Mucin Degradation Research Articles

A genetic system for Akkermansia muciniphila reveals a role for mucin foraging in gut colonization and host sterol biosynthesis gene expression.

Akkermansia muciniphila, a mucophilic member of the gut microbiota, protects its host against metabolic disorders. Because it is genetically intractable, the mechanisms underlying mucin metabolism, gut colonization and its impact on host physiology are not well understood. Here we developed and applied transposon mutagenesis to identify genes important for intestinal colonization and for the use of mucin. An analysis of transposon mutants indicated that de novo biosynthesis of amino acids was required for A. muciniphila growth on mucin medium and that many glycoside hydrolases are redundant. We observed that mucin degradation products accumulate in internal compartments within bacteria in a process that requires genes encoding pili and a periplasmic protein complex, which we term mucin utilization locus (MUL) genes. We determined that MUL genes were required for intestinal colonization in mice but only when competing with other microbes. In germ-free mice, MUL genes were required for A. muciniphila to repress genes important for cholesterol biosynthesis in the colon. Our genetic system for A. muciniphila provides an important tool with which to uncover molecular links between the metabolism of mucins, regulation of lipid homeostasis and potential probiotic activities.

Mucin glycans and their degradation by gut microbiota.

The human intestinal tract is inhabited by a tremendous number of microorganisms, which are collectively termed "the gut microbiota". The intestinal epithelium is covered with a dense layer of mucus that prevents penetration of the gut microbiota into underlying tissues of the host. Recent studies have shown that the maturation and function of the mucus layer are strongly influenced by the gut microbiota, and alteration in the structure and function of the gut microbiota is implicated in several diseases. Because the intestinal mucus layer is at a crucial interface between microbes and their host, its breakdown leads to gut bacterial invasion that can eventually cause inflammation and infection. The mucus is composed of mucin, which is rich in glycans, and the various structures of the complex carbohydrates of mucins can select for distinct mucosa-associated bacteria that are able to bind mucin glycans, and sometimes degrade them as a nutrient source. Mucin glycans are diverse molecules, and thus mucin glycan degradation is a complex process that requires a broad range of glycan-degrading enzymes. Because of the increased recognition of the role of mucus-associated microbes in human health, how commensal bacteria degrade and use host mucin glycans has become of increased interest. This review provides an overview of the relationships between the mucin glycan of the host and gut commensal bacteria, with a focus on mucin degradation.

Genome-wide multi-omics analysis reveals the nutrient-dependent metabolic features of mucin-degrading gut bacteria

ABSTRACT The prevalence and occurrence of mucin-degrading (MD) bacteria, such as Akkermansia muciniphila and Ruminococcus gnavus, is highly associated with human health and disease states. However, MD bacterial physiology and metabolism remain elusive. Here, we assessed functional modules of mucin catabolism, through a comprehensive bioinformatics-aided functional annotation, to identify 54 A. muciniphila genes and 296 R. gnavus genes. The reconstructed core metabolic pathways coincided with the growth kinetics and fermentation profiles of A. muciniphila and R. gnavus grown in the presence of mucin and its constituents. Genome-wide multi-omics analyses validated the nutrient-dependent fermentation profiles of the MD bacteria and identified their distinct mucolytic enzymes. The distinct metabolic features of the two MD bacteria induced differences in the metabolite receptor levels and inflammatory signals of the host immune cells. In addition, in vivo experiments and community-scale metabolic modeling demonstrated that different dietary intakes influenced the abundance of MD bacteria, their metabolic fluxes, and gut barrier integrity. Thus, this study provides insights into how diet-induced metabolic differences in MD bacteria determine their distinct physiological roles in the host immune response and the gut ecosystem.

