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

哺乳动物细胞的体外培养是帮助研究人员了解从疾病进展到宿主-微生物相互作用等广泛的细胞和多细胞过程的重要工具。然而，它的潜力受到哺乳动物细胞分析的分析工具的严重限制，很少有工具适合在其培养环境和长时间尺度内实时分析哺乳动物细胞。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，这种需求得到了加强，其中时空数据更加重要和丰富，但更难获取。新的报告细胞或传感器模式的设计可能在原位发挥作用，靶向特定蛋白质，放大信号，持续数天，并可再生，这将是哺乳动物细胞研究的变革。作为一个说明性的例子，这些工具可以帮助解开蛋白质的个体角色，这些蛋白质共同协调疾病相关的过程，如ECM重塑。我们建议设计一种工程细菌，可以报告上皮细胞分泌的酶的丰度，同时表现出基于正交和必需营养的严格控制的增殖。这一策略的特点是直接将大肠杆菌细胞引入哺乳动物细胞培养，直到最近才有可能在内在生物遏制方面取得进展。文献普遍表明，上皮细胞暴露于非致病性、非粘附性和非侵入性细菌中不会显著改变关键信号分子的表达。此外，虽然细菌可能具有某些感知某些哺乳动物蛋白质的固有能力，但这些系统的了解很少，范围有限。然而，最近在蛋白质工程和遗传电路设计方面的进展使得对几个小分子输入信号的响应成为可能。在这个研究项目中，我们利用我们过去在生物控制方面的工作，通过合成肌肉萎缩，重点是为我们独特的菌株配备创新的感知模式，以检测上皮细胞分泌的酶。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。除了在日常传代后努力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表达水平发生显着变化的细菌制度。我们的第二个目标是开发用于检测细胞外空间分泌酶的新报告。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和细菌靶向反应的设计。这项调查的预期结果是为阐明生物医学相关的更广泛的基本现象的技术平台的发展。

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.