PROJECT SUMMARY The human microbiome is the sum of microbes that live in or on the human body, and it contributes to both health and disease. Our previous work has established that nitric oxide (NO) generated by gut microbiota acts as a language of inter-species communication between the microbiome and its host by changing fundamental host functions. Altered gut microbiota has also been implicated as an important risk factor in the etiology of inflammatory bowel diseases such as Crohn’s disease (CD). While excess NO generated by overexpression of nitric oxide synthase (NOS) in the host gut has been observed in CD, the role of the NO derived from gut microbiota has not been investigated or considered. NO signals in large part by post-translationally modifying proteins via S-nitrosylation, the covalent attachment of NO to the thiol side-chain of specific cysteine residues to form S-nitrosothiols (SNOs), altering protein function. Here we will test the hypothesis that communication between gut microbiota and mammalian host via host protein S-nitrosylation impacts health in normal mice and in a mouse model of CD. To do this, we will first characterize the extent to which microbiota-derived NO mediates host S-nitrosylation of gut proteins including known CD-associated proteins, and demonstrate that host gut proteins are highly regulated by microbiotal-NO/SNO. Further, we will show that gut microbiota-derived NO is not limited to affecting just adjacent gut tissue but may have far-reaching systemic effects within the host, by identifying host organs beyond the gut where endogenous protein S-nitrosylation and consequently organ functions are impacted by gut microbiota-derived NO, in both healthy and CD mice. This will establish an organ- specific, gut microbial NO-dependent SNO-proteome atlas at baseline, to compare and identify alterations found in the SNO-proteome in the CD mouse model. This will also allow identification of specific host proteins in CD whose S-nitrosylation depends significantly on NO derived from gut microbiota, enabling investigation of the role of specific alterations in patients with CD. Additionally, the microbial-NO dependent S-nitrosylation signature in gut and beyond will be helpful towards the diagnosis and treatment of CD. Using our CD mouse model, we will also test the use of a specific class of aminoquinoline-based inhibitors that selectively target bacterial-NOSbut not mammalian-NOSsas a treatment option of CD. Furthermore, the establishment of this gut microbiota-NO- dependent SNO-proteome atlas in different major organs (gut, liver, heart, lung, kidney, brain) will be very useful in studying its perturbations across different mice models of human disease in the future. In addition, we will identify the mechanism(s) by which NO is transported from the gut to distant organs. The proposed work will, for the first time, determine: the effect of gut microbiota-derived NO on mammalian host physiology via S- nitrosylation, ...