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Identification of Rothia bacteria as gluten-degrading natural colonizers of the upper gastro-intestinal tract.

ABSTRACT: 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 ?-gliadin and 26-mer ?-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.

SUBMITTER: Zamakhchari M

PROVIDER: S-EPMC3177827 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Identification of Rothia bacteria as gluten-degrading natural colonizers of the upper gastro-intestinal tract.

Zamakhchari Maram M Wei Guoxian G Dewhirst Floyd F Lee Jaeseop J Schuppan Detlef D Oppenheim Frank G FG Helmerhorst Eva J EJ

PloS one 20110921 9

<h4>Background</h4>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.<h4>Methodology/principal find ...[more]

PMID: 21957450

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Project description:This study was aimed to isolate sinapine-degrading bacteria from the intestinal tract of laying hens and to identify the predominant bacteria. Thirty-week old healthy laying hens were killed, and the chyme in the digestive tract was inoculated into modified Czapek medium containing sinapin and cultivated at 37 °C for 10 days. The optical density (OD) values of the bacterial solutions at different cultivating times were detected by a spectrophotometric method. The predominant strains were identified by 16S rRNA gene analysis. We extracted the extracellular products of the predominant strains to determine the total protein using the Coomassie brilliant blue method, and to determine the activities of some extracellular enzymes using the agar plate diffusion method. Nine strains were isolated from the lower intestinal tract of laying hens. Among the 9 strains, 5 were from the ileum, 2 were from the ceca and 2 were from the jejunum. We could not isolate any strains from the upper intestinal tract, such as the stomach and duodenum. Eight of those 9 isolated strains were gram negative and one was gram positive. Strains YD-1 and YD-2 were better than other strains in their abilities to degrade sinapine. Strains YD-1 and YD-2 were identified as Escherichia coli and Klebsiella spp., respectively, by the 16S rRNA sequence analysis. The total protein level of the extracellular products was 1.213 g/L for YD-1 and 1.990 g/L for YD-2. Both extracellular products of YD-1 and YD-2 had the activities of protease, amylase and urease. This study confirmed that the primary site of sinapine degradation is in the lower intestinal tract of laying hens. The sinapine-degrading strains are mainly gram negative. Strains YD-1 and YD-2 are predominant in degrading sinapine and they belong to E. coli and Klebsiella spp., respectively. Both extracellular products of YD-1 and YD-2 contain protease, amylase and urease. Strain YD-2 is better than strain YD-1 in its ability to degrade sinapine.

Dataset Information

Identification of Rothia bacteria as gluten-degrading natural colonizers of the upper gastro-intestinal tract.

Publications

Identification of Rothia bacteria as gluten-degrading natural colonizers of the upper gastro-intestinal tract.

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