Modeling Dynamics of Human Gut Microbiota Derived from Gluten Metabolism: Obtention, Maintenance and Characterization of Complex Microbial Communities.

Western diets are rich in gluten-containing products, which are frequently poorly digested. The human large intestine harbors microorganisms able to metabolize undigested gluten fragments that have escaped digestion by human enzymatic activities. The aim of this work was obtaining and culturing complex human gut microbial communities derived from gluten metabolism to model the dynamics of healthy human large intestine microbiota associated with different gluten forms. For this purpose, stool samples from six healthy volunteers were inoculated in media containing predigested gluten or predigested gluten plus non-digested gluten. Passages were carried out every 24 h for 15 days in the same medium and community composition along time was studied via V3-V4 16S rDNA sequencing. Diverse microbial communities were successfully obtained. Moreover, communities were shown to be maintained in culture with stable composition for 14 days. Under non-digested gluten presence, communities were enriched in members of Bacillota, such as Lachnospiraceae, Clostridiaceae, Streptococcaceae, Peptoniphilaceae, Selenomonadaceae or Erysipelotrichaceae, and members of Actinomycetota, such as Bifidobacteriaceae and Eggerthellaceae. Contrarily, communities exposed to digested gluten were enriched in Pseudomonadota. Hence, this study shows a method for culture and stable maintenance of gut communities derived from gluten metabolism. This method enables the analysis of microbial metabolism of gluten in the gut from a community perspective.

The Human Digestive Tract Is Capable of Degrading Gluten from Birth.

The human gastrointestinal system has the capacity to metabolize dietary gluten. The capacity to degrade gliadin-derived peptide is present in humans from birth and increases during the first stages of life (up to 6-12 months of age). Fecal samples from 151 new-born and adult non-celiac disease (NCD) volunteers were collected, and glutenase and glianidase activities were evaluated. The capacity of total fecal proteins to metabolize 33-mer, 19-mer, and 13-mer gliadin peptides was also evaluated by high-performance liquid chromatography (HPLC). Feces from new-borns (meconium) showed glutenase and gliadinase activities, and peptidase activity against all three gliadin peptides. Maximal gluten degradative activity was observed in fecal samples from the youngest volunteers (0-12 months old). After the age of nine months, the gluten digestive capacity of gastrointestinal tract decreases and, from ±8 years old, individuals lose the ability to completely degrade toxic peptides. The gastrointestinal proteases involved in gluten digestion: elastase 2A, elastase 3B, and carboxipeptidase A1 are present from earlier stages of life. The human digestive tract contains the proteins capable of metabolizing gluten from birth, even before starting gluten intake. Humans are born with the ability to digest gluten and to completely degrade the potentially toxic gliadin-derived peptides (33-, 19-, and 13-mer).

Duodenal bacterial proteolytic activity determines sensitivity to dietary antigen through protease-activated receptor-2.

Microbe-host interactions are generally homeostatic, but when dysfunctional, they can incite food sensitivities and chronic diseases. Celiac disease (CeD) is a food sensitivity characterized by a breakdown of oral tolerance to gluten proteins in genetically predisposed individuals, although the underlying mechanisms are incompletely understood. Here we show that duodenal biopsies from patients with active CeD have increased proteolytic activity against gluten substrates that correlates with increased Proteobacteria abundance, including Pseudomonas. Using Pseudomonas aeruginosa producing elastase as a model, we show gluten-independent, PAR-2 mediated upregulation of inflammatory pathways in C57BL/6 mice without villus blunting. In mice expressing CeD risk genes, P. aeruginosa elastase synergizes with gluten to induce more severe inflammation that is associated with moderate villus blunting. These results demonstrate that proteases expressed by opportunistic pathogens impact host immune responses that are relevant to the development of food sensitivities, independently of the trigger antigen.

Lactobacilli Degrade Wheat Amylase Trypsin Inhibitors to Reduce Intestinal Dysfunction Induced by Immunogenic Wheat Proteins.

