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Assembly and development of the Pseudomonas aeruginosa biofilm matrix.

Ma L, Conover M, Lu H, Parsek MR, Bayles K, Wozniak DJ - PLoS Pathog. (2009)

Bottom Line: During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center.These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal.Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications.

View Article: PubMed Central - PubMed

Affiliation: Microbiology and Immunology, Wake Forest University Health Sciences, Winston-Salem, North Carolina, USA.

ABSTRACT
Virtually all cells living in multicellular structures such as tissues and organs are encased in an extracellular matrix. One of the most important features of a biofilm is the extracellular polymeric substance that functions as a matrix, holding bacterial cells together. Yet very little is known about how the matrix forms or how matrix components encase bacteria during biofilm development. Pseudomonas aeruginosa forms environmentally and clinically relevant biofilms and is a paradigm organism for the study of biofilms. The extracellular polymeric substance of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids, and proteins. Here, we directly visualize the product of the polysaccharide synthesis locus (Psl exopolysaccharide) at different stages of biofilm development. During attachment, Psl is anchored on the cell surface in a helical pattern. This promotes cell-cell interactions and assembly of a matrix, which holds bacteria in the biofilm and on the surface. Chemical dissociation of Psl from the bacterial surface disrupted the Psl matrix as well as the biofilm structure. During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center. At the dispersion stage, swimming cells appear in this matrix cavity. Dead cells and extracellular DNA (eDNA) are also concentrated in the Psl matrix-free area. Deletion of genes that control cell death and autolysis affects the formation of the matrix cavity and microcolony dispersion. These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal. Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications.

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Related in: MedlinePlus

How the Psl matrix maintains the biofilm architecture.Shown are sets of optical sectioned images acquired at different locations of the biofilm (position indicated on the top of each panel). DIC images are in gray. Green and red merged images are shown at the lower right corner for panels A and B. Bar, 5 µm for panels A and B, 10 µm for panels C and D. (A) Psl matrix (green, MOA-FITC staining, bottom left) in a multilayer biofilm (4 µm thickness) of WFPA801 (red, FM4-64 staining, upper left). (B) Psl matrix (green, MOA-FITC staining, bottom left) in a WFPA801 biofilm microcolony (FM4-64 staining, 24 µm thickness, upper left). The top-down view (square) and side view (rectangle) of 3D reconstituted images are shown, which reveals how the peripherally localized Psl matrix encases the bacteria in a mushroom-like microcolony. (C) The newly synthesized Psl matrix (red) covers the existing Psl matrix (green). The Psl matrix of WFPA801 biofilms was stained with MOA-FITC (green) at 40-h-growth and stained again by MOA-TRITC (red) at 60-h-growth. The large square image is a horizontal section at the top of microcolony. The blue line in the side view images marks the location of the section (rectangle). Green and gray merged images are at the lower right. Green and red merged images are at the right panel and the bottom middle of the middle panel.
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ppat-1000354-g004: How the Psl matrix maintains the biofilm architecture.Shown are sets of optical sectioned images acquired at different locations of the biofilm (position indicated on the top of each panel). DIC images are in gray. Green and red merged images are shown at the lower right corner for panels A and B. Bar, 5 µm for panels A and B, 10 µm for panels C and D. (A) Psl matrix (green, MOA-FITC staining, bottom left) in a multilayer biofilm (4 µm thickness) of WFPA801 (red, FM4-64 staining, upper left). (B) Psl matrix (green, MOA-FITC staining, bottom left) in a WFPA801 biofilm microcolony (FM4-64 staining, 24 µm thickness, upper left). The top-down view (square) and side view (rectangle) of 3D reconstituted images are shown, which reveals how the peripherally localized Psl matrix encases the bacteria in a mushroom-like microcolony. (C) The newly synthesized Psl matrix (red) covers the existing Psl matrix (green). The Psl matrix of WFPA801 biofilms was stained with MOA-FITC (green) at 40-h-growth and stained again by MOA-TRITC (red) at 60-h-growth. The large square image is a horizontal section at the top of microcolony. The blue line in the side view images marks the location of the section (rectangle). Green and gray merged images are at the lower right. Green and red merged images are at the right panel and the bottom middle of the middle panel.

