Anionic amino acids support hydrolysis of poly-β-(1,6)-N-acetylglucosamine exopolysaccharides by the biofilm dispersing glycosidase Dispersin B

Abstract

The exopolysaccharide poly- β -(1→6)- N -acetylglucosamine (PNAG) is a major structural determinant of bacterial biofilms responsible for persistent and nosocomial infections. The enzymatic dispersal of biofilms by PNAG-hydrolyzing glycosidase enzymes, such as Dispersin B (DspB), is a possible approach to treat biofilm-dependent bacterial infections. The cationic charge resulting from partial de- N -acetylation of native PNAG is critical for PNAG-dependent biofilm formation. We recently demonstrated that DspB has increased catalytic activity on de- N -acetylated PNAG oligosaccharides, but the molecular basis for this increased activity is not known. Here, we analyze the role of anionic amino acids surrounding the catalytic pocket of DspB in PNAG substrate recognition and hydrolysis using a combination of site-directed mutagenesis, activity measurements using synthetic PNAG oligosaccharide analogs, and in vitro biofilm dispersal assays. The results of these studies support a model in which bound PNAG is weakly associated with a shallow anionic groove on the DspB protein surface with recognition driven by interactions with the −1 GlcNAc residue in the catalytic pocket. An increased rate of hydrolysis for cationic PNAG was driven, in part, by interaction with D147 on the anionic surface. Moreover, we identified that a DspB mutant with improved hydrolysis of fully acetylated PNAG oligosaccharides correlates with improved in vitro dispersal of PNAG-dependent Staphylococcus epidermidis biofilms. These results provide insight into the mechanism of substrate recognition by DspB and suggest a method to improve DspB biofilm dispersal activity by mutation of the amino acids within the anionic binding surface.

Antibioflm effects of extracellular matrix degradative agents on the biofilm of different strains of multi-drug resistant Corynebacterium striatum

“…At the same time, five C. striatum strains with weak biofilm-forming ability were also positive with spaDEF genes, which indicates an indefinite relationship between biofilm formation abilities and expression of pili in C. striatum . For some well-known gram-positive bacteria (including S. aureus , and Bacillus subtilis ), the bacterial extracellular matrix has been recognized to be an essential determinant in mediating biofilm formation and participating in the protection process from external insults [ 21 , 22 ]. The extracellular matrix composition was similar among different bacteria, including EPA, proteins, and eDNA, all of which participated in the process of biofilm formation in different manners [ 21 , 23 – 25 ].…”

“…For some well-known gram-positive bacteria (including S. aureus , and Bacillus subtilis ), the bacterial extracellular matrix has been recognized to be an essential determinant in mediating biofilm formation and participating in the protection process from external insults [ 21 , 22 ]. The extracellular matrix composition was similar among different bacteria, including EPA, proteins, and eDNA, all of which participated in the process of biofilm formation in different manners [ 21 , 23 – 25 ]. Correspondingly, degradative agents directed against the above components were developed to deter biofilm formation, including dispersin B, proteinase K, and DNase I. Biofilm matrix degradation with enzymatic degradative agents toward polysaccharides, proteins, and nucleic acids paves an efficient way of controlling or treating infections caused by biofilm-producing bacterial pathogens [ 26 – 28 ].…”

Enzymatic dispersion of biofilms: An emerging biocatalytic avenue to combat biofilm-mediated microbial infections

“…129 The site of cleavage of the glycosidic bond (endo-or exo-) of the polysaccharide by Dispersin B depends on the nature of the substrate. [129][130][131] The active site architecture of the enzyme (PDB ID: 1YHT) 132 displays a conserved Asp-Glu dyad, where the Glu residue performs the general acid-base catalysis to hydrolyze the glycosidic bond through an oxazolinium intermediate (Figure 5A). The Asp residue assists the N-acetyl group for nucleophilic attack.…”

“…Binding of the polysaccharide substrate to the active site of Dispersin B is assisted by a set of conserved residues: one Glu, three Trp, and one Tyr ( 129 ). The site of cleavage of the glycosidic bond (endo- or exo-) of the polysaccharide by Dispersin B depends on the nature of the substrate ( 129 , 130 , 131 ). The active site architecture of the enzyme (Protein Data Bank [PDB] ID: 1YHT ) ( 132 ) displays a conserved Asp-Glu dyad, where the Glu residue performs the general acid-base catalysis to hydrolyze the glycosidic bond through an oxazolinium intermediate ( Fig.…”

The endoglycosidase activity of Dispersin B is mediated through electrostatic interactions with cationic poly‐β‐(1→6)‐N‐acetylglucosamine

“…Specifically, fully N ‐acetylated PNAG oligosaccharides (i.e. 1 ) are hydrolysed predominantly through sequential exo‐glycosidic bond cleavage at the non‐reducing end GlcNAc [31], which is enhanced by approximately three‐fold as a result of electrostatic interactions between DspB and substrates containing a cationic glucosamine (GlcN) in the +2 position relative to the site of cleavage [32]. Moreover, we found that in the presence of non‐reducing terminal GlcN (i.e.…”