In Silico and In Vitro Investigation of the Distribution and Expression of Key Genes in the Fucose Operon of Escherichia coli

Many gut bacteria degrade polysaccharides, providing nutritional advantages to their hosts. Fucose, a mucin degradation product, was suggested as a communication molecule between the resident microbiota and external pathogens. However, the precise role and variants of the fucose utilization pathway remain to be elucidated. Here, we computationally and experimentally investigated the fucose utilization operon of E. coli. While the operon is conserved among E. coli genomes, a variant pathway, in which an ABC transporter system replaces the fucose permease gene (fucP), was computationally identified in 50 out of 1058 genomes. Comparative genomics and subsystems analysis results were confirmed by polymerase chain reaction-based screening of 40 human E. coli isolates, which indicated the conservation of fucP in 92.5% of the isolates (vs. 7.5% of its suggested alternative, yjfF). The in silico predictions were confirmed by in vitro experiments comparing the growth of E. coli strains K12, BL21, and isogenic fucose-utilization K12 mutants. Additionally, fucP and fucI transcripts were quantified in E. coli K12 and BL21, after in silico analysis of their expression in 483 public transcriptomes. In conclusion, E. coli utilizes fucose by two pathway variants, with measurable transcriptional differences. Future studies will explore this variation’s impact on signaling and virulence.

Mucin utilization by gut microbiota: recent advances on characterization of key enzymes.

The gut microbiota interacts with the host through the mucus that covers and protects the gastrointestinal epithelium. The main component of the mucus are mucins, glycoproteins decorated with hundreds of different O-glycans. Some microbiota members can utilize mucin O-glycans as carbons source. To degrade these host glycans the bacteria express multiple carbohydrate-active enzymes (CAZymes) such as glycoside hydrolases, sulfatases and esterases which are active on specific linkages. The studies of these enzymes in an in vivo context have started to reveal their importance in mucin utilization and gut colonization. It is now clear that bacteria evolved multiple specific CAZymes to overcome the diversity of linkages found in O-glycans. Additionally, changes in mucin degradation by gut microbiota have been associated with diseases like obesity, diabetes, irritable bowel disease and colorectal cancer. Thereby understanding how CAZymes from different bacteria work to degrade mucins is of critical importance to develop new treatments and diagnostics for these increasingly prevalent health problems. This mini-review covers the recent advances in biochemical characterization of mucin O-glycan-degrading CAZymes and how they are connected to human health.

Differences in gut bacterial community composition between modern and slower-growing broiler breeder lines: Implications of growth selection on microbiome composition.

In the last century broiler chicken lines have undergone an extensive breeding regime aimed primarily at growth and high meat yield. It is not known if breeding has also resulted in a change to the broiler breeder's associated gut microbiota. Here we compared the gut microbiota of 37-week-old commercial Cobb breeding dams with dams from a broiler Legacy line which has not undergone selection since 1986. The dams from both lines were kept together in the same shed under the same management protocol from day of hatch to avoid additional confounders. We chose this age to allow significant bacterial exchange, thus avoiding exposure dependent artifacts and so that we could compare dams at the same developmental state of adulthood and peak laying performance. Significant differences in the composition of the cecum bacterial communities were found. Bacteria of the genus Akkermansia, implicated in mucin degradation and associated with host metabolic health, accounted for 4.98% ± 5.04% of the Cobb cecum community, but were mostly absent from the ceca of the Legacy line dams. Inversely, Legacy dams had higher levels of Clostridiales, Lactobacillales and Aeromonadales. These results show that breeding has resulted in a change in the gut microbiota composition, likely by changing the physiological conditions in the mucosa. It remains unclear if changes in gut microbiota composition are a part of the mechanism affecting growth or are a secondary result of other physiological changes accelerating growth. Therefore, the identification of these changes opens the door to further targeted research.

Characterization and Protective Properties of Lactic Acid Bacteria Intended to Be Used in Probiotic Preparation for Honeybees (Apis mellifera L.)-An In Vitro Study.

