Commensal Bacterium Rothia aeria Degrades and Detoxifies Gluten via a Highly Effective Subtilisin Enzyme.
Commensal Bacterium Rothia aeria Degrades and Detoxifies Gluten via a Highly Effective Subtilisin Enzyme.
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DOI：
10.3390/nu12123724
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发表时间：
2020-12-02
期刊：
影响因子：
5.9
通讯作者：
Helmerhorst EJ
中科院分区：
文献类型：
--
作者：
Wei G;Darwish G;Oppenheim FG;Schuppan D;Helmerhorst EJ
乳糜泻的特征是由小麦、大麦和黑麦中含有的麸质引发的慢性免疫介导的小肠炎症。 Rothia aeria 是一种口腔和上消化道的革兰氏阳性天然定植菌，能够在体外降解麸质并使其解毒。本研究的目的是评估活的和死的R. aeria 细菌的体外麸质降解活性，并分离R. aeria 麸质降解酶。方法：禁食过夜后，给 Balb/c 小鼠喂食 1 g 颗粒的标准饲料，其中含有 50% 小麦（和 4% 麦醇溶蛋白），含有或不含有 1.6 × 107 活 R. aeria 细菌。 2小时后，通过SDS-PAGE和免疫印迹评估胃内容物中的体内麸质降解，并使用R5麦醇溶蛋白ELISA测定评估免疫原性表位中和。通过 C18 色谱分离细菌细胞匀浆中的蛋白质，然后进行麦醇溶蛋白酶谱分析和切除条带的质谱分析，完成 R. aeria 酶的分离和鉴定。结果：与对照小鼠中消化的麸质相比，用 R. aeria 喂养的小鼠中，麦醇溶蛋白和免疫原性表位分别减少了 20% 和 33%。在乙醇中杀死 R. aeria 细菌并没有消除与细菌相关的酶活性。麸质降解酶被鉴定为 BAV86562.1，此处被鉴定为枯草杆菌蛋白酶家族的成员。结论：这项研究表明，R. aeria 可以作为活菌或死菌作为第一种用于体内麸质消化的益生菌，或者使用纯化的 R. aeria 酶，造福于麸质不耐受的患者群体。
Celiac disease is characterized by a chronic immune-mediated inflammation of the small intestine, triggered by gluten contained in wheat, barley, and rye. Rothia aeria, a gram-positive natural colonizer of the oral cavity and the upper digestive tract is able to degrade and detoxify gluten in vitro. The objective of this study was to assess gluten-degrading activity of live and dead R. aeria bacteria in vitro, and to isolate the R. aeria gluten-degrading enzyme. Methods: After an overnight fast, Balb/c mouse were fed a 1 g pellet of standard chow containing 50% wheat (and 4% gliadin) with or without 1.6 × 107 live R. aeria bacteria. After 2 h, in vivo gluten degradation was assessed in gastric contents by SDS-PAGE and immunoblotting, and immunogenic epitope neutralization was assessed with the R5 gliadin ELISA assay. R. aeria enzyme isolation and identification was accomplished by separating proteins in the bacterial cell homogenate by C18 chromatography followed by gliadin zymography and mass spectrometric analysis of excised bands. Results: In mice fed with R. aeria, gliadins and immunogenic epitopes were reduced by 20% and 33%, respectively, as compared to gluten digested in control mice. Killing of R. aeria bacteria in ethanol did not abolish enzyme activity associated with the bacteria. The gluten degrading enzyme was identified as BAV86562.1, here identified as a member of the subtilisin family. Conclusion: This study shows the potential of R. aeria to be used as a first probiotic for gluten digestion in vivo, either as live or dead bacteria, or, alternatively, for using the purified R. aeria enzyme, to benefit the gluten-intolerant patient population.
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