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Frontiers in Microbiology 2021Rhamnogalacturonan lyase (RGL) cleaves backbone α-1,4 glycosidic bonds between L-rhamnose and D-galacturonic acid residues in type I rhamnogalacturonan (RG-I) by...
Rhamnogalacturonan lyase (RGL) cleaves backbone α-1,4 glycosidic bonds between L-rhamnose and D-galacturonic acid residues in type I rhamnogalacturonan (RG-I) by β-elimination to generate RG oligosaccharides with various degrees of polymerization. Here, we cloned, expressed, purified and biochemically characterized two RGLs (Bo3128 and Bo4416) in the PL11 family from ATCC 8483. Bo3128 and Bo4416 displayed maximal activity at pH 9.5 and pH 6.5, respectively. Whereas the activity of Bo3128 could be increased 1.5 fold in the presence of 5 mM Ca, Bo4416 required divalent metal ions to show any enzymatic activity. Both of RGLs showed a substrate preference for RG-I compared to other pectin domains. Bo4416 and Bo3128 primarily yielded unsaturated RG oligosaccharides, with Bo3128 also producing them with short side chains, with yields of 32.4 and 62.4%, respectively. Characterization of both RGLs contribute to the preparation of rhamnogalacturonan oligosaccharides, as well as for the analysis of the fine structure of RG-I pectins.
PubMed: 35087500
DOI: 10.3389/fmicb.2021.799875 -
World Journal of Gastroenterology May 2021Gut microbiota dysbiosis is reportedly actively involved in autoimmune diseases such as type 1 diabetes mellitus (T1DM). However, the alterations in the gut microbiota... (Observational Study)
Observational Study
BACKGROUND
Gut microbiota dysbiosis is reportedly actively involved in autoimmune diseases such as type 1 diabetes mellitus (T1DM). However, the alterations in the gut microbiota and their correlation with fasting blood glucose (FBG) in Chinese children with T1DM remain unclear.
AIM
To investigate alterations in the gut microbiota in Chinese children with T1DM and their associations with clinical indicators.
METHODS
Samples from 51 children with T1DM and 47 age-matched and gender-matched healthy controls were obtained, to explore the structural and functional alterations in the fecal microbiota. The V3-V4 regions of the 16S rRNA gene were sequenced on a MiSeq instrument, and the association with FBG were analyzed.
RESULTS
We found that the bacterial diversity was significantly increased in the T1DM-associated fecal microbiota, and changes in the microbial composition were observed at different taxonomic levels. The T1DM-reduced differential taxa, such as ATCC8482, , , and , were negatively correlated with FBG, while the T1DM-enriched taxa, such as , group, , and , were positively correlated with FBG. ATCC8482, , the group, and , either alone or in combination, could be used as noninvasive diagnostic biomarkers to discriminate children with T1DM from healthy controls. In addition, the functional changes in the T1DM-associated fecal microbiota also suggest that these fecal microbes were associated with altered functions and metabolic activities, such as glycan biosynthesis and metabolism and lipid metabolism, which might play vital roles in the pathogenesis and development of T1DM.
CONCLUSION
Our present comprehensive investigation of the T1DM-associated fecal microbiota provides novel insights into the pathogenesis of the disease and sheds light on the diagnosis and treatment of T1DM.
Topics: Bacteroides; Child; China; Clostridiales; Diabetes Mellitus, Type 1; Dysbiosis; Gastrointestinal Microbiome; Humans; RNA, Ribosomal, 16S
PubMed: 34040330
DOI: 10.3748/wjg.v27.i19.2394 -
Journal of Clinical Microbiology Dec 1984Two members of the Bacteroides fragilis group, B. ovatus and B. thetaiotaomicron, are difficult to distinguish by biochemical methods. They are currently identified on...