BACKGROUND & AIMS: Wheat-related disorders, a spectrum of conditions induced by the ingestion of gluten-containing cereals, have been increasing in prevalence. Patients with celiac disease have gluten-specific immune responses, but the contribution of non-gluten proteins to symptoms in patients with celiac disease or other wheat-related disorders is controversial. METHODS: C57BL/6 (control), Myd88-/-, Ticam1-/-, and Il15-/- mice were placed on diets that lacked wheat or gluten, with or without wheat amylase trypsin inhibitors (ATIs), for 1 week. Small intestine tissues were collected and intestinal intraepithelial lymphocytes (IELs) were measured; we also investigated gut permeability and intestinal transit. Control mice fed ATIs for 1 week were gavaged daily with Lactobacillus strains that had high or low ATI-degrading capacity. Nonobese diabetic/DQ8 mice were sensitized to gluten and fed an ATI diet, a gluten-containing diet or a diet with ATIs and gluten for 2 weeks. Mice were also treated with Lactobacillus strains that had high or low ATI-degrading capacity. Intestinal tissues were collected and IELs, gene expression, gut permeability and intestinal microbiota profiles were measured. RESULTS: In intestinal tissues from control mice, ATIs induced an innate immune response by activation of Toll-like receptor 4 signaling to MD2 and CD14, and caused barrier dysfunction in the absence of mucosal damage. Administration of ATIs to gluten-sensitized mice expressing HLA-DQ8 increased intestinal inflammation in response to gluten in the diet. We found ATIs to be degraded by Lactobacillus, which reduced the inflammatory effects of ATIs. CONCLUSIONS: ATIs mediate wheat-induced intestinal dysfunction in wild-type mice and exacerbate inflammation to gluten in susceptible mice. Microbiome-modulating strategies, such as administration of bacteria with ATI-degrading capacity, may be effective in patients with wheat-sensitive disorders.

The human digestive tract has proteases capable of gluten hydrolysis.

OBJECTIVE: To identify, purify, and characterize the proteins responsible for glutenase activity in the feces of healthy subjects and patients with celiac disease (CD). METHODS: Sixteen subjects were included in this study; 8 were healthy with no known food intolerances, and 8 were treated CD patients on a gluten-free diet. Fecal samples were homogenized, and precipitated proteins were purified by chromatography. Glutenase activity was evaluated by bioassays, zymography, and high-performance liquid chromatography with immunogenic 33-mer, 19-mer, and 13-mer gliadin peptides. RESULTS: The gastrointestinal elastase 3B (CEL3B), elastase 2A (CEL2A), and carboxypeptidase A1 (CBPA1) enzymes degraded human gluten. These proteins fully hydrolyzed 13-mer and 19-mer gliadin peptides that trigger immune-mediated enteropathy in individuals genetically predisposed to CD and partially digested a 33-mer. Feces from patients with CD showed more glutenase activity than feces from individuals without CD (171-466% higher). Peptidase activity against the gliadin peptides also increased in patients with CD. CONCLUSION: The digestive tracts of patients with CD and healthy subjects have enzymatic machinery needed for gluten degradation. Patients with CD showed more gluten hydrolysis than did healthy individuals, although, in both cases, a fraction of 33-mer peptide remained intact. Gliadin peptides derived from gastrointestinal digestion, especially the 33-mer, can potentially be used by commensal microbiota from both CD-positive and CD-negative individuals, and differences in bacterial hydrolysis can modify its immunogenic capacity.

Gluten-degrading bacteria are present in the human small intestine of healthy volunteers and celiac patients.

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'.

Duodenal Bacteria From Patients With Celiac Disease and Healthy Subjects Distinctly Affect Gluten Breakdown and Immunogenicity.

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.

Diversity of the cultivable human gut microbiome involved in gluten metabolism: isolation of microorganisms with potential interest for coeliac disease.

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.

Coeliac disease screening in first-degree relatives on the basis of biopsy and genetic risk.

