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，其潜力受到已开发用于哺乳动物细胞分析的分析工具的严重限制，其中很少有工具适合于在其培养环境内和长时间尺度上实时分析哺乳动物细胞。随着该领域朝着包含动态细胞外基质（ECM）的更复杂的培养环境发展，这种需求得到了提高，其中时空数据更重要，更丰富，但更难访问。设计新的报告或传感器模式，可以潜在地在原位发挥作用，靶向特定的蛋白质，放大信号，持续多天，并再生，将是哺乳动物细胞研究的变革。作为一个说明性的例子，这样的工具可以帮助解开蛋白质的个体作用，这些蛋白质共同协调疾病相关的过程，如ECM重塑。我们提出了工程菌的设计，可以报告丰富的酶分泌的上皮细胞，同时表现出严格控制增殖的基础上正交和必需的营养素。这种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，并且仅在最近才可能在内在生物遏制方面取得进展。文献通常表明，上皮细胞暴露于非致病性、非粘附性和非侵入性的细菌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，蛋白质工程和遗传电路设计的最新进展已经能够对几种小分子输入信号做出响应。在这项研究计划中，我们利用我们过去在合成营养缺陷型生物遏制方面的工作，重点是为我们独特的菌株配备上皮细胞分泌的酶的创新传感模式。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，重点是建立长期稳态制度的可行性。除了每天传代后争取相对可预测的细菌和哺乳动物细胞比例外，我们还将鉴定不会导致我们选择研究的分泌蛋白表达水平发生显著变化的细菌方案。我们的第二个目标集中在开发新的报告，用于检测分泌的酶在细胞外空间。我们的第三个目标是研究将分泌蛋白丰度的信息传递回细菌细胞的方法，以及细菌靶向反应的设计。这项研究的预期成果是开发一个技术平台，用于阐明生物医学相关性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.