Gut microbiome-derived prolyl peptidases from Segatella copri and Stenotrophomonas maltophilia degrade immunogenic gliadin peptides and restore intestinal barrier integrity in a Celiac disease model

Celiac disease (CeD) is an autoimmune enteropathy triggered by gluten-derived peptides that resist gastrointestinal digestion, notably the proline-rich 33-mer and 11-mer gliadin epitopes. Here, we describe a rational, data-centric strategy to identify gut microbiome-derived prolyl peptidases capable of degrading these immunogenic peptides. Integrating metagenomic mining with structure-based in silico screening, we identified two novel enzymes PSP692 and PSP464 from Segatella copri and Stenotrophomonas maltophilia , respectively. Recombinant expression and enzymatic characterization confirmed their activity under physiologically relevant conditions: PSP692 efficiently degrades the 33-mer at pH 6, while PSP464 targets the 11-mer at pH 4. Functional assays using CaCo-2 monolayers demonstrated that enzymatic degradation of gliadin epitopes significantly restored expression of tight junction proteins (ZO-1 and Occludin), reduced IL-6 secretion, and improved barrier integrity. These findings establish a foundational strategy for the discovery of microbiome-derived glutenases and provide a compelling case for combinatorial enzyme therapy to mitigate gluten immunotoxicity in CeD and related disorders.