Investigating the Impact of Hydrogen Sulfide on the Mucosal Barrier through Development of a Hydrogen Sulfide Biosensor and Multiple Particle Tracking

Inflammatory bowels disease (IBD) is a gastrointestinal (GI) condition that afflicts more than 1% of adults. Hydrogen sulfide, a gaseous compound produced by host and microbial cells, has disputed effects on GI health. While some studies suggest therapeutic effects, others have shown cytotoxicity, mucosal barrier disruption, and promotion of an inflammatory cascade. Resolution of this dispute is hampered by the volatility and reactivity of sulfide, which renders traditional assays inaccurate, and incapable of providing a high-resolution view into spatial and temporal dynamics. To overcome this challenge, we have leveraged genetic sulfide regulation from Rhodobacter capsulatus to develop a fluorescent sulfide biosensor in E. coli. This system relies on enzymatic conversion of sulfide by sulfide:quinone oxidoreductase (Sqr) to polysulfides, which deactivate the transcriptional repressor SqrR. Through promoter engineering, we optimized the system to provide an operational range of 50 µM - 1 mM under aerobic conditions, but the system was unresponsive under anaerobic conditions due to quinone pool incompatibility. To facilitate sensing in the primarily anaerobic gut microenvironments, we isolated a previously uncharacterized Sqr from Wolinella succinogenes that enabled coupling of sulfide oxidation to nitrate and fumarate reduction. Concurrently, using in vitro Multiple Particle Tracking, we have shown that sulfide increases diffusivity in porcine mucus. Future work will integrate these two lines of experiment in a gut-on -chip system featuring primary intestinal stem cell monolayer growing on a vertical gel wall for cross-sectional analysis of mucus barriers, enhancing our understanding of the role of sulfide in GI disease.