Project Summary Hydrogen sulfide (H2S) is a redox-active signaling molecule that modulates electron transport and energy metabolism, and by largely unknown mechanisms, mediates neuro- and cardioprotection, vasodilation, the hypoxic response, protein translation, epigenetics, aging, and both patho- and physiological responses in colon. As H2S is also a respiratory poison at high doses, cells actively oxidize it to produce persulfides, sulfite, thiosulfate, (collectively referred to as reactive sulfur species), and sulfate. Oxidative modification of protein cysteine thiols to persulfides is postulated to be a primary mechanism by which H2S signals. The potential involvement of other reactive sulfur species in signaling is however, unknown. Studies in our laboratory have demonstrated that H2S impacts cellular redox metabolism via its dual effects on mitochondrial energetics, i.e. increasing electron flux at low, and inhibiting it at high concentrations. The highest exposure to exogenous H2S occurs in colon (0.2–2.4 mM) and is derived from gut resident microbiota, which synthesize sulfide. Preliminary data in our laboratory reveal that colonic epithelial cells quantitatively oxidize exogenous H2S to thiosulfate, and that the localization and expression levels of sulfide oxidation enzymes in murine colonocytes are strongly influenced by the presence or absence of sulfate reducing bacteria in the gut. I hypothesize that the dynamic interplay between host and microbial sulfur metabolites influences host metabolism and impacts longevity. Using Caenorhabditis elegans as a model organism, which has the full complement of orthologous genes involved in H2S biogenesis and oxidation found in humans and is readily amenable to genetic and dietary manipulation and to life-span analyses, I will test my hypothesis by addressing the following two aims. (i) I will characterize how alterations in H2S levels and reactive sulfur species impact organismal redox and histone modifications that are linked to longevity and stress response in C. elegans. H2S levels and its oxidative byproducts will be modulated by exogenous H2S, RNA interference knockdowns of host sulfide oxidation enzymes, and by diet, using Escherichia coli with deletions in specific genes involved in sulfur metabolism. I will assess how exogenous versus dietary H2S modulation affects organismal redox using the genetically encoded reduction-oxidation sensitive GRX1-roGFP2 sensor and track changes in histone methylation and acetylation status with reported links to redox, H2S availability, diet, and aging. (ii) I will investigate how differences in exogenous H2S exposure from air versus gut microbes affects the expression and localization of host sulfide oxidation enzymes, the abundance and type of reactive sulfur species, and their effect on lifespan. I will complement these studies with metabolomic and transcriptomic analyses to identify pathways that are impacted by H2S exposure. The successful completion...