Lactic acid bacteria (LAB) are widely used probiotics and offer promising prospects for increasing the viability of honeybees. Thus, the probiotic potential of 10 LAB strains was determined, which in our previous studies showed the most potent protective abilities. In the current study, we investigated various properties of probiotic candidates. The tested LAB strains varied in susceptibility to tested antibiotics. Isolates showed high viability in sugar syrups and gastrointestinal conditions. None of the LAB strains exhibited β-hemolytic activity, mutual antagonism, mucin degradation, hydrogen peroxide production capacity, or bile salt hydrolase (BSH) activity. Additionally, the cytotoxicity of LAB cell-free supernatants (CFS) was assessed, as well as the effect of CFS from P. pentosaceus 14/1 on the cytotoxicity of coumaphos and chlorpyrifos in the Caco-2 cell line. The viability of Caco-2 cells reached up to 89.81% in the presence of the highest concentration of CFS. Furthermore, LAB metabolites decreased the cytotoxicity of insecticides (up to 19.32%) thus demonstrating cytoprotective activity. All tested LAB strains produced lactic, acetic, and malonic acids. This research allowed the selection of the most effective LAB strains, in terms of probiosis, for future in vivo studies aimed at developing an ecologically protective biopreparation for honeybees.

Excessive consumption of mucin by over-colonized Akkermansia muciniphila promotes intestinal barrier damage during malignant intestinal environment.

Gut microbiota disorders damage the intestinal barrier, which causes intestinal disease. Thus, we screened the microbiota with significant changes using an in situ malignant colorectal cancer (CRC) model. Among the colonies with increased abundance, Akkermansia muciniphila (A. muciniphila) is known for its characteristic of breaking down mucin, which is an essential component of the intestinal barrier. The role of A. muciniphila remains controversial. To investigate the effect of excess A. muciniphila on the intestinal barrier, we established an over-colonized A. muciniphila mouse model by administering a live bacterial suspension after disrupting the original gut microbiome with antibiotics. The results showed that over-colonization of A. muciniphila decreased intestinal mucin content. The mRNA and protein expression levels of tight junction proteins also decreased significantly in the over-colonized A. muciniphila mouse model. Our findings reveal that excess colonization by A. muciniphila breaks the dynamic balance between mucin secretion and degradation, reduces the thickness of the intestinal mucus layer, and damages the intestinal barrier, which would eventually aggravate the development of colitis and CRC. These results will raise awareness about the safety of A. muciniphila serving as a probiotic.

Characterization of a highly conserved MUC5B-degrading protease, MdpL, from Limosilactobacillus fermentum.

MUC5B is the predominant glycoprotein in saliva and is instrumental in the establishment and maintenance of multi-species eubiotic biofilms in the oral cavity. Investigations of the aciduric Lactobacillaceae family, and its role in biofilms emphasizes the diversity across different genera of the proteolytic systems involved in the nutritional utilization of mucins. We have characterized a protease from Limosilactobacillus fermentum, MdpL (Mucin degrading protease from Limosilactobacillus) with a high protein backbone similarity with commensals that exploit mucins for attachment and nutrition. MdpL was shown to be associated with the bacterial cell surface, in close proximity to MUC5B, which was sequentially degraded into low molecular weight fragments. Mapping the substrate preference revealed multiple hydrolytic sites of proteins with a high O-glycan occurrence, although hydrolysis was not dependent on the presence of O-glycans. However, since proteolysis of immunoglobulins was absent, and general protease activity was low, a preference for glycoproteins similar to MUC5B in terms of glycosylation and structure is suggested. MdpL preferentially hydrolyzed C-terminally located hydrophobic residues in peptides larger than 20 amino acids, which hinted at a limited sequence preference. To secure proper enzyme folding and optimal conditions for activity, L. fermentum incorporates a complex system that establishes a reducing environment. The importance of overall reducing conditions was confirmed by the activity boosting effect of the added reducing agents L-cysteine and DTT. High activity was retained in low to neutral pH 5.5-7.0, but the enzyme was completely inhibited in the presence of Zn2+. Here we have characterized a highly conserved mucin degrading protease from L. fermentum. MdpL, that together with the recently discovered O-glycanase and O-glycoprotease enzyme groups, increases our understanding of mucin degradation and complex biofilm dynamics.

