About

Myles B. Poulin is an Associate Professor in the Department of Chemistry and Biochemistry at the University of Maryland. His research focuses on developing tools to study the biosynthesis of bacterial exopolysaccharides and their role in biofilm formation and bacterial infections. His laboratory employs a multidisciplinary approach that combines synthetic carbohydrate chemistry, enzymology, membrane protein biochemistry, and molecular biology to investigate the structure, biosynthesis, transport, and functions of exopolysaccharides that constitute the extracellular matrix of bacterial biofilm communities. His research projects are categorized into three main areas: developing tools to study the biosynthesis and export of bacterial exopolysaccharides and their role in biofilm formation; identifying and characterizing enzymes involved in the breakdown of bacterial biofilm exopolysaccharides and studying their role in biofilm dispersal; and creating chemical probes that disrupt exopolysaccharide biosynthesis to understand their role in biofilm assembly in vivo. This involves mechanism-based inhibitor design and transition state analog approaches based on kinetic isotope effect measurements to develop inhibitors of glycosyltransferase enzymes involved in exopolysaccharide biosynthesis.

Research topics

• Chemistry
• Biochemistry
• Biology
• Stereochemistry
• Microbiology

Selected publications

• Mapping protein–exopolysaccharide binding interaction in <i>Staphylococcus epidermidis</i> biofilms by live cell proximity labeling

  bioRxiv (Cold Spring Harbor Laboratory) · 2023-08-29 · 1 citations

  preprintOpen accessSenior authorCorresponding

  Abstract 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 epidermidis is 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 live S. epidermidis biofilms. 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 in S. epidermidis biofilms. 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.

  PublisherOA PDFDOI

• Direct Competitive Kinetic Isotope Effect Measurement Using Quantitative Whole Molecule MALDI-TOF Mass Spectrometry

  ChemRxiv · 2023-08-01 · 1 citations

  preprintOpen accessSenior author

  Kinetic isotope effect (KIE) measurements are a powerful tool to interrogate the microscopic steps in enzyme catalyzed reactions and can provide detailed information about transition state structures. However, the application of KIE measurements to study enzymatic reactions is not widely applied due to the tedious and complex analytical workflows required to measure KIEs with sufficient precession. Here, we report a method for the direct measurement of competitive KIEs using a whole molecule matrix assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS). Using isotope labeled internal standard introduced when quenching the enzyme reaction at multiple time points enables the simultaneous measurement of both the relative heavy/light isotope ratio R and fractional conversion F relative to the internal standard for each sample as the reaction progresses. We applied this approach to measure both [1'-13C]lactose and [6'-13C]lactose KIEs for the E. coli β-galactosidase (LacZ) catalyzed hydrolysis of lactose. This MALDI-TOF MS based KIE approach can measure enzymatic KIEs with precision comparable to those obtained using competitive radioisotope labelling, and NMR based approaches.

  PublisherOA PDFDOI

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

  FEBS Journal · 2022-09-09 · 12 citations

  articleOpen accessSenior authorCorresponding

  Bacterial biofilms consist of bacterial cells embedded within a self‐produced extracellular polymeric substance (EPS) composed of exopolysaccharides, extra cellular DNA, proteins and lipids. The enzyme Dispersin B (DspB) is a CAZy type 20 β‐hexosaminidase enzyme that catalyses the hydrolysis of poly‐ N ‐acetylglucosamine (PNAG), a major biofilm polysaccharide produced by a wide variety of biofilm‐forming bacteria. Native PNAG is partially de‐ N ‐acetylated, and the degree of deacetylation varies between species and dependent on the environment. We have previously shown that DspB is able to perform both endo‐ and exo‐glycosidic bond cleavage of PNAG depending on the de‐ N ‐acetylation patterns present in the PNAG substrate. Here, we used a combination of synthetic PNAG substrate analogues, site‐directed mutagenesis and in vitro biofilm dispersal assay to investigate the molecular basis for the endo‐glycosidic cleavage activity of DspB and the importance of this activity for dispersal of PNAG‐dependent Staphylococcus epidermidis biofilms. We found that D242 contributes to the endoglycosidase activity of DspB through electrostatic interactions with cationic substrates in the −2 binding site. A DspB D242N mutant was highly deficient in endoglycosidase activity while maintaining exoglycosidase activity. When used to disperse S. epidermidis biofilms, this DspB D242N mutant resulted in an increase in residual biofilm biomass after treatment when compared to wild‐type DspB. These results suggest that the de‐ N ‐acetylation of PNAG in S. epidermidis biofilms is not uniformly distributed and that the endoglycosidase activity of DspB is required for efficient biofilm dispersal.

