RESUMO
Gluten is the only known environmental factor that triggers celiac disease. Several studies have described an imbalance between the intestinal microbiota of different individuals based on diagnoses. Moreover, recent studies have suggested that human bacteria may play an important role in gluten hydrolysis. However, there has been no research focusing on the small intestine. This study aimed to characterize the adult small intestine microbiota possibly implicated in gluten hydrolysis. Duodenal biopsies from different diagnosed individuals were cultured in a gluten-containing medium, and the grown microbiota was analyzed by culture dependent/independent methods. Results showed that gluten-degrading bacteria can be found in the human small intestine. Indeed, 114 bacterial strains belonging to 32 species were isolated; 85 strains were able to grow in a medium containing gluten as the sole nitrogen source, 31 strains showed extracellular proteolytic activity against gluten protein and 27 strains showed peptidolytic activity towards the 33 mer peptide, an immunogenic peptide for celiac disease patients. We found that there are no differences based on the diagnosis, but each individual has its own population of gluten-hydrolyzing bacteria. These bacteria or their gluten-degrading enzymes could help to improve the quality of life of celiac disease patients'.
Assuntos
Bactérias/metabolismo , Doença Celíaca/microbiologia , Duodeno/microbiologia , Microbioma Gastrointestinal/fisiologia , Glutens/metabolismo , Intestino Delgado/microbiologia , Adulto , Idoso , Bactérias/efeitos dos fármacos , Doença Celíaca/fisiopatologia , Duodeno/efeitos dos fármacos , Duodeno/patologia , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Glutens/farmacologia , Voluntários Saudáveis , Humanos , Hidrólise , Masculino , Pessoa de Meia-Idade , Peptídeos/química , Peptídeos/imunologia , Peptídeos/farmacologiaRESUMO
BACKGROUND & AIMS: Partially degraded gluten peptides from cereals trigger celiac disease (CD), an autoimmune enteropathy occurring in genetically susceptible persons. Susceptibility genes are necessary but not sufficient to induce CD, and additional environmental factors related to unfavorable alterations in the microbiota have been proposed. We investigated gluten metabolism by opportunistic pathogens and commensal duodenal bacteria and characterized the capacity of the produced peptides to activate gluten-specific T-cells from CD patients. METHODS: We colonized germ-free C57BL/6 mice with bacteria isolated from the small intestine of CD patients or healthy controls, selected for their in vitro gluten-degrading capacity. After gluten gavage, gliadin amount and proteolytic activities were measured in intestinal contents. Peptides produced by bacteria used in mouse colonizations from the immunogenic 33-mer gluten peptide were characterized by liquid chromatography tandem mass spectrometry and their immunogenic potential was evaluated using peripheral blood mononuclear cells from celiac patients after receiving a 3-day gluten challenge. RESULTS: Bacterial colonizations produced distinct gluten-degradation patterns in the mouse small intestine. Pseudomonas aeruginosa, an opportunistic pathogen from CD patients, exhibited elastase activity and produced peptides that better translocated the mouse intestinal barrier. P aeruginosa-modified gluten peptides activated gluten-specific T-cells from CD patients. In contrast, Lactobacillus spp. from the duodenum of non-CD controls degraded gluten peptides produced by human and P aeruginosa proteases, reducing their immunogenicity. CONCLUSIONS: Small intestinal bacteria exhibit distinct gluten metabolic patterns in vivo, increasing or reducing gluten peptide immunogenicity. This microbe-gluten-host interaction may modulate autoimmune risk in genetically susceptible persons and may underlie the reported association of dysbiosis and CD.
