Search

Pel exopolysaccharide biosynthetic loci are phylogenetically widespread biofilm matrix determinants in bacteria. In Pseudomonas aeruginosa, Pel is crucial for cell-to-cell interactions and reducing susceptibility to antibiotic and mucolytic treatments. While genes encoding glycoside hydrolases have long been linked to biofilm exopolysaccharide biosynthesis, their physiological role in biofilm development is unclear. Here we demonstrate that the glycoside hydrolase activity of P. aeruginosa PelA decreases adherent biofilm biomass and is responsible for generating the low molecular weight secreted form of the Pel exopolysaccharide. We show that the generation of secreted Pel contributes to the biomechanical properties of the biofilm and decreases the virulence of P. aeruginosa in Caenorhabditis elegans and Drosophila melanogaster. Our results reveal that glycoside hydrolases found in exopolysaccharide biosynthetic systems can help shape the soft matter attributes of a biofilm and propose that secreted matrix components be referred to as matrix associated to better reflect their influence., (© 2023. The Author(s).)

Aspergillus fumigatus is a ubiquitous mold that can cause invasive pulmonary infections in immunocompromised patients. Within the lung, A. fumigatus forms biofilms that can enhance resistance to antifungals and immune defenses. Aspergillus biofilm formation requires the production of a cationic matrix exopolysaccharide, galactosaminogalactan (GAG). In this study, recombinant glycoside hydrolases (GH)s that degrade GAG were evaluated as antifungal agents in a mouse model of invasive aspergillosis. Intratracheal GH administration was well tolerated by mice. Pharmacokinetic analysis revealed that although GHs have short half-lives, GH prophylaxis resulted in reduced fungal burden in leukopenic mice and improved survival in neutropenic mice, possibly through augmenting pulmonary neutrophil recruitment. Combining GH prophylaxis with posaconazole treatment resulted in a greater reduction in fungal burden than either agent alone. This study lays the foundation for further exploration of GH therapy in invasive fungal infections. IMPORTANCE The biofilm-forming mold Aspergillus fumigatus is a common causative agent of invasive fungal airway disease in patients with a compromised immune system or chronic airway disease. Treatment of A. fumigatus infection is limited by the few available antifungals to which fungal resistance is becoming increasingly common. The high mortality rate of A. fumigatus-related infection reflects a need for the development of novel therapeutic strategies. The fungal biofilm matrix is in part composed of the adhesive exopolysaccharide galactosaminogalactan, against which antifungals are less effective. Previously, we demonstrated antibiofilm activity with recombinant forms of the glycoside hydrolase enzymes that are involved in galactosaminogalactan biosynthesis. In this study, prophylaxis with glycoside hydrolases alone or in combination with the antifungal posaconazole in a mouse model of experimental aspergillosis improved outcomes. This study offers insight into the therapeutic potential of combining biofilm disruptive agents to leverage the activity of currently available antifungals.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes both chronic and acute invasive infections. Galactosaminogalactan (GAG) is an integral component of the A. fumigatus biofilm matrix and a key virulence factor. GAG is a heterogeneous linear α-1,4-linked exopolysaccharide of galactose and GalNAc that is partially deacetylated after secretion. A cluster of five co-expressed genes has been linked to GAG biosynthesis and modification. One gene in this cluster, ega3, is annotated as encoding a putative α-1,4-galactosaminidase belonging to glycoside hydrolase family 114 (GH114). Herein, we show that recombinant Ega3 is an active glycoside hydrolase that disrupts GAG-dependent A. fumigatus and Pel polysaccharide-dependent Pseudomonas aeruginosa biofilms at nanomolar concentrations. Using MS and functional assays, we demonstrate that Ega3 is an endo-acting α-1,4-galactosaminidase whose activity depends on the conserved acidic residues, Asp-189 and Glu-247. X-ray crystallographic structural analysis of the apo Ega3 and an Ega3-galactosamine complex, at 1.76 and 2.09 Å resolutions, revealed a modified (β/α) 8 -fold with a deep electronegative cleft, which upon ligand binding is capped to form a tunnel. Our structural analysis coupled with in silico docking studies also uncovered the molecular determinants for galactosamine specificity and substrate binding at the -2 to +1 binding subsites. The findings in this study increase the structural and mechanistic understanding of the GH114 family, which has >600 members encoded by plant and opportunistic human pathogens, as well as in industrially used bacteria and fungi., (© 2019 Bamford et al.)