BACKGROUND: Serological markers of coeliac disease (CD) lack diagnostic value to identify mild histopathological lesions mainly in adults at risk of CD. AIMS: The aim of this study was to evaluate the usefulness of human leukocyte antigen (HLA)-DQ2/8 genotyping, followed by duodenal biopsy for the detection of CD in adult first-degree relatives (FDRs) of patients with CD. MATERIALS AND METHODS: Ninety-two adult DQ2/8 positive FDRs were consecutively included. A duodenal biopsy was offered irrespective of the serology result or associated symptoms. The clinical features, associated autoimmune diseases and biochemical parameters were recorded. RESULTS: Sixty-seven FDRs (mean age 34 years) underwent a duodenal biopsy. Histopathological alterations were found in 32 (48%) and showed the following stages: 12 Marsh I (18%), one Marsh II (1.5%), four Marsh IIIA (6%), five Marsh IIIB (7.5%) and 10 Marsh IIIC (15%). Positive serological markers were present in 17/67 (25%), with only one showing Marsh I and the remainder presenting some degree of duodenal atrophy (Marsh III). In addition, 33/67 (54%) had gastrointestinal symptoms, with dyspepsia being the most prevalent. The distribution of symptoms, anaemia and autoimmune disease was independent of the duodenal histopathological stage. Serology-based screening would diagnose 50% of the cases showing any degree of CD spectrum and miss 6% of the cases with mucosal atrophy. CONCLUSION: Adult FDRs of patients with CD can benefit from a screening strategy on the basis of HLA-DQ genotyping, followed by a duodenal biopsy. Gastrointestinal symptoms and lymphocytic enteritis are common findings that may benefit from a gluten-free diet.

Differences in faecal bacteria populations and faecal bacteria metabolism in healthy adults and celiac disease patients.

UNLABELLED: Differences in the intestinal microbiota between children and adults with celiac disease (CD) have been reported; however, differences between healthy adults and adults with CD have not been clearly demonstrated. The aim of this study was to evaluate the differences in the intestinal microbiota between adults with CD and healthy individuals. Microbial communities in faecal samples were evaluated by PCR-denaturing gradient gel electrophoresis (DGGE) and gas-liquid chromatography of short chain fatty acids (SCFAs). The study group included 10 untreated CD patients, 11 treated CD patients and 11 healthy adults (in normal gluten diet and in GFD). UPGMA clustered the dominant microbial communities of healthy individuals together and separated them from the dominant microbial communities of the untreated CD patients. Most of the dominant microbial communities of the treated CD patients clustered together with those of healthy adults. The treated CD patients showed a reduction in the diversity of Lactobacillus and Bifidobacterium species. The presence of Bifidobacterium bifidum was significantly higher in untreated CD patients than healthy adults. There was a significant difference between untreated CD patients and healthy adults, as well as between treated CD patients and healthy adults, regarding acetic acid, propionic acid, butyric acid, and total SCFAs. IN CONCLUSION: healthy adults have a different faecal microbiota from that of untreated CD patients. A portion of the treated CD patients displayed a restored "normal" microbiota. The treated CD patients significantly reduce the Lactobacillus and Bifidobacterium diversity. Healthy adults have a different faecal SCFAs content from that of CD patients.

Monitoring of gluten-free diet compliance in celiac patients by assessment of gliadin 33-mer equivalent epitopes in feces.

BACKGROUND: Certain immunotoxic peptides from gluten are resistant to gastrointestinal digestion and can interact with celiac-patient factors to trigger an immunologic response. A gluten-free diet (GFD) is the only effective treatment for celiac disease (CD), and its compliance should be monitored to avoid cumulative damage. However, practical methods to monitor diet compliance and to detect the origin of an outbreak of celiac clinical symptoms are not available. OBJECTIVE: We assessed the capacity to determine the gluten ingestion and monitor GFD compliance in celiac patients by the detection of gluten and gliadin 33-mer equivalent peptidic epitopes (33EPs) in human feces. DESIGN: Fecal samples were obtained from healthy subjects, celiac patients, and subjects with other intestinal pathologies with different diet conditions. Gluten and 33EPs were analyzed by using immunochromatography and competitive ELISA with a highly sensitive antigliadin 33-mer monoclonal antibody. RESULTS: The resistance of a significant part of 33EPs to gastrointestinal digestion was shown in vitro and in vivo. We were able to detect gluten peptides in feces of healthy individuals after consumption of a normal gluten-containing diet, after consumption of a GFD combined with controlled ingestion of a fixed amount of gluten, and after ingestion of <100 mg gluten/d. These methods also allowed us to detect GFD infringement in CD patients. CONCLUSIONS: Gluten-derived peptides could be sensitively detected in human feces in positive correlation with the amount of gluten intake. These techniques may serve to show GFD compliance or infringement and be used in clinical research in strategies to eliminate gluten immunotoxic peptides during digestion. This trial was registered at clinicaltrials.gov as NCT01478867.

