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

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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.