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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 &times, 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.

Gluten are proline- and glutamine-rich proteins present in wheat, barley, and rye and contain the immunogenic sequences that drive celiac disease (CD). Rothia mucilaginosa, an oral microbial colonizer, can cleave these gluten epitopes. The aim was to isolate and identify the enzymes and evaluate their potential as novel enzyme therapeutics for CD. The membrane-associated R. mucilaginosa proteins were extracted and separated by DEAE chromatography. Enzyme activities were monitored with paranitroanilide-derivatized and fluorescence resonance energy transfer (FRET) peptide substrates, and by gliadin zymography. Epitope elimination was determined in R5 and G12 ELISAs. The gliadin-degrading Rothia enzymes were identified by LC-ESI-MS/MS as hypothetical proteins ROTMU0001_0241 (C6R5V9_9MICC), ROTMU0001_0243 (C6R5W1_9MICC), and ROTMU0001_240 (C6R5V8_9MICC). A search with the Basic Local Alignment Search Tool revealed that these are subtilisin-like serine proteases belonging to the peptidase S8 family. Alignment of the major Rothia subtilisins indicated that all contain the catalytic triad with Asp (D), His (H), and Ser (S) in the D-H-S order. They cleaved succinyl-Ala-Ala-Pro-Phe-paranitroanilide, a substrate for subtilisin with Pro in the P2 position, as in Tyr-Pro-Gln and Leu-Pro-Tyr in gluten, which are also cleaved. Consistently, FRET substrates of gliadin immunogenic epitopes comprising Xaa-Pro-Xaa motives were rapidly hydrolyzed. The Rothia subtilisins and two subtilisins from Bacillus licheniformis, subtilisin A and the food-grade Nattokinase, efficiently degraded the immunogenic gliadin-derived 33-mer peptide and the immunodominant epitopes recognized by the R5 and G12 antibodies. This study identified Rothia and food-grade Bacillus subtilisins as promising new candidates for enzyme therapeutics in CD.

Immunogenic gluten proteins implicated in celiac disease (CD) largely resist degradation by human digestive enzymes. Here we pursued the isolation of gluten-degrading organisms from human feces, aiming at bacteria that would digest gluten under acidic conditions, as prevails in the stomach.Bacteria with gluten-degrading activities were isolated using selective gluten agar plates at pH 4.0 and 7.0. Proteins in concentrated bacterial cell sonicates were separated by diethylaminoethanol chromatography. Enzyme activity was monitored with chromogenic substrates and gliadin zymography. Elimination of major immunogenic gluten epitopes was studied with R5 and G12 enzyme-linked immunosorbent assays.Gliadin-degrading enzyme activities were observed for 43 fecal isolates, displaying activities in the ~150-200 and50 kDa regions. The active strains were identified as Pseudomonas aeruginosa. Gliadin degradation in gel was observed from pH 2.0 to 7.0. Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis identified the enzyme as pseudolysin (lasB), a metalloprotease belonging to the thermolysin (M4) family proteases. Its electrophoretic mobility in SDS-polyacrylamide gel electrophoresis and gliadin zymogram gels was similar to that of a commercial lasB preparation, with tendency of oligomerization. Pseudolysin eliminated epitopes recognized by the R5 antibody, while those detected by the G12 antibody remained intact, despite destruction of the nearby major T-cell epitope QPQLPY.Pseudolysin was identified as an enzyme cleaving gluten effectively at extremely low as well as near-neutral pH values. The potential to degrade gluten during gastric transport opens possibilities for its application as a novel therapeutic agent for the treatment of CD.