A gluten metabolism study in healthy individuals shows the presence of faecal glutenasic activity.

PURPOSE: To study the gluten metabolism in healthy individuals and its effect over the intestinal microbial activity. METHODS: The faeces of eleven healthy subjects were analysed under 4 diet regimens: their normal gluten diet, a strict gluten-free diet (GFD), a GFD with a supplemental intake of 9 g gluten/day and a GFD with a supplemental intake of 30 g gluten/day. Gluten content, faecal tryptic activity (FTA), short-chain fatty acids (SCFAs) and faecal glutenasic activity (FGA) were analysed in faecal samples. RESULTS: Faecal gluten contents, FTA, SCFAs and FGA varied significantly with different levels of gluten intake in the diet. When high gluten doses (30 g/day) were administered in the diet, SCFA concentrations (70.5 mmoles/kg faeces) were significantly different from those from the GFD period (33.8 mmoles/kg faeces) of the experiment. However, the FTA showed significant differences between the GFD (34 units) and the normal gluten-containing diet (60 units) and also between the GFD and the GFD + 30 g of gluten/day (67 units). When gluten was present in the diet, gluten was detected in the faeces, showing that at least a portion of the gluten ingested is eliminated in the large intestine, providing a substrate for intestinal microbial proteases. We have also shown the presence of faecal glutenasic activity that increased proportionally with the gluten intake in the diet, showing an enzymatic activity of 993 units in DSG, 2,063 units in DSG + 9 g and 6,090 units in DSG + 30 g. CONCLUSIONS: The activity of the intestinal microbiota is modified by gluten intake in the diet. The incorporation of gluten in the diet increases the activity of a gluten proteolytic activity in the faeces.

Differences of small intestinal bacteria populations in adults and children with/without celiac disease: effect of age, gluten diet, and disease.

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.

A preparative method for the purification of isopenicillin N from genetically blocked Acremonium chrysogenum strain TD189: studies on the degradation kinetics and storage conditions.

A protocol for preparative isopenicillin N (IPN) purification, a highly interesting and hitherto unavailable intermediate of the penicillin and cephalosporin biosynthetic pathway due to its high unstability, is described. Culture broths of Acremonium chrysogenum TD189, a strain blocked in cephalosporin biosynthesis that accumulates this metabolite, were treated with acetone and filtered though charcoal and a hydrophobic resin in a single step as tandem columns. The cleared broth was then lyophilized and passed though a Sephadex G-25 column. The last step was the purification to homogeneity of IPN in a semipreparative HPLC equipment and, optionally, a desalting step by Sephadex G-10 column. Once purified, a complete analysis of the stability of the compound and the conditions for its long-term storage was carried out. Our results suggest a first-order model for IPN decomposition for all the pH and temperature analyzed. IPN is more stable at neutral pH, and once lyophilized, can be stored under vacuum and -75 ° C with a half-life of 770 days.

Dynamics of non-conventional intraepithelial lymphocytes-NK, NKT, and γδ T-in celiac disease: relationship with age, diet, and histopathology.

BACKGROUND: Intraepithelial lymphocytes (IEL) are a heterogeneous population of lymphocytes raised in celiac disease (CD), whose role in CD pathogenesis remains to be defined. AIMS: To investigate how the age of diagnosis, diet, and the severity of the histological lesions are related to the changes observed in unconventional IEL populations. METHODS: Prospective analysis of 101 confirmed celiac patients from a single center, including 66 at diagnosis (45 children, 21 adults) and 112 non-celiac controls (12 children, 100 adults). IEL from duodenal biopsies were studied by six-color flow cytometry. The results were analyzed in relationship with age, diet (gluten intake), and histopathology (Marsh type). RESULTS: In comparison with respective age controls, both children and adult patients showed duodenal intraepithelial lymphocytosis with significant differences in every single non-conventional IEL population: CD3+ TCR γδ, NK (CD3-, CD16+, CD56+), NKT (CD3+, CD161+, CD56+), and iNKT (CD3+ Vα24) (P < 0.001 for all). Gluten intake was not only directly associated with severe atrophy, but also with decreased percentages of NK (P = 0.02), NKT (P = 0.003), and iNKT (P = 0.03). Changes in iNKT and γδ IEL were more marked in celiac children compared with celiac adults (P = 0.02 and 0.01, respectively). In contrast, increased CD3+ TCR γδ were diet- and Marsh grade-independent. CONCLUSIONS: The typical phenotypical profile of intraepithelial lymphocytosis in untreated pediatric and adult celiacs consists of increased CD3+ TCR γδ populations with decreased NK, NKT, and iNKT cells. NK, NKT, and iNKT IEL, but not γδ IEL, are dynamic populations associated with diet, age, and histopathology.