Mentions: Depending on conditions, biofilms can form either flat multilayer structures or microcolonies that have defined 3-dimensional arrangements. To visualize how the Psl matrix maintains the biofilm architecture, we studied the distribution of Psl in these two types of communities by optically sectioning MOA-stained biofilms. In a flat biofilm of WFPA801, Psl matrix was equally distributed and associated with bacterial cells (Figure 4A). However in a well-defined 3D microcolony structure, the Psl matrix was unevenly distributed. This was visualized in representative horizontal Z-images of a microcolony (Figure 4B, left and middle panels), a 3-D reconstruction of the microcolony (Figure 4B, right panel) or a series of 1 µm Z-stacks of the microcolony reconstructed into a movie file (Video S1). Here, enhanced lectin staining was observed in the periphery of each microcolony and reduced lectin staining was seen in the center of microcolonies (middle panel in Figure 4B). In the mushroom-like microcolonies, little Psl staining was detected in the lower center (the area from the microcolony center to the surface area, left panel of Figure 4B). This staining pattern resulted in a matrix-free cavity in the lower center of the mushroom-like microcolony. In some cases, such as that seen in the microcolony close to the substratum, there were fewer bacteria in the center than in the microcolony periphery (left panel, Figure 4B). However, at the top of the microcolony there was little difference in the density of cells in the center versus those in the periphery (Figure 4B, middle panel). This was observed by staining the microcolonies with either FM4-64 (Figure 4B) or when the cells expressed GFP (Video S1). A similar matrix distribution pattern was observed using biofilm-grown wild type PAO1, indicating the Psl staining pattern in biofilms was not due to Psl overproduction (data not shown). No Psl was detected in either multiple layer biofilms or microcolonies formed by the Δpsl strain WFPA800 (data not shown). Overall, our data shows that Psl surrounds the constituent cells in either a multilayer biofilm or a 3D-structured microcolony (see 3D view of the microcolony in the right panel of Figure 4B and the series of Z-stacks in Video S1).


Assembly and development of the Pseudomonas aeruginosa biofilm matrix.

Ma L, Conover M, Lu H, Parsek MR, Bayles K, Wozniak DJ - PLoS Pathog. (2009)

How the Psl matrix maintains the biofilm architecture.Shown are sets of optical sectioned images acquired at different locations of the biofilm (position indicated on the top of each panel). DIC images are in gray. Green and red merged images are shown at the lower right corner for panels A and B. Bar, 5 µm for panels A and B, 10 µm for panels C and D. (A) Psl matrix (green, MOA-FITC staining, bottom left) in a multilayer biofilm (4 µm thickness) of WFPA801 (red, FM4-64 staining, upper left). (B) Psl matrix (green, MOA-FITC staining, bottom left) in a WFPA801 biofilm microcolony (FM4-64 staining, 24 µm thickness, upper left). The top-down view (square) and side view (rectangle) of 3D reconstituted images are shown, which reveals how the peripherally localized Psl matrix encases the bacteria in a mushroom-like microcolony. (C) The newly synthesized Psl matrix (red) covers the existing Psl matrix (green). The Psl matrix of WFPA801 biofilms was stained with MOA-FITC (green) at 40-h-growth and stained again by MOA-TRITC (red) at 60-h-growth. The large square image is a horizontal section at the top of microcolony. The blue line in the side view images marks the location of the section (rectangle). Green and gray merged images are at the lower right. Green and red merged images are at the right panel and the bottom middle of the middle panel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654510&req=5

