Psl Polysaccharide Research Articles

Role of Pel and Psl polysaccharides in the response of Pseudomonas aeruginosa to environmental challenges: oxidative stress agents (UVA, H2O2, sodium hypochlorite) and its competitor Staphylococcus aureus.

Pseudomonas aeruginosa is a versatile bacterium capable of adapting to a wide range of stress factors, including solar UVA radiation (400-315 nm). High UVA doses produce lethal effects due to the action of reactive oxygen species. Sublethal UVA doses also induces oxidative damage, but, in addition, it triggers a variety of adaptive responses, including the overexpression of pelA and pslA genes in P. aeruginosa. These genes encode the synthesis of Pel and Psl, which are essential polysaccharides in biofilm formation. The present study analysed the role of Pel and Psl in the adaptive responses generated by exposure to low UVA doses, and their importance in the response to lethal doses of UVA, hydrogen peroxide (H2O2), and sodium hypochlorite, in both planktonic cells and submerged and air-liquid interface (ALI) biofilms. It also studied the roles of Pel and Psl in P. aeruginosa-Staphylococcus aureus interaction. The results demonstrate that the capacity of sublethal UVA exposure to increase cell hydrophobicity and cell attachment and generate cross-protection phenomena in P. aeruginosa depends on the presence of Pel and Psl. The study also shows that Pel and Psl have a key role in the tolerance to lethal doses of UVA radiation, sodium hypochlorite and H2O2, in both biofilms and planktonic cells. Finally, co-culture assays showed total inhibition of S. aureus growth in presence of P. aeruginosa. This phenomenon depends, at least in part, on the simultaneous presence of Pel and Psl in planktonic cells and biofilms, suggesting a relevant role of these polysaccharides in the interaction between these species.

Fluid dynamics and cell-bound Psl polysaccharide allows microplastic capture, aggregation and subsequent sedimentation by Pseudomonas aeruginosa in water.

SummaryDecades after incorporating plastics into consumer markets, research shows that these polymers have spread worldwide. Fragmentation of large debris leads to smaller particles, collectively called microplastics (MPs), which have become ubiquitous in aquatic environments. A fundamental aspect of understanding the implications of MP contamination on ecosystems is resolving the complex interactions of these artificial substrates with microbial cells. Using polystyrene microparticles as model polymers, we conducted an exploratory study where these interactions are quantitatively analyzed using an in vitro system consisting of single‐bacterial species capturing and aggregating MPs in water. Here we show that the production of Psl exopolysaccharide by Pseudomonas aeruginosa (PA) does not alter MPs colloidal stability but plays a key role in microspheres adhesion to the cell surface. Further aggregation of MPs by PA cells depends on bacterial mobility and the presence of sufficient flow to prevent rapid sedimentation of early MP‐PA assembles. Surprisingly, cells in MP‐PA aggregates are not in a sessile state despite the production of Psl, enhancing the motility of the aggregates by an order of magnitude relative to passive diffusion. The generated data could inform the creation of predictive models that accurately describe the dynamics and influence of bacterial growth on plastics debris.

Protective Liquid Crystal Nanoparticles for Targeted Delivery of PslG: A Biofilm Dispersing Enzyme.

The glycoside hydrolase, PslG, attacks and degrades the dominant Psl polysaccharide in the exopolymeric substance (EPS) matrix of Pseudomonas aeruginosa biofilms and is a promising therapy to potentiate the effect of antibiotics. However, the need for coadministration with an antibiotic and the potential susceptibility of PslG to proteolysis highlights the need for an effective delivery system. Here, we compared liposomes versus lipid liquid crystal nanoparticles (LCNPs) loaded with PslG and tobramycin as potential formulation approaches to (1) protect PslG from proteolysis, (2) trigger the enzyme's release in the presence of bacteria, and (3) improve the total antimicrobial effect in vitro and in vivo in a Caenorhabditis elegans infection model. LCNPs were an effective formulation strategy for PslG and tobramycin that better protected the enzyme against proteolysis, triggered and sustained the release of PslG, improved the antimicrobial effect by 10-100-fold, and increased the survival of C. elegans infected with P. aeruginosa. Digestible LCNPs had the advantage of triggering the enzyme's release in the presence of bacteria. However, compared to nondigestible LCNPs, negligible differences arose between the LCNPs' ability to protect PslG from proteolysis and potentiate the antimicrobial activity in combination with tobramycin. In C. elegans, the improved antimicrobial efficacy was comparable to tobramycin-LCNPs, although the PslG + tobramycin-LCNPs achieved a greater than 10-fold reduction in bacteria compared to the unformulated combination. Herewith, LCNPs are showcased as a promising protective delivery system for novel biofilm dispersing enzymes combined with antibiotics, enabling infection-directed therapy and improved performance.

