A bacterial pigment provides cross-species protection from H2O2- and neutrophil-mediated killing.

Bacterial infections are often polymicrobial. Pseudomonas aeruginosa and Staphylococcus aureus cause chronic co-infections, which are more problematic than mono-species infections. Understanding the mechanisms of their interactions is crucial for treating co-infections. Staphyloxanthin (STX), a yellow pigment synthesized by the S. aureus crt operon, promotes S. aureus resistance to oxidative stress and neutrophil-mediated killing. We found that STX production by S. aureus, either as surface-grown macrocolonies or planktonic cultures, was elevated when exposed to the P. aeruginosa exoproduct, 2-heptyl-4-hydroxyquinoline N-oxide (HQNO). This was observed with both mucoid and non-mucoid P. aeruginosa strains. The induction phenotype was found in a majority of P. aeruginosa and S. aureus clinical isolates examined. When subjected to hydrogen peroxide or human neutrophils, P. aeruginosa survival was significantly higher when mixed with wild-type (WT) S. aureus, compared to P. aeruginosa alone or with an S. aureus crt mutant deficient in STX production. In a murine wound model, co-infection with WT S. aureus, but not the STX-deficient mutant, enhanced P. aeruginosa burden and disease compared to mono-infection. In conclusion, we identified a role for P. aeruginosa HQNO mediating polymicrobial interactions with S. aureus by inducing STX production, which consequently promotes resistance to the innate immune effectors H2O2 and neutrophils. These results further our understanding of how different bacterial species cooperatively cause co-infections.

The Wsp system of Pseudomonas aeruginosa links surface sensing and cell envelope stress.

Surface sensing is a critical process that promotes the transition to a biofilm lifestyle. Several surface-sensing mechanisms have been described for a range of species, most involving surface appendages, such as flagella and pili. Pseudomonas aeruginosa uses the Wsp chemosensory-like signal transduction pathway to sense surfaces and promote biofilm formation. The methyl-accepting chemotaxis protein WspA recognizes an unknown surface-associated signal and initiates a phosphorylation cascade that activates the diguanylate cyclase WspR. We conducted a screen for Wsp-activating compounds and found that chemicals that impact the cell envelope induce Wsp signaling, increase intracellular c-di-GMP levels, and can promote surface attachment. To isolate the Wsp system from other P. aeruginosa surface-sensing systems, we heterologously expressed it in Escherichia coli and found it sufficient for sensing surfaces and the chemicals identified in our screen. Using well-characterized reporters for different E. coli cell envelope stress responses, we then determined that Wsp sensitivity overlapped with multiple E. coli cell envelope stress-response systems. Using mutational and CRISPRi analysis, we found that misfolded proteins in the periplasm appear to be a major stimulus of the Wsp system. Finally, we show that surface attachment appears to have an immediate, observable effect on cell envelope integrity. Collectively, our results provide experimental evidence that cell envelope stress represents an important feature of surface sensing in P. aeruginosa.

Binding of GTP to BifA is required for the production of Pel-dependent biofilms in Pseudomonas aeruginosa.

