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.     

The pel polysaccharide can serve a structural and protective role in the biofilm matrix of Pseudomonas aeruginosa

Bacterial extracellular polysaccharides are a key constituent of the extracellular matrix material of biofilms. Pseudomonas aeruginosa is a model organism for biofilm studies and produces three extracellular polysaccharides that have been implicated in biofilm development, alginate, Psl and Pel. Significant work has been conducted on the roles of alginate and Psl in biofilm development, however we know little regarding Pel. In this study, we demonstrate that Pel can serve two functions in biofilms. Using a novel assay involving optical tweezers, we demonstrate that Pel is crucial for maintaining cell-to-cell interactions in a PA14 biofilm, serving as a primary structural scaffold for the community. Deletion of pelB resulted in a severe biofilm deficiency. Interestingly, this effect is strain-specific. Loss of Pel production in the laboratory strain PAO1 resulted in no difference in attachment or biofilm development; instead Psl proved to be the primary structural polysaccharide for biofilm maturity. Furthermore, we demonstrate that Pel plays a second role by enhancing resistance to aminoglycoside antibiotics. This protection occurs only in biofilm populations. We show that expression of the pel gene cluster and PelF protein levels are enhanced during biofilm growth compared to liquid cultures. Thus, we propose that Pel is capable of playing both a structural and a protective role in P. aeruginosa biofilms.     

Polysaccharides serve as scaffold of biofilms formed by mucoid Pseudomonas 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.     

Role of exopolysaccharides in Pseudomonas aeruginosa biofilm formation and architecture

Pseudomonas aeruginosa is an opportunistic human pathogen and has been established as a model organism to study bacterial biofilm formation. At least three exopolysaccharides (alginate, Psl, and Pel) contribute to the formation of biofilms in this organism. Here mutants deficient in the production of one or more of these polysaccharides were generated to investigate how these polymers interactively contribute to biofilm formation. Confocal laser scanning microscopy of biofilms formed in flow chambers showed that mutants deficient in alginate biosynthesis developed biofilms with a decreased proportion of viable cells than alginate-producing strains, indicating a role of alginate in viability of cells in biofilms. Alginate-deficient mutants showed enhanced extracellular DNA (eDNA)-containing surface structures impacting the biofilm architecture. PAO1 ΔpslA Δalg8 overproduced Pel, and eDNA showing meshwork-like structures presumably based on an interaction between both polymers were observed. The formation of characteristic mushroom-like structures required both Psl and alginate, whereas Pel appeared to play a role in biofilm cell density and/or the compactness of the biofilm. Mutants producing only alginate, i.e., mutants deficient in both Psl and Pel production, lost their ability to form biofilms. A lack of Psl enhanced the production of Pel, and the absence of Pel enhanced the production of alginate. The function of Psl in attachment was independent of alginate and Pel. A 30% decrease in Psl promoter activity in the alginate-overproducing MucA-negative mutant PDO300 suggested inverse regulation of both biosynthesis operons. Overall, this study demonstrated that the various exopolysaccharides and eDNA interactively contribute to the biofilm architecture of P. aeruginosa.     

Analysis of Pseudomonas aeruginosa conditional psl variants reveals roles for the psl polysaccharide in adhesion and maintaining biofilm structure postattachment

The ability to form biofilms in the airways of people suffering from cystic fibrosis is a critical element of Pseudomonas aeruginosa pathogenesis. The 15-gene psl operon encodes a putative polysaccharide that plays an important role in biofilm initiation in nonmucoid P. aeruginosa strains. Biofilm initiation by a P. aeruginosa PAO1 strain with disruption of pslA and pslB (DeltapslAB) was severely compromised, indicating that psl has a role in cell-surface interactions. In this study, we investigated the adherence properties of this DeltapslAB mutant using biotic surfaces (epithelial cells and mucin-coated surfaces) and abiotic surfaces. Our results showed that psl is required for attachment to a variety of surfaces, independent of the carbon source. To study the potential roles of Psl apart from attachment, we generated a psl-inducible P. aeruginosa strain (Deltapsl/p(BAD)-psl) by replacing the psl promoter region with araC-p(BAD), so that expression of psl could be controlled by addition of arabinose. Analysis of biofilms formed by the Deltapsl/p(BAD)-psl strain indicated that expression of the psl operon is required to maintain the biofilm structure at steps postattachment. Overproduction of the Psl polysaccharide led to enhanced cell-surface and intercellular adhesion of P. aeruginosa. This translated into significant changes in the architecture of the biofilm. We propose that Psl has an important role in P. aeruginosa adhesion, which is critical for initiation and maintenance of the biofilm structure.     