Two members of the Bacteroides fragilis group, B. ovatus and B. thetaiotaomicron, are difficult to distinguish by biochemical methods. They are currently identified on the basis of their variable ability to ferment salicin. We studied a method of identification for these two species by using cell lysis by bacteriophages. A total of 38 bacteriophages were used to distinguish the two species. Identification by bacteriophages was compared with species identification by prereduced anaerobically sterilized biochemical testing with salicin as the differentiating test. A total of 215 clinical isolates biochemically identified as B. ovatus or B. thetaiotaomicron were tested. A total of 100% of the strains identified as B. ovatus by bacteriophages produced strong acid in salicin (pH less than or equal to 5.4). However, 40% of the strains identified as B. thetaiotaomicron by bacteriophages also produced strong acid in salicin, and an additional 39% produced weak acid (pH 5.5 to 5.7). This study demonstrates that salicin fermentation is an inadequate test for the differentiation of B. ovatus and B. thetaiotaomicron.
Topics: Bacteriophage Typing; Bacteriophages; Bacteroides
PubMed: 6520221
DOI: 10.1128/jcm.20.6.1122-1125.1984 -
Frontiers in Bioengineering and... 2020Human colon microbiota, composed of hundreds of different species, is closely associated with several health conditions. Controlled cultivation and up-to-date...
Human colon microbiota, composed of hundreds of different species, is closely associated with several health conditions. Controlled cultivation and up-to-date analytical methods make possible the systematic evaluation of the underlying mechanisms of complex interactions between the members of microbial consortia. Information on reproducing fecal microbial consortia can be used for various clinical and biotechnological applications. In this study, chemostat and changestat cultures were used to elucidate the effects of the physiologically relevant range of dilution rates on the growth and metabolism of adult fecal microbiota. The dilution rate was kept either at = 0.05 or = 0.2 1/h in chemostat cultures, while gradually changing from 0.05 to 0.2 1/h in the A-stat and from 0.2 to 0.05 1/h in the De-stat. Apple pectin as a substrate was used in the chemostat experiments and apple pectin or birch xylan in the changestat experiments, in the presence of porcine mucin in all cases. The analyses were comprised of HPLC for organic acids, UPLC for amino acids, GC for gas composition, 16S-rDNA sequencing for microbial composition, and growth parameter calculations. It was shown that the abundance of most bacterial taxa was determined by the dilution rate on both substrates. , , and were prevalent within the whole range of dilution rates. and Ruminococcaceae UCG-013 were significantly enriched at = 0.05 1/h, while , Lachnospiraceae unclassified and clearly preferred = 0.2 1/h. In the chemostat cultures, the production of organic acids and gases from pectin was related to the dilution rate. The ratio of acetate, propionate and butyrate was 5:2:1 ( = 0.05 1/h) and 14:2:1 ( = 0.2 1/h). It was shown that the growth rate-related characteristics of the fecal microbiota were concise in both directions between = 0.05 and 0.2 1/h. Reproducible adaptation of the fecal microbiota was shown in the continuous culture with a changing dilution rate: changestat. Consortia cultivation is a promising approach for research purposes and several biotechnological applications, including the production of multi-strain probiotics and fecal transplantation mixtures.
PubMed: 32117913
DOI: 10.3389/fbioe.2020.00024 -
Applied and Environmental Microbiology Oct 2023Humans consume alginate in the form of seaweed, food hydrocolloids, and encapsulations, making the digestion of this mannuronic acid (M) and guluronic acid (G) polymer...