Food for thought-The link between Clostridioides difficile metabolism and pathogenesis.

Clostridioides difficile (C. difficile) is an opportunistic pathogen that leads to antibiotic-associated diarrhoea and is a leading cause of morbidity and mortality worldwide. Antibiotic usage is the main risk factor leading to C. difficile infection (CDI), as a dysbiotic gut environment allows colonisation and eventual pathology manifested by toxin production. Although colonisation resistance is mediated by the action of secondary bile acids inhibiting vegetative outgrowth, nutrient competition also plays a role in preventing CDI as the gut microbiota compete for nutrient niches inhibiting C. difficile growth. C. difficile is able to metabolise carbon dioxide, the amino acids proline, hydroxyproline, and ornithine, the cell membrane constituent ethanolamine, and the carbohydrates trehalose, cellobiose, sorbitol, and mucin degradation products as carbon and energy sources through multiple pathways. Zinc sequestration by the host response mediates metabolic adaptation of C. difficile by perhaps signalling an inflamed gut allowing it to acquire abundant nutrients. Persistence within the gut environment is also mediated by the by-products of metabolism through the production of p-cresol, which inhibit gut commensal species growth promoting dysbiosis. This review aims to explore and describe the various metabolic pathways of C. difficile, which facilitate its survival and pathogenesis within the colonised host gut.

The impact of gut microbiota changes on the intestinal mucus barrier in burned mice: a study using 16S rRNA and metagenomic sequencing.

The gut microbiota is a complex ecosystem that plays a critical role in human health and disease. However, the relationship between gut microbiota and intestinal damage caused by burns is not well understood. The intestinal mucus layer is crucial for maintaining intestinal homeostasis and providing a physiological barrier against bacterial invasion. This study aims to investigate the impact of gut microbiota on the synthesis and degradation of intestinal mucus after burns and explore potential therapeutic targets for burn injury. A modified histopathological grading system was employed to investigate the effects of burn injury on colon tissue and the intestinal mucus barrier in mice. Subsequently, 16S ribosomal RNA sequencing was used to analyze alterations in the gut microbiota at days 1-10 post-burn. Based on this, metagenomic sequencing was conducted on samples collected at days 1, 5 and 10 to investigate changes in mucus-related microbiota and explore potential underlying mechanisms. Our findings showed that the mucus barrier was disrupted and that bacterial translocation occurred on day 3 following burn injury in mice. Moreover, the gut microbiota in mice was significantly disrupted from days 1 to 3 following burn injury, but gradually recovered to normal as the disease progressed. Specifically, there was a marked increase in the abundance of symbiotic and pathogenic bacteria associated with mucin degradation on day 1 after burns, but the abundance returned to normal on day 5. Conversely, the abundance of probiotic bacteria associated with mucin synthesis changed in the opposite direction. Further analysis revealed that after a burn injury, bacteria capable of degrading mucus may utilize glycoside hydrolases, flagella and internalins to break down the mucus layer, while bacteria that synthesize mucus may help restore the mucus layer by promoting the production of short-chain fatty acids. Burn injury leads to disruption of colonic mucus barrier and dysbiosis of gut microbiota. Some commensal and pathogenic bacteria may participate in mucin degradation via glycoside hydrolases, flagella, internalins, etc. Probiotics may provide short-chain fatty acids (particularly butyrate) as an energy source for stressed intestinal epithelial cells, promote mucin synthesis and accelerate repair of mucus layer.

