PRO-INFLAMMATORY MOLECULAR AND INFLAMMATORY MECHANISMS OF SULFUR METABOLISM IN IBD-RELEVANT CLOSTRIDIA SPECIES

Abstract

Abstract Sulfur metabolism is emerging as a signature of IBD gut microbiota. Overrepresentation of sulfur-reducing bacteria (SRB) in IBD results in SRB-derived epithelial toxic H2S production that can overwhelm the body’s detoxification capacity, leading to impaired cellular respiration by inhibiting oxygen binding to mitochondrial cytochrome-c-oxidase. Butyrate potently inhibits SRBs and H2S, yet IBD patients have reduced short chain fatty acid (SCFA) production. More critically, H2S blocks butyrate oxidation, the primary energy source of colonocytes; butyrate oxidation deficiency is a defining characteristic of IBD. Since cysteine is the preferred substrate for H2S production by SRBs, a cysteine-rich environment provided by either a high protein diet or local intestinal mucus degradation promotes ideal conditions for SRB establishment and proliferation. SRBs can catabolize other sulfur-containing compounds critical for immune homeostasis and cellular health, such as taurine-conjugated bile acids and the “master antioxidant” glutathione, leading to further toxic H2S production. However, the molecular underpinnings of sulfur metabolism by specific bacterial genera is understudied in IBD. Results: Using a combination of in-vivo and in-vitro screening to detect the relative induction of interleukin 10 (IL-10) and interferon g (IFNg) by 19 resident bacterial strains isolated from a healthy human donor, we identified 4 bacterial strains that induce a low IL-10/IFNg ratio. These 4 strains (low group), but not 3 bacterial strains that induce a high IL-10/IFNg ratio, induce colitis in selectively colonized gnotobiotic Il10-/- mice (Fig.1A). Two of these 4 disease-inducing strains, Clostridium perfringens (A12) and Clostridium bolteae (B6), produce high concentrations of H2S in monoassociated mice (Fig.1B). In-vitro H2S production by these strains is dependent on cysteine (Fig.1C). C. perfringens and C. bolteae each induce colitis in monoassociated Il10-/- mice (Fig.1D). We are dissecting the sulfur metabolic pathways in C. perfringens and C. bolteae and their contribution to inflammatory processes by interrupting key genes predicted to contribute to H2S production, cysteine catabolism and bile acid metabolism. We will use these mutants in both in-vitro and in-vivo Il10 -/- gnotobiotic mice models to characterize their metabolic and inflammatory profiles. We have created several mutants using Targetron gene editing, including the dissimilatory sulfate reductase (Δdsr), a putative sulfonate membrane transporter (ΔssuA), anaerobic sulfite reductase (ΔasrA) and bile salt hydrolase (Δbsh). Conclusions: H2S producing bacterial strains can induce experimental colitis. Our planned mechanistic studies will determine the metabolic routes for H2S production by specific aggressive bacteria to guide novel therapeutic or dietary interventions to improve IBD prognosis.