Biochemical characterization of bifunctional enzymatic activity of a recombinant protein (Bp0469) from Blautia producta ATCC 27340 and its role in the utilization of arabinogalactan oligosaccharides.

Human consumption of larch arabinogalactan has a significant effect on enhancing probiotic microflora in the gut, and it also promotes the production of short-chain fatty acids. Bacterial members of Lachnospiraceae family are important and play significant roles in maintaining our gut health. However, it is less known about biochemistry of members of this family by which they utilize non-cellulosic fiber in the gut. For enhancing this understanding, we studied that B. producta ATCC 27340 grew on arabinogalactan oligosaccharides (AGOs) as compared to polysaccharide form of arabinogalactan. Recombinant protein (Bp0469) was heterologously expressed in Escherichia coli BL21 (DE3) and revealed the optimum pH and temperature at 7.4 in phosphate buffer and 45 °C, respectively. Catalytic efficiency of recombinant Bp0469 for p-nitrophenyl (pNP)-α-L-arabinofuranoside was about half of pNP-ß-D-galactopyranoside. It also cleaved natural substrates (lactose, arabinobiose and 3-O-(ß-d-galactopyranosyl)-d-galactopyranose) and characterized AGOs in this study. Based on genomic, structural models, and biochemical characteristics, identified Bp0469 is a peculiar enzyme with two distinct domains that cleave α1-5 linked arabinobiose and ß-D-Galp-1-3/4 linkages. Overall, the study enhances the knowledge on nutritional perspective of B. producta ATCC 27340 for thriving on non-cellulosic biomass, and identified enzyme can also be used for producing industrial important AGOs.

Effect of dextransucrase antibodies on biofilm formation and certain cariogenic activities in Streptococcus mutans.

Introduction. Dextransucrase produced by Streptococcus mutans plays a vital role in the formation of dental caries by synthesizing exopolysaccharides from sucrose, which helps in the attachment of microbes to the tooth surface, causing caries. Exploring antibody production against S. mutans antigens could be an effective method to protect against dental caries.Hypothesis. Dextransucrase antibodies may help in the prevention of caries formation by inhibiting essential cariogenic factors.Aims. The aim of this study was to investigate the effects of dextransucrase antibodies on biofilm formation and certain associated cariogenic factors of S. mutans.Methodology. Dextransucrase was purified from culture of S. mutans. The antisera against the enzyme were raised in rabbits. The effect of dextransucrase antibodies on biofilm formation was studied using scanning electron microscopy, fluorescence microscopy and quantitative real-time polymerase chain reaction. The effects of the antibodies on associated cariogenic factors were examined using established methods. The cross-reactivity of antibodies with human lung, liver, heart, thyroid and kidney tissues was evaluated by immunohistochemistry.Results. Our findings showed impaired biofilm formation in S. mutans in the presence of dextransucrase antibodies. Genes associated with biofilm formation such as gtfB, gtfC, brpA, relA, Smu.630 and vicK were downregulated (50-97 %) by dextransucrase antibodies in S. mutans. The adherence of S. mutans to glass surface was reduced by 58 % and hydrophobicity was reduced by 55.2 % in the presence of the antibodies compared to the controls. Immunohistochemistry studies revealed no cross-reactivity of human tissues with dextransucrase antibodies.Conclusions. These findings suggest that antibodies raised against dextransucrase exhibit a profound inhibitory effect on biofilm formation and vital cariogenic factors of S. mutans, which supports the contention that dextransucrase could be a promising antigen to study for its anticariogenic potential.

Biochemical Basis of Xylooligosaccharide Utilisation by Gut Bacteria.

Xylan is one of the major structural components of the plant cell wall. Xylan present in the human diet reaches the large intestine undigested and becomes a substrate to species of the gut microbiota. Here, we characterised the capacity of Limosilactobacillus reuteri and Blautia producta strains to utilise xylan derivatives. We showed that L. reuteri ATCC 53608 and B. producta ATCC 27340 produced ß-D-xylosidases, enabling growth on xylooligosaccharide (XOS). The recombinant enzymes were highly active on artificial (p-nitrophenyl ß-D-xylopyranoside) and natural (xylobiose, xylotriose, and xylotetraose) substrates, and showed transxylosylation activity and tolerance to xylose inhibition. The enzymes belong to glycoside hydrolase family 120 with Asp as nucleophile and Glu as proton donor, as shown by homology modelling and confirmed by site-directed mutagenesis. In silico analysis revealed that these enzymes were part of a gene cluster in L. reuteri but not in Blautia strains, and quantitative proteomics identified other enzymes and transporters involved in B. producta XOS utilisation. Based on these findings, we proposed a model for an XOS metabolism pathway in L. reuteri and B. producta strains. Together with phylogenetic analyses, the data also revealed the extended xylanolytic potential of the gut microbiota.

