Background: H₂S imbalances in the intestinal tract trigger Crohn's disease (CD), a chronic inflammatory gastrointestinal disorder characterized by microbiota dysbiosis and barrier dysfunction. However, a comprehensive understanding of H₂S generation in the gut, and the contributions of both microbiota and host to systemic H₂S levels in CD, remain to be elucidated. This investigation aimed to enhance comprehension regarding the sulfidogenic potential of both the human host and the gut microbiota. Results: Our analysis of a treatment-naive CD cohorts' fecal metagenomic and biopsy metatranscriptomic data revealed reduced expression of host endogenous H₂S generation genes alongside increased abundance of microbial exogenous H₂S production genes in correlation with CD. While prior studies focused on microbial H₂S production via dissimilatory sulfite reductases, our metagenomic analysis suggests the assimilatory sulfate reduction (ASR) pathway is a more significant contributor in the human gut, given its high prevalence and abundance. Subsequently, we validated our hypothesis experimentally by generating ASR-deficient E. coli mutants ∆cysJ and ∆cysM through the deletion of sulfite reductase and L-cysteine synthase genes. This alteration significantly affected bacterial sulfidogenic capacity, colon epithelial cell viability, and colonic mucin sulfation, ultimately leading to colitis in murine model. Further study revealed that gut microbiota degrade sulfopolysaccharides and assimilate sulfate to produce H₂S via the ASR pathway, highlighting the role of sulfopolysaccharides in colitis and cautioning against their use as food additives. Conclusions: Our study significantly advances understanding of microbial sulfur metabolism in the human gut, elucidating the complex interplay between diet, gut microbiota, and host sulfur metabolism. We highlight the microbial ASR pathway as an overlooked endogenous H₂S producer and a potential therapeutic target for managing CD.

To better understand the interplay of diet and gut microbiota in Crohn’s disease (CD), taking advantage of a newly-onset treatment naïve CD cohort. We focus on phenylacetylglutamine (PAGln), a diet-derived meta-organismal prothrombotic metabolite. We collected fecal and serum samples from a CD cohort (n=136) and healthy controls (n=126) prior to treatment, and quantified serum PAGln using LC-MS/MS. Diet was assessed using food-frequency questionnaires. Mice (C57BL/6) were fed high/low protein diets and administered DSS to examine plasma PAGly, thrombosis potential, and colitis severity. PAGly or saline was administered to DSS-induced colitis mice, colitis severity and colonic tissue gene expression were examined. P-selectin and CD40L expression were determined in human platelet-rich plasma (n=5-6) after exposure to platelet agonists following PAGln priming. Bioinformatic analysis and bacterial culturing identified the main contributor of PAGln in CD. PAGln, a meta-organismal prothrombotic metabolite, is associated with CD. Administration of PAGly exacerbated colitis in mouse model and upregulated coagulation-related biological processes. Antiplatelet medicine, dipyridamole, attenuated PAGly enhanced colitis susceptibility. PAGln enhanced platelet activation and CD40L expression in platelet rich plasma ex vivo. Further study revealed that high dietary protein intake and increased abundance of phenylacetic acid (PAA)-producing Proteobacteria mediated by phenylpyruvate decarboxylase act in concert to cause the elevated PAGln levels in CD patients. Taken together, ppdc-carrying Proteobacteria generated PAGln from dietary protein is associated with CD and exacerbates colitis possibly via platelet-induced coagulation and inflammation These results suggest that PAGln is a potential early diagnostic marker and therapeutic target of CD.

Bacterial trimethylamine N‐oxide (TMAO) demethylase, Tdm, carries out an unusual oxygen‐independent demethylation reaction, resulting in the formation of dimethylamine and formaldehyde. In this study, site‐directed mutagenesis, homology modelling and metal analyses by inorganic mass spectrometry have been applied to gain insight into metal stoichiometry and underlying catalytic mechanism of Tdm of Methylocella silvestris BL2. Herein, we demonstrate that active Tdm has 1 molar equivalent of Zn²⁺ and 1 molar equivalent of non‐haem Fe²⁺. We further investigated Zn²⁺‐ and Fe²⁺‐binding sites through homology modelling and site‐directed mutagenesis and found that Zn²⁺ is coordinated by a 3‐sulfur‐1‐O motif. An aspartate residue (D198) likely bridges Fe²⁺ and Zn²⁺ centres, either directly or indirectly via H‐bonding through a neighbouring H₂O molecule. H276 contributes to Fe²⁺ binding, mutation of which results in an inactive enzyme, and the loss of iron, but not zinc. Site‐directed mutagenesis of Tdm also led to the identification of three hydrophobic aromatic residues likely involved in substrate coordination (F259, Y305, W321), potentially through a cation–π interaction. Furthermore, a crossover experiment using a substrate analogue gave direct evidence that a trimethylamine‐alike intermediate was produced during the Tdm catalytic cycle, suggesting TMAO has a dual role of being both a substrate and an oxygen donor for formaldehyde formation. Together, our results provide novel insight into the role of Zn²⁺ and Fe²⁺ in the catalysis of TMAO demethylation by this unique oxygen‐independent enzyme.

The peroxisome proliferator-activated receptor- (PPAR) plays a key role in lipid metabolism and energy combustion. Chronic activation of PPAR in rodents leads to the development of hepatocellular carcinomas. The ability of PPAR to induce expression of its target genes depends on Mediator, an evolutionarily conserved complex of cofactors and, in particular, the subunit 1 (Med1) of this complex. Here, we report the identification and characterization of PPAR-interacting cofactor (PRIC)-295 (PRIC295), a novel coactivator protein, and show that it interacts with the Med1 and Med24 subunits of the Mediator complex. PRIC295 contains 10 LXXLL signature motifs that facilitate nuclear receptor binding and interacts with PPAR and five other members of the nuclear receptor superfamily in a ligand-dependent manner. PRIC295 enhances the transactivation function of PPAR, PPAR, and ER. These data demonstrate that PRIC295 interacts with nuclear receptors such as PPAR and functions as a transcription coactivator underin vitroconditions and may play an important role in mediating the effectsin vivoas a member of the PRIC complex with Med1 and Med24.