Why Symptoms Linger in Quiescent Crohn's Disease: Investigating the Impact of Sulfidogenic Microbes and Sulfur Metabolic Pathways

Introduction: Even in the absence of inflammation, persistent symptoms in patients with Crohn's disease (CD) are prevalent and worsen quality of life. We previously demonstrated enrichment in sulfidogenic microbes in quiescent CD patients with (qCD+S) vs. without persistent GI symptoms (qCD-S). Thus, we hypothesized that sulfur metabolic pathways would be enriched in stool while differentially abundant microbes would be associated with important sulfur-metabolic pathways in qCD+S. Methods: We performed a multi-center observational study nested within SPARC IBD. Quiescent inflammation was defined by fecal calprotectin level < 150 mcg/g. Persistent symptoms were defined by CD-PRO2. Active CD (aCD) and non-IBD diarrhea-predominant irritable bowel syndrome (IBS-D) were included as controls. Results: Thirty-nine patients with qCD+S, 274 qCD-S, 21 aCD, and 40 IBS-D underwent paired shotgun metagenomic sequencing and untargeted metabolomic profiling. The fecal metabolome in qCD+S was significantly different relative to qCD-S and IBS-D but not aCD. Patients with qCD+S were enriched in sulfur-containing amino acid pathways, including cysteine and methionine, as well as serine, glycine, and threonine. Glutathione and nicotinate/nicotinamide pathways were also enriched in qCD+S relative to qCD-S, suggestive of mitochondrial dysfunction, a downstream target of H₂S signaling. Multi-omic integration demonstrated that enriched microbes in qCD+S were associated with important sulfur-metabolic pathways. Bacterial sulfur-metabolic genes, including CTH, isfD, sarD, and asrC, were dysregulated in qCD+S. Finally, sulfur metabolites with and without sulfidogenic microbes showed good accuracy in predicting presence of qCD+S. Discussion: Microbial-derived sulfur pathways and downstream mitochondrial function are perturbed in qCD+S, which implicate H₂S signaling in the pathogenesis of this condition. Future studies will determine whether targeting H₂S pathways results in improved quality of life in qCD+S.