The roles of biofilm matrix polysaccharide Psl in mucoid Pseudomonas aeruginosa biofilms

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.     

Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production

Exopolysaccharides contribute significantly to attachment and biofilm formation in the opportunisitc pathogen Pseudomonas aeruginosa . The Psl polysaccharide, which is synthesized by the p olysaccharide s ynthesis l ocus ( psl ), is required for biofilm formation in non-mucoid strains that do not rely on alginate as the principal biofilm polysaccharide. In-frame deletion and complementation studies of individual psl genes revealed that 11 psl genes, pslACDEFGHIJKL , are required for Psl production and surface attachment. We also present the first structural analysis of the psl -dependent polysaccharide, which consists of a repeating pentasaccharide containing d -mannose, d -glucose and l -rhamnose:

In addition, we identified the sugar nucleotide precursors involved in Psl generation and demonstrated the requirement for GDP- d -mannose, UDP- d -glucose and dTDP- l -rhamnose in Psl production and surface attachment. Finally, genetic analyses revealed that wbpW restored Psl production in a pslB mutant and pslB promoted A-band LPS synthesis in a wbpW mutant, indicating functional redundancy and overlapping roles for these two enzymes. The structural and genetic data presented here provide a basis for further investigation of the Psl proteins and potential roles for Psl in the biology and pathogenesis of 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.     

Pseudomonas aeruginosa Psl is a galactose- and mannose-rich exopolysaccharide

The Pseudomonas aeruginosa polysaccharide synthesis locus (psl) is predicted to encode an exopolysaccharide which is critical for biofilm formation. Here we used chemical composition analyses and mannose- or galactose-specific lectin staining, followed by confocal laser scanning microscopy and electron microscopy, to show that Psl is a galactose-rich and mannose-rich exopolysaccharide.     

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

Decades 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.     

The Extracellular Matrix Component Psl Provides Fast-Acting Antibiotic Defense in Pseudomonas aeruginosa Biofilms

Bacteria within biofilms secrete and surround themselves with an extracellular matrix, which serves as a first line of defense against antibiotic attack. Polysaccharides constitute major elements of the biofilm matrix and are implied in surface adhesion and biofilm organization, but their contributions to the resistance properties of biofilms remain largely elusive. Using a combination of static and continuous-flow biofilm experiments we show that Psl, one major polysaccharide in the Pseudomonas aeruginosa biofilm matrix, provides a generic first line of defense toward antibiotics with diverse biochemical properties during the initial stages of biofilm development. Furthermore, we show with mixed-strain experiments that antibiotic-sensitive “non-producing” cells lacking Psl can gain tolerance by integrating into Psl-containing biofilms. However, non-producers dilute the protective capacity of the matrix and hence, excessive incorporation can result in the collapse of resistance of the entire community. Our data also reveal that Psl mediated protection is extendible to E. coli and S. aureus in co-culture biofilms. Together, our study shows that Psl represents a critical first bottleneck to the antibiotic attack of a biofilm community early in biofilm development.     

The Pel and Psl polysaccharides provide Pseudomonas aeruginosa structural redundancy within the biofilm matrix

Extracellular polysaccharides comprise a major component of the biofilm matrix. Many species that are adept at biofilm formation have the capacity to produce multiple types of polysaccharides. Pseudomonas aeruginosa produces at least three extracellular polysaccharides, alginate, Pel and Psl, that have been implicated in biofilm development. Non-mucoid strains can use either Pel or Psl as the primary matrix structural polysaccharide. In this study, we evaluated a range of clinical and environmental P.aeruginosa isolates for their dependence on Pel and Psl for biofilm development. Mutational analysis demonstrates that Psl plays an important role in surface attachment for most isolates. However, there was significant strain-to-strain variability in the contribution of Pel and Psl to mature biofilm structure. This analysis led us to propose four classes of strains based upon their Pel and Psl functional and expression profiles. Our data also suggest that Pel and Psl can serve redundant functions as structural scaffolds in mature biofilms. We propose that redundancy could help preserve the capacity to produce a biofilm when exopolysaccharide genes are subjected to mutation. To test this, we used PAO1, a common lab strain that primarily utilizes Psl in the matrix. As expected, a psl mutant strain initially produced a poor biofilm. After extended cultivation, we demonstrate that this strain acquired mutations that upregulated expression of the Pel polysaccharide, demonstrating the utility of having a redundant scaffold exopolysaccharide. Collectively, our studies revealed both unique and redundant roles for two distinct biofilm exopolysaccharides.     