Beyond the Wall: Exopolysaccharides in the Biofilm Lifestyle of Pathogenic and Beneficial Plant-Associated Pseudomonas.

The formation of biofilms results from a multicellular mode of growth, in which bacteria remain enwrapped by an extracellular matrix of their own production. Many different bacteria form biofilms, but among the most studied species are those that belong to the Pseudomonas genus due to the metabolic versatility, ubiquity, and ecological significance of members of this group of microorganisms. Within the Pseudomonas genus, biofilm studies have mainly focused on the opportunistic human pathogen Pseudomonas aeruginosa due to its clinical importance. The extracellular matrix of P. aeruginosa is mainly composed of exopolysaccharides, which have been shown to be important for the biofilm architecture and pathogenic features of this bacterium. Notably, some of the exopolysaccharides recurrently used by P. aeruginosa during biofilm formation, such as the alginate and polysaccharide synthesis loci (Psl) polysaccharides, are also used by pathogenic and beneficial plant-associated Pseudomonas during their interaction with plants. Interestingly, their functions are multifaceted and seem to be highly dependent on the bacterial lifestyle and genetic context of production. This paper reviews the functions and significance of the exopolysaccharides produced by plant-associated Pseudomonas, particularly the alginate, Psl, and cellulose polysaccharides, focusing on their equivalents produced in P. aeruginosa within the context of pathogenic and beneficial interactions.

Biological role of EPS from Pseudomonas syringae pv. syringae UMAF0158 extracellular matrix, focusing on a Psl-like polysaccharide

Pseudomonas syringae is a phytopathogenic model bacterium that is used worldwide to study plant–bacteria interactions and biofilm formation in association with a plant host. Within this species, the syringae pathovar is the most studied due to its wide host range, affecting both, woody and herbaceous plants. In particular, Pseudomonas syringae pv. syringae (Pss) has been previously described as the causal agent of bacterial apical necrosis on mango trees. Pss exhibits major epiphytic traits and virulence factors that improve its epiphytic survival and pathogenicity in mango trees. The cellulose exopolysaccharide has been described as a key component in the development of the biofilm lifestyle of the P. syringae pv. syringae UMAF0158 strain (PssUMAF0158). PssUMAF0158 contains two additional genomic regions that putatively encode for exopolysaccharides such as alginate and a Psl-like polysaccharide. To date, the Psl polysaccharide has only been studied in Pseudomonas aeruginosa, in which it plays an important role during biofilm development. However, its function in plant-associated bacteria is still unknown. To understand how these exopolysaccharides contribute to the biofilm matrix of PssUMAF0158, knockout mutants of genes encoding these putative exopolysaccharides were constructed. Flow-cell chamber experiments revealed that cellulose and the Psl-like polysaccharide constitute a basic scaffold for biofilm architecture in this bacterium. Curiously, the Psl-like polysaccharide of PssUMAF0158 plays a role in virulence similar to what has been described for cellulose. Finally, the impaired swarming motility of the Psl-like exopolysaccharide mutant suggests that this exopolysaccharide may play a role in the motility of PssUMAF0158 over the mango plant surface.

Fusobacterium nucleatum Interaction with Pseudomonas aeruginosa Induces Biofilm-Associated Antibiotic Tolerance via Fusobacterium Adhesin A.

Respiratory infections with Pseudomonas aeruginosa or Fusobacterium nucleatum are associated with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) and failure in antibiotic treatment. However, the impact of these dual-species interactions on the severity of chronic obstructive pulmonary disease (COPD) and biofilm antibiotic susceptibility remains poorly understood. This study demonstrated that F.nucleatum frequently coexisted with P.aeruginosa in the respiratory tract, and the number of F.nucleatum was negatively correlated with the lung function of AECOPD patients. The coculture of P.aeruginosa and F.nucleatum promoted bacterial proliferation and induced antibiotic tolerance through the formation of a dense biofilm surrounded by excessive Pel and Psl polysaccharides. Moreover, Fusobacterium adhesin A (FadA), rather than F. nucleatum spent medium, induced antibiotic tolerance of the P.aeruginosa biofilm. These results indicate that F.nucleatum is a biomarker of lung function decline in AECOPD patients and interacts with P.aeruginosa in vitro to resist antibiotics via FadA, which would be a potential anti-infective target of these dual-species infection.

