(264g) Engineering Intestinal Sulfur Metabolism for Understanding Host-Microbiome Interactions

Microbial metabolism in the human gut is increasingly linked with a range of host diseases. The native microbiota utilizes host-derived inorganic sulfur as terminal electron acceptors, and ferments sulfur containing-amino acids. Both processes produce hydrogen sulfide (H2S), a gaseous compound that accumulates to millimolar levels in the colon. H2S has been studied for its potential role in inflammatory bowel disease (IBD) and colon cancer, with conflicting evidence supporting both a pro- and anti-inflammatory role. These controversies are due to the concentration-dependent effects of H2S, which could not be captured in previous work due to experimental difficulties associated with accurately dosing and measuring this volatile, reactive compound. Traditional methods of studying H2S include high-concentration bolus injections in animals and in vitro, which do not represent i) slower, consistent production by native microbiota that are ii) proximally located (100s of microns) to the intestinal epithelium, which lead to local concentration gradients. Here, we overcome these challenges by using metabolically engineered strains as a continuous, localized, and titratable source of H2S in a humanized gut-on-chip system. This platform mimics the in vivo environment and enables accurate re-creation of host-microbe interactions for investigating the concentration-dependent effects of H2S on human intestinal tissue.

To do so, we engineered Escherichia coli to produce H2S from L-cysteine. E. coli has multiple native cysteine desulfidases (Cdl) which catalyze the conversion of cysteine to H2S. Through a combination of gene knockouts, modulated expression of heterologous and native Cdl homologs, and transporter engineering, we generated a library of strains capable of titrating H2S across the gut physiological range (0.1-3.7 mM) in Hungate tubes. We then used these engineered bacteria to control H2S levels in a gut microphysiological system (gut-on-a-chip) supportive of the co-culture of microbes and human intestinal cells, specially engineered to maintain H2S gas tension. The engineered strains colonized the intestinal environment in the chip and were metabolically active for two days, producing H2S across a sixteen-fold range and inducing changes in host gene expression and sulfur metabolism in an H2S concentration-dependent manner. We demonstrated microbial H2S production is more consistent and reproducible than small molecule sulfide donors, which is advantageous for understanding concentration-dependent effects. These results [1] validate a platform for studying the mechanisms underlying microbe-host interactions and demonstrate the practical use of metabolic engineering in disease research.

Since Cdl is widely distributed in the gut microbiota, we next aimed to engineer strains to use a sulfur source not as readily degraded by native microbes, both for the production of H2S, and for sequestration of excess levels. To identify potential stable sulfur sources, we tested the conversion rate of several different sulfur compounds to H2Sin ex vivo rat colon enrichments, resulting in one compound (S1) which was not metabolized. Using native and heterologous genes, we then engineered E. coli to convert S1 into H2S, resulting in a specific production rate of 0.3 mM H2S/hr-OD600. Next, we engineered a synthetic pathway to sequester H2S by converting it into S1. The best strain, containing a four-step pathway consumed H2S at a specific rate of 1 mM H2S/hr-OD600, which our calculations suggest is sufficiently rapid to modify intestinal sulfide levels at cell densities that have been achieved in previous studies. In summary, this work demonstrate the potential of metabolically engineered strains to enhance our understanding of how microbial metabolites impact host health, and sets the stage for potential use of these strains in a clinical context.

References:

[1] Hayes JA, Lunger AW, Sharma AS, Fernez MT, Carrier RL, Koppes AN, Koppes R, Woolston BM. Engineered bacteria titrate hydrogen sulfide and induce concentration-dependent effects on the host in a gut microphysiological system. Cell Rep. 2023 Dec 26;42(12):113481. doi: 10.1016/j.celrep.2023.113481. Epub 2023 Nov 18. PMID: 37980564; PMCID: PMC10791167.