In vitro safety assessment of electrohydrodynamically encapsulated Lactiplantibacillus plantarum CRD7 and Lacticaseibacillus rhamnosus CRD11 for probiotics use

The current study aimed to validate the safety of electrohydrodynamically encapsulated Lactiplantibacillus plantarum CRD7 and Lacticaseibacillus rhamnosus CRD11 in accordance with guidelines of FAO/WHO and ICMR/DBT. In vitro assays such as mucin degradation, hemolysis of blood cells, antimicrobial susceptibility pattern, possession of virulence factors, biogenic amine, and ammonia production were assessed. In results, the cross-streak and co-culture techniques revealed that CRD7 and CRD11 were compatible in vitro. Upon visual inspection through scanning electron and fluorescence microscopy, the integrity of bacterial cell membrane was confirmed even after the encapsulation process. CRD7 and CRD11 were non-hemolytic and showed negative responses to gelatinase, urease, and DNase activities. Non-mucinolytic activity of CRD7 and CRD11 was verified by measuring cell growth rate (p < 0.05) in different modified media followed by SDS-PAGE. High-performance liquid chromatography analysis revealed that both the strains did not produce biogenic amines (putrescine, cadaverine, histamine, and tyramine). Neither of the Lactobacillus strains produced ammonia after growing in BHI broth for 5 days at 37 °C. L-lactate production was highest (p < 0.05) in CRD11 (8.83 g/L), followed by CRD7 (8.16 g/L), whereas the lowest (p < 0.05) D-lactate was registered for encapsulated CRD11 (0.33 g/L) and CRD7 (0.49 g/L). The antibiogram profile determined through minimum inhibitory concentration showed that CRD7 and CRD11 were sensitive to key antibiotics suggested by EFSA except for gentamycin and kanamycin. PCR data on virulence genes demonstrated that both strains were safe for probiotic use. Moreover, CRD7 and CRD11 strains caused insignificant (p > 0.05) changes in the cell viability of Caco-2 cells as estimated by MTT (98.94–99.50%) and NR uptake (95.42–97.03%) assays and showed sensitivity to human serum. According to the results of these evaluated attributes, it is concluded that L. plantarum CRD7 and L. rhamnosus CRD11 are safe, non-toxic to human epithelial cells, and thus may be potentially suitable for various food/feed applications.

Genome-centric investigation of the potential succession pattern in gut microbiota and altered functions under high-protein diet

Excessive intake of protein has been considered as a factor leading to intestinal microecological disorder, but why and how intestinal microbes change under the high-protein diet (HPD) have yet to be fully elucidated. Here, we performed 16S rRNA gene amplicon sequencing and metagenomic sequencing on contents of cecum, colon and feces from two groups of mice with standard diet (SD) and HPD. And then the microbial alteration of composition and function were deeply analyzed by using several statistical models and bioinformatic methods. Among the three niches, the microbes in the colon are observed to show the most significant change with lower alpha-diversity and higher beta-diversity after HPD. In addition, this alteration of microbial structure may be related to the replacement process and co-occurring community. Most species are also enriched or impoverished in the colon during this process. After analyzing the functional genes related to protein and carbohydrate hydrolysis in different niches, we found that the carbon source provided by poor carbohydrates compared with the rich protein may be the potential factor driving the enrichment of mucin degraders and desulphaters in the colon under HPD. Therefore, our study provided a new insight to understand the underlying mechanism of HPD affecting intestinal health from the perspective of microbial functional ecology.

In Vitro Probiotic Characterization and Safety Assessment of Lactic Acid Bacteria Isolated from Raw Milk of Japanese-Saanen Goat (Capra hircus)

Two novel probiotic strains of lactic acid bacteria were successfully isolated from the raw milk of dairy Japanese-Saanen goats. Selection criteria for positive candidates were grown on de Man-Rogosa-Sharpe or M17 selective medium at 30, 35, or 42 °C anaerobically, and characterized based on Gram reaction, catalase test, and tolerance to low pH and bile salts. Among the 101 isolated positive candidates, two strains, YM2-1 and YM2-3, were selected and identified as Lacticaseibacillus rhamnosus using 16S rDNA sequence similarity. Culture supernatants of the two strains exhibited antipathogenic activity against Salmonella enterica subsp. enterica serovar. Typhimurium, Shigella sonnei, methicillin-resistant Staphylococcus aureus, methicillin-sensitive Staphylococcus aureus, Listeria monocytogenes, and Escherichia coli O157. The antipathogenic activities were retained to some extent after neutralization, indicating the presence of antipathogenic substances other than organic acids in the culture supernatants. The two strains were sensitive with coincidental minimum inhibition concentrations (indicated in the parentheses hereafter) to ampicillin (0.25 μg/mL), chloramphenicol (4 μg/mL), gentamycin (4 μg/mL), kanamycin (64 μg/mL), streptomycin (16 μg/mL), and tetracycline (4 μg/mL). Furthermore, the two strains were resistant to clindamycin (16 μg/mL) and erythromycin (4 μg/mL). In addition, both YM2-1 and YM2-3 strains showed less unfavorable activities, including bile acid bioconversion, carcinogenic-related enzymes, mucin degradation, plasminogen activation, and hemolysis, than the detection limits of in vitro evaluation methods used in this study. In summary, L. rhamnosus YM2-1 and YM2-3 are highly safe and promising probiotic strains applicable in the dairy industry, and were first isolated from the raw milk of Japanese-Saanen goats.