Duodenal biopsy may be avoided when high transglutaminase antibody titers are present.

AIM: To evaluate the predictive value of tissue transglutaminase (tTG) antibodies for villous atrophy in adult and pediatric populations to determine if duodenal biopsy can be avoided. METHODS: A total of 324 patients with celiac disease (CD; 97 children and 227 adults) were recruited prospectively at two tertiary centers. Human IgA class anti-tTG antibody measurement and upper gastrointestinal endoscopy were performed at diagnosis. A second biopsy was performed in 40 asymptomatic adults on a gluten-free diet (GFD) and with normal tTG levels. RESULTS: Adults showed less severe histopathology (26% vs 63%, P < 0.0001) and lower tTG antibody titers than children. Levels of tTG antibody correlated with Marsh type in both populations (r = 0.661, P < 0.0001). Multiple logistic regression revealed that only tTG antibody was an independent predictor for Marsh type 3 lesions, but clinical presentation type and age were not. A cut-off point of 30 U tTG antibody yielded the highest area under the receiver operating characteristic curve (0.854). Based on the predictive value of this cut-off point, up to 95% of children and 53% of adults would be correctly diagnosed without biopsy. Despite GFDs and decreased tTG antibody levels, 25% of the adults did not recover from villous atrophy during the second year after diagnosis. CONCLUSION: Strongly positive tTG antibody titers might be sufficient for CD diagnosis in children. However, duodenal biopsy cannot be avoided in adults because disease presentation and monitoring are different.

Transcriptional upregulation of four genes of the lysine biosynthetic pathway by homocitrate accumulation in Penicillium chrysogenum: homocitrate as a sensor of lysine-pathway distress.

The lysine biosynthetic pathway has to supply large amounts of alpha-aminoadipic acid for penicillin biosynthesis in Penicillium chrysogenum. In this study, we have characterized the P. chrysogenum L2 mutant, a lysine auxotroph that shows highly increased expression of several lysine biosynthesis genes (lys1, lys2, lys3, lys7). The L2 mutant was found to be deficient in homoaconitase activity since it was complemented by the Aspergillus nidulans lysF gene. We have cloned a gene (named lys3) that complements the L2 mutation by transformation with a P. chrysogenum genomic library, constructed in an autonomous replicating plasmid. The lys3-encoded protein showed high identity to homoaconitases. In addition, we cloned the mutant lys3 allele from the L2 strain that showed a G(1534) to A(1534) point mutation resulting in a Gly(495) to Asp(495) substitution. This mutation is located in a highly conserved region adjacent to two of the three cysteine residues that act as ligands to bind the iron-sulfur cluster required for homoaconitase activity. The L2 mutant accumulates homocitrate. Deletion of the lys1 gene (homocitrate synthase) in the L2 strain prevented homocitrate accumulation and reverted expression levels of the four lysine biosynthesis genes tested to those of the parental prototrophic strain. Homocitrate accumulation seems to act as a sensor of lysine-pathway distress, triggering overexpression of four of the lysine biosynthesis genes.

Age-related clinical, serological, and histopathological features of celiac disease.