Rothia mucilaginosa, a natural microbial inhabitant of the oral cavity, cleaves gluten (gliadin) proteins at regions that are resistant to degradation by mammalian enzymes. The aim of this study was to investigate to what extent the R. mucilaginosa cell-associated enzymes abolish gliadin immunogenic properties. Degradation of total gliadins and highly immunogenic gliadin 33-mer or 26-mer peptides was monitored by SDS-PAGE and RP-HPLC, and fragments were sequenced by liquid chromatography and electrospray ionization tandem mass spectrometer (LC-ESI-MS/MS). Peptide deamidation by tissue transglutaminase (TG2), a critical step in rendering the fragments more immunogenic, was assessed by TG2-mediated cross-linking to monodansyl cadaverine (MDC), and by a +1-Da mass difference by LC-ESI-MS. Survival of potential immunogenic gliadin epitopes was determined by use of the R5 antibody-based ELISA. R. mucilaginosa-associated enzymes cleaved gliadins, 33-mer and 26-mer peptides into smaller fragments. TG2-mediated cross-linking showed a perfect inverse relationship with intact 33-mer and 26-mer peptide levels, and major degradation fragments showed a slow rate of MDC cross-linking of 6.18 ± 2.20 AU/min compared with 97.75 ± 10.72 and 84.17 ± 3.25 AU/min for the intact 33-mer and 26-mer, respectively, which was confirmed by reduced TG2-mediated deamidation of the fragments in mass spectrometry. Incubation of gliadins with Rothia cells reduced R5 antibody binding by 20, 82, and 97% after 30 min, 2 h, and 5 h, respectively, which was paralleled by reduced reactivity of enzyme-treated 33-mer and 26-mer peptides in the R5 competitive ELISA. Our broad complementary approach to validate gluten degrading activities qualifies R. mucilaginosa-associated enzymes as promising tools to neutralize T cell immunogenic properties for treatment of celiac disease.

To investigate the susceptibility of selected bacteria as well as Streptococcus mutans biofilm to MUC7 peptides and compare the activities with those of other known antimicrobial peptides.MIC and MBC of peptides for S. mutans, Escherichia coli, Streptococcus gordonii, Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Pseudomonas aeruginosa were determined using the microdilution method. For S. mutans, the effects of the peptides on the kinetics of growth inhibition, time-killing, and on biofilm formation and reduction were also examined. For biofilm studies, polystyrene microtitre plates, Calgary Biofilm Device (CBD) and hydroxylapatite (HA) discs, along with Crystal Violet and Alamar Blue dyes, and/or EM observations, were employed.S. mutans was the most susceptible to all peptides tested (MICs of 9.4-25.0 microM), compared with the other species (MICs of 3.1-100 microM). MUC7 peptides (except MUC7-12-mer-L4) exerted 2-fold higher activity against S. mutans than Hsn5-12-mer and magainin-II, and faster killing of S. mutans than Hsn5-12-mer. The MUC7 peptides also had an effect on S. mutans biofilm. On the polystyrene plates, they suppressed the biofilm formation, with MBIC(50) of 6.25-12.5 microM, and reduced the 1 day developed biofilm in a batch culture, with MBRC(50) of 25-50 microM. On the CBD pegs, the viabilities of the biofilm were suppressed by95% in the presence of MUC7 peptides at 4x MIC (50 microM). One day developed biofilm viabilities were inhibited by 49-75%. On HA, the formation of biofilm (as observed by EM) was also considerably reduced.MUC7 peptides present somewhat preferential antimicrobial activity against S. mutans. They also have an effect on in vitro formation and reduction of the preformed S. mutans biofilm.

