656 Rothia Bacteria as Gluten-Degrading Natural Colonizers of the Oral Cavity

Friday, March 23, 2012: 8 a.m. - 9:30 a.m.
Presentation Type: Oral Session
G. WEI1, M. ZAMAKHCHARI1, F.E. DEWHIRST2, D. SCHUPPAN3, F.G. OPPENHEIM1, and E. HELMERHORST4, 1Goldman School of Dental Medicine, Boston University, Boston, MA, 2Forsyth Institute, Cambridge, MA, 3Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Harvard University, Boston, MA, 4Dept. of Periodontology & Oral Biol, Boston University, Boston, MA
Gluten proteins are prominent constituents of barley, wheat and rye. They have an unusual amino acid composition and are difficult to digest by mammalian proteolytic enzymes. The protease-resistant domains contain multiple immunogenic epitopes causing celiac disease in genetically pre-disposed individuals.

Objective: To identify novel sources of gluten-digesting microbial enzymes from the oral cavity, representing the upper gastro-intestinal tract, with the potential to neutralize gluten epitopes.

Method: 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.

Result: Rothia mucilaginosa and Rothia aeria were identified as gluten-degrading strains in the oral cavity. Gliadins as well as immunogenic 33-mer and 26-mer peptides (250 µg/ml) added to Rothia cell suspensions (OD620 1.2) were degraded. Cleavage occured 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). The enzymes also demonstrated activity in the presence of EDTA, which suggest that the enzymes appears to be non-metalloproteases.

Conclusion: 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 and lead to novel and effective strategies to combat celiac disease.

This abstract is based on research that was funded entirely or partially by an outside source: NIH grants A1087803, DE 05672, DE07652, DK 07254, A1078385, and DE016937

Keywords: Enzymes, Gluten, Immunology, Microbiology and Saliva
See more of: Infection
See more of: Microbiology / Immunology