Fragmentation of Immunogenic Gluten Domains by Oral Microorganisms

Dietary gluten are proteins that are difficult to digest and in genetically predisposed subjects can cause celiac disease. Certain immunogenic gluten domains are completely resistant to the major human digestive enzymes trypsin, chymotrypsin and pepsin. We recently demonstrated that oral microorganisms in dental plaque produce gluten-degrading enzymes. Objectives: to characterize these enzymes with respect to activity and cleavage site specificity. Methods: Aliquots of suspensions of whole saliva and dental plaque were cultured on wheat gluten-limited agar plates. Individual colonies were sub-cultured on Brucella agar plates to purity. Suspensions of selected pure bacterial strains (OD620=1.2) were evaluated for gliadin degradation in-solution and in-gel (zymography), and for enzymatic activities directed at the immunogenic gliadin domains (33-mer and 26-mer, 250 μg/ml). Protease specificities were assessed by sequencing the proteolytic fragments by LC-ESI-MS/MS and by measuring hydrolysis of gliadin sequence-mimicking synthetic enzyme substrates: Z-YPQ-pNA, Z-QQP-pNA, Z-PPF-pNA, Z-PFP-pNA and Z-LPY-pNA Results: With the selective plating strategy employed several pure oral microbial strains were obtained that were capable of growth on gluten-limited agar. Gliadin zymography of selected strains showed enzymatic activities in the 70-75 kD region. The 33-mer and 26-mer domains were degraded completely in 5h. Structural analysis of the fragments generated indicated prominent cleavage activities after YPQ and LPY, consistent with hydrolysis of the corresponding synthetic substrates Z-YPQ-pNA and Z-LPY-pNA. Conclusions: While the human digestive enzyme system seemingly lacks the capacity to neutralize immunogenic gluten domains implicated in celiac disease, such activities are present in the oral microbial proteasome. The role of gluten-degrading bacteria in the digestion of wheat products will be further explored from a clinical and therapeutic perspective. Supported by NIH grants DE18132, AI087803, DE05672 and DE07652.