Ghassan Darwish

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.

Celiac disease (CeD) affects about 1% of most world populations. It presents a wide spectrum of clinical manifestations, ranging from minor symptoms to mild or severe malabsorption, and it may be associated with a wide variety of autoimmune diseases. CeD is triggered and maintained by the ingestion of gluten proteins from wheat and related grains. Gluten peptides that resist gastrointestinal digestion are antigenically presented to gluten specific T cells in the intestinal mucosa via HLA-DQ2 or HLA-DQ8, the necessary genetic predisposition for CeD. To date, there is no effective or approved treatment for CeD other than a strict adherence to a gluten-free diet, which is difficult to maintain in professional or social environments. Moreover, many patients with CeD have active disease despite diet adherence due to a high sensitivity to traces of gluten. Therefore, safe pharmacological treatments that complement the gluten-free diet are urgently needed. Oral enzyme therapy, employing gluten-degrading enzymes, is a promising therapeutic approach. A prerequisite is that such enzymes are active under gastro-duodenal conditions, quickly neutralize the T cell activating gluten peptides and are safe for human consumption. Several enzymes including prolyl endopeptidases, cysteine proteases and subtilisins can cleave the human digestion-resistant gluten peptides in vitro and in vivo. Examples are several prolyl endopeptidases from bacterial sources, subtilisins from Rothia bacteria that are natural oral colonizers and synthetic enzymes with optimized gluten-degrading activities. Without exception, these enzymes must cleave the otherwise unusual glutamine and proline-rich domains characteristic of antigenic gluten peptides. Moreover, they should be stable and active in both the acidic environment of the stomach and under near neutral pH in the duodenum. This review focuses on those enzymes that have been characterized and evaluated for the treatment of CeD, discussing their origin and activities, their clinical evaluation and challenges for therapeutic application. Novel developments include strategies like enteric coating and genetic modification to increase enzyme stability in the digestive tract.