During infection, the fungal pathogen Aspergillus fumigatus forms biofilms that enhance its resistance to antimicrobials and host defenses. An integral component of the biofilm matrix is galactosaminogalactan (GAG), a cationic polymer of α-1,4-linked galactose and partially deacetylated N -acetylgalactosamine (GalNAc). Recent studies have shown that recombinant hydrolase domains from Sph3, an A. fumigatus glycoside hydrolase involved in GAG synthesis, and PelA, a multifunctional protein from Pseudomonas aeruginosa involved in Pel polysaccharide biosynthesis, can degrade GAG, disrupt A. fumigatus biofilms, and attenuate fungal virulence in a mouse model of invasive aspergillosis. The molecular mechanisms by which these enzymes disrupt biofilms have not been defined. We hypothesized that the hydrolase domains of Sph3 and PelA (Sph3 h and PelA h , respectively) share structural and functional similarities given their ability to degrade GAG and disrupt A. fumigatus biofilms. MALDI-TOF enzymatic fingerprinting and NMR experiments revealed that both proteins are retaining endo-α-1,4- N- acetylgalactosaminidases with a minimal substrate size of seven residues. The crystal structure of PelA h was solved to 1.54 Å and structure alignment to Sph3 h revealed that the enzymes share similar catalytic site residues. However, differences in the substrate-binding clefts result in distinct enzyme-substrate interactions. PelA h hydrolyzed partially deacetylated substrates better than Sph3 h , a finding that agrees well with PelA h 's highly electronegative binding cleft versus the neutral surface present in Sph3 h Our insight into PelA h 's structure and function necessitate the creation of a new glycoside hydrolase family, GH166, whose structural and mechanistic features, along with those of GH135 (Sph3), are reported here.

Pseudomonas aeruginosa is an opportunistic, nosocomial bacterial pathogen that forms persistent infections due to the formation of protective communities, known as biofilms. Once the biofilm is formed, the bacteria embedded within it are recalcitrant to antimicrobial treatment and host immune defenses. Moreover, the presence of biofilms in wounds is correlated with chronic infection and delayed healing. The current standard of care for chronic wound infections typically involves physical disruption of the biofilm via debridement and subsequent antimicrobial treatment. The glycoside hydrolases PelA h and PslG h have been demonstrated in vitro to disrupt biofilm integrity through degradation of the key biofilm matrix exopolysaccharides Pel and Psl, respectively. Herein, we demonstrate that PslG h hydrolase therapy is a promising strategy for controlling P. aeruginosa wound infections. Hydrolase treatment of P. aeruginosa biofilms resulted in increased antibiotic efficacy and penetration into the biofilm. PslG h treatment of P. aeruginosa biofilms also improved innate immune activity leading to greater complement deposition, neutrophil phagocytosis, and neutrophil reactive oxygen species production. Furthermore, when P. aeruginosa -infected wounds were treated with a combination of PslG h and tobramycin, we observed an additive effect leading to greater bacterial clearance than treatments of tobramycin or PslG h alone. This study demonstrates that PelA h and PslG h have promising therapeutic potential and that PslG h may aid in the treatment of P. aeruginosa wound infections., (Copyright © 2019 American Society for Microbiology.)

Bacterial colonization and biofilm formation on surfaces are typically mediated by the deposition of exopolysaccharides and conditioning protein layers. Pseudomonas aeruginosa is a nosocomial opportunistic pathogen that utilizes strain-specific exopolysaccharides such as Psl, Pel or alginate for both initial surface attachment and biofilm formation. To generate surfaces that resist P. aeruginosa colonization, we covalently bound a Psl-specific glycoside hydrolase (PslG h ) to several, chemically-distinct surfaces using amine functionalization (APTMS) and glutaraldehyde (GDA) linking. In situ quartz crystal microbalance (QCM) experiments and fluorescence microscopy demonstrated a complete lack of Psl adsorption on the PslG h -bound surfaces. Covalently-bound PslG h was also found to significantly reduce P. aeruginosa surface attachment and biofilm formation over extended growth periods (8 days). The PslG h surfaces showed a ∼99.9% (∼3-log) reduction in surface associated bacteria compared to control (untreated) surfaces, or those treated with inactive enzyme. This work demonstrates a non-eluting 'bioactive' surface that specifically targets a mechanism of cell adhesion, and that surface-bound glycoside hydrolase can significantly reduce surface colonization of bacteria through local, continuous enzymatic degradation of exopolysaccharide (Psl). These results have significant implications for the surface design of medical devices to keep bacteria in a planktonic state, and therefore susceptible to antibiotics and antimicrobials., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Poly-β(1,6)-N-acetyl-D-glucosamine (PNAG) is a major biofilm component of many pathogenic bacteria. The production, modification, and export of PNAG in Escherichia coli and Bordetella species require the protein products encoded by the pgaABCD operon. PgaB is a two-domain periplasmic protein that contains an N-terminal deacetylase domain and a C-terminal PNAG binding domain that is critical for export. However, the exact function of the PgaB C-terminal domain remains unclear. Herein, we show that the C-terminal domains of Bordetella bronchiseptica PgaB (PgaBBb) and E. coli PgaB (PgaBEc) function as glycoside hydrolases. These enzymes hydrolyze purified deacetylated PNAG (dPNAG) from Staphylococcus aureus, disrupt PNAG-dependent biofilms formed by Bordetella pertussis, Staphylococcus carnosus, Staphylococcus epidermidis, and E. coli, and potentiate bacterial killing by gentamicin. Furthermore, we found that PgaBBb was only able to hydrolyze PNAG produced in situ by the E. coli PgaCD synthase complex when an active deacetylase domain was present. Mass spectrometry analysis of the PgaB-hydrolyzed dPNAG substrate showed a GlcN-GlcNAc-GlcNAc motif at the new reducing end of detected fragments. Our 1.76 Å structure of the C-terminal domain of PgaBBb reveals a central cavity within an elongated surface groove that appears ideally suited to recognize the GlcN-GlcNAc-GlcNAc motif. The structure, in conjunction with molecular modeling and site directed mutagenesis led to the identification of the dPNAG binding subsites and D474 as the probable catalytic acid. This work expands the role of PgaB within the PNAG biosynthesis machinery, defines a new glycoside hydrolase family GH153, and identifies PgaB as a possible therapeutic agent for treating PNAG-dependent biofilm infections.