Priestia flexa KS1: A new bacterial strain isolated from human faeces implicated in mucin degradation.

The human gut acts as a habitat for diverse microbial communities, including mucin utilizers that play a significant role in host health and diseases. In this study, a gram-positive, rod-shaped mucin degrading bacterium was isolated from human faeces that belonged to the Priestia flexa species. Priestia isolate was analyzed for mucin-degrading ability and found that the KS1 strain could grow on mucin as the sole carbon source. The experimental results of the mucolytic zone around the colony and a 58% decrease in carbohydrate concentration confirmed the ability of Priestia to degrade mucin. The intracellular and extracellular glycosidase assay data supported the above results suggesting the ability of P. flexa to produce glycan hydrolysis enzymes that convert complex mucin oligosaccharide chains into simple glycans. The survival ability of the KS1 strain in simulated gastrointestinal conditions revealed that it could tolerate low pH (≥ 50% cell viability at pH 1.0) and 0.5% bile salt concentration (≥ 85% cell viability). The strain showed low hydrophobicity towards n-hexadecane (26.51 ± 0.92%) and xylene (21.71 ± 0.54%). Moreover, the KS1 culture was resistant to cefixime, clavulanic acid/ceftazidime, nafallin, methicillin, trimethoprim, kanamycin, and nalidixic antibiotic. Our results highlight the isolation of P. flexa KS1 strain that degrade mucin under in vitro conditions and show its better acclimatization within the GI environment. Further studies are required to unearth the molecular mechanisms involved in the degradation of mucin oligosaccharides in the human gut, advancing our understanding of health and disease.

Chicken embryos are a valuable model for the selection of Bacillus subtilis for probiotic purposes.

Bacillus subtilis (BS) has been used as an excellent probiotic; however, some BS strains seem to be opportunist pathogens or do not present inhibitory effects in the pathogenic bacteria, so the characterization of BS strains for use in animals is mandatory. This study aimed to select nonpathogenic strains of BS, which can inhibit Salmonella spp., avian pathogenic Escherichia coli (APEC), and Campylobacter jejuni (CJ) using a chicken embryo as a model. We tested nine (9) strains of BS isolated from several sources (named A to I) in in vitro by tests of mucin degradation activity, haemolytic activity, apoptosis, and necrosis in fibroblasts from chickens. After the in vitro test, we tested the remaining seven (7) strains (strains A to G) in a chicken embryo (CE) as an in vivo model and target animal. We inoculated 3 log CFU/CE of each strain via allantoic fluid at the 10th day postincubation (DPI). Each treatment group consisted of eight CEs. At the 17th DPI we checked CE mortality, gross lesions, CE weight, and whether BS strains were still viable. To perform the cytokine, total protein, albumin, and reactive C protein analysis, we collected the CE blood from the allantoic vessel and intestine fragments in the duodenum portion for histomorphometric analysis. After the results in CEs, we tested the inhibition capacity of the selected BS strains for diverse strains of Salmonella Heidelberg(SH), S. Typhimurium (ST), S. Enteritidis(SE), S. Minnesota(SM), S. Infantis(SI), Salmonella var. monophasic (SVM),APEC and C. jejuni. After the in vitro trial (mucin degradation activity, haemolytic activity, apoptosis, and necrosis), we removed two (2) strains (H and I) that showed β-haemolysis, mucin degradation, and/or high apoptosis and necrosis effects. Although all strains of BS were viable in CEs at the 17th DPI, we removed four (4) strains (A, B, D, F) once they led to the highest mortality in CEs or a high albumin/protein ratio. C. jejuni inoculated with strain G had greater weight than the commercial strain, which could be further used for egg inoculation with benefits to the CE. From the tests in CEs, we selected the strains C, E, and G for their ability to inhibit pathogenic strains of relevant foodborne pathogens. We found that the inhibition effect was strain dependent. In general, strains E and/or G presented better or similar results than commercial control strains in the inhibition of SH, ST, SI, APEC, and two (2) strains of CJ. In this study, we selected BS strains C, E and G due to their in vitro and in vivo safety and beneficial effects. In addition, we emphasize the value of CE as an in vivo experimental model for assessing BS's safety and possible benefits for poultry and other animals.