Untethering and Degradation of the Polysaccharide Matrix Are Essential Steps in the Dispersion Response of Pseudomonas aeruginosa Biofilms.

Biofilms are multicellular aggregates of bacteria that are encased in an extracellular matrix. The biofilm matrix of Pseudomonas aeruginosa PAO1 is composed of eDNA, proteins, and the polysaccharides Pel and Psl. This matrix is thought to be degraded during dispersion to liberate cells from the biofilms, with dispersion being apparent not only by single cells escaping from the biofilm but also leaving behind eroded or hollowed-out biofilm. However, little is known of the factors involved in matrix degradation. Here, we focused on the glycoside hydrolases PelA and PslG. We demonstrate that induction of pelA but not pslG expression resulted in dispersion. As Psl is tethered to the matrix adhesin CdrA, we furthermore explored the role of CdrA in dispersion. cdrA mutant biofilms were hyperdispersive, while lapG mutant biofilms were impaired in dispersion in response to glutamate and nitric oxide, indicating the presence of the surface-associated matrix protein CdrA impedes the dispersion response. In turn, insertional inactivation of cdrA enabled pslG-induced dispersion. Lowering of the intracellular c-di-GMP level via induction of PA2133 encoding a phosphodiesterase was not sufficient to induce dispersion by wild-type strains and strains overexpressing pslG, indicating that pslG-induced dispersion is independent of c-di-GMP modulation and, likely, LapG.IMPORTANCEPseudomonas aeruginosa forms multicellular aggregates or biofilms encased in a matrix. We show for the first time here that dispersion by P. aeruginosa requires the endogenous expression of pelA and pslG, leading to the degradation of both Pel and Psl polysaccharides, with PslG-induced dispersion being CdrA dependent. The findings suggested that endogenously induced Psl degradation is a sequential process, initiated by untethering of CdrA-bound Psl or CdrA-dependent cell interactions to enable Psl degradation and ultimately, dispersion. Untethering likely involves CdrA release in a manner independent of c-di-GMP modulation and thus LapG. Our findings not only provide insight into matrix degrading factors contributing to dispersion but also identify key steps in the degradation of structural components of the P. aeruginosa biofilm matrix.

The Pseudomonas aeruginosa PSL Polysaccharide Is a Social but Noncheatable Trait in Biofilms.

ABSTRACTExtracellular polysaccharides are compounds secreted by microorganisms into the surrounding environment, and they are important for surface attachment and maintaining structural integrity within biofilms. The social nature of many extracellular polysaccharides remains unclear, and it has been suggested that they could function as either cooperative public goods or as traits that provide a competitive advantage. Here, we empirically tested the cooperative nature of the PSL polysaccharide, which is crucial for the formation of biofilms in Pseudomonas aeruginosa. We show that (i) PSL is not metabolically costly to produce; (ii) PSL provides population-level benefits in biofilms, for both growth and antibiotic tolerance; (iii) the benefits of PSL production are social and are shared with other cells; (iv) the benefits of PSL production appear to be preferentially directed toward cells which produce PSL; (v) cells which do not produce PSL are unable to successfully exploit cells which produce PSL. Taken together, this suggests that PSL is a social but relatively nonexploitable trait and that growth within biofilms selects for PSL-producing strains, even when multiple strains are on a patch (low relatedness at the patch level).

Revisiting interrelated effects of extracellular polysaccharides during biofouling of reverse osmosis membranes: Viscoelastic properties and biofilm enhanced osmotic pressure