Impact of the exopolysaccharides Pel and Psl on the initial adhesion of Pseudomonas aeruginosa to sand

In this study, the impact of the exopolysaccharides Pel and Psl on the cell surface electron donor-electron acceptor (acid-base) properties and adhesion to quartz sand was investigated by using Pseudomonas aeruginosa PAO1 and its isogenic EPS-mutant strains Δpel, Δpsl and Δpel/Δpsl. The microbial adhesion to hydrocarbon (MATH) test and titration results showed that both Pel and Psl contribute to the surface hydrophobicity of the cell. The results of contact angle measurement, however, showed no correlation with the cell surface hydrophobicity measured by the MATH test and the titration method. Packed-bed column experiments indicated that the exopolysaccharides Pel and Psl are involved in the initial cell attachment to the sand surface and the extent of their impact is dependent on the ionic strength (IS) of the solution. Overall, the Δpel/Δpsl double mutant had the lowest adhesion coefficient to sand compared with the wild-type PAO1, the Δpel mutant and the Δpsl mutant. It is hypothesized that in addition to bacterial surface hydrophobicity and DLVO forces, other factors, eg steric repulsion caused by extracellular macromolecules, and cell surface appendages (flagella and pili) also contribute significantly to the interaction between the cell surface and a sand grain.     

Sedimentation-equilibrium studies of the polysaccharide components of Pseudomonas aeruginosa

Mild acid hydrolysis of lipopolysaccharide antigens from seven different serotype strains antigen immunotypes nos. 1–7 [in the classification of Fisher, M. W., Devlin, H. B. & Gnabasik, F. J. (1969) J. Bacteriol. 98 , 835–836] of Pseudomonas aeruginosa gave polysaccharide components of high molecular weight, which were isolated by gel filtration and dialysis. These components were examined by ultracentrifugation at equilibrium with the Rayleigh interferometric optical system. The partial specific volumes were calculated from densities obtained by using a mechanical oscillator. The average molecular weights (M _n, M _w, and M _z) were calculated and compared to evaluate the polydispersity of the polysaccharides. The nonideality was investigated by varying the rotor speed, the height of the solution column, and the concentrations of the polysaccharide fractions. The molar masses were found to range from 14,000 for the polysaccharide from immunotype two to 24,000 for that from immunotype one, when extrapolated to zero rotor speed and solution column height.     

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.     

Effect of polysaccharide interactions on antibiotic susceptibility of Pseudomonas aeruginosa

The relative viscosity of Pseudomonas aeruginosa alginate was shown to increase markedly when combined with mucin, Ca²⁺ ions and the exopolysaccharide from Pseudomonas cepacia. The presence of such a heterodisperse polysaccharide solution significantly reduced the diffusion and hence antimicrobial activity of tobramycin and to a lesser extent ciprofloxacin against Ps. aeruginosa by factors of 90 and 2.5-fold respectively over a 5 h incubation period. The clinical implications of these results are discussed in relation to cystic fibrosis.     

Immunosuppressive components of extracellular polysaccharide from Pseudomonas aeruginosa clinical strains

The immunosuppressive activity of lipopolysaccharide (LPS) contained in filtrates of newly isolated P. aeruginosa cultures was studied. The experimental model of delayed hypersensitivity to non-bacterial antigen in CBA mice and gel filtration through Sephadex G-200 were used. In addition to the already known LPS component with direct immunosuppressive action and having a mol.wt. of 150-800 kD, a new component was detected. It was found to be in inactive state and could be activated by redox treatment, thus becoming capable of inducing capacity for immunosuppression in Escherichia coli. This component had a mol.wt. of 50-70 kD and lost its activity after heating.     

Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa

The slime polysaccharides produced by Pseudomonas aeruginosa isolated from a variety of human infections were investigated. Slime production in culture seemed optimal when adequate amounts of carbohydrate were present and under conditions of either high osmotic pressure or inadequate protein supply. The polysaccharides produced by the organisms were similar to each other, to the slime of Azotobacter vinelandii, and to seaweed alginic acids. They were composed of beta-1,4-linked d-mannuronic acid residues and variable amounts of its 5-epimer l-guluronic acid. All bacterial polymers contained o-acetyl groups which are absent in the alginates. The polysaccharides differed considerably in the ratio of mannuronic to guluronic acid content and in the number of o-acetyl groups. The particular composition of the slime was not found to be characteristic for the disease process from which the mucoid variants of P. aeruginosa were obtained.     