BACKGROUND: Celiac disease (CD) is a common disorder in children and adults. However, limited data are available when comparing differences between both populations. AIMS: To prospectively evaluate and compare the clinical and histological features present at diagnosis in a cohort of celiac children and adults. METHODS: Consecutive new cases diagnosed between 2000 and 2006 were prospectively included (66 children and 54 adults). The clinical spectrum was categorized in two groups: (a) typical (malabsorption, chronic diarrhea, or failure to thrive) and (b) oligosymptomatic (abdominal pain, anemia, hypertransaminasemia, or screening in risk groups or in relatives). The histological results were divided into mild (i.e., Marsh I, II, and IIIA) and severe (i.e., Marsh IIIB, IIIC). In all cases, the human antitissue transglutaminase IgA antibodies (TTGA) were determined. RESULTS: Overall, a female/male ratio (2.6:1) was observed. This ratio was significantly higher in adults (5.7:1) than in children (1.6:1) (P= 0.009). Typical symptoms were present in 62.5% children versus 31% adults (P= 0.01). The average time to diagnosis after the appearance of symptoms was 7.6 months for children and 90 months for adults (P < 0.001). TTGA levels were higher in children and correlated with age (P < 0.001) and with the degree of villous atrophy (P < 0.001). Histological analysis revealed a marked atrophy in 86% children versus 52% adults (P < 0.001). The degree of villous atrophy was inversely correlated with age (P < 0.001). Classic symptoms were also associated with more severe villous atrophy. CONCLUSIONS: At initial diagnosis, CD shows age-related differences, which consist of more evident clinical and histological features in children. Furthermore, IgA TTGA levels correlate both with the degree of villous atrophy and with the patient's age.

Deacetylcephalosporin C production in Penicillium chrysogenum by expression of the isopenicillin N epimerization, ring expansion, and acetylation genes.

Penicillium chrysogenum npe6 lacking isopenicillin N acyltransferase activity is an excellent host for production of different beta-lactam antibiotics. We have constructed P. chrysogenum strains expressing cefD1, cefD2, cefEF, and cefG genes cloned from Acremonium chrysogenum. Northern analysis revealed that the four genes were expressed in P. chrysogenum. The recombinant strains TA64, TA71, and TA98 secreted significant amounts of deacetylcephalosporin C, but cephalosporin C was not detected in the culture broths. DAC-acetyltransferase activity was found in all transformants containing the cefG gene. HPLC analysis of cell extracts showed that transformant TA64, TA71, and TA98 accumulate intracellularly deacetylcephalosporin C and, in the last strain (TA98), also cephalosporin C. Mass spectra analysis confirmed that transformant TA98 synthesize true deacetylcephalosporin C and cephalosporin C. Even when accumulated intracellularly, cephalosporin C was not found in the culture broth.

Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N-acyltransferase.

A gene, phl, encoding a phenylacetyl-CoA ligase was cloned from a phage library of Penicillium chrysogenum AS-P-78. The presence of five introns in the phl gene was confirmed by reverse transcriptase-PCR. The phl gene encoded an aryl-CoA ligase closely related to Arabidopsis thaliana 4-coumaroyl-CoA ligase. The Phl protein contained most of the amino acids defining the aryl-CoA (4-coumaroyl-CoA) ligase substrate-specificity code and differed from acetyl-CoA ligase and other acyl-CoA ligases. The phl gene was not linked to the penicillin gene cluster. Amplification of phl in an autonomous replicating plasmid led to an 8-fold increase in phenylacetyl-CoA ligase activity and a 35% increase in penicillin production. Transformants containing the amplified phl gene were resistant to high concentrations of phenylacetic acid (more than 2.5 g/l). Disruption of the phl gene resulted in a 40% decrease in penicillin production and a similar reduction of phenylacetyl-CoA ligase activity. The disrupted mutants were highly susceptible to phenylacetic acid. Complementation of the disrupted mutants with the phl gene restored normal levels of penicillin production and resistance to phenylacetic acid. The phenylacetyl-CoA ligase encoded by the phl gene is therefore involved in penicillin production, although a second aryl-CoA ligase appears to contribute partially to phenylacetic acid activation. The Phl protein lacks a peptide-carrier-protein domain and behaves as an aryl-capping enzyme that activates phenylacetic acid and transfers it to the isopenicillin N acyltransferase. The Phl protein contains the peroxisome-targeting sequence that is also present in the isopenicillin N acyltransferase. The peroxisomal co-localization of these two proteins indicates that the last two enzymes of the penicillin pathway form a peroxisomal functional complex.