Humans consume alginate in the form of seaweed, food hydrocolloids, and encapsulations, making the digestion of this mannuronic acid (M) and guluronic acid (G) polymer of key interest for human health. To increase knowledge on alginate degradation in the gut, a gene catalog from human feces was mined for potential alginate lyases (ALs). The predicted ALs were present in nine species of the Bacteroidetes phylum, of which two required supplementation of an -acting AL, expected to mimic cross-feeding in the gut. However, only a new isolate grew on alginate. Whole-genome sequencing of this alginate-utilizing isolate suggested that it is a new strain harboring a polysaccharide utilization locus (PUL) containing three ALs of families: PL6, PL17, and PL38. The PL6 degraded polyG to oligosaccharides of DP 1-3, and PL17 released 4,5-unsaturated monouronate from polyM. PL38 degraded both alginates, polyM, polyG, and polyMG, in -mode; hence, it was assumed to deliver oligosaccharide substrates for PL6 and PL17, corresponding well with synergistic action on alginate. PL17 and PL38 crystal structures, determined at 1.61 and 2.11 Å, respectively, showed (α/α)-barrel + anti-parallel β-sheet and (α/α)-barrel folds, distinctive for these PL families. PL17 had a more open active site than the two homologous structures. PL38 was very similar to the structure of an uncharacterized PL38, albeit with a different triad of residues possibly interacting with substrate in the presumed active site tunnel. Altogether, the study provides unique functional and structural insights into alginate-degrading lyases of a PUL in a human gut bacterium.IMPORTANCEHuman ingestion of sustainable biopolymers calls for insight into their utilization in our gut. Seaweed is one such resource with alginate, a major cell wall component, used as a food hydrocolloid and for encapsulation of pharmaceuticals and probiotics. Knowledge is sparse on the molecular basis for alginate utilization in the gut. We identified a new strain from human feces that grew on alginate and encoded three alginate lyases in a gene cluster. PL6 and PL17 show complementary specificity toward guluronate (G) and mannuronate (M) residues, releasing unsaturated oligosaccharides and monouronic acids. PL38 produces oligosaccharides degraded by PL6 and PL17 from both alginates, G-, M-, and MG-substrates. Enzymatic and structural characterization discloses the mode of action and synergistic degradation of alginate by these alginate lyases. Other bacteria were cross-feeding on alginate oligosaccharides produced by an endo-acting alginate lyase. Hence, there is an interdependent community in our guts that can utilize alginate.
Topics: Humans; Alginates; Bacteria; Oligosaccharides; Polysaccharide-Lyases; Substrate Specificity
PubMed: 37791757
DOI: 10.1128/aem.01185-23 -
Scientific Reports Oct 2020Yeast mannan (YM) is an indigestible water-soluble polysaccharide of the yeast cell wall, with a notable prebiotic effect on the intestinal microbiota. We previously...
Yeast mannan (YM) is an indigestible water-soluble polysaccharide of the yeast cell wall, with a notable prebiotic effect on the intestinal microbiota. We previously reported that YM increased Bacteroides thetaiotaomicron abundance in in vitro rat faeces fermentation, concluding that its effects on human colonic microbiota should be investigated. In this study, we show the effects of YM on human colonic microbiota and its metabolites using an in vitro human faeces fermentation system. Bacterial 16S rRNA gene sequence analysis showed that YM administration did not change the microbial diversity or composition. Quantitative real-time PCR analysis revealed that YM administration significantly increased the relative abundance of Bacteroides ovatus and B. thetaiotaomicron. Moreover, a positive correlation was observed between the relative ratio (with or without YM administration) of B. thetaiotaomicron and B. ovatus (r = 0.92), suggesting that these bacteria utilise YM in a coordinated manner. In addition, YM administration increased the production of acetate, propionate, and total short-chain fatty acids. These results demonstrate the potential of YM as a novel prebiotic that selectively increases B. thetaiotaomicron and B. ovatus and improves the intestinal environment. The findings also provide insights that might be useful for the development of novel functional foods.
Topics: Bacteroides; Colon; Functional Food; Gastrointestinal Microbiome; Humans; Mannans; Prebiotics; Species Specificity; Yeasts
PubMed: 33060635
DOI: 10.1038/s41598-020-74379-0 -
Microbiology Spectrum Dec 2021A multigene polysaccharide utilization locus (PUL) encoding enzymes and surface carbohydrate (glycan)-binding proteins (SGBPs) was recently identified in prominent...