ppat-1000354-g004: How the Psl matrix maintains the biofilm architecture.Shown are sets of optical sectioned images acquired at different locations of the biofilm (position indicated on the top of each panel). DIC images are in gray. Green and red merged images are shown at the lower right corner for panels A and B. Bar, 5 µm for panels A and B, 10 µm for panels C and D. (A) Psl matrix (green, MOA-FITC staining, bottom left) in a multilayer biofilm (4 µm thickness) of WFPA801 (red, FM4-64 staining, upper left). (B) Psl matrix (green, MOA-FITC staining, bottom left) in a WFPA801 biofilm microcolony (FM4-64 staining, 24 µm thickness, upper left). The top-down view (square) and side view (rectangle) of 3D reconstituted images are shown, which reveals how the peripherally localized Psl matrix encases the bacteria in a mushroom-like microcolony. (C) The newly synthesized Psl matrix (red) covers the existing Psl matrix (green). The Psl matrix of WFPA801 biofilms was stained with MOA-FITC (green) at 40-h-growth and stained again by MOA-TRITC (red) at 60-h-growth. The large square image is a horizontal section at the top of microcolony. The blue line in the side view images marks the location of the section (rectangle). Green and gray merged images are at the lower right. Green and red merged images are at the right panel and the bottom middle of the middle panel.
Mentions: Depending on conditions, biofilms can form either flat multilayer structures or microcolonies that have defined 3-dimensional arrangements. To visualize how the Psl matrix maintains the biofilm architecture, we studied the distribution of Psl in these two types of communities by optically sectioning MOA-stained biofilms. In a flat biofilm of WFPA801, Psl matrix was equally distributed and associated with bacterial cells (Figure 4A). However in a well-defined 3D microcolony structure, the Psl matrix was unevenly distributed. This was visualized in representative horizontal Z-images of a microcolony (Figure 4B, left and middle panels), a 3-D reconstruction of the microcolony (Figure 4B, right panel) or a series of 1 µm Z-stacks of the microcolony reconstructed into a movie file (Video S1). Here, enhanced lectin staining was observed in the periphery of each microcolony and reduced lectin staining was seen in the center of microcolonies (middle panel in Figure 4B). In the mushroom-like microcolonies, little Psl staining was detected in the lower center (the area from the microcolony center to the surface area, left panel of Figure 4B). This staining pattern resulted in a matrix-free cavity in the lower center of the mushroom-like microcolony. In some cases, such as that seen in the microcolony close to the substratum, there were fewer bacteria in the center than in the microcolony periphery (left panel, Figure 4B). However, at the top of the microcolony there was little difference in the density of cells in the center versus those in the periphery (Figure 4B, middle panel). This was observed by staining the microcolonies with either FM4-64 (Figure 4B) or when the cells expressed GFP (Video S1). A similar matrix distribution pattern was observed using biofilm-grown wild type PAO1, indicating the Psl staining pattern in biofilms was not due to Psl overproduction (data not shown). No Psl was detected in either multiple layer biofilms or microcolonies formed by the Δpsl strain WFPA800 (data not shown). Overall, our data shows that Psl surrounds the constituent cells in either a multilayer biofilm or a 3D-structured microcolony (see 3D view of the microcolony in the right panel of Figure 4B and the series of Z-stacks in Video S1).

Bottom Line: During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center.These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal.Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications.

View Article: PubMed Central - PubMed

Affiliation: Microbiology and Immunology, Wake Forest University Health Sciences, Winston-Salem, North Carolina, USA.

ABSTRACT
Virtually all cells living in multicellular structures such as tissues and organs are encased in an extracellular matrix. One of the most important features of a biofilm is the extracellular polymeric substance that functions as a matrix, holding bacterial cells together. Yet very little is known about how the matrix forms or how matrix components encase bacteria during biofilm development. Pseudomonas aeruginosa forms environmentally and clinically relevant biofilms and is a paradigm organism for the study of biofilms. The extracellular polymeric substance of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids, and proteins. Here, we directly visualize the product of the polysaccharide synthesis locus (Psl exopolysaccharide) at different stages of biofilm development. During attachment, Psl is anchored on the cell surface in a helical pattern. This promotes cell-cell interactions and assembly of a matrix, which holds bacteria in the biofilm and on the surface. Chemical dissociation of Psl from the bacterial surface disrupted the Psl matrix as well as the biofilm structure. During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center. At the dispersion stage, swimming cells appear in this matrix cavity. Dead cells and extracellular DNA (eDNA) are also concentrated in the Psl matrix-free area. Deletion of genes that control cell death and autolysis affects the formation of the matrix cavity and microcolony dispersion. These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal. Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications.

Show MeSH
Related in: MedlinePlus