Karmarkar_2026_Microbiol.Spectr__e0321425

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, metagenome-based 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 from Segatella copri and PSP464 from Stenotrophomonas maltophilia. Recombinant expression, purification, and 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 cell line, both in bi- and tri-dimensional assays, demonstrated that degradation of gliadin peptides by PSP692 and PSP464 significantly restored the 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 both a compelling case and a methodology for data-driven discovery of functional enzymes that degrade immunogenic gliadin peptides, with translational potential as adjunct therapies in CeD and gluten-related disorders. IMPORTANCE: Celiac disease affects 1.4% of the global population, and, as of date, a gluten-free diet (GFD) is the only therapy available. Adherence to GFD is difficult, and inadvertent exposure to gluten still occurs. To address this, various approaches are utilized to develop adjuvant therapies. These include recombinant enzymes that, to date, have been discovered by serendipity. We have outlined and validated a method to identify enzymes with potential from metagenomic data, which will also be validated experimentally.