(160v) Development of a Transcriptional Biosensor for Interrogating the Role of Hydrogen Sulfide in IBD Onset

As of 2015, Inflammatory Bowels Disease (IBD) affects 1.3% of the adult population in the United States1. The onset of IBD is thought to be partly mediated by the gut microbiota. One specific metabolite produced by gut microbes that has been implicated in IBD development is hydrogen sulfide (H2S). However, the role of H2S is largely disputed: some evidence suggests a role in inflammation, inhibition of mitochondrial function, and degradation of mucus through reduction of disulfide bonds in mucin, while other work suggest a beneficial role in wound healing and as an antioxidant2,3. We hypothesize that the seemingly contradictory effects of H2S in previous work may reflect a concentration dependence that could not be captured previously due to the severe technical difficulty of accurately dosing and measuring this highly reactive, volatile compound in the complex gut environment. To overcome this challenge and more precisely define the role of H2S in IBD etiology, we are developing a H2S transcriptional biosensor. It has previously been demonstrated that the repressor CstR from Staphylococcus aureus responds to persulfides4. Further, CstR was used as a transcriptional H2S biosensor by coupling it with the enzyme sulfide-quinone reductase (Sqr), which produces persuflide from H2S5. In order to improve the sensitivity and dynamic range of the sensor to the level that would be required for in vivo work, we first developed a computational kinetic model to identify critical determinants of performance. Next, to experimentally engineer these determinants, we introduced CstR and Sqr on companion plasmids, and optimized transcriptional and translational components of expression while modifying the repressor binding domain to maintain low levels of ‘leaky’ expression. A collection of upstream transcriptional enhancing elements were screened, the –35 and –10 boxes were mutated, and a consensus ribosome binding site was inserted. The best combinatorial product of these changes was chosen, and we then optimized the repressor binding site orientation and spacing. Results from these ongoing optimization efforts will be presented, and set the stage for an improved sensor that will allows for spatially resolved sulfide measurements for in vivo applications. The development of this tool will permit coupling this with known sulfide-producing strains to interrogate the impact of sulfide on mucus barrier integrity and epithelial health via a gut-on-a-chip model. Overall, this work will enable a greater mechanistic understanding of the pathology of the disease in the context of the gut microbiota and may inform new treatment strategies.

References:

1. “Data and Statistics.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 11 Aug. 2020, www.cdc.gov/ibd/data-statistics.htm.

1. Wallace JL, Motta JP, Buret AG. Hydrogen sulfide: an agent of stability at the microbiome-mucosa interface. Am J Physiol Gastrointest Liver Physiol. 2018 Feb 1;314(2):G143-G149. doi: 10.1152/ajpgi.00249.2017. Epub 2017 Oct 12. PMID: 29025733; PMCID: PMC5866422.

1. Guo FF, Yu TC, Hong J, Fang JY. Emerging Roles of Hydrogen Sulfide in Inflammatory and Neoplastic Colonic Diseases. Front Physiol. 2016;7:156. Published 2016 May 3. doi:10.3389/fphys.2016.00156

1. Luebke JL, Shen J, Bruce KE, Kehl-Fie TE, Peng H, Skaar EP, Giedroc DP. The CsoR-like sulfurtransferase repressor (CstR) is a persulfide sensor in Staphylococcus aureus. Mol Microbiol. 2014 Dec;94(6):1343-60. doi: 10.1111/mmi.12835. Epub 2014 Nov 17. PMID: 25318663; PMCID: PMC4264537.

1. “Synthetic Gene Circuits Enable Escherichia Coli To Use Endogenous H2S as a Signaling Molecule for Quorum Sensing.” doi:10.1021/acssynbio.9b00210.s001.