Commensal Bacterium Rothia aeria Degrades and Detoxifies Gluten via a Highly Effective Subtilisin Enzyme.

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.

Gluten Degrading Enzymes for Treatment of Celiac Disease.

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.

Pharmaceutically modified subtilisins withstand acidic conditions and effectively degrade gluten in vivo.

Detoxification of gluten immunogenic epitopes is a promising strategy for the treatment of celiac disease. Our previous studies have shown that these epitopes can be degraded in vitro by subtilisin enzymes derived from Rothia mucilaginosa, a natural microbial colonizer of the oral cavity. The challenge is that the enzyme is not optimally active under acidic conditions as encountered in the stomach. We therefore aimed to protect and maintain subtilisin-A enzyme activity by exploring two pharmaceutical modification techniques: PEGylation and Polylactic glycolic acid (PLGA) microencapsulation. PEGylation of subtilisin-A (Sub-A) was performed by attaching methoxypolyethylene glycol (mPEG, 5 kDa). The PEGylation protected subtilisin-A from autolysis at neutral pH. The PEGylated Sub-A (Sub-A-mPEG) was further encapsulated by PLGA. The microencapsulated Sub-A-mPEG-PLGA showed significantly increased protection against acid exposure in vitro. In vivo, gluten immunogenic epitopes were decreased by 60% in the stomach of mice fed with chow containing Sub-A-mPEG-PLGA (0.2 mg Sub-A/g chow) (n = 9) compared to 31.9% in mice fed with chow containing unmodified Sub-A (n = 9). These results show that the developed pharmaceutical modification can protect Sub-A from auto-digestion as well as from acid inactivation, thus rendering the enzyme more effective for applications in vivo.

A Role for Salivary Peptides in the Innate Defense Against Enterotoxigenic Escherichia coli.

Background: Diarrheal disease from enterotoxigenic Escherichia coli (ETEC) causes significant worldwide morbidity and mortality in young children residing in endemic countries and is the leading cause of traveler's diarrhea. As ETEC enters the body through the oral cavity and cotransits the digestive tract with salivary components, we hypothesized that the antimicrobial activity of salivary proteins might extend beyond the oropharynx into the proximal digestive tract. Results: Here, we show that the salivary peptide histatin-5 binds colonization factor antigen I pili, thereby blocking adhesion of ETEC to intestinal epithelial cells. Mechanistically, we demonstrate that histatin-5 stiffens the typically dynamic pili, abolishing their ability to function as spring-like shock absorbers, thereby inhibiting colonization within the turbulent vortices of chyme in the gastrointestinal tract. Conclusions: Our data represent the first report of a salivary component exerting specific antimicrobial activity against an enteric pathogen and suggest that histatin-5 and related peptides might be exploited for prophylactic and/or therapeutic uses. Numerous viruses, bacteria, and fungi traverse the oropharynx to cause disease, so there is considerable opportunity for various salivary components to neutralize these pathogens prior to arrival at their target organ. Identification of additional salivary components with unexpectedly broad antimicrobial spectra should be a priority.

The association between coeliac disease and periodontitis: Results from NHANES 2009-2012.

AIM: To investigate whether coeliac disease (CD) was associated with periodontitis among a nationally representative sample of US adults. MATERIALS AND METHODS: The National Health and Nutrition Examination Survey (NHANES) 2009-2012 enrolled 6,661 subjects with full-mouth periodontal examination and serological testing for antitissue transglutaminase (tTg) and antiendomysial (EMA) antibodies. CD was defined as (i) self-reported physician diagnosis while on a gluten-free diet; or (ii) tTg levels >10.0 U/ml and positive EMA results. Positive serology without self-reported diagnosis was defined as undiagnosed CD (UdxCD). Periodontitis was defined according to the CDC/AAP definition. Multivariable linear and logistic models were used to regress the mean probing depth (PD) or attachment loss (AL) outcomes across CD categories (none, diagnosed and undiagnosed). RESULTS: The prevalence of moderate/severe periodontitis and diagnosed/undiagnosed CD was 40% and 0.74%, respectively. Mean AL was lower among those with CD although results were not statistically significant (p = .67). The odds of periodontitis among individuals with diagnosed and undiagnosed CD were: 0.5(0.22, 1.16) and 0.62(0.1, 3.75), respectively. Mean PD levels among those without CD or with diagnosed or undiagnosed CD were 1.49 ± 0.02, 1.36 ± 0.11 and 1.31 ± 0.11 (p = .03). CONCLUSION: CD is associated with modestly lower levels of mean PD but was not associated with mean AL or periodontitis. Larger studies are necessary to enhance precision and strengthen conclusions.

Salivary Gluten Degradation and Oral Microbial Profiles in Healthy Individuals and Celiac Disease Patients.

