Dietary Effect of Padina boergesenii on Growth, Immune Response, and Disease Resistance Against Pseudomonas aeruginosa in Cirrhinus mrigala.

In the aquatic environment, seaweeds have the potential to be renewable resources. The current study was designed to assess the impact of seaweed Padina boergesenii incorporated into a basal diet at various concentrations. The phytoconstituents of the seaweeds were characterised by gas chromatography-mass spectrometry. Diets were designed to include elevated levels of 0.5%, 2.5%, 4.5%, and 6.5% of seaweed meal. Significant differences in Cirrhinus mrigala fed with P. boergesenii incorporated into the basal diet for 45 days. The growth parameters (weight gain, specific growth rate), biochemical constituents, and immunological assays were observed. The extract fraction demonstrated effective inhibitory activity against Pseudomonas aeruginosa. As a result, this research suggests that extracts of the seaweed P. boergesenii contain potential bioactive compounds with significant antibiotic activity.

Structural and functional analysis of de-N-acetylase PgaB from periodontopathogen Aggregatibacter actinomycetemcomitans.

The oral pathogen Aggregatibacter actinomycetemcomitans uses pga gene locus for the production of an exopolysaccharide made up of a linear homopolymer of ß-1,6-N-acetyl-d-glucosamine (PGA). An enzyme encoded by the pgaB of the pga operon in A. actinomycetemcomitans is a de-N-acetylase, which is used to alter the PGA. The full length enzyme (AaPgaB) and the N-terminal catalytic domain (residues 25-290, AaPgaBN) from A. actinomycetemcomitans were cloned, expressed and purified. The enzymatic activities of the AaPgaB enzymes were determined using 7-acetoxycoumarin-3-carboxylic acid as the substrate. The AaPgaB enzymes displayed significantly lower de-N-acetylase activity compared with the activity of the deacetylase PdaA from Bacillus subtilis, a member of the CE4 family of enzymes. To delineate the differences in the activity and the active site architecture, the structure of AaPgaBN was determined. The AaPgaBN structure has two metal ions in the active site instead of one found in other CE4 enzymes. Based on the crystal structure comparisons among the various CE4 enzymes, two residues, Q51 and R271, were identified in AaPgaB, which could potentially affect the enzyme activity. Of the two mutants generated, Q51E and R271K, the variant Q51E showed enhanced activity compared with AaPgaB, validating the requirement that an activating aspartate residue in the active site is essential for higher activity. In summary, our study provides the first structural evidence for a di-nuclear metal site at the active site of a member of the CE4 family of enzymes, evidence that AaPgaBN is catalytically active and that mutant Q51E exhibits higher de-N-acetylase activity.

Surface display of Aggregatibacter actinomycetemcomitans autotransporter Aae and dispersin B hybrid act as antibiofilm agents.

Among the various proteins expressed by the periodontopathogen Aggregatibacter actinomycetemcomitans, two proteins play important roles for survival in the oral cavity. The autotransporter Aae facilitates the attachment of the pathogen to oral epithelial cells, which act as a reservoir, while the biofilm-degrading glycoside hydrolase dispersin B facilitates the movement of daughter cells from the mature biofilm to a new site. The objective of this study was to use the potential of these two proteins to control biofilms. To this end, we generated a hybrid construct between the Aae C-terminal translocating domain and dispersin B, and mobilized it into Escherichia coli Rosetta (DE3) pLysS cells. Immunofluorescence analysis of the modified E. coli cells confirmed the presence of dispersin B on the surface. Further, the membrane localization of the displayed dispersin B was confirmed with Western blot analysis. The integrity of the E. coli cells displaying the dispersin B was confirmed through FACS analysis. The hydrolytic activity of the surface-displayed dispersin B was confirmed by using 4-methylumbelliferyl-ß-d-glucopyranoside as the substrate. The detachment ability of the dispersin B surface-displaying E. coli cells was shown using Staphylococcus epidermidis and Actinobacillus pleuropneumoniae biofilms in a microtiter assay. We concluded that the Aae ß-domain is sufficient to translocate foreign enzymes in the native folded form and that the method of Aae-mediated translocation of surface displayed enzymes might be useful for control of biofilms.

Modeling and biochemical analysis of the activity of antibiofilm agent Dispersin B.

