Discovery of a Novel and Rich Source of Gluten-Degrading Microbial Enzymes in the Oral Cavity

BACKGROUND. Celiac disease is a T cell mediated-inflammatory enteropathy caused by the ingestion of gluten in genetically predisposed individuals carrying HLA-DQ2 or HLA-DQ8. The immunogenic gliadin epitopes, containing multiple glutamine and proline residues, are largely resistant to degradation by gastric and intestinal proteases. Salivary microorganisms however exhibit glutamine endoprotease activity, discovered towards glutamine- and proline-rich salivary proteins. The aim was to explore if gliadins can serve as substrates for oral microbial enzymes. METHODOLOGY/PRINCIPAL FINDINGS. Proteolytic activity in suspended dental plaque was studied towards a) gliadin-derived paranitroanilide(pNA)-linked synthetic enzyme substrates b) a mixture of natural gliadins and c) synthetic highly immunogenic gliadin peptides (33-mer of a2-gliadin and 26-mer of ?-gliadin). In addition, gliadin zymography was conducted to obtain the approximate molecular weights and pH activity profiles of the gliadin-degrading oral enzymes and liquid iso-electric focusing was performed to establish overall enzyme iso-electric points. Plaque bacteria efficiently hydrolyzed Z-YPQ-pNA, Z-QQP-pNA, Z-PPF-pNA and Z-PFP-pNA, with Z-YPQ-pNA being most rapidly cleaved. Gliadin immunogenic domains were extensively degraded in the presence of oral bacteria. Gliadin zymography revealed that prominent enzymes exhibit molecular weights >70 kD and are active over a broad pH range from 3 to 10. Liquid iso-electric focusing indicated that most gliadin-degrading enzymes are acidic in nature with iso-electric points between 2.5 and 4.0. CONCLUSIONS/SIGNIFICANCE. This is the first reported evidence for gluten-degrading microorganisms associated with the upper gastro-intestinal tract. Such microorganisms may play a hitherto unappreciated role in the digestion of dietary gluten and thus protection from celiac disease in subjects at risk.National Institutes of Health (AI087803, DE18132, DK073254, AI078385, DE05672, DE7652

Identification of Rothia Bacteria as Gluten-Degrading Natural Colonizers of the Upper Gastro-Intestinal Tract

Gluten proteins, prominent constituents of barley, wheat and rye, cause celiac disease in genetically predisposed subjects. Gluten is notoriously difficult to digest by mammalian proteolytic enzymes and the protease-resistant domains contain multiple immunogenic epitopes. The aim of this study was to identify novel sources of gluten-digesting microbial enzymes from the upper gastro-intestinal tract with the potential to neutralize gluten epitopes.Oral microorganisms with gluten-degrading capacity were obtained by a selective plating strategy using gluten agar. Microbial speciations were carried out by 16S rDNA gene sequencing. Enzyme activities were assessed using gliadin-derived enzymatic substrates, gliadins in solution, gliadin zymography, and 33-mer α-gliadin and 26-mer γ-gliadin immunogenic peptides. Fragments of the gliadin peptides were separated by RP-HPLC and structurally characterized by mass spectrometry. Strains with high activity towards gluten were typed as Rothia mucilaginosa and Rothia aeria. Gliadins (250 µg/ml) added to Rothia cell suspensions (OD(620) 1.2) were degraded by 50% after ∼30 min of incubation. Importantly, the 33-mer and 26-mer immunogenic peptides were also cleaved, primarily C-terminal to Xaa-Pro-Gln (XPQ) and Xaa-Pro-Tyr (XPY). The major gliadin-degrading enzymes produced by the Rothia strains were ∼70-75 kDa in size, and the enzyme expressed by Rothia aeria was active over a wide pH range (pH 3-10).While the human digestive enzyme system lacks the capacity to cleave immunogenic gluten, such activities are naturally present in the oral microbial enzyme repertoire. The identified bacteria may be exploited for physiologic degradation of harmful gluten peptides

