Biofilm formation is a process in which microbial cells aggregate to form collectives that are embedded in a self-produced extracellular matrix. Bacillus subtilis is a Gram-positive bacterium that is ...used to dissect the mechanisms controlling matrix production and the subsequent transition from a motile planktonic cell state to a sessile biofilm state. The collective nature of life in a biofilm allows emergent properties to manifest, and B. subtilis biofilms are linked with novel industrial uses as well as probiotic and biocontrol processes. In this Review, we outline the molecular details of the biofilm matrix and the regulatory pathways and external factors that control its production. We explore the beneficial outcomes associated with biofilms. Finally, we highlight major advances in our understanding of concepts of microbial evolution and community behaviour that have resulted from studies of the innate heterogeneity of biofilms.
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 (Sph3h and PelAh, 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 PelAh was solved to 1.54 Å and structure alignment to Sph3h revealed that the enzymes share similar catalytic site residues. However, differences in the substrate-binding clefts result in distinct enzyme-substrate interactions. PelAh hydrolyzed partially deacetylated substrates better than Sph3h, a finding that agrees well with PelAh's highly electronegative binding cleft versus the neutral surface present in Sph3h. Our insight into PelAh'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.
The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus. Deletion of a gene encoding a putative deacetylase, Agd3, leads to ...defects in GAG deacetylation, biofilm formation, and virulence. Here, we show that Agd3 deacetylates GAG in a metal-dependent manner, and is the founding member of carbohydrate esterase family CE18. The active site is formed by four catalytic motifs that are essential for activity. The structure of Agd3 includes an elongated substrate-binding cleft formed by a carbohydrate binding module (CBM) that is the founding member of CBM family 87. Agd3 homologues are encoded in previously unidentified putative bacterial exopolysaccharide biosynthetic operons and in other fungal genomes.
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.
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.
Poly-β-1,6-N-acetyl-D-glucosamine (PNAG) is an exopolysaccharide produced by a wide variety of medically important bacteria. Poly-glucosamine subunit B (PgaB) is responsible for the de—N-acetylation ...of PNAG, a process required for polymer export and biofilm formation. PgaB is located in the periplasm and likely bridges the inner membrane synthesis and outer membrane export machinery. Here, we present structural, functional, and molecular simulation data that suggest PgaB associates with PNAG continuously during periplasmic transport. We show that the association of PgaB's N- and C-terminal domains forms a cleft required for the binding and de—N-acetylation of PNAG. Molecular dynamics (MD) simulations of PgaB show a binding preference for N-acetylglucosamine (GlcNAc) to the N-terminal domain and glucosammonium to the C-terminal domain. Continuous ligand binding density is observed that extends around PgaB from the N-terminal domain active site to an electronegative groove on the C-terminal domain that would allow for a processive mechanism. PgaB's C-terminal domain (PgaB 310—672 ) directly binds PNAG oligomers with dissociation constants of ∼1—3 mM, and the structures of PgaB 310—672 in complex with β-1,6-(GlcNAc) 6 , GlcNAc, and glucosamine reveal a unique binding mode suitable for interaction with de—N-acetylated PNAG (dPNAG). Furthermore, PgaB 310—672 contains a β-hairpin loop (βHL) important for binding PNAG that was disordered in previous PgaB 42—655 structures and is highly dynamic in the MD simulations. We propose that conformational changes in PgaB 310—672 mediated by the βHL on binding of PNAG/dPNAG play an important role in the targeting of the polymer for export and its release.
Microbial growth often occurs within multicellular communities called biofilms, where cells are enveloped by a protective extracellular matrix. Bacillus subtilis serves as a model organism for ...biofilm research and produces two crucial secreted proteins, BslA and TasA, vital for biofilm matrix formation. BslA exhibits surface-active properties, spontaneously self-assembling at hydrophobic/hydrophilic interfaces to form an elastic protein film, which renders B. subtilis biofilm surfaces water-repellent. TasA is traditionally considered a fiber-forming protein with multiple matrix-related functions. In our current study, we investigate whether TasA also possesses interfacial properties and whether it has any impact on BslA’s ability to form an interfacial protein film. Our research demonstrates that TasA indeed exhibits interfacial activity, partitioning to hydrophobic/hydrophilic interfaces, stabilizing emulsions, and forming an interfacial protein film. Interestingly, TasA undergoes interface-induced restructuring similar to BslA, showing an increase in β-strand secondary structure. Unlike BslA, TasA rapidly reaches the interface and forms nonelastic films that rapidly relax under pressure. Through mixed protein pendant drop experiments, we assess the influence of TasA on BslA film formation, revealing that TasA and other surface-active molecules can compete for interface space, potentially preventing BslA from forming a stable elastic film. This raises a critical question: how does BslA self-assemble to form the hydrophobic “raincoat” observed in biofilms in the presence of other potentially surface-active species? We propose a model wherein surface-active molecules, including TasA, initially compete with BslA for interface space. However, under lateral compression or pressure, BslA retains its position, expelling other molecules into the bulk. This resilience at the interface may result from structural rearrangements and lateral interactions between BslA subunits. This combined mechanism likely explains BslA’s role in forming a stable film integral to B. subtilis biofilm hydrophobicity.