Lacticaseibacillus rhamnosus CA15 (DSM 33960) as a Candidate Probiotic Strain for Human Health.

Lactobacilli with probiotic properties have emerged as promising tools for both the prevention and treatment of vaginal dysbiosis. The present study aimed to study the in vitro probiotic potential of the Lacticaseibacillus rhamnosus CA15 (DSM 33960) strain isolated from a healthy vaginal ecosystem. The strain was evaluated for both functional (antagonistic activity against pathogens; H2O2, organic acid, and lactic acid production; antioxidant and anti-inflammatory activities; ability to adhere to intestinal mucus and to both CaCo-2 and VK7/E6E7 cell lines; exopolysaccharide production; surface properties; and ability to survive during gastrointestinal transit) and safety (hemolytic, DNase, and gelatinase activities; mucin degradation ability; production of biogenic amines; and resistance to antimicrobials) characteristics. Data revealed that the tested strain was able to antagonize a broad spectrum of vaginal pathogens. In addition, the adhesion capacity to both vaginal and intestinal cell lines, as well as anti-inflammatory and antioxidant activities, was detected. The ability of the Lacticaseibacillus rhamnosus CA15 (DSM 33960) strain to survive under harsh environmental conditions occurring during the gastrointestinal passage suggests its possible oral delivery. Thus, in vitro data highlighted interesting probiotic properties of the CA15 (DSM 33960) strain, which could represent a valuable candidate for in vivo vaginal infections treatment.

Nucleotide binding as an allosteric regulatory mechanism for Akkermansia muciniphila β-N-acetylhexosaminidase Am2136

ABSTRACT β-N-acetylhexosaminidases (EC3.2.1.52), which belong to the glycosyl hydrolase family GH20, are important enzymes for oligosaccharides modification. Numerous microbial β-N-acetylhexosaminidases have been investigated for applications in biology, biomedicine and biotechnology. Akkermansia muciniphila is an anaerobic intestinal commensal bacterium which possesses specific β-N-acetylhexosaminidases for gut mucosal layer colonization and mucin degradation. In this study, we assessed the in vitro mucin glycan cleavage activity of the A. muciniphila β-N-acetylhexosaminidase Am2136 and demonstrated its ability that hydrolyzing the β-linkages joining N-acetylglucosamine to a wide variety of aglycone residues, which indicated that Am2136 may be a generalist β-N-acetylhexosaminidase. Structural and enzyme activity assay experiments allowed us to probe the essential function of the inter-domain interactions in β23-β33. Importantly, we revealed that the hydrolysis activity of Am2136 was enhanced by nucleotides. We further speculated that this activation mechanism might be associated with the conformational motions between domain III and IV. To our knowledge, this is the first report of nucleotide effector regulated β-N-acetylhexosaminidase, to reveal its novel biological functions. These findings contribute to understanding the distinct properties within the GH20 family and lay a certain foundation to develop controllable glycan hydrolyzing catalysts. Abbreviations: OD600 - optical cell densities at 600 nm; LB - Luria–Bertani; IPTG - isopropyl β-D-1-thiogalactopyranoside; PMSF - phenylmethanesulfonyl fluoride; rmsd - root mean square deviation; GlcNAc - N-acetyl-β-D-glucosamine; GalNAc - N-acetyl-β-D-galactosamine; Gal - galactose

Mucin-degrading gut commensals isolated from healthy faecal donor suppress intestinal epithelial inflammation and regulate tight junction barrier function.