A multigene polysaccharide utilization locus (PUL) encoding enzymes and surface carbohydrate (glycan)-binding proteins (SGBPs) was recently identified in prominent members of in the human gut and characterized in Bacteroides ovatus. This PUL-encoded system specifically targets mixed-linkage β1,3-1,4-glucans, a group of diet-derived carbohydrates that promote a healthy microbiota and have potential as prebiotics. The BoSGBP-A protein encoded by the gene is a SusD-like protein that plays a key role in the PUL's specificity and functionality. Here, we perform a detailed analysis of the molecular determinants underlying carbohydrate binding by BoSGBP-A, combining carbohydrate microarray technology with quantitative affinity studies and a high-resolution X-ray crystallography structure of the complex of BoSGBP-A with a β1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward β1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of β1,3 linkages. The interaction is defined by a 41-Å-long extended binding site that accommodates the oligosaccharide in a mode distinct from that of previously described bacterial β1,3-1,4-glucan-binding proteins. In addition to the shape complementarity mediated by CH-π interactions, a complex hydrogen bonding network complemented by a high number of key ordered water molecules establishes additional specific interactions with the oligosaccharide. These support the twisted conformation of the β-glucan backbone imposed by the β1,3 linkages and explain the dependency on the oligosaccharide chain length. We propose that the specificity of the PUL conferred by BoSGBP-A to import long β1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows to outcompete bacteria that lack this PUL for utilization of β1,3-1,4-glucans. With the knowledge of bacterial gene systems encoding proteins that target dietary carbohydrates as a source of nutrients and their importance for human health, major efforts are being made to understand carbohydrate recognition by various commensal bacteria. Here, we describe an integrative strategy that combines carbohydrate microarray technology with structural studies to further elucidate the molecular determinants of carbohydrate recognition by BoSGBP-A, a key protein expressed at the surface of Bacteroides ovatus for utilization of mixed-linkage β1,3-1,4-glucans. We have mapped at high resolution interactions that occur at the binding site of BoSGBP-A and provide evidence for the role of key water-mediated interactions for fine specificity and affinity. Understanding at the molecular level how commensal bacteria, such as prominent members of , can differentially utilize dietary carbohydrates with potential prebiotic activities will shed light on possible ways to modulate the microbiome to promote human health.
Topics: Bacterial Proteins; Bacteroides; Binding Sites; Carrier Proteins; Dietary Carbohydrates; Gastrointestinal Microbiome; Glucans; Humans; Membrane Proteins; Oligosaccharides; Periplasm
PubMed: 34817219
DOI: 10.1128/Spectrum.01826-21 -
Biomolecules May 2021Hypertriglyceridemia-associated acute pancreatitis (HTGAP) is linked with increased severity and morbidity. Intestinal flora plays an important role in the progression... (Observational Study)
Observational Study
Hypertriglyceridemia-associated acute pancreatitis (HTGAP) is linked with increased severity and morbidity. Intestinal flora plays an important role in the progression of acute pancreatitis (AP). However, pathogenetic association between gut microbiota and HTGAP remains unknown. In this study, we enrolled 30 HTGAP patients and 30 patients with AP that is evoked by other causes. The V3-V4 regions of 16S rRNA sequences of the gut microbiota were analyzed. Clinical characteristics, microbial diversity, taxonomic profile, microbiome composition, microbiological phenotype, and functional pathways were compared between the two groups. Our results showed that the HTGAP group had a higher proportion of severe AP (46.7% vs. 20.0%), organ failure (56.7% vs. 30.0%), and a longer hospital stay (18.0 days vs. 6.5 days). HTGAP group also had poorer microbial diversity, higher abundances of and , but lower abundances of , , and as compared with non-HTGAP group. Correlation analysis revealed that gut bacterial taxonomic and functional changes were linked with local and systemic complications, ICU admission, and mortality. This study revealed that alterations of gut microbiota were associated with disease severity and poor prognosis in HTGAP patients, indicating a potential pathophysiological link between gut microbiota and hypertriglyceridemia related acute pancreatitis.