In addition to the effect of extracellular polysaccharides on biofilms viscoelastic properties and their related integrity and cohesion, their presence in biofilms of RO membranes can affect biofilm porosity and associated electrolytes diffusion. In order to elucidate how polysaccharides can affect both biofilm viscoelasticity and back diffusion of electrolytes on RO membranes, three bacterial strains of Pseudomonas aeruginosa PAO1, differentially expressing the Psl polysaccharide, were used as biofouling agents. During the biofouling stage, similar permeate flux decline was observed for all strains, while elevation in salt passage was lower for the Psl-deletion mutant biofilm. After the biofilm washing stage, as shear rate was elevated, permeate flux recovery was significantly higher and a decrease in salt passage was evident, in the Psl-deletion mutant biofilm. In contrast, in Psl containing biofilms, an elevation in salt passage was evident after the washing stage. Salt passage results, CLSM analysis and the intrinsic mass transfer coefficient kd, provided us with an overall range of biofouling layer’s porosity between 0.25 and 0.35. Lastly, the higher amounts of biomass of Psl containing biofilms persisted on the RO membranes after the washing stage, corroborated with both EPS viscoelastic properties measured by QCM-D and biofilm elasticity measured by AFM. This study highlights mechanistically, the EPS effects on biofouling of RO membranes, mediated by biofilm mechanical properties as well as ion transport.

Viscoelastic Properties of Extracellular Polymeric Substances Can Strongly Affect Their Washing Efficiency from Reverse Osmosis Membranes.

The role of the viscoelastic properties of biofouling layers in their removal from the membrane was studied. Model fouling layers of extracellular polymeric substances (EPS) originated from microbial biofilms of Pseudomonas aeruginosa PAO1 differentially expressing the Psl polysaccharide were used for controlled washing experiments of fouled RO membranes. In parallel, adsorption experiments and viscoelastic modeling of the EPS layers were conducted in a quartz crystal microbalance with dissipation (QCM-D). During the washing stage, as shear rate was elevated, significant differences in permeate flux recovery between the three different EPS layers were observed. According to the amount of organic carbon remained on the membrane after washing, the magnitude of Psl production provides elevated resistance of the EPS layer to shear stress. The highest flux recovery during the washing stage was observed for the EPS with no Psl. Psl was shown to elevate the layer's shear modulus and shear viscosity but had no effect on the EPS adhesion to the polyamide surface. We conclude that EPS retain on the membrane as a result of the layer viscoelastic properties. These results highlight an important relation between washing efficiency of fouling layers from membranes and their viscoelastic properties, in addition to their adhesion properties.

SiaA/D Interconnects c-di-GMP and RsmA Signaling to Coordinate Cellular Aggregation of Pseudomonas aeruginosa in Response to Environmental Conditions.

Pseudomonas aeruginosa has emerged as an important opportunistic human pathogen that is often highly resistant to eradication strategies, mediated in part by the formation of multicellular aggregates. Cellular aggregates may occur attached to a surface (biofilm), at the air-liquid interface (pellicle), or as suspended aggregates. Compared to surface attached communities, knowledge about the regulatory processes involved in the formation of suspended cell aggregates is still limited. We have recently described the SiaA/D signal transduction module that regulates macroscopic cell aggregation during growth with, or in the presence of the surfactant SDS. Targets for SiaA/D mediated regulation include the Psl polysaccharide, the CdrAB two-partner secretion system and the CupA fimbriae. While the global regulators c-di-GMP and RsmA are known to inversely coordinate cell aggregation and regulate the expression of several adhesins, their potential impact on the expression of the cupA operon remains unknown. Here, we investigated the function of SiaA (a putative ser/thr phosphatase) and SiaD (a di-guanylate cyclase) in cupA1 expression using transcriptional reporter fusions and qRT-PCR. These studies revealed a novel interaction between the RsmA posttranscriptional regulatory system and SiaA/D mediated macroscopic aggregation. The RsmA/rsmY/Z system was found to affect macroscopic aggregate formation in the presence of surfactant by impacting the stability of the cupA1 mRNA transcript and we reveal that RsmA directly binds to the cupA1 leader sequence in vitro. We further identified that transcription of the RsmA antagonist rsmZ is controlled in a SiaA/D dependent manner during growth with SDS. Finally, we found that the siaD transcript is also under regulatory control of RsmA and that overproduction of RsmA or the deletion of siaD results in decreased cellular cyclic di-guanosine monophosphate (c-di-GMP) levels quantified by a transcriptional reporter, demonstrating that SiaA/D connects c-di-GMP and RsmA/rsmY/Z signaling to reciprocally regulate cell aggregation in response to environmental conditions.