  PublisherOA PDFDOI

• Regulation of Biofilm Exopolysaccharide Production by Cyclic Di-Guanosine Monophosphate

  Frontiers in Microbiology · 2021-09-10 · 75 citations

  reviewOpen access1st authorCorresponding

  Many bacterial species in nature possess the ability to transition into a sessile lifestyle and aggregate into cohesive colonies, known as biofilms. Within a biofilm, bacterial cells are encapsulated within an extracellular polymeric substance (EPS) comprised of polysaccharides, proteins, nucleic acids, lipids, and other small molecules. The transition from planktonic growth to the biofilm lifecycle provides numerous benefits to bacteria, such as facilitating adherence to abiotic surfaces, evasion of a host immune system, and resistance to common antibiotics. As a result, biofilm-forming bacteria contribute to 65% of infections in humans, and substantially increase the energy and time required for treatment and recovery. Several biofilm specific exopolysaccharides, including cellulose, alginate, Pel polysaccharide, and poly- N -acetylglucosamine (PNAG), have been shown to play an important role in bacterial biofilm formation and their production is strongly correlated with pathogenicity and virulence. In many bacteria the biosynthetic machineries required for assembly of these exopolysaccharides are regulated by common signaling molecules, with the second messenger cyclic di-guanosine monophosphate (c - di-GMP) playing an especially important role in the post-translational activation of exopolysaccharide biosynthesis. Research on treatments of antibiotic-resistant and biofilm-forming bacteria through direct targeting of c-di-GMP signaling has shown promise, including peptide-based treatments that sequester intracellular c-di-GMP. In this review, we will examine the direct role c-di-GMP plays in the biosynthesis and export of biofilm exopolysaccharides with a focus on the mechanism of post-translational activation of these pathways, as well as describe novel approaches to inhibit biofilm formation through direct targeting of c-di-GMP.

  PublisherOA PDFDOI

• Multifunctional fluorescent probes for high-throughput characterization of hexosaminidase enzyme activity

  Bioorganic Chemistry · 2021-11-29 · 14 citations

  articleSenior authorCorresponding
  PublisherDOI

• Development of a colorimetric assay for high throughput screening of poly-β-D-(1-6)-<i>N</i>-acetyl-glucosamine specific glycosidase activity

  2020-04-30

  preprint
  PublisherDOI

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

  Journal of Biological Chemistry · 2020-12-23 · 26 citations

  articleOpen accessSenior authorCorresponding

  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.

  PublisherOA PDFDOI

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

  bioRxiv (Cold Spring Harbor Laboratory) · 2020-08-06 · 3 citations

  preprintOpen accessSenior authorCorresponding

  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 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.

  PublisherOA PDFDOI

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

  Chemistry - A European Journal · 2020-06-27 · 12 citations

  articleSenior authorCorresponding

  Glycosidase enzymes that hydrolyze the biofilm exopolysaccharide poly-β-(1→6)-N-acetylglucosamine (PNAG) are critical tools to study biofilm and potential therapeutic biofilm dispersal agents. Function-driven metagenomic screening is a powerful approach for the discovery of new glycosidase but requires sensitive assays capable of distinguishing between the desired enzyme and functionally related enzymes. Herein, we report the synthesis of a colorimetric PNAG disaccharide analogue whose hydrolysis by PNAG glycosidases results in production of para-nitroaniline that can be continuously monitored at 410 nm. The assay is specific for enzymes capable of hydrolyzing PNAG and not related β-hexosaminidase enzymes with alternative glycosidic linkage specificities. This analogue enabled development of a continuous colorimetric assay for detection of PNAG hydrolyzing enzyme activity in crude E. coli cell lysates and suggests that this disaccharide probe will be critical for establishing the functional screening of metagenomic DNA libraries.

  PublisherDOI

• Development of a colorimetric assay for high throughput screening of poly-β-D-(1-6)-<i>N</i>-acetyl-glucosamine specific glycosidase activity

  2020-04-30

  preprint
  PublisherDOI

Frequent coauthors

• Todd L. Lowary

  Institute of Biological Chemistry, Academia Sinica

  25 shared

• Vern L. Schramm

  Albert Einstein College of Medicine

  13 shared

• Alexandra P. Breslawec

  University of Maryland, College Park

  8 shared

• Shaochi Wang

  First Affiliated Hospital of Zhengzhou University

  8 shared

• Emily J. Parker

  Monash University

  6 shared

• Quan Du

  Westlake University

  5 shared

• Crystal Li

  5 shared

• Harald Nothaft

  University of Alberta

  4 shared

Labs

• Myles B. PoulinPI

Education

• PhD Chemistry, Chemistry

  University of Alberta

  2012

Awards & honors

• NSF CAREER award (2020)
• Dennis Shields Postdoctoral Research Prize, Albert Einstein…
• NSERC Postdoctoral Fellow, Albert Einstein College of Medici…
• NSERC Alexander Graham Bell Canada Graduate Scholarship, Uni…
• President’s Doctoral Prize of Distinction, University of Alb…