Engineering Probiotic and Commensal Bacteria for Detection and Elimination of Disease Biomarkers Through Human Gastrointestinal Tract

Abstract

Human gastrointestinal (GI) tract is a long hollow tube, connecting mouth, esophagus, stomach, small intestine, large intestine, and anus. Two main functions of GI tract are food digestion and nutrient absorption. Moreover, it is the largest immune organ because the lumen of this hollow tube has the largest surface area for the contact between the outside world and internal body environment. This provides an extensive interface for colonization of microorganisms, referred as gut microbiota. Probiotic and commensal bacteria, which are important components of this microbial community, have profound effects on human health, including immunity, metabolism, and gut-brain axis. Thus, these organisms can be utilized to develop next-generation microbiota-based therapeutics. In this dissertation, we focused on harnessing probiotic and commensal Escherichia coli strains as novel therapeutics for hyperuricemia and Crohn’s disease. Particularly, we reprogrammed these bacteria via genetic engineering for detection and elimination of disease biomarkers through human GI tract. In the first project, our goal was to reprogram probiotic and commensal E. coli strains to develop a novel system for accurate diagnosis and effective long-term management of hyperuricemia. To achieve this goal, we constructed (i) a synthetic promoter sequence that can detect the changes in uric acid in a dose-dependent manner, (ii) a uric acid degradation module that can eliminate the excess uric acid in the intestine, and (iii) an in vitro model with human intestinal cell line, Caco-2, that provided a versatile tool to study uric acid transport and degradation. In the second project, our goal was to build a bioreporter monitoring early stages of Crohn’s disease, which is characterized by elevated IFN-γ levels in the intestine. To achieve this goal, we investigated the potential of two signal transduction systems in commensal E. coli for IFN-γ detection: (i) optimization of a previously developed OmpA-OprF chimeric protein- and pspA-dependent biosensor, and (ii) development of a GacS/GacA- and rsmZ-dependent biosensor, which is adapted from Pseudomonas aeruginosa PAO1. These synthetic gene circuits and signaling systems provided an essential contribution for in situ detection and elimination of human disease biomarkers through GI tract.