Celiac disease (CD) is a chronic immune-mediated enteropathy induced by dietary gluten in genetically predisposed individuals. Saliva harbors the second highest bacterial load of the gastrointestinal (GI) tract after the colon. We hypothesized that enzymes produced by oral bacteria may be involved in gluten processing in the intestine and susceptibility to celiac disease. The aim of this study was to investigate salivary enzymatic activities and oral microbial profiles in healthy subjects versus patients with classical and refractory CD. Stimulated whole saliva was collected from patients with CD in remission (n = 21) and refractory CD (RCD; n = 8) and was compared to healthy controls (HC; n = 20) and subjects with functional GI complaints (n = 12). Salivary gluten-degrading activities were monitored with the tripeptide substrate Z-Tyr-Pro-Gln-pNA and the α-gliadin-derived immunogenic 33-mer peptide. The oral microbiome was profiled by 16S rRNA-based MiSeq analysis. Salivary glutenase activities were higher in CD patients compared to controls, both before and after normalization for protein concentration or bacterial load. The oral microbiomes of CD and RCD patients showed significant differences from that of healthy subjects, e.g., higher salivary levels of lactobacilli (P < 0.05), which may partly explain the observed higher gluten-degrading activities. While the pathophysiological link between the oral and gut microbiomes in CD needs further exploration, the presented data suggest that oral microbe-derived enzyme activities are elevated in subjects with CD, which may impact gluten processing and the presentation of immunogenic gluten epitopes to the immune system in the small intestine.IMPORTANCE Ingested gluten proteins are the triggers of intestinal inflammation in celiac disease (CD). Certain immunogenic gluten domains are resistant to intestinal proteases but can be hydrolyzed by oral microbial enzymes. Very little is known about the endogenous proteolytic processing of gluten proteins in the oral cavity. Given that this occurs prior to gluten reaching the small intestine, such enzymes are likely to contribute to the composition of the gluten digest that ultimately reaches the small intestine and causes CD. We demonstrated that endogenous salivary protease activities are incomplete, likely liberating peptides from larger gluten proteins. The potentially responsible microbes were identified. The study included refractory CD patients, who have been studied less with regard to CD pathogenesis.

Identification of food-grade subtilisins as gluten-degrading enzymes to treat celiac disease.

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.

Despite sequence homologies to gluten, salivary proline-rich proteins do not elicit immune responses central to the pathogenesis of celiac disease.

Celiac disease (CD) is an inflammatory disorder triggered by ingested gluten, causing immune-mediated damage to the small-intestinal mucosa. Gluten proteins are strikingly similar in amino acid composition and sequence to proline-rich proteins (PRPs) in human saliva. On the basis of this feature and their shared destination in the gastrointestinal tract, we hypothesized that salivary PRPs may modulate gluten-mediated immune responses in CD. Parotid salivary secretions were collected from CD patients, refractory CD patients, non-CD patients with functional gastrointestinal complaints, and healthy controls. Structural similarities of PRPs with gluten were probed with anti-gliadin antibodies. Immune responses to PRPs were investigated toward CD patient-derived peripheral blood mononuclear cells and in a humanized transgenic HLA-DQ2/DQ8 mouse model for CD. Anti-gliadin antibodies weakly cross-reacted with the abundant salivary amylase but not with PRPs. Likewise, the R5 antibody, recognizing potential antigenic gluten epitopes, showed negligible reactivity to salivary proteins from all groups. Inflammatory responses in peripheral blood mononuclear cells were provoked by gliadins whereas responses to PRPs were similar to control levels, and PRPs did not compete with gliadins in immune stimulation. In vivo, PRP peptides were well tolerated and nonimmunogenic in the transgenic HLA-DQ2/DQ8 mouse model. Collectively, although structurally similar to dietary gluten, salivary PRPs were nonimmunogenic in CD patients and in a transgenic HLA-DQ2/DQ8 mouse model for CD. It is possible that salivary PRPs play a role in tolerance induction to gluten early in life. Deciphering the structural basis for the lack of immunogenicity of salivary PRPs may further our understanding of the toxicity of gluten.

Identification of Pseudolysin (lasB) as an Aciduric Gluten-Degrading Enzyme with High Therapeutic Potential for Celiac Disease.

OBJECTIVES: 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. METHODS: 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. RESULTS: Gliadin-degrading enzyme activities were observed for 43 fecal isolates, displaying activities in the ~150-200 and <50 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. CONCLUSIONS: 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.

Salivary proline-rich proteins and gluten: Do structural similarities suggest a role in celiac disease?