The intestinal epithelium surface is covered by a layer of mucus that harbors a complex and dynamic population of bacteria termed gut microbiota. In particular, some gut bacteria have the ability to degrade the mucin glycan for nutritional sources. However, the bacterial diversity of mucin-degrading bacteria in human gut microbiota and their role in the gut remains unclear. In this study, we characterized the diversity of mucin-degrading bacteria in the human gut microbiota by an established cultivation-based molecular profiling method. The results showed the gut commensals having the mucin degrading ability were widely distributed in the gut microbiota and were more abundant than previously thought. In addition, many previously uncharacterized mucin degraders were isolated from faecals samples, suggesting the mucin-degrading gut commensals were underappreciated. To gain a better understanding of the interaction between these mucin-degrading gut commensals and the host, the effect of the commensals on intestinal epithelial cells were examined, and the results revealed that the commensals (8 Bacteroides spp., 2 Parabacteroides spp, Akkermanisa muciniphila and Bifidobacterial dentium) incited low level of inflammatory response (IL-8 and TNF-α) but suppressed the inflammatory response induced by E. coli through downregulating the NF-κB pathway. The presence of gut commensals also showed potential in enhancing the epithelial tight junction (TJ) barrier function through regulating the mRNA expression of TJ protein genes such as Zo-1, Occludin, Claudin-1 and E-cadherin. Furthermore, the presence of commensal bacteria P. distasonis, B. thetaiotaomicron and A. muciniphila completely or partly restored the pro-inflammatory cytokine IL-1β induced TJ barrier disruption. In conclusion, these findings indicate that mucin-degrading gut commensals were widely distributed in the gut microbiota and showed anti-inflammatory effect against pathogen infection and potential in modulating the epithelial barrier function.

Bacteroides vulgatus SNUG 40005 Restores Akkermansia Depletion by Metabolite Modulation

Weight loss and exercise intervention have been reported to increase the interaction between Bacteroides spp and Akkermansiamuciniphila (Am), although the underlying mechanisms and consequences of the interaction remain unknown. Using a healthy Korean twin cohort (n= 582), we analyzed taxonomic associations with host body mass index. B vulgatus strains were isolated from mice and human subjects to investigate the strain-specific effect of B vulgatus SNUG 40005 (Bvul) on obesity. The mechanisms underlying Am enrichment by Bvul administration were investigated by multiple experiments: (1) invitro cross-feeding experiments, (2) construction of Bvul mutants with the N-acetylglucosaminidase gene knocked out, and (3) invivovalidation cohorts with different metabolites. Finally, metabolite profiling in mouse and human fecal samples was performed. An interaction between Bvul and Am was observed in lean subjects but was disrupted in obese subjects. The administration of Bvul to mice fed a high-fat diet decreased body weight, insulin resistance, and gut permeability. In particular, Bvul restored the abundance of Am, which decreased significantly after a long-term high-fat diet. A cross-feeding analysis of Am with cecal contents or Bvul revealed that Am enrichment was attributed to metabolites produced during mucus degradation by Bvul. The metabolome profile of mouse fecal samples identified N-acetylglucosamine ascontributing to Am enrichment, which was confirmed by invitro and invivo experiments. Metabolite network analysis of the twin cohort found that lysine serves as a bridge between N-acetylglucosamine, Bvul, and Am. Strain-specific microbe-microbe interactions modulate the mucosal environment via metabolites produced during mucin degradation in the gut.