The Pel exopolysaccharide is one of the most mechanistically conserved and phylogenetically diverse bacterial biofilm matrix determinants. Pel is a major contributor to the structural integrity of Pseudomonas aeruginosa biofilms, and its biosynthesis is regulated by the binding of cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP) to the PelD receptor. c-di-GMP is synthesized from two molecules of guanosine triphosphate (GTP) by diguanylate cyclases with GGDEF domains and degraded by phosphodiesterases with EAL or HD-GYP domains. As the P. aeruginosa genome encodes 43 c-di-GMP metabolic enzymes, one way signaling specificity can be achieved is through direct interaction between specific enzyme-receptor pairs. Here, we show that the inner membrane hybrid GGDEF-EAL enzyme, BifA, directly interacts with PelD via its cytoplasmic HAMP, GGDEF, and EAL domains. Despite having no catalytic function, the degenerate active site motif of the BifA GGDEF domain (GGDQF) has retained the ability to bind GTP with micromolar affinity. Mutations that abolish GTP binding result in increased biofilm formation but stable global c-di-GMP levels. Our data suggest that BifA forms a dimer in solution and that GTP binding induces conformational changes in dimeric BifA that enhance the BifA-PelD interaction and stimulate its phosphodiesterase activity, thus reducing c-di-GMP levels and downregulating Pel biosynthesis. Structural comparisons between the dimeric AlphaFold2 model of BifA and the structures of other hybrid GGDEF-EAL proteins suggest that the regulation of BifA by GTP may occur through a novel mechanism.IMPORTANCEc-di-GMP is the most common cyclic dinucleotide used by bacteria to regulate phenotypes such as motility, biofilm formation, virulence factor production, cell cycle progression, and cell differentiation. While the identification and initial characterization of c-di-GMP metabolic enzymes are well established, our understanding of how these enzymes are regulated to provide signaling specificity remains understudied. Here we demonstrate that the inactive GGDEF domain of BifA binds GTP and regulates the adjacent phosphodiesterase EAL domain, ultimately downregulating Pel-dependent P. aeruginosa biofilm formation through an interaction with PelD. This discovery adds to the growing body of literature regarding how hybrid GGDEF-EAL enzymes are regulated and provides additional precedence for studying how direct interactions between c-di-GMP metabolic enzymes and effectors result in signaling specificity.

Extracellular DNA enhances biofilm integrity and mechanical properties of mucoid Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most common biofilm-forming pathogens responsible for lung infections of individuals with cystic fibrosis (CF). P. aeruginosa becomes tolerant to antimicrobials in the biofilm state and is difficult to treat. Production of extracellular polymeric substances (EPS), such as alginate and extracellular DNA (eDNA), can allow adherence to abiotic and biotic surfaces, antimicrobial evasion, and resilience to environmental pressures. Alginate-producing mucoid variants of P. aeruginosa are frequently isolated from CF airway samples and are associated with worsening patient outcomes. While eDNA is a major structural component of nonmucoid P. aeruginosa biofilms, the potential role of eDNA in mucoid biofilms is unclear. Here, we investigate how eDNA contributes to clinical mucoid biofilm physiology and integrity. We predicted that eDNA plays a structural and mechanical role in mucoid biofilms. To test this, we quantified biofilm eDNA in mucoid biofilms and used microscopy and rheology to visualize eDNA and detect changes in biofilm structure and mechanics upon DNaseI treatment. We showed that biofilm eDNA abundance is diverse across clinical mucoid strains and observed a temporal increase in foci of eDNA within intact mucoid biofilms. Increased cell dispersal and reduced biomass were also observed following DNaseI treatment of mucoid biofilms. Degradation of eDNA also impacted the mechanical integrity of mucoid biofilms by increasing the stiffness and decreasing the cohesion of the biofilm. These findings advance our understanding of clinical mucoid P. aeruginosa biofilms and facilitate the development of new approaches to target biofilms by exploiting the functions of EPS components. IMPORTANCE Understanding the role of eDNA in mucoid Pseudomonas aeruginosa biofilms will lead to therapeutic strategies that combat the biophysical and structural function of EPS for the eradication of bacteria in mucoid biofilms during chronic infections. This knowledge can be used to further identify unknown matrix component interactions within pathogenic biofilm-forming clinical isolates.

The Pseudomonas aeruginosa homeostasis enzyme AlgL clears the periplasmic space of accumulated alginate during polymer biosynthesis.

Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of chronic infection in the lungs of individuals with cystic fibrosis. After colonization, P. aeruginosa often undergoes a phenotypic conversion to mucoidy, characterized by overproduction of the alginate exopolysaccharide. This conversion is correlated with poorer patient prognoses. The majority of genes required for alginate synthesis, including the alginate lyase, algL, are located in a single operon. Previous investigations of AlgL have resulted in several divergent hypotheses regarding the protein's role in alginate production. To address these discrepancies, we determined the structure of AlgL and, using multiple sequence alignments, identified key active site residues involved in alginate binding and catalysis. In vitro enzymatic analysis of active site mutants highlights R249 and Y256 as key residues required for alginate lyase activity. In a genetically engineered P. aeruginosa strain where alginate biosynthesis is under arabinose control, we found that AlgL is required for cell viability and maintaining membrane integrity during alginate production. We demonstrate that AlgL functions as a homeostasis enzyme to clear the periplasmic space of accumulated polymer. Constitutive expression of the AlgU/T sigma factor mitigates the effects of an algL deletion during alginate production, suggesting that an AlgU/T-regulated protein or proteins can compensate for an algL deletion. Together, our study demonstrates the role of AlgL in alginate biosynthesis, explains the discrepancies observed previously across other P. aeruginosa ΔalgL genetic backgrounds, and clarifies the existing divergent data regarding the function of AlgL as an alginate degrading enzyme.

Elevated exopolysaccharide levels in Pseudomonas aeruginosa flagellar mutants have implications for biofilm growth and chronic infections.

Pseudomonas aeruginosa colonizes the airways of cystic fibrosis (CF) patients, causing infections that can last for decades. During the course of these infections, P. aeruginosa undergoes a number of genetic adaptations. One such adaptation is the loss of swimming motility functions. Another involves the formation of the rugose small colony variant (RSCV) phenotype, which is characterized by overproduction of the exopolysaccharides Pel and Psl. Here, we provide evidence that the two adaptations are linked. Using random transposon mutagenesis, we discovered that flagellar mutations are linked to the RSCV phenotype. We found that flagellar mutants overexpressed Pel and Psl in a surface-contact dependent manner. Genetic analyses revealed that flagellar mutants were selected for at high frequencies in biofilms, and that Pel and Psl expression provided the primary fitness benefit in this environment. Suppressor mutagenesis of flagellar RSCVs indicated that Psl overexpression required the mot genes, suggesting that the flagellum stator proteins function in a surface-dependent regulatory pathway for exopolysaccharide biosynthesis. Finally, we identified flagellar mutant RSCVs among CF isolates. The CF environment has long been known to select for flagellar mutants, with the classic interpretation being that the fitness benefit gained relates to an impairment of the host immune system to target a bacterium lacking a flagellum. Our new findings lead us to propose that exopolysaccharide production is a key gain-of-function phenotype that offers a new way to interpret the fitness benefits of these mutations.

Mucoid Pseudomonas aeruginosa Can Produce Calcium-Gelled Biofilms Independent of the Matrix Components Psl and CdrA.

Biofilms are aggregates of microorganisms embedded in an extracellular matrix comprised largely of exopolysaccharides (EPSs), nucleic acids, and proteins. Pseudomonas aeruginosa is an opportunistic human pathogen that is also a model organism for studying biofilms in the laboratory. Here, we define a novel program of biofilm development used by mucoid (alginate-overproducing) P. aeruginosa in the presence of elevated calcium. Calcium cations cross-link negatively charged alginate polymers, resulting in individual cells being suspended in an alginate gel. The formation of this type of structurally distinct biofilm is not reliant on the canonical biofilm EPS components Psl and Pel or the matrix protein CdrA. We also observed that mucoid P. aeruginosa biofilm cells do not have the typical elevated levels of the secondary messenger cyclic di-GMP (c-di-GMP), as expected of biofilm cells, nor does the overproduction of alginate rely on high c-di-GMP. This contrasts with nonmucoid biofilms in which the production of the matrix components Psl, Pel, and CdrA is positively regulated by elevated c-di-GMP. We further demonstrate that calcium-gelled alginate biofilms impede the penetration of the antibiotic tobramycin, thus protecting the biofilm community from antibiotic-mediated killing. Finally, we show that bacterial aggregates with a dispersed cell arrangement like laboratory-grown calcium-alginate biofilm structures are present in explanted cystic fibrosis (CF) lung samples. Our findings illustrate the diverse nature of biofilm formation and structure in P. aeruginosa. IMPORTANCE The opportunistic pathogen Pseudomonas aeruginosa produces a complex biofilm matrix comprised of exopolysaccharides (EPSs), nucleic acids, and proteins. P. aeruginosa biofilm formation canonically depends on a variable combination of the exopolysaccharides Psl and Pel and the matrix protein CdrA. We demonstrate that mucoid P. aeruginosa, which overproduces the EPS alginate, possesses an entirely alternate and calcium-dependent method of biofilm formation. These mucoid biofilm structures do not require Psl, Pel, or CdrA, and they display a unique organization of individually suspended cells similar to bacterial aggregates observed in cystic fibrosis airways. Furthermore, calcium-gelled mucoid biofilms impede the penetration and killing action of the antibiotic tobramycin, illustrating their potential clinical significance. Our findings highlight the compositional and structural variety of P. aeruginosa biofilm aggregates.