Enzyme immobilization on biomaterial surfaces for prevention of pseudomonas aeruginosa biofilms

Event Abstract Back to Event Enzyme immobilization on biomaterial surfaces for prevention of pseudomonas aeruginosa biofilms Dalal Asker1, 2, Tarek Awad1, Perrin Baker3, Lynne Howell3, 4 and Benjamin Hatton1 1 University of Toronto, Materials Science and Engineering, Canada 2 Alexandria University, Food Science & Technology Department, Egypt 3 Hospital for Sick Children, Canada 4 University of Toronto, Dept. of Biochemistry, Canada Introduction: Implanted devices are at significant risk of developing bacterial biofilm-associated infections. Approximately 60% of all hospital-acquired infections (HAIs) are the result of biofilm formation on implanted medical devices and the bacterium Pseudomonas aeruginosa is one of the most prevalent isolated pathogens. Bacterial biofilms are communities of bacteria encased within a protective extracellular matrix. Exopolysaccharides (EPS) are the predominant component of biofilm matrix for many bacteria, contributing to initial adhesion, architecture, and resistance. Bacteria in biofilm are highly tolerant to antibiotics and can evade the host immune system. Recently we identified and produced several recombinant glycoside hydrolases that selectively target and hydrolyze biofilm EPS. Two of these enzymes - PslGh, and PelAh - hydrolyze the Psl and Pel polysaccharides from P. aeruginosa, respectively, and were found to be effective in solution at disrupting the biofilms from lab, clinical and environmental isolates. Current antibiotic therapy and antimicrobial surfaces are ineffective against biofilm infections. Therefore, we aim to use surface-immobilized enzymes as a means of preventing biofilm formation on implanted medical devices such as endotracheal tubes. Materials and Methods: We tested the immobilization of PslGh onto polymer biomaterial surfaces as a means of preventing biofilm growth for P. aeruginosa in long term static and flow culture. The enzyme was covalently bound with glutaraldehyde cross-linking to glass and PDMS surfaces and its attachment confirmed by ATR-FTIR and contact angle measurements. To evaluate anti-biofilm efficacy, our biomaterial surfaces were incubated in P. aeruginosa culture that overexpresses the Psl polysaccharide for varying duration. Fluorescence microscopy and fluorescent dyes (SYTOX Green) were used and for quantitative analysis, we used turbidity and crystal violet staining methods. Results and Discussion: The covalently-bound PslGh inhibited both cell attachment and biofilm formation of the P. aeruginosa (Psl-dependent) cells (Fig 1a), compared with the control (i.e., no enzyme) or a catalytically inactive enzyme (Fig. 1b) over a 24 hour period. This indicates that the anti-biofilm activity of PslGh is directly related its catalytic activity. The covalently-immobilized PslGh enzyme was also effective at preventing biofilm formation for an 8 day static culture. Quantitative analysis using crystal violet straining indicated significant anti-biofilm activity for the immobilized PslG, as compared with the control sample. Conclusion: These results indicate the high specificity of PslGh in targeting the Psl exopolysacchride in P. aeruginosa biofilms, which appears to greatly inhibit bacterial colonization and biofilm growth, even when the enzyme is covalently-immobilized on the polymer surface. Figure 1. Inhibition of P. aeruginosa (Psl-dependent) biofilm attachment and formation by covalently-attached PslGh on PDMS samples. (a) untreated PDMS; (b) catalytically-inactive mutant PslGh (mPslGh) covalently immobilized on PDMS; and (c) PslGh covalently immobilized on PDMS. Images were observed with 20X dry objectives. Scale bars, 40 µm. Keywords: Bacteria, Implant, biofunctionalization, bioactive interface, enzyme Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: New Frontier Oral Topic: Interfacial phenomena Citation: Asker D, Awad T, Baker P, Howell L and Hatton B (2016). Enzyme immobilization on biomaterial surfaces for prevention of pseudomonas aeruginosa biofilms. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00131 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Dalal Asker Tarek Awad Perrin Baker Lynne Howell Benjamin Hatton Google Dalal Asker Tarek Awad Perrin Baker Lynne Howell Benjamin Hatton Google Scholar Dalal Asker Tarek Awad Perrin Baker Lynne Howell Benjamin Hatton PubMed Dalal Asker Tarek Awad Perrin Baker Lynne Howell Benjamin Hatton Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Characterization of the Pseudomonas aeruginosa Glycoside Hydrolase PslG Reveals That Its Levels Are Critical for Psl Polysaccharide Biosynthesis and Biofilm Formation

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.