Isolation and characterization of oral microorganisms degrading dietary gluten

PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact [email protected] (DScD) --Boston University, Henry M. Goldman School of Dental Medicine, 2011 (Department of Periodontology and Oral Biology).Includes bibliography: leaves 88-97.Introduction: Gluten contains domains that are resistant to digestion by human digestive enzymes. These gliadin digestion-resistant domains (33-mer and 26-mer) elicit an immune response in genetically predisposed individuals causing celiac disease. Oral microbial proteases exhibit cleavage activity towards the frequently tripeptide is also abundant in dietary gluten including glutenins and gliadins. Gluten contains domains that are resistant to digestion by human digestive enzymes. These gliadin digestion-resistant domains (33-mer and 26-mer) elicit an immune response in genetically predisposed individuals causing celiac disease. Based on the observed similarity in amino acid sequence between basic protein-rich proteins and gluten, we hypothesized that oral microbial proteases involved in cleaving salivary basic proline-rich proteins may also degrade gluten. The goal of this study was to isolate and characterize gluten degrading microorganisms from the oral cavity, to establish overall proteolytic cleavage site specificities and to make efforts towards the isolation of the gluten-degrading enzymes. Materials and methods: Aliquots of whole saliva and supragingival plaque suspensions were plated on gluten agar and bacteria were sub-cultured on Brucella agar plates to purity. Suspensions of selected pure bacterial strains were tested for protease activities towards mixed gliadins as well as gliadin-derived 33-mer and 26-mer peptides. Enzymatic cleavage site specificities were investigated using five synthetic gliadin tripeptide analogs derivatized with paranitroanilide. pH activity and inhibitor profiIes were determined by monitoring hydrolysis of one of the substrates (Z-YPQ-pNA). Bacteria showing enzymatic cleavage activity of interest were speciated by 16S rDNA analysis. Gliadin-degrading enzyme(s) produced buy one of the identified bacteria, R. mucilaginosa were partially purified by DEAE Ion-exchange chromatography followed by hydrophobic interaction chromatography and Mono Q Ion-exchange chromatography. Results: Dental plaque microorganisms were capable of cleaving mixed gliadins as well as 33-mer and 26-mer peptides,which were resistant to degradation by the mammalian gastrointestinal enzymes trypsin, chymotrypsin and pepsin. Among the tripeptide substrates Z-YPQ-pNA was most efficiently cleaved by dental plaque bacteria. Longer incubations also led to hydrolysis of Z-QQP-pNA, Z-PPF-pNA and Z-PFP-pNA, Gliadin-degrading proteases in dental plaque were active over a wide pH range (3-10). Using a selective agar approach oral gluten degrading bacteria was isolated. Aerobic strains identified were Rothia mucillaginosa and Rohia ot 188; anaerobic strains found were Bifidobacterium Bifidobacerium dentium Gliadin zymography of isolated oral microbial strains revealed prominent gliadin-degrading protease activities in Rothia species. The approximate molecular weight of the gliadin-degrading enzyme was 75kD. Rothia bacteria cleaved Z-YPQ-pNA and Z-LPY-pNA but not Z-QQP-pNA, Z-PPF-pNA and Z-PFP-pNA. The gliadin- degrading enzyme(s) of R. aeria were tentatively classified as a serine-protease(s) based on strong inhibition by PMFS and AEBSF. Its enzymes were not active at neutral to basic pH values. Partial isolation of the gliadin degrading enzyme(S) was achieved separating the enzymes with cleavage specificities towards Z-YPQ-pNA or Z- LPY pNA. ConcIusion: This is the first reported evidence for gluten-degrading microorganisms to be part of the natural oral microflora. 1t is very well possible that oral microorganisms play an unappreciated role in the digestion of gluten and might be implicated in gluten-related enteropathies such as celiac disease. Furthermore, therapeutic applications of the enzymes identified can be envisioned