Interbacterial Antagonism Mediated by a Released Polysaccharide.

Pseudomonas aeruginosa and Staphylococcus aureus are two common pathogens causing chronic infections in the lungs of people with cystic fibrosis (CF) and in wounds, suggesting that these two organisms coexist in vivo. However, P. aeruginosa utilizes various mechanisms to antagonize S. aureus when these organisms are grown together in vitro. Here, we suggest a novel role for Psl in antagonizing S. aureus growth. Psl is an exopolysaccharide that exists in both cell-associated and cell-free forms and is important for biofilm formation in P. aeruginosa. When grown in planktonic coculture with a P. aeruginosa psl mutant, S. aureus had increased survival compared to when it was grown with wild-type P. aeruginosa. We found that cell-free Psl was critical for the killing, as purified cell-free Psl was sufficient to kill S. aureus. Transmission electron microscopy of S. aureus treated with Psl revealed disrupted cell envelopes, suggesting that Psl causes S. aureus cell lysis. This was independent of known mechanisms used by P. aeruginosa to antagonize S. aureus. Cell-free Psl could also promote S. aureus killing during growth in in vivo-like conditions. We also found that Psl production in P. aeruginosa CF clinical isolates positively correlated with the ability to kill S. aureus. This could be a result of P. aeruginosa coevolution with S. aureus in CF lungs. In conclusion, this study defines a novel role for P. aeruginosa Psl in killing S. aureus, potentially impacting the coexistence of these two opportunistic pathogens in vivo. IMPORTANCE Pseudomonas aeruginosa and Staphylococcus aureus are two important opportunistic human pathogens commonly coisolated from clinical samples. However, P. aeruginosa can utilize various mechanisms to antagonize S. aureus in vitro. Here, we investigated the interactions between these two organisms and report a novel role for P. aeruginosa exopolysaccharide Psl in killing S. aureus. We found that cell-free Psl could kill S. aureus in vitro, possibly by inducing cell lysis. This was also observed in conditions reflective of in vivo scenarios. In accord with this, Psl production in P. aeruginosa clinical isolates positively correlated with their ability to kill S. aureus. Together, our data suggest a role for Psl in affecting the coexistence of P. aeruginosa and S. aureus in vivo.

The Sia System and c-di-GMP Play a Crucial Role in Controlling Cell-Association of Psl in Planktonic P. aeruginosa.

Many bacterial species use the secondary messenger, c-di-GMP, to promote the production of biofilm matrix components. In Pseudomonas aeruginosa, c-di-GMP production is stimulated upon initial surface contact and generally remains high throughout biofilm growth. Transcription of several gene clusters, including the Sia signal transduction system, are induced in response to high cellular levels of c-di-GMP. The output of this system is SiaD, a diguanylate cyclase whose activity is induced in the presence of the detergent SDS. Previous studies demonstrated that Sia-mediated cellular aggregation is a key feature of P. aeruginosa growth in the presence of SDS. Here, we show that the Sia system is important for producing low levels of c-di-GMP when P. aeruginosa is growing planktonically. In addition, we show that Sia activity is important for maintaining cell-associated Psl in planktonic populations. We also demonstrate that Sia mutant strains have reduced cell-associated Psl and a surface attachment-deficient phenotype. The Sia system also appears to posttranslationally impact cell-associated Psl levels. Collectively, our findings suggest a novel role for the Sia system and c-di-GMP in planktonic populations by regulating levels of cell-associated Psl.

