Isolation and characterization of oral microorganisms degrading dietary gluten
Zamakhchari, Maram Fouad
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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.
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 firstname.lastname@example.org.Thesis (DScD) --Boston University, Henry M. Goldman School of Dental Medicine, 2011 (Department of Periodontology and Oral Biology).Includes bibliography: leaves 88-97.
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