Gluten-Degrading Microbes in Saliva: Novel Therapeutic Perspectives for Celiac Disease

Introduction: Celiac disease is a T cell mediated-inflammatory enteropathy caused by dietary gluten. Certain immunogenic domains in gluten are completely resistant to the major human digestive enzymes. Deamidation of gluten by transglutaminase-2 in the lamina propria greatly enhances their binding to antigen presenting cells, provoking an inflammatory immune response in susceptible individuals. One therapeutic strategy focuses on enzymes that degrade gluten into non-immunogenic peptides. We recently discovered that the oral microbiome is a novel and rich source of such enzymes. Objectives: 1) isolate the gluten-degrading oral microorganisms, 2) determine enzymatic cleavage site specificities, 3) investigate the effect of gluten degradation on transglutaminase recognition. Methods: Gluten degrading microbes were isolated from human saliva and supragingival plaque on gluten agar plates. Degradation of gliadins, representing the most immunogenic fraction of gluten was tested by zymography, SDS-PAGE, or RP-HPLC. Enzymatic cleavage site specificities were determined with gliadin-derived synthetic tripeptides. Recognition of gliadin peptides in the degradation mixture by human recombinant transglutaminas-2 was measured by crosslinking to monodansylcadaverine. Results: Selected oral microorganisms, identified as Rothia species, effectively hydrolyzed the gluten-derived synthetic peptides after YPQ and LPY. They greatly degraded gliadin in gel and in solution after 2-hours, and cleaved immunogenic gliadin peptide domains 26-mer and 33-mer into smaller fragments after 30-minutes and 8-hours respectively. A perfect correlation was observed between degradation and loss of cross-linking, showing 50% reductions after 10.5 and 10.2-minutes, respectively, for 26-mer and 4.9 and 4.3-hours, respectively, for 33-mer. Conclusion: While the human digestive enzymes are unable to cleave immunogenic gluten peptides implicated in celiac disease, such activities are present in the oral microbiome. Enzymatic abolishment of transglutaminase-mediated deamidation is a first indication for gluten epitope neutralization. The Rothia enzymes open promising new avenues in the search for novel therapies to neutralize the deleterious effects of gluten in celiac disease patients.