Pseudomonas aeruginosa PAO1 exopolysaccharides are important for mixed species biofilm community development and stress tolerance.

Pseudomonas aeruginosa PAO1 produces three polysaccharides, alginate, Psl, and Pel that play distinct roles in attachment and biofilm formation for monospecies biofilms. Considerably less is known about their role in the development of mixed species biofilm communities. This study has investigated the roles of alginate, Psl, and Pel during biofilm formation of P. aeruginosa in a defined and experimentally informative mixed species biofilm community, consisting of P. aeruginosa, Pseudomonas protegens, and Klebsiella pneumoniae. Loss of the Psl polysaccharide had the biggest impact on the integration of P. aeruginosa in the mixed species biofilms, where the percent composition of the psl mutant was significantly lower (0.06%) than its wild-type (WT) parent (2.44%). In contrast, loss of the Pel polysaccharide had no impact on mixed species biofilm development. Loss of alginate or its overproduction resulted in P. aeruginosa representing 8.4 and 18.11%, respectively, of the mixed species biofilm. Dual species biofilms of P. aeruginosa and K. pneumoniae were not affected by loss of alginate, Pel, or Psl, while the mucoid P. aeruginosa strain achieved a greater biomass than its parent strain. When P. aeruginosa was grown with P. protegens, loss of the Pel or alginate polysaccharides resulted in biofilms that were not significantly different from biofilms formed by the WT PAO1. In contrast, overproduction of alginate resulted in biofilms that were comprised of 35–40% of P. aeruginosa, which was significantly higher than the WT (5–20%). Loss of the Psl polysaccharide significantly reduced the percentage composition of P. aeruginosa in dual species biofilms with P. protegens (<1%). Loss of the Psl polysaccharide significantly disrupted the communal stress resistance of the three species biofilms. Thus, the polysaccharide composition of an individual species significantly impacts mixed species biofilm development and the emergent properties of such communities.

Catheter-Associated Urinary Tract Infection by Pseudomonas aeruginosa Is Mediated by Exopolysaccharide-Independent Biofilms

Pseudomonas aeruginosa is an opportunistic human pathogen that is especially adept at forming surface-associated biofilms. P. aeruginosa causes catheter-associated urinary tract infections (CAUTIs) through biofilm formation on the surface of indwelling catheters. P. aeruginosa encodes three extracellular polysaccharides, PEL, PSL, and alginate, and utilizes the PEL and PSL polysaccharides to form biofilms in vitro; however, the requirement of these polysaccharides during in vivo infections is not well understood. Here we show in a murine model of CAUTI that PAO1, a strain harboring pel, psl, and alg genes, and PA14, a strain harboring pel and alg genes, form biofilms on the implanted catheters. To determine the requirement of exopolysaccharide during in vivo biofilm infections, we tested isogenic mutants lacking the pel, psl, and alg operons and showed that PA14 mutants lacking these operons can successfully form biofilms on catheters in the CAUTI model. To determine the host factor(s) that induces the ΔpelD mutant to form biofilm, we tested mouse, human, and artificial urine and show that urine can induce biofilm formation by the PA14 ΔpelD mutant. By testing the major constituents of urine, we show that urea can induce a pel-, psl-, and alg-independent biofilm. These pel-, psl-, and alg-independent biofilms are mediated by the release of extracellular DNA. Treatment of biofilms formed in urea with DNase I reduced the biofilm, indicating that extracellular DNA supports biofilm formation. Our results indicate that the opportunistic pathogen P. aeruginosa utilizes a distinct program to form biofilms that are independent of exopolysaccharides during CAUTI.

A spider web strategy of type IV pili‐mediated migration to build a fibre‐like Psl polysaccharide matrix in Pseudomonas aeruginosa biofilms

Bacterial motilities participate in biofilm development. However, it is unknown how/if bacterial motility affects formation of the biofilm matrix. Psl polysaccharide is a key biofilm matrix component of Pseudomonas aeruginosa. Here we report that type IV pili (T4P)-mediated bacterial migration leads to the formation of a fibre-like Psl matrix. Deletion of T4P in wild type and flagella-deficient strains results in loss of the Psl-fibres and reduction of biofilm biomass in flow cell biofilms as well as pellicles at air-liquid interface. Bacteria lacking T4P-driven twitching motility including those that still express surface T4P are unable to form the Psl-fibres. Formation of a Psl-fibre matrix is critical for efficient biofilm formation, yet does not require flagella and polysaccharide Pel or alginate. The Psl-fibres are likely formed by Psl released from bacteria during T4P-mediated migration, a strategy similar to spider web formation. Starvation can couple Psl release and T4P-driven twitching motility. Furthermore, a radial-pattern Psl-fibre matrix is present in the middle of biofilms, a nutrient-deprived region. These imply a plausible model for how bacteria respond to nutrient-limited local environment to build a polysaccharide-fibre matrix by T4P-dependent bacterial migration strategy. This strategy may have general significance for bacterial survival in natural and clinical settings.