Galactosaminogalactan and Pel are cationic heteropolysaccharides produced by the opportunistic pathogens Aspergillus fumigatus and Pseudomonas aeruginosa , respectively. These exopolysaccharides both contain 1,4-linked N -acetyl-d-galactosamine and play an important role in biofilm formation by these organisms. Proteins containing glycoside hydrolase domains have recently been identified within the biosynthetic pathway of each exopolysaccharide. Recombinant hydrolase domains from these proteins (Sph3 h from A. fumigatus and PelA h from P. aeruginosa ) were found to degrade their respective polysaccharides in vitro. We therefore hypothesized that these glycoside hydrolases could exhibit antibiofilm activity and, further, given the chemical similarity between galactosaminogalactan and Pel, that they might display cross-species activity. Treatment of A. fumigatus with Sph3 h disrupted A. fumigatus biofilms with an EC 50 of 0.4 nM. PelA h treatment also disrupted preformed A. fumigatus biofilms with EC 50 values similar to those obtained for Sph3 h In contrast, Sph3 h was unable to disrupt P. aeruginosa Pel-based biofilms, despite being able to bind to the exopolysaccharide. Treatment of A. fumigatus hyphae with either Sph3 h or PelA h significantly enhanced the activity of the antifungals posaconazole, amphotericin B, and caspofungin, likely through increasing antifungal penetration of hyphae. Both enzymes were noncytotoxic and protected A549 pulmonary epithelial cells from A. fumigatus -induced cell damage for up to 24 h. Intratracheal administration of Sph3 h was well tolerated and reduced pulmonary fungal burden in a neutropenic mouse model of invasive aspergillosis. These findings suggest that glycoside hydrolases can exhibit activity against diverse microorganisms and may be useful as therapeutic agents by degrading biofilms and attenuating virulence., Competing Interests: Conflict of interest statement: A patent has been filed describing the utility of the glycoside hydrolases as antibiofilm therapeutics (CA2951152 A1, WO2015184526 A1). B.D.S., P.B., N.C.B., P.L.H., and D.C.S. are listed as inventors.

Bacterial biofilms present a significant medical challenge because they are recalcitrant to current therapeutic regimes. A key component of biofilm formation in the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharides Pel and Psl, which are involved in the formation and maintenance of the structural biofilm scaffold and protection against antimicrobials and host defenses. Given that the glycoside hydrolases PelAh and PslGh encoded in the pel and psl biosynthetic operons, respectively, are utilized for in vivo exopolysaccharide processing, we reasoned that these would provide specificity to target P. aeruginosa biofilms. Evaluating these enzymes as potential therapeutics, we demonstrate that these glycoside hydrolases selectively target and degrade the exopolysaccharide component of the biofilm matrix. PelAh and PslGh inhibit biofilm formation over a 24-hour period with a half maximal effective concentration (EC50) of 69.3 ± 1.2 and 4.1 ± 1.1 nM, respectively, and are capable of disrupting preexisting biofilms in 1 hour with EC50 of 35.7 ± 1.1 and 12.9 ± 1.1 nM, respectively. This treatment was effective against clinical and environmental P. aeruginosa isolates and reduced biofilm biomass by 58 to 94%. These noncytotoxic enzymes potentiated antibiotics because the addition of either enzyme to a sublethal concentration of colistin reduced viable bacterial counts by 2.5 orders of magnitude when used either prophylactically or on established 24-hour biofilms. In addition, PelAh was able to increase neutrophil killing by ~50%. This work illustrates the feasibility and benefits of using bacterial exopolysaccharide biosynthetic glycoside hydrolases to develop novel antibiofilm therapeutics.

