A Colorimetric Assay to Enable High‐Throughput Identification of Biofilm Exopolysaccharide‐Hydrolyzing Enzymes

Wang, Shaochi; Breslawec, Alexandra P.; Li, Crystal; Poulin, Myles B.

doi:10.1002/chem.202002475

Cited by 7 publications

(5 citation statements)

References 50 publications

(66 reference statements)

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“…To evaluate the role of anionic amino acids on PNAG substrate recognition and turnover, we mutated residues D147, D245 and E248 to the corresponding asparagine or glutamine residue or to an alanine. We also analyzed the activity of a D183A catalytic site mutant that has been previously shown to be inactive for hydrolysis of colorimetric PNAG substrate analogs (30,47). The effect of these mutations on DspB specificity was evaluated by analyzing reaction progress curves for the breakdown of synthetic PNAG trisaccharide analogs 1-4 as a function of time.…”

Section: Mutation Of Anionic Amino Acids Reveals Their Functional Rolmentioning

confidence: 99%

“…Recombinant Dispersin B (residues 16-381, DspBwt) from A. actinomycetemcomitans was prepared as described previously (33). Plasmids for expression of DspBD183A were prepared as previously described (47). Plasmids for expression of DspBD147N, DspBD245N, DspBE248Q, and DspBE248A mutants were prepared by Quikchange site-directed mutagenesis using the primer pairs outlined in Table 1.…”

Section: Protein Productionmentioning

confidence: 99%

See 1 more Smart Citation

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

Breslawec

Wang

et al. 2021

Journal of Biological Chemistry

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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.

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Section: Mutation Of Anionic Amino Acids Reveals Their Functional Rolmentioning

confidence: 99%

Section: Protein Productionmentioning

confidence: 99%

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

Breslawec

Wang

et al. 2021

Journal of Biological Chemistry

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“…1A), hereafter referred to as APEX2-DspB. This DspBE184Q mutant has been shown to lack PNAG hydrolase activity [35][36][37] but retains the ability to bind PNAG and has previously been genetically fused to enhanced green fluorescent protein (eGFP) for fluorescent labeling of PNAG within live biofilms. 38,39 APEX2 was chosen over other peroxidase enzymes due to its small size and monomeric nature, improving the likelihood that the fusion protein would effectively penetrate and bind to PNAG within S. epidermidis biofilms.…”

Section: Resultsmentioning

confidence: 99%

Mapping protein–exopolysaccharide binding interaction inStaphylococcus epidermidisbiofilms by live cell proximity labeling

Vo,

Hong,

Stepler

et al. 2023

Preprint

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Bacterial biofilms consist of cells encased in an extracellular polymeric substance (EPS) composed of exopolysaccharides, extracellular DNA, and proteins that are critical for cell–cell adhesion and protect the cells from environmental stress, antibiotic treatments, and the host immune response. Degrading EPS components or blocking their production have emerged as promising strategies for prevention or dispersal of bacterial biofilms, but we still have little information about the specific biomolecular interactions that occur between cells and EPS components and how those interactions contribute to biofilm production.Staphylococcus epidermidisis a leading cause of nosocomial infections as a result of producing biofilms that use the exopolysaccharide poly- (1→6)-β-N-acetylglucosamine (PNAG) as a major structural component. In this study, we have developed a live cell proximity labeling approach combined with quantitative mass spectrometry-based proteomics to map the PNAG interactome of liveS. epidermidisbiofilms. Through these measurements we discovered elastin-binding protein (EbpS) as a major PNAG-interacting protein. Using live cell binding measurements, we found that the lysin motif (LysM) domain of EbpS specifically binds to PNAG present inS. epidermidisbiofilms. Our work provides a novel method for the rapid identification of exopolysaccharide-binding proteins in live biofilms that will help to extend our understanding of the biomolecular interactions that are required for bacterial biofilm formation.

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Section: Mutation Of Anionic Amino Acids Reveals Their Functional Rolmentioning

confidence: 99%

The role of anionic amino acids in hydrolysis of poly-β-(1,6)-N-acetylglucosamine exopolysaccharides by the biofilm dispersing glycosidase Dispersin B

Breslawec

Wang

et al. 2020

Preprint

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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 with de-N-acetylated PNAG oligosaccharides; however, there is still little known about the molecular interaction required for DspB to bind native de-N-acetylated PNAG polysaccharides. 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.

show abstract

A Colorimetric Assay to Enable High‐Throughput Identification of Biofilm Exopolysaccharide‐Hydrolyzing Enzymes

Cited by 7 publications

References 50 publications

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

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

Mapping protein–exopolysaccharide binding interaction inStaphylococcus epidermidisbiofilms by live cell proximity labeling

The role of anionic amino acids in hydrolysis of poly-β-(1,6)-N-acetylglucosamine exopolysaccharides by the biofilm dispersing glycosidase Dispersin B

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