advancements in protein engineering and genetic circuit design have enabled responses to several small molecule input signals. In this research program, we leverage our past work on biological containment by synthetic auxotrophy with a focus on equipping our unique strains with innovative sensing modalities for enzymes secreted by epithelial cells. Our first aim explores the introduction of these bacterial strains directly to mammalian cell culture, with a focus on the feasibility of establishing a long-term steady-state regime. In addition to striving for relatively predictable ratios of bacterial and mammalian cells after daily passaging, we will identify bacterial regimes that do not result in significant changes in the expression level of the secreted proteins we choose to study. Our second aim focuses on development of new reporters for detection of secreted enzymes in the extracellular space. Our third aim investigates ways to relay information about secreted protein abundance back to the bacterial cell and the design of targeted responses by bacteria. Expected outcomes of this investigation are the development of a technology platform for elucidating broader fundamental phenomena of biomedical relevance.