Engineering E. coli Nissle 1917 to advance and facilitate its use in biomedical applications

Abstract

The concept of engineered living therapeutics refers to the utilization and reprogramming of living cells for a wide range of medical applications. Currently, most examples focus on engineering microbes for use in the human gut, with many choosing the strain E. coli Nissle 1917 (EcN) as a chassis organism due to its safety profile, probiotic properties, and the availability of compatible genetic tools. The number of proposed applications continues to increase, supported by our growing understanding of the human microbiome and advances in the field of synthetic biology. In this dissertation, I describe the development of new tools to further facilitate EcN engineering, as well as propose a novel approach for the use of engineered EcN to combat infectious disease. We engineered and characterized two cryptic plasmids endogenous to EcN – pMUT1 and pMUT2. By inserting and testing several heterologous functional and regulatory components, we demonstrated the pMUT plasmids can be used as synthetic biology vectors in EcN. The removal of native pMUTs required the adaptation and optimization of existing plasmid curing techniques, and in the process shed light on the importance of the toxin-antitoxin system in pMUT2 for plasmid retention. Notably, we show that the engineered pMUTs can be used to express proteins in vivo and are retained during transit through the gastrointestinal tract without administration of antibiotics. Next, we utilized EcN to display a network of functionalized protein fibers designed to bind and sequester pathogens. Building on the previously developed BIND platform, we used curli fibers, the main proteinaceous components of E. coli biofilms, fused to nanobodies – small antibody fragments derived from camelids. We demonstrate binding to several enteric pathogens, including enteropathogenic E. coli, Shigella flexneri and Cryptosporidium parvum, as well as the potent bacterial toxin Stx2. The work also describes the generation and testing of new nanobodies targeting several antigens associated with pathogenic E. coli strains. Overall, the work described herein advances EcN engineering by providing novel genetic tools for its use in research and in the clinic, as well as demonstrating novel therapeutic functionalities, contributing to the fields of synthetic biology and engineered living therapeutics.