Objectives MUC7 12-mer (RKSYKCLHKRCR), a cationic peptide derived from human salivary MUC7 mucin, exhibits potent in vitro antifungal activity, as determined by killing assays in phosphate buffer. In this study we examined the MUC7 12-mer antifungal activity alone or in combination with other antifungal agents in LYM medium (modified RPMI 1640). Methods Antifungal activities of MUC7 12-mer and other compounds against several fungal strains were first measured by MIC and minimum fungicidal concentration (MFC) tests using broth microdilution assay. The viability of Candida albicans and Cryptococcus neoformans were also determined by killing assays and time kinetics of peptide-mediated killing. Antifungal activities of MUC7 12-mer in combination with other compounds [histatin-5 (Hsn5) 12-mer: AKRHHGYKRKFH, amphotericin B or miconazole] against C. albicans and C. neoformans were determined by chequerboard assays and confirmed by killing assays. Toxicities of individual compounds were determined by haemolytic assays. Results MICs and MFCs of MUC7 12-mer ranged from 3.13 to 6.25 mg/L for most of the strains tested, and were, in most cases, comparable to those of amphotericin B and miconazole (0.78-6.25 mg/L). ED(50) values of MUC7 12-mer and Hsn5 12-mer were 7.1 and 7.4 micro M (or 11.2 and 11.6 mg/L), respectively, for C. albicans; and 1.2 and 1.1 micro M (or 1.9 and 1.7 mg/L), respectively, for C. neoformans. The killing of C. albicans and C. neoformans was achieved after 30 and 10 min exposure to the peptides, respectively. Combinations of MUC7 12-mer and Hsn5 12-mer, and of MUC7 12-mer and miconazole have a synergic antifungal effect on C. neoformans, with a fractional inhibitory concentration index (FICI) of 0.37 and 0.25, respectively; and a slightly lower than synergic effect on C. albicans, with a FICI of 0.63 and 0.56, respectively. In addition, using human erythrocytes, the two salivary peptides showed low levels of haemolytic activity. Conclusions This study suggests that MUC7 12-mer and Hsn5 12-mer peptides may be suitable candidates for use in combination antifungal therapy.

Aims: To determine the expediency of a microtitre assay system for establishing, quantifying and antimicrobial testing of two representative oral pathogens. Methods and Results:Streptococcus mutans and Porphyromonas gingivalis were used. Morphological characteristics of the attached population were evaluated. Biofilm growth was evaluated spectrophotometrically (undisturbed and 1 N NaOH dissipated biofilm). The minimum concentration of chlorhexidine gluconate that inhibited biofilm growth was determined. Growth of the biofilms was successfully monitored by direct optical density measurements or those re-suspended in 1 N NaOH. The latter was necessary when glucans were present in Strep. mutans biofilms. The minimum concentration of chlorhexidine gluconate that inhibited biofilm growth was 1·25 µg ml−1 for both species. The attached bacteria exhibited common biofilm characteristics. Significance and Impact of the Study: The assay system developed was especially useful for monitoring the growth of adherent Strep. mutans in the presence of glucans, which is particularly significant for the study of anti-plaque chemicals.

Background 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. Methodology/Principal Findings 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. Strains with high activity towards gluten were typed as Rothia mucilaginosa and Rothia aeria. Gliadins (250 µg/ml) added to Rothia cell suspensions (OD620 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). Conclusion/Significance 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.

Coeliac disease is characterized by intestinal inflammation caused by gluten, proteins which are widely contained in the Western diet. Mammalian digestive enzymes are only partly capable of cleaving gluten, and fragments remain that induce toxic responses in patients with coeliac disease. We found that the oral microbiome is a novel and rich source of gluten-degrading organisms. Here we report on the isolation and characterization of the cultivable resident oral microbes that are capable of cleaving gluten, with special emphasis on the immunogenic domains. Bacteria were obtained by a selective culturing approach and enzyme activities were characterized by: (i) hydrolysis of paranitroanilide-derivatized gliadin-derived tripeptide substrates; (ii) gliadin degradation in-gel (gliadin zymography); (iii) gliadin degradation in solution; (iv) proteolysis of the highly immunogenic α-gliadin-derived 33-mer peptide. For selected strains pH activity profiles were determined. The culturing strategy yielded 87 aerobic and 63 anaerobic strains. Species with activity in at least two of the four assays were typed as: Rothia mucilaginosa HOT-681, Rothia aeria HOT-188, Actinomyces odontolyticus HOT-701, Streptococcus mitis HOT-677, Streptococcus sp. HOT-071, Neisseria mucosa HOT-682 and Capnocytophaga sputigena HOT-775, with Rothia species being active in all four assays. Cleavage specificities and substrate preferences differed among the strains identified. The approximate molecular weights of the enzymes were ~75 kD (Rothia spp.), ~60 kD (A. odontolyticus) and ~150 kD (Streptococcus spp.). In conclusion, this study identified new gluten-degrading microorganisms in the upper gastrointestinal tract. A cocktail of the most active oral bacteria, or their isolated enzymes, may offer promising new treatment modalities for coeliac disease.