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Interaction between extracellular lipase LipA and the polysaccharide alginate of Pseudomonas aeruginosa.

Tielen P, Kuhn H, Rosenau F, Jaeger KE, Flemming HC, Wingender J - BMC Microbiol. (2013)

Bottom Line: This effect was influenced by the chemical properties of the alginate molecules and was enhanced by the presence of O-acetyl groups in the alginate chain.This represents a physiological advantage for the cells.Especially in the biofilm lifestyle, the enzyme is retained near the cell surface, with the catalytic centre exposed towards the substrate and is protected from denaturation and proteolytic degradation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Aquatic Microbiology, University of Duisburg-Essen, Faculty of Chemistry, Biofilm Centre, Essen, Germany. p.tielen@tu-bs.de

ABSTRACT

Background: As an opportunistic human pathogen Pseudomonas aeruginosa is able to cause acute and chronic infections. The biofilm mode of life significantly contributes to the growth and persistence of P. aeruginosa during an infection process and mediates the pathogenicity of the bacterium. Within a biofilm mucoid strains of P. aeruginosa simultaneously produce and secrete several hydrolytic enzymes and the extracellular polysaccharide alginate. The focus of the current study was the interaction between extracellular lipase LipA and alginate, which may be physiologically relevant in biofilms of mucoid P. aeruginosa.

Results: Fluorescence microscopy of mucoid P. aeruginosa biofilms were performed using fluorogenic lipase substrates. It showed a localization of the extracellular enzyme near the cells. A microtiter plate-based binding assay revealed that the polyanion alginate is able to bind LipA. A molecular modeling approach showed that this binding is structurally based on electrostatic interactions between negatively charged residues of alginate and positively charged amino acids of the protein localized opposite of the catalytic centre. Moreover, we showed that the presence of alginate protected the lipase activity by protection from heat inactivation and from degradation by the endogenous, extracellular protease elastase LasB. This effect was influenced by the chemical properties of the alginate molecules and was enhanced by the presence of O-acetyl groups in the alginate chain.

Conclusion: We demonstrate that the extracellular lipase LipA from P. aeruginosa interacts with the polysaccharide alginate in the self-produced extracellular biofilm matrix of P. aeruginosa via electrostatic interactions suggesting a role of this interaction for enzyme immobilization and accumulation within biofilms. This represents a physiological advantage for the cells. Especially in the biofilm lifestyle, the enzyme is retained near the cell surface, with the catalytic centre exposed towards the substrate and is protected from denaturation and proteolytic degradation.

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Visualization of lipase activity in biofilms of P. aeruginosa. Membrane filter biofilms (PIA + Ca2+, 24 h, 36°C) of the parent strain P. aeruginosa SG81, the lipA overexpression strain SG81lipA+, the lipA defect mutant SG81ΔlipA and their corresponding complementation strain SG81ΔlipA::lipA were stained using the lipase substrate ELF®-97-palmitate. Shown are CLSM micrographs (optical section in the vertical middle of the biofilms) at a 400-fold magnification. For cell staining SYTO 9 (green) were used. Lipase activity, red; cells, green; overlay, yellow. The bars indicate 20 μm.
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Figure 1: Visualization of lipase activity in biofilms of P. aeruginosa. Membrane filter biofilms (PIA + Ca2+, 24 h, 36°C) of the parent strain P. aeruginosa SG81, the lipA overexpression strain SG81lipA+, the lipA defect mutant SG81ΔlipA and their corresponding complementation strain SG81ΔlipA::lipA were stained using the lipase substrate ELF®-97-palmitate. Shown are CLSM micrographs (optical section in the vertical middle of the biofilms) at a 400-fold magnification. For cell staining SYTO 9 (green) were used. Lipase activity, red; cells, green; overlay, yellow. The bars indicate 20 μm.

Mentions: In order to visualize lipase activity in situ, fluorescence micrographs of biofilms of the mucoid P. aeruginosa strain SG81 and its derivates were made using the fluorigenic lipase substrate ELF®-97-palmitate (Figure 1). An emulsion of the water insoluble ELF-97®-palmitate was prepared using sodium desoxycholate and gum arabic for emulsification and stabilisation of the substrate according to the well-established method for lipase activity determination with pNPP as a substrate [45]. Biofilms were grown on agar medium (PIA) supplemented with 0.1 M CaCl2 for stabilization of the biofilm matrix, since Ca2+ ions enhance the mechanical stability of P. aeruginosa biofilms by complexing the polyanion alginate [25,28,46]. This facilitates the treatment of the biofilms necessary for activity staining and subsequent observation by confocal laser scanning microscopy (CLSM).