PURPOSE: Gluten proteins, the culprits in celiac disease (CD), show striking similarities in primary structure with human salivary proline-rich proteins (PRPs). Both are enriched in proline and glutamine residues that often occur consecutively in their sequences. We investigated potential differences in the spectrum of salivary PRPs in health and CD. EXPERIMENTAL DESIGN: Stimulated salivary secretions were collected from CD patients, patients with refractory CD, patients with gastrointestinal complaints but no CD, and healthy controls. PRP isoforms/peptides were characterized by anionic and SDS-PAGE, PCR, and LC-ESI-MS. RESULTS: The gene frequencies of the acidic PRP isoforms PIF, Db, Pa, PRP1, and PRP2 did not differ between groups. At the protein level, PRPs peptides showed minor group differences, but these could not differentiate the CD and/or refractory CDs groups from the controls. CONCLUSIONS AND CLINICAL RELEVANCE: This extensive study established that salivary PRPs, despite similarity to gluten proteins, show no apparent correlation with CD and thus will not serve as diagnostic markers for the disease. The structural basis for the tolerance to the gluten-like PRP proteins in CD is worthy of further exploration and may lead to the development of gluten-like analogs lacking immunogenicity that could be used therapeutically.

Effect of Rothia mucilaginosa enzymes on gliadin (gluten) structure, deamidation, and immunogenic epitopes relevant to celiac disease.

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.

Uncovering the molecular networks in periodontitis.

Periodontitis is a complex immune-inflammatory disease that results from a preestablished infection in gingiva, mainly due to Gram-negative bacteria that colonize deeper in gingival sulcus and latter periodontal pocket. Host inflammatory and immune responses have both protective and destructive roles. Although cytokines, prostaglandins, and proteases struggle against microbial burden, these molecules promote connective tissue loss and alveolar bone resorption, leading to several histopathological changes, namely destruction of periodontal ligament, deepening of periodontal pocket, and bone loss, which can converge to attain tooth loss. Despite the efforts of genomics, transcriptomics, proteomics/peptidomics, and metabolomics, there is no available biomarker for periodontitis diagnosis, prognosis, and treatment evaluation, which could assist on the established clinical evaluation. Nevertheless, some genes, transcripts, proteins and metabolites have already shown a different expression in healthy subjects and in patients. Though, so far, 'omics approaches only disclosed the host inflammatory response as a consequence of microbial invasion in periodontitis and the diagnosis in periodontitis still relies on clinical parameters, thus a molecular tool for assessing periodontitis lacks in current dental medicine paradigm. Saliva and gingival crevicular fluid have been attracting researchers due to their diagnostic potential, ease, and noninvasive nature of collection. Each one of these fluids has some advantages and disadvantages that are discussed in this review.

High-resolution high-performance liquid chromatography with electrospray ionization mass spectrometry and tandem mass spectrometry characterization of a new isoform of human salivary acidic proline-rich proteins named Roma-Boston Ser22 (Phos) → Phe variant.

During a survey of human saliva by a top-down reversed-phase high-performance liquid chromatography with electrospray ionization mass spectrometry approach, two proteins eluting at 27.4 and 28.4 min, with average masses of 15 494 ± 1 and 11 142 ± 1 Da, were detected in a subject from Boston. The Δmass value (4352 Da) of the two proteins was similar to the difference in mass values between intact (150 amino acids, [a.a.]) and truncated acidic proline-rich proteins (aPRPs; 106 a.a.) suggesting an a.a. substitution in the first 106 residues resulting in a strong reduction in polarity, since under the same experimental conditions aPRPs eluted at ∼22.5 min (intact) and 23.5 min (truncated forms). Manual inspection of the high-resolution high-performance liquid chromatography with electrospray ionization tandem mass spectra of the truncated isoform showed the replacement of the phosphorylated Ser-22 in PRP-3 with a Phe residue. Inspection of the tandem mass spectra of the intact isoform confirmed the substitution, which is allowed by the code transition TCT→TTT and is in agreement with the dramatic increase in elution time. The isoform was also detected in two other subjects, one from Boston (unrelated to the previous) and one from Rome. For this reason we propose to name this variant PRP-1 (PRP-3) RB (Roma-Boston) Ser22 (phos)→Phe.

Experimental Strategy to Discover Microbes with Gluten-degrading Enzyme Activities.

Gluten proteins contained in the cereals barley, rye and wheat cause an inflammatory disorder called celiac disease in genetically predisposed individuals. Certain immunogenic gluten domains are resistant to degradation by mammalian digestive enzymes. Enzymes with the ability to target such domains are potentially of clinical use. Of particular interest are gluten-degrading enzymes that would be naturally present in the human body, e.g. associated with resident microbial species. This manuscript describes a selective gluten agar approach and four enzyme activity assays, including a gliadin zymogram assay, designed for the selection and discovery of novel gluten-degrading microorganisms from human biological samples. Resident and harmless bacteria and/or their derived enzymes could potentially find novel applications in the treatment of celiac disease, in the form of a probiotic agent or as a dietary enzyme supplement.

Anti-candidal activity of genetically engineered histatin variants with multiple functional domains.