Topics: Adult; Aged; Bacteria; DNA, Bacterial; DNA, Ribosomal; Female; Gastrointestinal Microbiome; Humans; Hypertriglyceridemia; Length of Stay; Male; Middle Aged; Pancreatitis; Phylogeny; Prognosis; Prospective Studies; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Severity of Illness Index
PubMed: 34066441
DOI: 10.3390/biom11050695 -
Surgical Case Reports Jun 2022Appendicectomy is generally a minimally invasive surgery, after which postoperative complications such as acute respiratory distress syndrome (ARDS) are rare. We...
BACKGROUND
Appendicectomy is generally a minimally invasive surgery, after which postoperative complications such as acute respiratory distress syndrome (ARDS) are rare. We describe a case of ARDS due to sepsis caused by Bacteroides ovatus after appendicectomy.
CASE PRESENTATION
A man in his 60 s presented to our hospital with a chief complaint of right lower quadrant abdominal pain. He was diagnosed with acute appendicitis and underwent emergency laparoscopic appendicectomy. Cefmetazole was administered as a perioperative antibacterial drug. Postoperatively, the abdominal findings improved. However, on postoperative day three, bloody sputum and respiratory distress were observed. We performed thoracoabdominal computed tomography (CT) and observed bilateral pleural effusion and mottled frosted glass shadows extending to both lung fields. ARDS was diagnosed. We treated the patient with steroids and sivelestat sodium and switched the antibacterial drug to meropenem. The patient's general condition improved. After the patient was treated, Bacteroides ovatus was isolated from preoperative blood culture, which was resistant to cefmetazole.
CONCLUSIONS
We encountered a case in which ARDS due to sepsis was caused by Bacteroides ovatus after acute appendicectomy. Blood culture to isolate the causative organism and determine its antimicrobial sensitivity after commencement of empiric antibiotics is important even in common diseases, such as acute appendicitis.
PubMed: 35718841
DOI: 10.1186/s40792-022-01475-w -
Frontiers in Oncology 2020Although increasing evidences showed a correlation between cholecystectomy and the prevalence rate of colorectal cancer (CRC), and shed light on gut microbiota in...
Although increasing evidences showed a correlation between cholecystectomy and the prevalence rate of colorectal cancer (CRC), and shed light on gut microbiota in colorectal pathogenesis, only a few studies focused on microbial alterations after cholecystectomy, and its sequent role in carcinogenesis and progression of CRC has not been reported. Thus, we aimed to investigate the bacterial alterations and tried to clarify their clinical significance. 104 subjects were enrolled and divided into post-cholecystectomy patients (PC, = 52) and healthy controls (HC, = 52). To investigate the bacterial role in carcinogenesis, PC patients were further separated into preCA_CRC (patients with precancerous lesions and/or CRC, = 9) and non-CA (patients without precancerous lesions and CRC, = 43) based on the histopathology. Qualified stool samples were collected for 16S rRNA gene sequencing to analyze the bacterial profile. Our data showed noteworthy compositional and abundant alterations of bacterial microbiota in PC patients, characterized as , and remarkably increased; , and significantly decreased. Additionally, the duration after cholecystectomy was the critical factor that affected bacterial composition. Machine learning-based analysis showed a pivotal role of in discriminating PC from HC subjects and involving in the progression of CRC. The bacterial dysbiosis may associate with CRC in PC patients, and the duration after cholecystectomy was highlighted as an important factor. Altered bacterial microbiota was likely to play a pivotal role in related-disease in the long-term follow-up of PC patients.
PubMed: 32903396
DOI: 10.3389/fonc.2020.01418