A key component of colonization, biofilm formation, and protection of the opportunistic human pathogen Pseudomonas aeruginosa is the biosynthesis of the exopolysaccharide Psl. Composed of a pentameric repeating unit of mannose, glucose, and rhamnose, the biosynthesis of Psl is proposed to occur via a Wzx/Wzy-dependent mechanism. Previous genetic studies have shown that the putative glycoside hydrolase PslG is essential for Psl biosynthesis. To understand the function of this protein, the apo-structure of the periplasmic domain of PslG (PslG(31-442)) and its complex with mannose were determined to 2.0 and 1.9 Å resolution, respectively. Despite a domain architecture and positioning of catalytic residues similar to those of other family 39 glycoside hydrolases, PslG(31-442) exhibits a unique 32-Å-long active site groove that is distinct from other structurally characterized family members. PslG formed a complex with two mannose monosaccharides in this groove, consistent with binding data obtained from intrinsic tryptophan fluorescence. PslG was able to catalyze the hydrolysis of surface-associated Psl, and this activity was abolished in a E165Q/E276Q double catalytic variant. Surprisingly, P. aeruginosa variants with these chromosomal mutations as well as a pslG deletion mutant were still capable of forming Psl biofilms. However, overexpression of PslG in a pslG deletion background impaired biofilm formation and resulted in less surface-associated Psl, suggesting that regulation of this enzyme is important during polysaccharide biosynthesis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Aspergillus fumigatus is the most virulent species within the Aspergillus genus and causes invasive infections with high mortality rates. The exopolysaccharide galactosaminogalactan (GAG) contributes to the virulence of A. fumigatus. A co-regulated five-gene cluster has been identified and proposed to encode the proteins required for GAG biosynthesis. One of these genes, sph3, is predicted to encode a protein belonging to the spherulin 4 family, a protein family with no known function. Construction of an sph3-deficient mutant demonstrated that the gene is necessary for GAG production. To determine the role of Sph3 in GAG biosynthesis, we determined the structure of Aspergillus clavatus Sph3 to 1.25 Å. The structure revealed a (β/α)8 fold, with similarities to glycoside hydrolase families 18, 27, and 84. Recombinant Sph3 displayed hydrolytic activity against both purified and cell wall-associated GAG. Structural and sequence alignments identified three conserved acidic residues, Asp-166, Glu-167, and Glu-222, that are located within the putative active site groove. In vitro and in vivo mutagenesis analysis demonstrated that all three residues are important for activity. Variants of Asp-166 yielded the greatest decrease in activity suggesting a role in catalysis. This work shows that Sph3 is a glycoside hydrolase essential for GAG production and defines a new glycoside hydrolase family, GH135., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The biofilm matrix is a fortress; sheltering bacteria in a protective and nourishing barrier that allows for growth and adaptation to various surroundings. A variety of different components are found within the matrix including water, lipids, proteins, extracellular DNA, RNA, membrane vesicles, phages, and exopolysaccharides. As part of its biofilm matrix, Pseudomonas aeruginosa is genetically capable of producing three chemically distinct exopolysaccharides – alginate, Pel, and Psl – each of which has a distinct role in biofilm formation and immune evasion during infection. The polymers are produced by highly conserved mechanisms of secretion, involving many proteins that span both the inner and outer bacterial membranes. Experimentally determined structures, predictive modelling of proteins whose structures are yet to be solved, and structural homology comparisons give us insight into the molecular mechanisms of these secretion systems, from polymer synthesis to modification and export. Here, we review recent advances that enhance our understanding of P. aeruginosa multiprotein exopolysaccharide biosynthetic complexes, and how the glycoside hydrolases/lyases within these systems have been commandeered for antimicrobial applications. [ABSTRACT FROM AUTHOR]

[Display omitted] Biofilm-dispersing enzymes degrade the extracellular polymeric matrix surrounding bacterial biofilms, disperse the microbial community and increase their susceptibility to antibiotics and immune cells. Challenges for the clinical translation of biofilm-dispersing enzymes involve their susceptibility to denaturation, degradation, and clearance upon administration in vivo. Drug delivery systems aim to overcome these limitations through encapsulation, stabilization and protection from the exterior environment, thereby maintaining the enzymatic activity. Smart drug delivery systems offer target specificity, releasing payloads at the site of infection while minimizing unnecessary systemic exposure. This review highlights critical advances of biofilm-dispersing enzymes as a novel therapeutic approach for biofilm-associated infections. We explore how smart, bio-responsive delivery systems overcome the limiting factors of biofilm-dispersing enzymes and summarize the key systems designed. This review will guide future developments, focusing on utilizing selective and specific therapies in a targeted fashion to meet the unmet therapeutic needs of biofilm infections. [ABSTRACT FROM AUTHOR]