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

Eva J Helmerhorst,Frank G Oppenheim,Detlef Schuppan,Maram Zamakhchari,Frank R Deleo

doi:10.1371/journal.pone.0013264

Eva J Helmerhorst, Frank G Oppenheim + Show 3 more

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https://doi.org/10.1371/journal.pone.0013264

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Abstract

BackgroundCeliac 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 FindingsProteolytic 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 α2-gliadin and 26-mer of γ-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/SignificanceThis 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.

Highlights

The gastrointestinal (GI) tract consists of distinct but connected anatomical regions, comprising the oral cavity, the oesophagus, the stomach, the small and the large intestine[1]
Each search result was validated by manual inspection of the acquired MS/MS spectra to ensure that the fragment ions (e.g. b and y ions) were above background level and that three consecutive b- and y-ions we found for each identified peptide
One of the therapeutic strategies to counteract or prevent the deleterious effect of these minor amounts of dietary gluten focuses on proteolytic enzymes to aid in their degradation, preventing their antigenic presentation to and activation of intestinal T cells [24,25]

Summary

Introduction

The gastrointestinal (GI) tract consists of distinct but connected anatomical regions, comprising the oral cavity, the oesophagus, the stomach, the small and the large intestine[1]. The proximal region, the oral cavity, provides a rich environment for bacterial colonization as it contains a variety of different habitats and ecological niches. Ingested proteins are in part degraded by host proteolytic enzymes such as pepsin, elastase, carboxypeptidase, trypsin, and chymotrypsin converting them into oligopeptides and single amino acids which can enter the enterocyte via selective transporters and be further metabolized or transported [8]. This mechanism is contingent upon the susceptibility of the substrate proteins to digestion by host enzymes. The aim was to explore if gliadins can serve as substrates for oral microbial enzymes

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