The human bodily defense system includes a wide variety of innate antimicrobial proteins. Histatins are small molecular weight proteins produced by the human salivary glands that exhibit antifungal and antibacterial activities. While evolutionarily old salivary proteins such as mucins and proline-rich proteins contain large regions of tandem repeats, relatively young proteins like histatins do not contain such repeated domains. Anticipating that domain duplications have a functional advantage, we genetically engineered variants of histatin 3 with one, two, three, or four copies of the functional domain by PCR and splice overlap. The resulting proteins, designated reHst3 1-mer, reHist3 2-mer, reHis3 3-mer and reHist3 4-mer, exhibited molecular weights of 4,062, 5,919, 7,777, and 9,634 Da, respectively. The biological activities of these constructs were evaluated in fungicidal assays toward Candida albicans blastoconidia and germinated cells. The antifungal activities per mole of protein increased concomitantly with the number of functional domains present. This increase, however, was higher than could be anticipated from the molar concentration of functional domains present in the constructs. The demonstrated increase in antifungal activity may provide an evolutionary explanation why such domain multiplication is a frequent event in human salivary proteins.

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

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 (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). 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.

The antifungal activity of human parotid secretion is species-specific.

Candida albicans is the major fungal colonizer of the oral cavity and causes oral candidiasis in immunocompromised patient populations. While antifungal proteins in saliva have been identified and the virulence factors of C. albicans have been well studied, little is known about the role saliva plays in the preferential colonization of the oral cavity by C. albicans. We report that the fungistatic activity of human parotid secretion toward six C. albicans strains is considerably lower than towards nine non-C. albicans fungal species (average IC50 values >1000 mg/l and <70 mg/l, respectively). The species-specific activity of parotid secretion suggests that saliva may play a determining role in oral fungal colonization patterns.

Influence of histatin 5 on Candida albicans mitochondrial protein expression assessed by quantitative mass spectrometry.

Individual aspects of the mode of action of histatin 5, a human salivary antifungal protein, have been partially elucidated, but the mechanism likely involves a complex set of events that have not been characterized. Previous evidence points toward histatin-induced alterations in mitochondrial function. The purpose of the present study was to verify and quantify changes in the mitochondrial proteome of Candida albicans treated with histatin 5. Cell killing was determined by plating and differential protein expression levels in the mitochondrial samples were determined by quantitative proteomics approaches employing mTRAQ and ICAT labeling and Western blotting. Relative quantitation ratios were established for 144 different proteins. Up-regulated mitochondrial proteins were predominantly involved in genome maintenance and gene expression, whereas proteins that constitute the respiratory enzyme complexes were mostly down-regulated. The differential expression of ATP synthase gamma chain and elongation factor 1-alpha were confirmed by Western blotting by comparison to levels of cytochrome c which were unchanged upon histatin treatment. The mTRAQ and ICAT proteomics results suggest that key steps in the histatin 5 antifungal mechanism involve a bioenergetic collapse of C. albicans, caused essentially by a decrease in mitochondrial ATP synthesis.

Discovery of a novel and rich source of gluten-degrading microbial enzymes in the oral cavity.

BACKGROUND: Celiac 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 FINDINGS: Proteolytic 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/SIGNIFICANCE: This 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.

Mass spectrometric identification of key proteolytic cleavage sites in statherin affecting mineral homeostasis and bacterial binding domains.

Human salivary statherin inhibits both primary and secondary calcium phosphate precipitation and, upon binding to hydroxyapatite, associates with a variety of oral bacteria. These functions, crucial in the maintenance of tooth enamel integrity, are located in defined regions within the statherin molecule. Proteases associated with saliva, however, cleave statherin effectively, and it is of importance to determine how statherin functional domains are affected by these events. Statherin was isolated from human parotid secretion by zinc precipitation and purified by reversed-phase high performance liquid chromatography (RP-HPLC). To characterize the proteolytic process provoked by oral proteases, statherin was incubated with whole saliva and fragmentation was monitored by RP-HPLC. The early formed peptides were structurally characterized by reversed phase liquid chromatography electrospray-ionization tandem mass spectrometry. Statherin was degraded 3.6× faster in whole saliva than in whole saliva supernatant. The main and primary cleavage sites were located in the N-terminal half of statherin, specifically after Arg(9), Arg(10), and Arg(13); after Phe(14) and Tyr(18); and after Gly(12), Gly(15), Gly(17) and Gly(19) while the C-terminal half of statherin remained intact. Whole saliva protease activities separated the charged N-terminus from the hydrophobic C-terminus, negatively impacting on full length statherin functions comprising enamel lubrication and inhibition of primary calcium phosphate precipitation. Cryptic epitopes for bacterial binding residing in the C-terminal domain were likewise affected. The full characterization of the statherin peptides generated facilitates the elucidation of their novel functional roles in the oral and gastro-intestinal environment.