Engineering bacterial multicellular structures for therapeutic applications - PROJECT SUMMARY The long-term goal of this R35 research plan is to define the engineering principles to rationally design programmable bioengineered multicellular structures (ProBioMS) in prokaryotic cells. Modeled after natural probiotic strains, ProBioMS are promising probiotic-based therapeutics. Naturally aggregating into multicellular structures, probiotic strains exert a curative effect because they colonize specific bodily compartments and remain viable over time. By designing their engineered principles, ProBioMS can use a variety of existing probiotics and reach the macroscale, thus enabling the development of novel treatments such as anticavity pastes, vaginal creams, and wound dressings. To design effective therapeutic strategies, the most critical aspect of ProBioMS assembly is the regulation of their size, as it determines tissue adherence and the clearance rate from bodily cavities. ProBioMS are formed by genetically engineering cells to display self-assembling binding peptides that mediate cell-cell interactions. The strength of peptide self-assembling on the cell surface will determine the degree of intercellular interactions and, thus, the size of ProBioMS. The genetic tools currently available for de-novo ProBioMS design only allow for their assembly on the microscopic scale, thus limiting the applications this technology can support. Preliminary findings from the PI demonstrate that surface-displayed peptides that form multivalent interactions allow for macroscopic ProBioMS assembly. This interdisciplinary five- year research plan is centered on the hypothesis that multivalent interaction-forming peptides can be leveraged to control the size of ProBioMS over several length scales, from a few cells to the macroscopic scale. This proposal is articulated around three main broad projects. The first effort aims to discover and characterize novel genetic tools to assemble ProBioMS. For this purpose, we will initially focus on a subset of peptides with different degrees of self-assembly. The characteristics observed in this first subset of peptides can be leveraged in the future to identify additional peptides able to mediate the formation of ProBioMS with a defined size. A second project will uncover the design rules of cell surface display to control the density of peptides on the cell surface. This project will also add quantitative information to the under-characterized display mechanisms. Ultimately, we will define innovative experimental methods and a computational framework for generating the engineering principles that govern ProBioMS size, and, in the future, transfer these principles to relevant probiotic strains to design tailored therapeutics. Overall, this MIRA proposal is significant because it will generate novel genetic tools and engineering principles that can be applied to virtually any probiotics, enabling biomedical applications targeting a variety of bodily compartments and diseases. This R35 program supports the mission of NIGMS on engineering and technology development. It also broadly aligns with the NOSI “Synthetic Biology for Biomedical Applications - NOT-EB-23-002” because it lays the foundation for creating a new class of biological materials made of living cells.