Role of polysaccharides in Pseudomonas aeruginosa biofilm development

During the past decade, there has been a renewed interest in using Pseudomonas aeruginosa as a model system for biofilm development and pathogenesis. Since the biofilm matrix represents a crucial interface between the bacterium and the host or its environment, considerable effort has been expended to acquire a more complete understanding of the matrix composition. Here, we focus on recent developments regarding the roles of alginate, Psl, and Pel polysaccharides in the biofilm matrix.     

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.     

Pseudomonas biofilm matrix composition and niche biology

Biofilms are a predominant form of growth for bacteria in the environment and in the clinic. Critical for biofilm development are adherence, proliferation, and dispersion phases. Each of these stages includes reinforcement by, or modulation of, the extracellular matrix. Pseudomonas aeruginosa has been a model organism for the study of biofilm formation. Additionally, other Pseudomonas species utilize biofilm formation during plant colonization and environmental persistence. Pseudomonads produce several biofilm matrix molecules, including polysaccharides, nucleic acids, and proteins. Accessory matrix components shown to aid biofilm formation and adaptability under varying conditions are also produced by pseudomonads. Adaptation facilitated by biofilm formation allows for selection of genetic variants with unique and distinguishable colony morphology. Examples include rugose small-colony variants and wrinkly spreaders (WS), which over produce Psl/Pel or cellulose, respectively, and mucoid bacteria that over produce alginate. The well-documented emergence of these variants suggests that pseudomonads take advantage of matrix-building subpopulations conferring specific benefits for the entire population. This review will focus on various polysaccharides as well as additional Pseudomonas biofilm matrix components. Discussions will center on structure-function relationships, regulation, and the role of individual matrix molecules in niche biology.     

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 .     

Low concentrations of ethanol stimulate biofilm and pellicle formation in Pseudomonas aeruginosa

Biofilms are communities of surface-attached microbial cells that resist environmental stresses. In this study, we found that low concentrations of ethanol increase biofilm formation in Pseudomonas aeruginosa PAO1 but not in a mutant of it lacking both Psl and Pel exopolysaccharides. Low concentrations of ethanol also increased pellicle formation at the air–liquid interface.     

Pseudomonas aeruginosa biofilm: Potential therapeutic targets

Pseudomonas aeruginosa is a gram-negative pathogen that has become an important cause of infection, especially in patients with compromised host defense mechanisms. It is frequently related to nosocomial infections such as pneumonia, urinary tract infections (UTIs) and bacteremia. The biofilm formed by the bacteria allows it to adhere to any surface, living or non-living and thus Pseudomonal infections can involve any part of the body. Further, the adaptive and genetic changes of the micro-organisms within the biofilm make them resistant to all known antimicrobial agents making the Pseudomonal infections complicated and life threatening. Pel, Psl and Alg operons present in P. aeruginosa are responsible for the biosynthesis of extracellular polysaccharide which plays an important role in cell–cell and cell–surface interactions during biofilm formation. Understanding the bacterial virulence which depends on a large number of cell-associated and extracellular factors is essential to know the potential drug targets for future studies. Current novel methods like small molecule based inhibitors, phytochemicals, bacteriophage therapy, photodynamic therapy, antimicrobial peptides, monoclonal antibodies and nanoparticles to curtail the biofilm formed by P. aeruginosa are being discussed in this review.     

Identification of psl, a locus encoding a potential exopolysaccharide that is essential for Pseudomonas aeruginosa PAO1 biofilm formation

Bacteria inhabiting biofilms usually produce one or more polysaccharides that provide a hydrated scaffolding to stabilize and reinforce the structure of the biofilm, mediate cell-cell and cell-surface interactions, and provide protection from biocides and antimicrobial agents. Historically, alginate has been considered the major exopolysaccharide of the Pseudomonas aeruginosa biofilm matrix, with minimal regard to the different functions polysaccharides execute. Recent chemical and genetic studies have demonstrated that alginate is not involved in the initiation of biofilm formation in P. aeruginosa strains PAO1 and PA14. We hypothesized that there is at least one other polysaccharide gene cluster involved in biofilm development. Two separate clusters of genes with homology to exopolysaccharide biosynthetic functions were identified from the annotated PAO1 genome. Reverse genetics was employed to generate mutations in genes from these clusters. We discovered that one group of genes, designated psl, are important for biofilm initiation. A PAO1 strain with a disruption of the first two genes of the psl cluster (PA2231 and PA2232) was severely compromised in biofilm initiation, as confirmed by static microtiter and continuous culture flow cell and tubing biofilm assays. This impaired biofilm phenotype could be complemented with the wild-type psl sequences and was not due to defects in motility or lipopolysaccharide biosynthesis. These results implicate an as yet unknown exopolysaccharide as being required for the formation of the biofilm matrix. Understanding psl-encoded exopolysaccharide expression and protection in biofilms will provide insight into the pathogenesis of P. aeruginosa in cystic fibrosis and other infections involving biofilms.     