The mold Aspergillus fumigatus causes invasive infection in immunocompromised patients. Recently, galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetylgalactosamine ...(GalNAc), was identified as a virulence factor required for biofilm formation. The molecular mechanisms underlying GAG biosynthesis and GAG-mediated biofilm formation were unknown. We identified a cluster of five coregulated genes that were dysregulated in GAG-deficient mutants and whose gene products share functional similarity with proteins that mediate the synthesis of the bacterial biofilm exopolysaccharide poly-(β1-6)-N-acetyl-D-glucosamine (PNAG). Bioinformatic analyses suggested that the GAG cluster gene agd3 encodes a protein containing a deacetylase domain. Because deacetylation of N-acetylglucosamine residues is critical for the function of PNAG, we investigated the role of GAG deacetylation in fungal biofilm formation. Agd3 was found to mediate deacetylation of GalNAc residues within GAG and render the polysaccharide polycationic. As with PNAG, deacetylation is required for the adherence of GAG to hyphae and for biofilm formation. Growth of the Δagd3 mutant in the presence of culture supernatants of the GAG-deficient Δuge3 mutant rescued the biofilm defect of the Δagd3 mutant and restored the adhesive properties of GAG, suggesting that deacetylation is an extracellular process. The GAG biosynthetic gene cluster is present in the genomes of members of the Pezizomycotina subphylum of the Ascomycota including a number of plant-pathogenic fungi and a single basidiomycete species,Trichosporon asahii, likely a result of recent horizontal gene transfer. The current study demonstrates that the production of cationic, deacetylated exopolysaccharides is a strategy used by both fungi and bacteria for biofilm formation.
This study sheds light on the biosynthetic pathways governing the synthesis of galactosaminogalactan (GAG), which plays a key role in A. fumigatus virulence and biofilm formation. We find that bacteria and fungi use similar strategies to synthesize adhesive biofilm exopolysaccharides. The presence of orthologs of the GAG biosynthetic gene clusters in multiple fungi suggests that this exopolysaccharide may also be important in the virulence of other fungal pathogens. Further, these studies establish a molecular mechanism of adhesion in which GAG interacts via charge-charge interactions to bind to both fungal hyphae and other substrates. Finally, the importance of deacetylation in the synthesis of functional GAG and the extracellular localization of this process suggest that inhibition of deacetylation may be an attractive target for the development of novel antifungal therapies.
The bacterium Pseudomonas aeruginosa can colonize the airways of patients with chronic lung disease. Within the lung, P. aeruginosa forms biofilms that can enhance resistance to antibiotics and ...immune defenses. P. aeruginosa biofilm formation is dependent on the secretion of matrix exopolysaccharides, including Pel and Psl. In this study, recombinant glycoside hydrolases (GHs) that degrade Pel and Psl were evaluated alone and in combination with antibiotics in a mouse model of P. aeruginosa infection. Intratracheal GH administration was well tolerated by mice. Pharmacokinetic analysis revealed that, although GHs have short half-lives, administration of two GHs in combination resulted in increased GH persistence. Combining GH prophylaxis and treatment with the antibiotic ciprofloxacin resulted in greater reduction in pulmonary bacterial burden than that with either agent alone. This study lays the foundation for further exploration of GH therapy in bacterial 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 (Sph3h from A. fumigatus and PelAh 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 Sph3h disrupted A. fumigatus biofilms with an EC50 of 0.4 nM. PelAh treatment also disrupted preformed A. fumigatus biofilms with EC50 values similar to those obtained for Sph3h. In contrast, Sph3h was unable to disrupt P. aeruginosa Pel-based biofilms, despite being able to bind to the exopolysaccharide. Treatment of A. fumigatus hyphae with either Sph3h or PelAh 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 Sph3h 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.