Biofilm formation is a co‐operative behaviour, where microbial cells become embedded in an extracellular matrix. This biomolecular matrix helps manifest the beneficial or detrimental outcome mediated ...by the collective of cells. Bacillus subtilis is an important bacterium for understanding the principles of biofilm formation. The protein components of the B. subtilis matrix include the secreted proteins BslA, which forms a hydrophobic coat over the biofilm, and TasA, which forms protease‐resistant fibres needed for structuring. TapA is a secreted protein also needed for biofilm formation and helps in vivo TasA‐fibre formation but is dispensable for in vitro TasA‐fibre assembly. We show that TapA is subjected to proteolytic cleavage in the colony biofilm and that only the first 57 amino acids of the 253‐amino acid protein are required for colony biofilm architecture. Through the construction of a strain which lacks all eight extracellular proteases, we show that proteolytic cleavage by these enzymes is not a prerequisite for TapA function. It remains unknown why TapA is synthesised at 253 amino acids when the first 57 are sufficient for colony biofilm structuring; the findings do not exclude the core conserved region of TapA having a second role beyond structuring the B. subtilis colony biofilm.
In the Bacillus subtilis biofilm matrix, TapA undergoes proteolytic cleavage. TapA secretion is aided by the action of a signal peptidease called SipW. An unknown protease results in the removal of the C‐terminus and the serine exoprotease Vpr cleaves the N‐terminus. Although proteolytic processing was not essential for TapA activity, it was found that a minimal functional form (yellow) was sufficient to facilitate rugose biofilm architecture.
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.
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.
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.
The pathways governing biosynthesis of the Aspergillus fumigatus exopolysaccharide galactosaminogalactan are poorly understood.
The structure of Sph3 revealed a (β/α)8 barrel fold. The enzyme hydrolyzes galactosaminogalactan and is required for the synthesis of this exopolysaccharide.
Sph3 is a glycoside hydrolase (GH) whose activity is essential for galactosaminogalactan biosynthesis.
Sph3 defines a new glycoside hydrolase superfamily, GH family 135.
The soil bacterium
is a model organism to investigate the formation of biofilms, the predominant form of microbial life. The secreted protein BslA self-assembles at the surface of the biofilm to give ...the
biofilm its characteristic hydrophobicity. To understand the mechanism of BslA self-assembly at interfaces, here we built a molecular model based on the previous BslA crystal structure and the crystal structure of the BslA paralogue YweA that we determined. Our analysis revealed two conserved protein-protein interaction interfaces supporting BslA self-assembly into an infinite 2-dimensional lattice that fits previously determined transmission microscopy images. Molecular dynamics simulations and in vitro protein assays further support our model of BslA elastic film formation, while mutagenesis experiments highlight the importance of the identified interactions for biofilm structure. Based on this knowledge, YweA was engineered to form more stable elastic films and rescue biofilm structure in
deficient strains. These findings shed light on protein film assembly and will inform the development of BslA technologies which range from surface coatings to emulsions in fast-moving consumer goods.
Bacterial surface (S) layers are paracrystalline arrays of protein assembled on the bacterial cell wall that serve as protective barriers and scaffolds for housekeeping enzymes and virulence factors. ...The attachment of S-layer proteins to the cell walls of the Bacillus cereus sensu lato, which includes the pathogen Bacillus anthracis, occurs through noncovalent interactions between their S-layer homology domains and secondary cell wall polysaccharides. To promote these interactions, it is presumed that the terminal N-acetylmannosamine (ManNAc) residues of the secondary cell wall polysaccharides must be ketal-pyruvylated. For a few specific S-layer proteins, the O-acetylation of the penultimate N-acetylglucosamine (GlcNAc) is also required. Herein, we present the X-ray crystal structure of the SLH domain of the major surface array protein Sap from B. anthracis in complex with 4,6-O-ketal-pyruvyl-β-ManNAc-(1,4)-β-GlcNAc-(1,6)-α-GlcN. This structure reveals for the first time that the conserved terminal SCWP unit is the direct ligand for the SLH domain. Furthermore, we identify key binding interactions that account for the requirement of 4,6-O-ketal-pyruvyl-ManNAc while revealing the insignificance of the O-acetylation on the GlcNAc residue for recognition by Sap.