乳酸锌和氟化亚锡对铜绿假单胞菌、鲍曼不动杆菌和变异链球菌生物被膜的抑制作用

乳酸锌(Zn lactate·3H_2O)和氟化亚锡(SnF_2)常作为牙膏中的活性物质添加剂用来预防龋齿及口腔生物被膜的形成。文中评估了Zn lactate·3H_2O和SnF_2对铜绿假单胞菌、鲍曼不动杆菌和变异链球菌生物被膜的作用。对铜绿假单胞菌PAO1生物被膜的抑制实验证实乳酸锌和氟化亚锡都具有抑制其生物被膜的功能,联用效果尤佳。乳酸锌通过干扰胞外多糖基质网的形成起作用,而氟化亚锡则可以明显降低生物被膜的生物量。更为重要的是,工作浓度的两种化合物联用几乎可以完全抑制3种实验菌株生物被膜的形成。     

假单胞菌生物被膜核心胞外多糖生物合成系统研究进展

胞外多糖是假单胞菌生物被膜的重要组成部分,能增强菌体对外界环境、抗菌剂和宿主防御的耐受性.假单胞菌能产生3种与生物被膜形成密切相关的核心胞外多糖:褐藻胶、Psl和Pel,它们在细菌细胞中的合成和转运分别依赖对应的褐藻胶、Psl和Pel生物合成系统.因此,本综述系统全面地总结了假单胞菌3种胞外多糖生物合成系统结构生物学的...     

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.     

Role of extracellular matrix components in the formation of biofilms and their contribution to the biocontrol activity of Pseudomonas chlororaphis PCL1606

Pseudomonas chlororaphis PCL1606 (PcPCL1606) displays plant-colonizing features and exhibits antagonistic traits against soil-borne phytopathogenic fungi. Biofilm formation could be relevant for the PcPCL1606 lifestyle, and in this study the role of some putative extracellular matrix components (EMC; Fap-like fibre, alginate and Psl-like polysaccharides) in the biofilm architecture and biocontrol activity of this bacterium were determined. EMC such as the Fap-like fibre and alginate polysaccharide play secondary roles in biofilm formation in PcPCL1606, because they are not fundamental to its biofilm architecture in flow cell chamber, but synergistically they have shown to favour bacterial competition during biofilm formation. Conversely, studies on Psl-like polysaccharide have revealed that it may contain mannose, and that it is strongly involved in the PcPCL1606 biofilm architecture and niche competition. Furthermore, the Fap-like fibre and Psl-like exopolysaccharide play roles in early surface attachment and contribute to biocontrol activity against the white root rot disease caused by Rosellinia necatrix in avocado plants. These results constitute the first report regarding the study of the extracellular matrix of the PcPCL1606 strain and highlight the importance of a putative Fap-like fibre and Psl-like exopolysaccharide produced by PcPCL1606 in the biofilm formation process and interactions with the host plant root.     

Biofilm formation by Pseudomonas aeruginosa wild type,flagella and type IV pili mutants

Biofilm formation by Gfp-tagged Pseudomonas aeruginosa PAO1 wild type, flagella and type IV pili mutants in flow chambers irrigated with citrate minimal medium was characterized by the use of confocal laser scanning microscopy and comstat image analysis. Flagella and type IV pili were not necessary for P. aeruginosa initial attachment or biofilm formation, but the cell appendages had roles in biofilm development, as wild type, flagella and type IV pili mutants formed biofilms with different structures. Dynamics and selection during biofilm formation were investigated by tagging the wild type and flagella/type IV mutants with Yfp and Cfp and performing time-lapse confocal laser scanning microscopy in mixed colour biofilms. The initial microcolony formation occurred by clonal growth, after which wild-type P. aeruginosa bacteria spread over the substratum by means of twitching motility. The wild-type biofilms were dynamic compositions with extensive motility, competition and selection occurring during development. Bacterial migration prevented the formation of larger microcolonial structures in the wild-type biofilms. The results are discussed in relation to the current model for P. aeruginosa biofilm development.