Bacteria in a biofilm are enmeshed in a self-synthesized extracellular polysaccharide matrix (PGA), which is a linear polymer of beta(1,6)-linked N-acetylglucosamine (GlcNAc) residues. Dispersin B (DspB), a soluble glycoside hydrolase produced by the periodontal pathogen Actinobacillus actinomycetemcomitans degrades PGA. The enzyme DspB is an alpha/beta TIM-barrel protein and belongs to family 20 glycosyl hydrolases members. The enzyme activity of DspB with regard to its substrate specificity towards beta(1,6)-linked GlcNAc polymers and its endo/exo character was investigated through ligand docking and the hydrolysis of synthetic oligosaccharides. Ligand docking analysis suggested that beta(1,6)-linked GlcNAc oligosaccharide bound to the active site better that beta(1,4)-linked GlcNAc oligosaccharide. Our combined results indicate that DspB is an exo-acting enzyme that hydrolyzes beta(1,6)-linked N-acetylglucosamine oligomers.

Detachment and killing of Aggregatibacter actinomycetemcomitans biofilms by dispersin B and SDS.

The periodontopathogen Aggregatibacter actinomycetemcomitans forms tenacious biofilms on abiotic surfaces in vitro. The objective of the present study was to measure the susceptibility of A. actinomycetemcomitans biofilms to detachment and killing by the anionic surfactant sodium dodecyl sulfate (SDS). We found that biofilms formed by a wild-type strain were resistant to detachment by SDS. In contrast, biofilms formed by an isogenic mutant strain that was deficient in the production of PGA (poly-N-acetyl-glucosamine), a biofilm matrix polysaccharide, were sensitive to detachment by SDS. Pre-treatment of wild-type biofilms with dispersin B, a PGA-degrading enzyme, rendered them sensitive to detachment by SDS and resulted in a > 99% increase in SDS-mediated cell killing. We concluded that PGA protects A. actinomycetemcomitans cells from detachment and killing by SDS. Dispersin B and SDS may be useful agents for treating chronic infections caused by A. actinomycetemcomitans and other PGA-producing bacteria.

Synergistic activity of dispersin B and cefamandole nafate in inhibition of staphylococcal biofilm growth on polyurethanes.

Antibiotic therapies to eradicate medical device-associated infections often fail because of the ability of sessile bacteria, encased in their exopolysaccharide matrix, to be more drug resistant than planktonic organisms. In the last two decades, several strategies to prevent microbial adhesion and biofilm formation on the surfaces of medical devices, based mainly on the use of antiadhesive, antiseptic, and antibiotic coatings on polymer surfaces, have been developed. More recent alternative approaches are based on molecules able to interfere with quorum-sensing phenomena or to dissolve biofilms. Interestingly, a newly purified beta-N-acetylglucosaminidase, dispersin B, produced by the gram-negative periodontal pathogen Actinobacillus actinomycetemcomitans, is able to dissolve mature biofilms produced by Staphylococcus epidermidis as well as some other bacterial species. Therefore, in this study, we developed new polymeric matrices able to bind dispersin B either alone or in combination with an antibiotic molecule, cefamandole nafate (CEF). We showed that our functionalized polyurethanes could adsorb a significant amount of dispersin B, which was able to exert its hydrolytic activity against the exopolysaccharide matrix produced by staphylococcal strains. When microbial biofilms were exposed to both dispersin B and CEF, a synergistic action became evident, thus characterizing these polymer-dispersin B-antibiotic systems as promising, highly effective tools for preventing bacterial colonization of medical devices.

Structural analysis of dispersin B, a biofilm-releasing glycoside hydrolase from the periodontopathogen Actinobacillus actinomycetemcomitans.

Bacteria in a biofilm are enmeshed in a self-synthesized extracellular polysaccharide matrix that holds the bacteria together in a mass and firmly attaches the bacterial mass to the underlying surface. A major component of the extracellular polysaccharide matrix in several phylogenetically diverse bacteria is PGA, a linear polymer of N-acetylglucosamine residues in beta(1,6)-linkage. PGA is produced by the Gram-negative periodontopathogen Actinobacillus actinomycetemcomitans as well as by the Gram-positive device-associated pathogen Staphylococcus epidermidis. We recently reported that A.actinomycetemcomitans produces a soluble glycoside hydrolase named dispersin B, which degrades PGA. Here, we present the crystal structure of dispersin B at 2.0A in complex with a glycerol and an acetate ion at the active site. The enzyme crystallizes in the orthorhombic space group C222(1) with cell dimensions a=41.02A, b=86.13A, c=185.77A. The core of the enzyme consists a (beta/alpha)(8) barrel topology similar to other beta-hexosaminidases but significant differences exist in the arrangement of loops hovering in the vicinity of the active site. The location and interactions of the glycerol and acetate moieties in conjunction with the sequence analysis suggest that dispersin B cleaves beta(1,6)-linked N-acetylglucosamine polymer using a catalytic machinery similar to other family 20 hexosaminidases which cleave beta(1,4)-linked N-acetylglucosamine residues.