Bordetella pertussis and Bordetella bronchiseptica are the causative agents of whooping cough in humans and a variety of respiratory diseases in animals, respectively. Bordetella species produce an ...exopolysaccharide, known as the Bordetella polysaccharide (Bps), which is encoded by the bpsABCD operon. Bps is required for Bordetella biofilm formation, colonization of the respiratory tract, and confers protection from complement-mediated killing. In this report, we have investigated the role of BpsB in the biosynthesis of Bps and biofilm formation by B. bronchiseptica. BpsB is a two-domain protein that localizes to the periplasm and outer membrane. BpsB displays metal- and length-dependent deacetylation on poly-β-1,6-N-acetyl-d-glucosamine (PNAG) oligomers, supporting previous immunogenic data that suggests Bps is a PNAG polymer. BpsB can use a variety of divalent metal cations for deacetylase activity and showed highest activity in the presence of Ni2+ and Co2+. The structure of the BpsB deacetylase domain is similar to the PNAG deacetylases PgaB and IcaB and contains the same circularly permuted family four carbohydrate esterase motifs. Unlike PgaB from Escherichia coli, BpsB is not required for polymer export and has unique structural differences that allow the N-terminal deacetylase domain to be active when purified in isolation from the C-terminal domain. Our enzymatic characterizations highlight the importance of conserved active site residues in PNAG deacetylation and demonstrate that the C-terminal domain is required for maximal deacetylation of longer PNAG oligomers. Furthermore, we show that BpsB is critical for the formation and complex architecture of B. bronchiseptica biofilms.
Background: The Bordetella polysaccharide (Bps) is involved in Bordetella biofilm formation.
Results: BpsB is a periplasmic metal-dependent poly-β-1,6-N-acetyl-d-glucosamine (PNAG) deacetylase that has unique structural and functional features from known PNAG deacetylases.
Conclusion: BpsB-dependent deacetylation of Bps is required for Bordetella bronchiseptica biofilm formation.
Significance: Deacetylated Bps is a key component for the structural complexity of Bordetella biofilms.
Exopolysaccharides are required for the development and integrity of biofilms produced by a wide variety of bacteria. In staphylococci, partial de-N-acetylation of the exopolysaccharide ...poly-β-1,6-N-acetyl-d-glucosamine (PNAG) by the extracellular protein IcaB is required for biofilm formation. To understand the molecular basis for PNAG de-N-acetylation, the structure of IcaB from Ammonifex degensii (IcaBAd) has been determined to 1.7 Å resolution. The structure of IcaBAd reveals a (β/α)7 barrel common to the family four carbohydrate esterases (CE4s) with the canonical motifs circularly permuted. The metal dependence of IcaBAd is similar to most CE4s showing the maximum rates of de-N-acetylation with Ni2+, Co2+, and Zn2+. From docking studies with β-1,6-GlcNAc oligomers and structural comparison to PgaB from Escherichia coli, the Gram-negative homologue of IcaB, we identify Arg-45, Tyr-67, and Trp-180 as key residues for PNAG binding during catalysis. The absence of these residues in PgaB provides a rationale for the requirement of a C-terminal domain for efficient deacetylation of PNAG in Gram-negative species. Mutational analysis of conserved active site residues suggests that IcaB uses an altered catalytic mechanism in comparison to other characterized CE4 members. Furthermore, we identified a conserved surface-exposed hydrophobic loop found only in Gram-positive homologues of IcaB. Our data suggest that this loop is required for membrane association and likely anchors IcaB to the membrane during polysaccharide biosynthesis. The work presented herein will help guide the design of IcaB inhibitors to combat biofilm formation by staphylococci.IcaB is a poly-β-1,6-N-acetyl-d-glucosamine (PNAG) deacetylase required for polysaccharide intercellular adhesion-dependent biofilm formation by staphylococci.
The structure of Ammonifex degensii IcaB has been determined and its catalytic mechanism and localization characterized.
IcaB is a membrane-associated PNAG deacetylase that uses an altered catalytic mechanism relative to other family 4 carbohydrate esterases.
First structural characterization of a Gram-positive PNAG deacetylase.
The mold
and bacterium
form biofilms in the airways of individuals with cystic fibrosis. Biofilm formation by
depends on the self-produced cationic exopolysaccharide galactosaminogalactan (GAG), ...while
biofilms can contain the cationic exopolysaccharide Pel. GAG and Pel are rendered cationic by deacetylation mediated by either the secreted deacetylase Agd3 (
) or the periplasmic deacetylase PelA (
). Given the similarities between these polymers, the potential for biofilm interactions between these organisms were investigated.
were observed to adhere to
hyphae in a GAG-dependent manner and to GAG-coated coverslips of
biofilms. In biofilm adherence assays, incubation of
with
culture supernatants containing de-
-acetylated GAG augmented the formation of adherent
biofilms, increasing protection against killing by the antibiotic colistin. Fluorescence microscopy demonstrated incorporation of GAG within
biofilms, suggesting that GAG can serve as an alternate biofilm exopolysaccharide for this bacterium. In contrast, Pel-containing bacterial culture supernatants only augmented the formation of adherent
biofilms when antifungal inhibitory molecules were removed. This study demonstrates biofilm interaction via exopolysaccharides as a potential mechanism of co-operation between these organisms in chronic lung disease.
Biofilms are complex communities of microbes that are bound by an extracellular macromolecular matrix produced by the residents. Biofilms are the predominant form of microbial life in the natural ...environment and although they are the leading cause of chronic infections, they are equally deeply connected to our ability to bioremediate waste and toxic materials. Here we highlight the emergent properties of biofilm communities and explore notable biofilms before concluding by providing examples of their major impact on our health and both natural and built environments.
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