Transduction of a freshwater microbial community by a new Pseudomonas aeruginosa generalized transducing phage,UT1

A pseudolysogenic, generalized transducing bacteriophage, UT1, isolated from a natural freshwater habitat, is capable of mediating the transfer of both chromosomal andplasmid DNA between strains of Pseudomonas aeruginosa. Several chromosomal alleles from three different P. aeruginosa strains were found to transduce at frequencies from 10^-8 to 10^-10 transductants per PFU at multiplicities of infection (MOD between 0.1 and 1. Transduction frequencies of certain alleles increased up to 1000-fold as MOIs were decreased to 0.01. UT1 is also capable of transducing plasmid DNA to indigenous populations of microorganisms in natural lake-water environments. Data obtained in this study suggest that environmentally endemic bacteriophages such as UT1 are formidable transducers of naturally occurring microbial communities. It should be possible to develop model systems to test transduction in freshwater environments using components derived exclusively from these environments.     

Effect of polysaccharide interactions on antibiotic susceptibility of Pseudomonas aeruginosa.

The relative viscosity of Pseudomonas aeruginosa alginate was shown to increase markedly when combined with mucin, Ca2+ ions and the exopolysaccharide from Pseudomonas cepacia. The presence of such a heterodisperse polysaccharide solution significantly reduced the diffusion and hence antimicrobial activity of tobramycin and to a lesser extent ciprofloxacin against Ps. aeruginosa by factors of 90 and 2.5-fold respectively over a 5 h incubation period. The clinical implications of these results are discussed in relation to cystic fibrosis.     

Structure of an extracellular cross-reactive polysaccharide from Pseudomonas aeruginosa immunotype 4

A neutral small molecular mass (approximately 6.5 kDa) polysaccharide comprising a pentasaccharide repeat unit was isolated from culture supernatants of Pseudomonas aeruginosa immunotype 4. The polysaccharide had a pentasaccharide repeating unit as follows (formula; see text) where Rha is rhamnose. The structure was determined using acid hydrolysis, solvolysis with anhydrous hydrogen fluoride, methylation analysis, and 1H and 13C nuclear magnetic resonance spectroscopy including nuclear Overhauser enhancement experiments. The polysaccharide bound antibody raised to the lipopolysaccharide of the seven P. aeruginosa Fisher-Devlin immunotype strains. Inhibition assays demonstrated the presence of a serologically similar polysaccharide in supernatants of these strains. Affinity-purified antibody to the polysaccharide bound to lipopolysaccharide and whole cells of the immunotype strains of P. aeruginosa in a Western immunoblot and colony blot assay, respectively. This polysaccharide seems to contain an antigenic determinant present in the core of the P. aeruginosa lipopolysaccharide or may represent another minor polysaccharide substituent on the lipopolysaccharide in addition to the O side chain.     

Cell death in Pseudomonas aeruginosa biofilm development

Bacteria growing in biofilms often develop multicellular, three-dimensional structures known as microcolonies. Complex differentiation within biofilms of Pseudomonas aeruginosa occurs, leading to the creation of voids inside microcolonies and to the dispersal of cells from within these voids. However, key developmental processes regulating these events are poorly understood. A normal component of multicellular development is cell death. Here we report that a repeatable pattern of cell death and lysis occurs in biofilms of P. aeruginosa during the normal course of development. Cell death occurred with temporal and spatial organization within biofilms, inside microcolonies, when the biofilms were allowed to develop in continuous-culture flow cells. A subpopulation of viable cells was always observed in these regions. During the onset of biofilm killing and during biofilm development thereafter, a bacteriophage capable of superinfecting and lysing the P. aeruginosa parent strain was detected in the fluid effluent from the biofilm. The bacteriophage implicated in biofilm killing was closely related to the filamentous phage Pf1 and existed as a prophage within the genome of P. aeruginosa. We propose that prophage-mediated cell death is an important mechanism of differentiation inside microcolonies that facilitates dispersal of a subpopulation of surviving cells.