PelX is a UDP-N-acetylglucosamine C4-epimerase involved in Pel polysaccharide-dependent biofilm formation.

Pel is a GalNAc-rich bacterial polysaccharide that contributes to the structure and function of Pseudomonas aeruginosa biofilms. The pelABCDEFG operon is highly conserved among diverse bacterial species, and Pel may therefore be a widespread biofilm determinant. Previous annotation of pel gene clusters has helped us identify an additional gene, pelX, that is present adjacent to pelABCDEFG in >100 different bacterial species. The pelX gene is predicted to encode a member of the short-chain dehydrogenase/reductase (SDR) superfamily, but its potential role in Pel-dependent biofilm formation is unknown. Herein, we have used Pseudomonas protegens Pf-5 as a model to elucidate PelX function as Pseudomonas aeruginosa lacks a pelX homologue in its pel gene cluster. We found that P. protegens forms Pel-dependent biofilms; however, despite expression of pelX under these conditions, biofilm formation was unaffected in a ΔpelX strain. This observation led us to identify a pelX paralogue, PFL_5533, which we designate here PgnE, that appears to be functionally redundant to pelX In line with this, a ΔpelX ΔpgnE double mutant was substantially impaired in its ability to form Pel-dependent biofilms. To understand the molecular basis for this observation, we determined the structure of PelX to 2.1 Å resolution. The structure revealed that PelX resembles UDP-GlcNAc C4-epimerases. Using 1H NMR analysis, we show that PelX catalyzes the epimerization between UDP-GlcNAc and UDP-GalNAc. Our results indicate that Pel-dependent biofilm formation requires a UDP-GlcNAc C4-epimerase that generates the UDP-GalNAc precursors required by the Pel synthase machinery for polymer production.

The Versatile Pseudomonas aeruginosa Biofilm Matrix Protein CdrA Promotes Aggregation through Different Extracellular Exopolysaccharide Interactions.

Pseudomonas aeruginosa is an important pathogen that causes chronic infections that involve multicellular aggregates called biofilms. Within biofilms, bacteria are surrounded in a protective extracellular matrix of proteins, exopolysaccharides (EPS), and DNA. A key P. aeruginosa matrix protein is an extracellular adhesin called CdrA, which promotes aggregation by binding to the EPS Psl and via CdrA-CdrA interactions. We hypothesized that because of its ability to bind Psl, CdrA would be important only for strains that use Psl as the primary EPS (e.g., the laboratory strain PAO1). Thus, we predicted that cdrA might be dispensable for biofilm formation by strains that do not utilize Psl (e.g., the laboratory strain PA14). Instead, we observed that cdrA deletion strains exhibited biofilm defects, regardless of their EPS dependencies. We screened a panel of clinical and environmental P. aeruginosa isolates for the presence of the cdrA allele and production of CdrA protein. All isolates that we tested contained the cdrA allele, and these alleles had minimal sequence variation compared to the reference PAO1 cdrA gene. Additionally, all isolates except one produced detectable CdrA protein. We investigated the possible mechanisms of CdrA-promoted biofilm formation in these strains where Psl is not dominant, and we discovered that CdrA binds to Pel. Although Psl and Pel chemical structures are distinct, this appears to be a specific interaction, since previous work has shown that CdrA binds discriminately to other EPS. Our findings provide new understanding of biofilm formation across P. aeruginosa isolates and emphasize the versatility of CdrA.IMPORTANCE Depending upon the strain, Pseudomonas aeruginosa can use different exopolysaccharides (e.g., Psl, Pel, and alginate) to build its biofilm matrix. Previously, we demonstrated that the biofilm matrix protein CdrA binds to Psl, promoting biofilm formation and aggregate stability. As such, it was thought that CdrA might be important for biofilm assembly only in strains that rely upon Psl. However, past studies indicated that CdrA can interact with monosaccharides not present in Psl, including N-acetylglucosamine, a constituent of another EPS called Pel. We discovered that CdrA also binds to Pel and promotes biofilm formation by strains in which Psl is not dominant. Thus, our findings suggest that CdrA plays a common role as a biofilm matrix cross-linker across P. aeruginosa isolates with different EPS.