Antibodies generated against dextransucrase exhibit potential anticariostatic properties in Streptococcus mutans.

Streptococcus mutans is a common principal causative agent of dental caries. In this communication, we describe that the antibodies raised against purified dextransucrase effectively inhibited the growth of S. mutans. The purified enzyme showed 58-fold enrichment, 17.5% yield and a specific activity of 3.96 units/mg protein. Purified IgG fraction of the antibody showed significant affinity with the antigenic protein. Immunotritation of the enzyme with dextransucrase antibody showed a gradual increase in inhibition of dextransucrase activity. The growth of S. mutans was also inhibited by 85% in the presence of 28 µg of IgG fraction of the antibody. Antibodies also impaired glucosyltransferase activity (72.8%) and biofilm formation by 92.6% in S. mutans. Western blot analysis revealed no cross reactivity with the various tissues of mice, rat, rabbit and humans. Dot blot analysis showed little reactivity with Lactobacillus acidophilus and Staphylococcus aureus and there was no reactivity with other bacterial strains like Enterococcus faecalis, Escherichia coli and Salmonella typhimurium. These findings suggest that antibody raised against dextransucrase exhibit inhibitory effects on the growth of S. mutans and biofilm formation with no reactivity with various mammalian tissues, thus it could be an effective anticariogenic agent.

Bioconjugation of InGaP quantum dots for molecular sensing.

Fluorescence-based molecular sensing and cellular imaging are commonly carried out with the application of organic dyes. Quantum dots (QDs) are now recognized as better tools because they are brighter, size tunable, and more photostable than dyes. Most of the proposed QD-based biosensing systems involve elements of known toxicity. The present work reports the functionalization of biocompatible InGaP/ZnS core-shell QDs with anti-bovine serum albumin (anti-BSA) to exploit them as fluorescent probes for antigen detection. Successful bioconjugation was characterized with the absorption and emission spectra showing blue shifts of around 40 and 30 nm, respectively. Gel electrophoresis and particle size distribution studies further confirmed the mass increment of QDs after their functionalization with anti-BSA. Surface plasmon resonance spectrometry has been used to study the affinity of QD-(anti-BSA) probes for bovine serum albumin (BSA). Photoluminescence quenching of the developed probe is observed in the presence of BSA.

Role of glutathione in ethanol stress tolerance in yeast Pachysolen tannophilus.

The effect of glutathione enrichment and depletion on the survival of Pachysolen tannophilus after ethanol stress was investigated. In this work, we verified that both control and glutathione deficient yeast cells were much more oxidized after ethanol stress. Depletion of cellular glutathione enhanced the sensitivity to ethanol and suppressed the adaptation. Incubation of the cell with amino acids constituting glutathione (GIu, Cys, Gly) increased the intracellular glutathione content, and subsequently the cell acquired resistance against ethanol. The level of reactive oxygen species, protein carbonyl, and lipid peroxidation in glutathione enriched groups were also studied. These results strongly suggest that intracellular glutathione plays an important role in the adaptive response in P. tannophilus to ethanol induced oxidative stress.

Implications of sterol structure for membrane lipid composition, fluidity and phospholipid asymmetry in Saccharomyces cerevisiae.

Sterols are essential components of the plasma membrane in eukaryotic cells. Nystatin-resistant erg mutants were used in the present study to investigate the in vitro effects of altered sterol structure on membrane lipid composition, fluidity, and asymmetry of phospholipids. Quantitative analyses of the wild type and mutants erg2, erg3 and erg6 revealed that mutants have lower sterol (free)-to-phospholipid molar ratios than the wild type. Phosphatidylcholine content was decreased in erg2 and erg3 mutants; however, it was increased in erg6 strains as compared to normals. Phosphatidylserine content was increased in the erg6 mutant only. Fluorescence anisotropy decreased with temperature in both probes, and was lower for mutants than for the wild type, suggesting an increased freedom in rotational movement due to decreased membrane order. Investigation of changes in the aminophospholipid transbilayer distribution using two chemical probes, trinitrobenzene sulfonic acid and fluorescamine, revealed that the amounts of phosphatidylethanolamine derivatized by these probes were quite similar in both the wild type and various erg strains. The present findings suggest that adaptive responses in yeast cells with altered sterol structure are possibly manifested through changes in membrane lipid composition and fluidity, and not through transbilayer rearrangement of aminophospholipids.

Role of selenium supplementation and heat stress on trehalose and glutathione content in Saccharomyces cerevisiae.

The role of selenium (Se) supplementation on glutathione, a potent intracellular redox buffer, and trehalose, a well-known stress protectant molecule, was studied. The amount of glutathione decreased significantly while that of trehalose showed a minor decrease in the cells grown in Se-supplemented medium. After heat shock, glutathione content diminished further, whereas that of trehalose increased significantly in control and Se-supplemented cells. These findings suggest the importance of trehalose as an antioxidant molecule.