Polysaccharides serve as scaffold of biofilms formed by mucoidPseudomonas aeruginosa

Chronic lung infection by mucoid Pseudomonas aeruginosa is one of the major pathologic features in patients with cystic fibrosis. Mucoid P.aeruginosa is notorious for its biofilm forming capability and resistance to immune attacks. In this study, the roles of extracellular polymeric substances from biofilms formed by mucoid P.aeruginosa were investigated. Alginate is not an essential structure component for mucoid P.aeruginosa biofilms. Genetic studies revealed that Pel and Psl polysaccharides serve as essential scaffold and mediate macrocolony formation in mucoid P.aeruginosa biofilms. The Psl polysaccharide is more important than Pel polysaccharide in mucoid P.aeruginosa biofilm structure maintenance and phagocytosis resistance. The polysaccharides were further found to protect mucoid P.aeruginosa strain from host immune clearance in a mouse model of acute lung infection.

The roles of biofilm matrix polysaccharide Psl in mucoidPseudomonas aeruginosabiofilms

The opportunistic pathogen Pseudomonas aeruginosa causes life-threatening, persistent infections in patients with cystic fibrosis (CF). Persistence is attributed to the ability of these bacteria to form structured communities (biofilms). Biofilms rely on an extracellular polymeric substances matrix to maintain structure. Psl exopolysaccharide is a key matrix component of nonmucoid biofilms, yet the role of Psl in mucoid biofilms is unknown. In this report, using a variety of mutants in a mucoid P.aeruginosa background, we found that deletion of Psl-encoding genes dramatically decreased their biofilm formation ability, indicating that Psl is also a critical matrix component of mucoid biofilms. Our data also suggest that the overproduction of alginate leads to mucoid biofilms, which occupy more space, whereas Psl-dependent biofilms are densely packed. These data suggest that Psl polysaccharide may have significant contributions in biofilm persistence in patients with CF and may be helpful for designing therapies for P.aeruginosa CF infection.

Pseudomonas aeruginosa Psl polysaccharide reduces neutrophil phagocytosis and the oxidative response by limiting complement-mediated opsonization: Psl and the innate immune response towards P. aeruginosa

Pseudomonas aeruginosa Psl polysaccharide reduces neutrophil phagocytosis and the oxidative response by limiting complement-mediated opsonization: Psl and the innate immune response towards P. aeruginosa

Pseudomonas aeruginosa Psl polysaccharide reduces neutrophil phagocytosis and the oxidative response by limiting complement-mediated opsonization

Pseudomonas aeruginosa causes chronic lung infections in the airways of cystic fibrosis (CF) patients. Psl is an extracellular polysaccharide expressed by non-mucoid P. aeruginosa strains, which are believed to be initial colonizers. We hypothesized that Psl protects P. aeruginosa from host defences within the CF lung prior to their conversion to the mucoid phenotype. We discovered that serum opsonization significantly increased the production of reactive oxygen species (ROS) by neutrophils exposed to a psl-deficient mutant, compared with wild-type (WT) and Psl overexpressing strains (Psl(++)). Psl-deficient P. aeruginosa were internalized and killed by neutrophils and macrophages more efficiently than WT and Psl(++) variants. Deposition of complement components C3, C5 and C7 was significantly higher on psl-deficient strains compared with WT and Psl(++) bacteria. In an in vivo pulmonary competition assay, there was a 4.5-fold fitness advantage for WT over psl-deficient P. aeruginosa. Together, these data show that Psl inhibits efficient opsonization, resulting in reduced neutrophil ROS production, and decreased killing by phagocytes. This provides a survival advantage in vivo. Since phagocytes are critical in early recognition and control of infection, therapies aimed at Psl could improve the quality of life for patients colonized with P. aeruginosa.