Pel Polysaccharide Biosynthesis Requires an Inner Membrane Complex Comprised of PelD, PelE, PelF, and PelG.

The Pel polysaccharide is a structural component of the extracellular matrix of Pseudomonas aeruginosa biofilms. Recent analyses suggest that Pel production proceeds via a synthase-dependent polysaccharide secretion pathway, which in Gram-negative bacteria is defined by an outer membrane ß-barrel porin, a periplasmic tetratricopeptide repeat-containing scaffold protein, and an inner membrane-embedded synthase. Polymerization is catalyzed by the glycosyltransferase domain of the synthase component of these systems, which is allosterically regulated by cyclic 3',5'-dimeric GMP (c-di-GMP). However, while the outer membrane and periplasmic components of the Pel system have been characterized, the inner membrane complex required for Pel polymerization has yet to be defined. To address this, we examined over 500 pel gene clusters from diverse species of Proteobacteria This analysis identified an invariant set of four syntenic genes, three of which, pelD, pelE, and pelG, are predicted to reside within the inner membrane, while the fourth, pelF, encodes a glycosyltransferase domain. Using a combination of gene deletion analysis, subcellular fractionation, coimmunoprecipitation, and bacterial two-hybrid assays, we provide evidence for the existence of an inner membrane complex of PelD, PelE, and PelG. Furthermore, we show that this complex interacts with PelF in order to facilitate its localization to the inner membrane. Mutations that abolish c-di-GMP binding to the known receptor domain of PelD had no effect on complex formation, suggesting that c-di-GMP binding stimulates Pel production through quaternary structural rearrangements. Together, these data provide the first experimental evidence of an inner membrane complex involved in Pel polysaccharide production.IMPORTANCE The exopolysaccharide Pel plays an important role in bacterial cell-cell interactions, surface adhesion, and protection against certain antibiotics. We identified invariant pelDEFG gene clusters in over 500 diverse proteobacterial species. Using Pseudomonas aeruginosa, we demonstrate that PelD, PelE, PelF, and PelG form a complex at the inner membrane and propose that this complex represents the previously unidentified Pel polysaccharide synthase, which is responsible for Pel polymerization and transport across the cytoplasmic membrane. We show that the formation of this complex is independent of cyclic 3',5'-dimeric GMP (c-di-GMP) binding to the receptor PelD. Collectively, these data establish the widespread Pel apparatus as a member of the synthase-dependent pathway of polysaccharide biosynthetic systems and broaden the architectural diversity of already-established bacterial polysaccharide synthases.

Pseudomonas aeruginosa rugose small-colony variants evade host clearance, are hyper-inflammatory, and persist in multiple host environments.

Pseudomonas aeruginosa causes devastating infections in immunocompromised individuals. Once established, P. aeruginosa infections become incredibly difficult to treat due to the development of antibiotic tolerant, aggregated communities known as biofilms. A hyper-biofilm forming clinical variant of P. aeruginosa, known as a rugose small-colony variant (RSCV), is frequently isolated from chronic infections and is correlated with poor clinical outcome. The development of these mutants during infection suggests a selective advantage for this phenotype, but it remains unclear how this phenotype promotes persistence. While prior studies suggest RSCVs could survive by evading the host immune response, our study reveals infection with the RSCV, PAO1ΔwspF, stimulated an extensive inflammatory response that caused significant damage to the surrounding host tissue. In both a chronic wound model and acute pulmonary model of infection, we observed increased bacterial burden, host tissue damage, and a robust neutrophil response during RSCV infection. Given the essential role of neutrophils in P. aeruginosa-mediated disease, we investigated the impact of the RSCV phenotype on neutrophil function. The RSCV phenotype promoted phagocytic evasion and stimulated neutrophil reactive oxygen species (ROS) production. We also demonstrate that bacterial aggregation and TLR-mediated pro-inflammatory cytokine production contribute to the immune response to RSCVs. Additionally, RSCVs exhibited enhanced tolerance to neutrophil-produced antimicrobials including H2O2 and the antimicrobial peptide LL-37. Collectively, these data indicate RSCVs elicit a robust but ineffective neutrophil response that causes significant host tissue damage. This study provides new insight on RSCV persistence, and indicates this variant may have a critical role in the recurring tissue damage often associated with chronic infections.