Assuntos
Doença Celíaca/imunologia , Doença Celíaca/microbiologia , Duodeno/microbiologia , Glutens/imunologia , Glutens/metabolismo , Fenômenos Imunogenéticos , Animais , Translocação Bacteriana , Estudos de Casos e Controles , Doença Celíaca/genética , Humanos , Lactobacillus/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Pseudomonas aeruginosa/fisiologia , Linfócitos T/imunologiaRESUMO
Coeliac disease (CD) is an immune-mediated enteropathy resulting from exposure to gluten in genetically predisposed individuals. Gluten proteins are partially digested by human proteases generating immunogenic peptides that cause inflammation in patients carrying HLA-DQ2 and DQ8 genes. Although intestinal dysbiosis has been associated with patients with CD, bacterial metabolism of gluten has not been studied in depth thus far. The aim of this study was to analyse the metabolic activity of intestinal bacteria associated with gluten intake in healthy individuals, CD patients and first-degree relatives of CD patients. Faecal samples belonging to twenty-two untreated CD patients, twenty treated CD patients, sixteen healthy volunteers on normal diet, eleven healthy volunteers on gluten-free diet (GFD), seventy-one relatives of CD patients on normal diet and sixty-nine relatives on GFD were tested for several proteolytic activities, cultivable bacteria involved in gluten metabolism, SCFA and the amount of gluten in faeces. We detected faecal peptidasic activity against the gluten-derived peptide 33-mer. CD patients showed differences in faecal glutenasic activity (FGA), faecal tryptic activity (FTA), SCFA and faecal gluten content with respect to healthy volunteers. Alterations in specific bacterial groups metabolising gluten such as Clostridium or Lactobacillus were reported in CD patients. Relatives showed similar parameters to CD patients (SCFA) and healthy volunteers (FTA and FGA). Our data support the fact that commensal microbial activity is an important factor in the metabolism of gluten proteins and that this activity is altered in CD patients.
Assuntos
Doença Celíaca/dietoterapia , Glutens/administração & dosagem , Glutens/metabolismo , Ácido Acético/metabolismo , Actinobacteria/isolamento & purificação , Actinobacteria/metabolismo , Adolescente , Adulto , Alelos , Ácido Butírico/metabolismo , Caproatos/metabolismo , Dieta Livre de Glúten , Fezes/química , Firmicutes/isolamento & purificação , Firmicutes/metabolismo , Antígenos HLA-DQ/metabolismo , Voluntários Saudáveis , Humanos , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Pessoa de Meia-Idade , Ácidos Pentanoicos/metabolismo , Propionatos/metabolismo , Proteobactérias/isolamento & purificação , Proteobactérias/metabolismo , Adulto JovemRESUMO
Gluten, a common component in the human diet, is capable of triggering coeliac disease pathogenesis in genetically predisposed individuals. Although the function of human digestive proteases in gluten proteins is quite well known, the role of intestinal microbiota in the metabolism of proteins is frequently underestimated. The aim of this study was the isolation and characterisation of the human gut bacteria involved in the metabolism of gluten proteins. Twenty-two human faecal samples were cultured with gluten as the principal nitrogen source, and 144 strains belonging to 35 bacterial species that may be involved in gluten metabolism in the human gut were isolated. Interestingly, 94 strains were able to metabolise gluten, 61 strains showed an extracellular proteolytic activity against gluten proteins, and several strains showed a peptidasic activity towards the 33-mer peptide, an immunogenic peptide in patients with coeliac disease. Most of the strains were classified within the phyla Firmicutes and Actinobacteria, mainly from the genera Lactobacillus, Streptococcus, Staphylococcus, Clostridium and Bifidobacterium. In conclusion, the human intestine exhibits a large variety of bacteria capable of utilising gluten proteins and peptides as nutrients. These bacteria could have an important role in gluten metabolism and could offer promising new treatment modalities for coeliac disease.
Assuntos
Bactérias/classificação , Bactérias/metabolismo , Glutens/metabolismo , Intestinos/microbiologia , Microbiota , Actinobacteria/metabolismo , Adulto , Bactérias/enzimologia , Bactérias/isolamento & purificação , Biodiversidade , Doença Celíaca/microbiologia , Meios de Cultura , Fezes/microbiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeo Hidrolases/metabolismo , Adulto JovemRESUMO
BACKGROUND: Scientific evidence has revealed microecological changes in the intestinal tract of celiac infants. The objective of this work is the study of bacterial differences in the upper small intestine in both adults (healthy, untreated celiac disease [CD], and CD treated with a gluten-free diet) and children (healthy and untreated CD). METHODS: Intestinal bacterial communities were identified by 16S rRNA gene sequencing of DNA extracted from duodenal biopsies. RESULTS: Analysis of the sequences from adults and children showed that this niche was colonized by bacteria affiliated mainly with three phyla: Firmicutes, Proteobacteria, and Bacteroidetes. In total, 89 different genera were identified in adults and 46 in children. Bacterial richness was significantly lower in the children than in the adults. A global principal component analysis of the bacterial communities of both healthy and untreated CD patient groups (including both children and adults) revealed a strong effect of age in principal component 1--clustering all adults and children separately--and a possible effect of the disease in adults with untreated patients clustering separately. CONCLUSIONS: There are bacterial differences in the upper small intestine between untreated children CD patients and untreated CD adults due to age. There are bacterial differences in the upper small bacteria microbiota between treated and untreated CD adults due to treatment with a gluten-free diet.