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Theses Doctoral

Engineered bacteria for the modulation of intestinal physiology, inflammation, and behavior along the microbiome-gut-brain axis

Cusimano, Frank Anthony

Bacteria in the gastrointestinal tract play an important role in intestinal motility, inflammation, homeostasis, and behavior. Bacteria, through the natural synthesis of neuroactive compounds and secondary metabolites, can modulate the host immune system and communicate with the host along the signaling pathway along the gut-brain axis. Here, we functionally design, develop, test, and characterize a platform for the study of microbial-host interactions using advancements in the field of synthetic biology.

First, we describe the engineering of Escherichia coli Nissle to biosynthesize serotonin within the mammalian gut using a native-plasmid optimized approach. Serotonin is crucial for neurotransmission throughout the body and may be playing a role in microbial gut-brain communication. In the gastrointestinal tract, serotonin regulates intestinal motility, cell turnover, intestinal inflammation, and gastrointestinal homeostasis. Upon serial daily oral gavages, our engineered bacterium populates a murine colon to produce serotonin locally in the mucosa layers along the epithelial lining. Changes in host physiology were observed including decreased gastrointestinal motility, increased colonic Muc2 expression, induction of host TPH2, responsible for serotonin biosynthesis in enteric neurons, and upregulation of serotonin receptors HTR3, HTR4, and HTR7 in the colon. Behavioral tests revealed a statistically significant decrease in anxiety and depression in stress-induced environments in mice treated with the engineered bacterium. This work suggests that gut bacteria engineered to modulate host gut-brain axis may have both scientific and clinical uses to study microbial-host interactions and treat gastrointestinal and behavioral mood disorders in humans.

Second, we engineered bacteria to produce exogenous butyrate and other SCFAs in the murine gut. Short chain fatty acids (SCFAs) play an important role in intestinal homeostasis, fluid dynamics, inflammation, oxidative stress, and intestinal hypersensitivity and motility. With this development, we characterized the effects of our butyrate-producing bacteria on a high-fat diet and DSS-induced colitis model within the colon. Although energetically burdensome to produce, our strains produced butyrate in the colon at higher density in an actively inflamed colitis model. After 14 days of oral administration, our engineered strain (EcN:B) increased the colon length of normal wild-type mice, in high fat fed mice, and in mice with recovering and actively inflamed DSS-induced colitis. EcN:B increased mucosal barrier thickness, upregulated gene expression of the barrier integrity markers Cldn1, Ocln, Zo1, and altered crypt and villus height during inflammation recovery. Furthermore, as butyrate is known to induce Foxp3+ Regulatory T cells, we saw a 13.01% percent increase in Foxp3+ cells in the colon of mice fed our engineered bacteria. This work suggests that synthetic gut bacteria engineered to produce short chain fatty acids may have future clinical uses to treat patients with inflammatory bowel disease including Crohn’s and Colitis with future potential to serve as a therapeutic for irritable bowel syndrome, idiopathic constipation, obesity, and colorectal cancer.

This platform, with the use of synthetic biology to natively engineer Escherichia coli Nissle to produce bioactive compounds in the distal gastrointestinal tract, creates a framework for future characterization of bacterial-host communication and future microbial-based therapeutics.

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More About This Work

Academic Units
Nutritional and Metabolic Biology
Thesis Advisors
Wang, Harris H.
Degree
Ph.D., Columbia University
Published Here
October 30, 2019