Oligomeric lipoprotein PelC guides Pel polysaccharide export across the outer membrane of Pseudomonas aeruginosa.

Secreted polysaccharides are important functional and structural components of bacterial biofilms. The opportunistic pathogen Pseudomonas aeruginosa produces the cationic exopolysaccharide Pel, which protects bacteria from aminoglycoside antibiotics and contributes to biofilm architecture through ionic interactions with extracellular DNA. A bioinformatics analysis of genome databases suggests that gene clusters for Pel biosynthesis are present in >125 bacterial species, yet little is known about how this biofilm exopolysaccharide is synthesized and exported from the cell. In this work, we characterize PelC, an outer membrane lipoprotein essential for Pel production. Crystal structures of PelC from Geobacter metallireducens and Paraburkholderia phytofirmans coupled with structure-guided disulfide cross-linking in P. aeruginosa suggest that PelC assembles into a 12- subunit ring-shaped oligomer. In this arrangement, an aromatic belt in proximity to its lipidation site positions the highly electronegative surface of PelC toward the periplasm. PelC is structurally similar to the Escherichia coli amyloid exporter CsgG; however, unlike CsgG, PelC does not possess membrane-spanning segments required for polymer export across the outer membrane. We show that the multidomain protein PelB with a predicted C-terminal ß-barrel porin localizes to the outer membrane, and propose that PelC functions as an electronegative funnel to guide the positively charged Pel polysaccharide toward an exit channel formed by PelB. Together, our findings provide insight into the unique molecular architecture and export mechanism of the Pel apparatus, a widespread exopolysaccharide secretion system found in environmental and pathogenic bacteria.

Microbial glycoside hydrolases as antibiofilm agents with cross-kingdom activity.

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.

Treatment with the Pseudomonas aeruginosa Glycoside Hydrolase PslG Combats Wound Infection by Improving Antibiotic Efficacy and Host Innate Immune Activity.

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 PelAh and PslGh 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 PslGh 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. PslGh 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 PslGh and tobramycin, we observed an additive effect leading to greater bacterial clearance than treatments of tobramycin or PslGh alone. This study demonstrates that PelAh and PslGh have promising therapeutic potential and that PslGh may aid in the treatment of P. aeruginosa wound infections.

Psl trails guide exploration and microcolony formation in Pseudomonas aeruginosa biofilms.

Bacterial biofilms are surface-associated, multicellular, morphologically complex microbial communities. Biofilm-forming bacteria such as the opportunistic pathogen Pseudomonas aeruginosa are phenotypically distinct from their free-swimming, planktonic counterparts. Much work has focused on factors affecting surface adhesion, and it is known that P. aeruginosa secretes the Psl exopolysaccharide, which promotes surface attachment by acting as 'molecular glue'. However, how individual surface-attached bacteria self-organize into microcolonies, the first step in communal biofilm organization, is not well understood. Here we identify a new role for Psl in early biofilm development using a massively parallel cell-tracking algorithm to extract the motility history of every cell on a newly colonized surface. By combining this technique with fluorescent Psl staining and computer simulations, we show that P. aeruginosa deposits a trail of Psl as it moves on a surface, which influences the surface motility of subsequent cells that encounter these trails and thus generates positive feedback. Both experiments and simulations indicate that the web of secreted Psl controls the distribution of surface visit frequencies, which can be approximated by a power law. This Pareto-type behaviour indicates that the bacterial community self-organizes in a manner analogous to a capitalist economic system, a 'rich-get-richer' mechanism of Psl accumulation that results in a small number of 'elite' cells becoming extremely enriched in communally produced Psl. Using engineered strains with inducible Psl production, we show that local Psl concentrations determine post-division cell fates and that high local Psl concentrations ultimately allow elite cells to serve as the founding population for initial microcolony development.