Interaction between extracellular lipase LipA and the polysaccharide alginate of Pseudomonas aeruginosa.

Tielen P, Kuhn H, Rosenau F, Jaeger KE, Flemming HC, Wingender J - BMC Microbiol. (2013)

Visualization of lipase activity in biofilms of P. aeruginosa. Membrane filter biofilms (PIA + Ca2+, 24 h, 36°C) of the parent strain P. aeruginosa SG81, the lipA overexpression strain SG81lipA+, the lipA defect mutant SG81ΔlipA and their corresponding complementation strain SG81ΔlipA::lipA were stained using the lipase substrate ELF®-97-palmitate. Shown are CLSM micrographs (optical section in the vertical middle of the biofilms) at a 400-fold magnification. For cell staining SYTO 9 (green) were used. Lipase activity, red; cells, green; overlay, yellow. The bars indicate 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3733896&req=5

Figure 1: Visualization of lipase activity in biofilms of P. aeruginosa. Membrane filter biofilms (PIA + Ca2+, 24 h, 36°C) of the parent strain P. aeruginosa SG81, the lipA overexpression strain SG81lipA+, the lipA defect mutant SG81ΔlipA and their corresponding complementation strain SG81ΔlipA::lipA were stained using the lipase substrate ELF®-97-palmitate. Shown are CLSM micrographs (optical section in the vertical middle of the biofilms) at a 400-fold magnification. For cell staining SYTO 9 (green) were used. Lipase activity, red; cells, green; overlay, yellow. The bars indicate 20 μm.
Mentions: In order to visualize lipase activity in situ, fluorescence micrographs of biofilms of the mucoid P. aeruginosa strain SG81 and its derivates were made using the fluorigenic lipase substrate ELF®-97-palmitate (Figure 1). An emulsion of the water insoluble ELF-97®-palmitate was prepared using sodium desoxycholate and gum arabic for emulsification and stabilisation of the substrate according to the well-established method for lipase activity determination with pNPP as a substrate [45]. Biofilms were grown on agar medium (PIA) supplemented with 0.1 M CaCl2 for stabilization of the biofilm matrix, since Ca2+ ions enhance the mechanical stability of P. aeruginosa biofilms by complexing the polyanion alginate [25,28,46]. This facilitates the treatment of the biofilms necessary for activity staining and subsequent observation by confocal laser scanning microscopy (CLSM).

Bottom Line: This effect was influenced by the chemical properties of the alginate molecules and was enhanced by the presence of O-acetyl groups in the alginate chain.This represents a physiological advantage for the cells.Especially in the biofilm lifestyle, the enzyme is retained near the cell surface, with the catalytic centre exposed towards the substrate and is protected from denaturation and proteolytic degradation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Aquatic Microbiology, University of Duisburg-Essen, Faculty of Chemistry, Biofilm Centre, Essen, Germany. p.tielen@tu-bs.de

ABSTRACT

Background: As an opportunistic human pathogen Pseudomonas aeruginosa is able to cause acute and chronic infections. The biofilm mode of life significantly contributes to the growth and persistence of P. aeruginosa during an infection process and mediates the pathogenicity of the bacterium. Within a biofilm mucoid strains of P. aeruginosa simultaneously produce and secrete several hydrolytic enzymes and the extracellular polysaccharide alginate. The focus of the current study was the interaction between extracellular lipase LipA and alginate, which may be physiologically relevant in biofilms of mucoid P. aeruginosa.

Results: Fluorescence microscopy of mucoid P. aeruginosa biofilms were performed using fluorogenic lipase substrates. It showed a localization of the extracellular enzyme near the cells. A microtiter plate-based binding assay revealed that the polyanion alginate is able to bind LipA. A molecular modeling approach showed that this binding is structurally based on electrostatic interactions between negatively charged residues of alginate and positively charged amino acids of the protein localized opposite of the catalytic centre. Moreover, we showed that the presence of alginate protected the lipase activity by protection from heat inactivation and from degradation by the endogenous, extracellular protease elastase LasB. This effect was influenced by the chemical properties of the alginate molecules and was enhanced by the presence of O-acetyl groups in the alginate chain.

Conclusion: We demonstrate that the extracellular lipase LipA from P. aeruginosa interacts with the polysaccharide alginate in the self-produced extracellular biofilm matrix of P. aeruginosa via electrostatic interactions suggesting a role of this interaction for enzyme immobilization and accumulation within biofilms. This represents a physiological advantage for the cells. Especially in the biofilm lifestyle, the enzyme is retained near the cell surface, with the catalytic centre exposed towards the substrate and is protected from denaturation and proteolytic degradation.

Show MeSH
Related in: MedlinePlus