PelA and PelB proteins form a modification and secretion complex essential for Pel polysaccharide-dependent biofilm formation in Pseudomonas aeruginosa.

The pellicle (PEL) polysaccharide is synthesized by the opportunistic pathogen Pseudomonas aeruginosa and is an important biofilm constituent critical for bacterial virulence and persistence. PEL is a cationic polymer that promotes cell-cell interactions within the biofilm matrix through electrostatic interactions with extracellular DNA. Translocation of PEL across the outer membrane is proposed to occur via PelB, a membrane-embedded porin with a large periplasmic domain predicted to contain 19 tetratricopeptide repeats (TPRs). TPR-containing domains are typically involved in protein-protein interactions, and we therefore sought to determine whether PelB serves as a periplasmic scaffold that recruits other components of the PEL secretion apparatus. In this study, we show that the TPR domain of PelB interacts with PelA, an enzyme with PEL deacetylase and hydrolase activities. Structure determination of PelB TPRs 8-11 enabled us to design systematic deletions of individual TPRs and revealed that repeats 9-14, which are required for the cellular localization of PelA with PelB are also essential for PEL-dependent biofilm formation. Copurification experiments indicated that the interaction between PelA and PelB is direct and that the deacetylase activity of PelA increases and its hydrolase activity decreases when these proteins interact. Combined, our results indicate that the TPR-containing domain of PelB localizes PelA to the PEL secretion apparatus within the periplasm and that this may allow for efficient deacetylation of PEL before its export from the cell.

Oligoribonuclease is a central feature of cyclic diguanylate signaling in Pseudomonas aeruginosa.

The second messenger cyclic diguanylate (c-di-GMP) controls diverse cellular processes among bacteria. Diguanylate cyclases synthesize c-di-GMP, whereas it is degraded by c-di-GMP-specific phosphodiesterases (PDEs). Nearly 80% of these PDEs are predicted to depend on the catalytic function of glutamate-alanine-leucine (EAL) domains, which hydrolyze a single phosphodiester group in c-di-GMP to produce 5'-phosphoguanylyl-(3',5')-guanosine (pGpG). However, to degrade pGpG and prevent its accumulation, bacterial cells require an additional nuclease, the identity of which remains unknown. Here we identify oligoribonuclease (Orn)-a 3'→5' exonuclease highly conserved among Actinobacteria, Beta-, Delta- and Gammaproteobacteria-as the primary enzyme responsible for pGpG degradation in Pseudomonas aeruginosa cells. We found that a P. aeruginosa Δorn mutant had high intracellular c-di-GMP levels, causing this strain to overexpress extracellular polymers and overproduce biofilm. Although recombinant Orn degraded small RNAs in vitro, this enzyme had a proclivity for degrading RNA oligomers comprised of two to five nucleotides (nanoRNAs), including pGpG. Corresponding with this activity, Δorn cells possessed highly elevated pGpG levels. We found that pGpG reduced the rate of c-di-GMP degradation in cell lysates and inhibited the activity of EAL-dependent PDEs (PA2133, PvrR, and purified recombinant RocR) from P. aeruginosa. This pGpG-dependent inhibition was alleviated by the addition of Orn. These data suggest that elevated levels of pGpG exert product inhibition on EAL-dependent PDEs, thereby increasing intracellular c-di-GMP in Δorn cells. Thus, we propose that Orn provides homeostatic control of intracellular pGpG under native physiological conditions and that this activity is fundamental to c-di-GMP signal transduction.

Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix.

Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel's chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel's sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.