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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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Lectin staining results of surface attached bacteria; Psl is anchored on the bacteria cell surface in a helical pattern.(A) MOA-FITC (green) and membrane stain FM4-64 (red) double staining of WFPA801 cells. The green image depicts the staining of helical structures around the cell surface as indicated in the inset. The white arrow points out the division site. (B) An optical section of HHA-FITC stained WFPA801 cells without (1) or with (1') deconvolution. (C) HHA-FITC and MOA-TRITC (white) double staining of WFPA801 cells. (D) HHA-FITC stained PAO1 cells. In all panels, green and red signals are merged images of the FITC-lectins and FM4-64 stained cells. The gray signal represents DIC images while green with gray are merged images of the FITC-lectins with DIC images. Scale bars, 1 µm.
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ppat-1000354-g001: Lectin staining results of surface attached bacteria; Psl is anchored on the bacteria cell surface in a helical pattern.(A) MOA-FITC (green) and membrane stain FM4-64 (red) double staining of WFPA801 cells. The green image depicts the staining of helical structures around the cell surface as indicated in the inset. The white arrow points out the division site. (B) An optical section of HHA-FITC stained WFPA801 cells without (1) or with (1') deconvolution. (C) HHA-FITC and MOA-TRITC (white) double staining of WFPA801 cells. (D) HHA-FITC stained PAO1 cells. In all panels, green and red signals are merged images of the FITC-lectins and FM4-64 stained cells. The gray signal represents DIC images while green with gray are merged images of the FITC-lectins with DIC images. Scale bars, 1 µm.

Mentions: To visualize Psl on the bacterial cell surface and to detect Psl exopolysaccharide during the attachment stage of biofilm development, we used fluorescently labeled lectins MOA (from Marasmium oreades agglutinin) or HHA (from Hippeastrum hybrid) to stain individual P. aeruginosa cells. The lectins MOA and HHA detect the galactose and mannose structure in Psl, respectively, and we previously demonstrated that they specifically stained Psl [21]. The cells were allowed to attach to a glass surface keeping Psl in its native state on the bacterial surface. When surface-attached P. aeruginosa WFPA801 (Psl-overproducing strain) cells were stained by FITC-MOA, we found that Psl was associated with the bacterial cell surface. More interestingly, most cells had a helical-like patterned fluorescence signal (green in Figure 1A). Such patterns were only seen with the MOA stained Psl and not with FM4-64 (red in Figure 1A) that stained the cell membrane. These helical patterns were observed on dividing (Figure 1A) as well as non-dividing cells (Figure 1B and 1C). The helical pattern of Psl on the cell surface was also seen with HHA staining (green, Figure 1B). Moreover, FITC-HHA and TRITC-MOA double staining of WFPA801 cells showed a similar helical pattern (compare green with white images in Figure 1C).


Assembly and development of the Pseudomonas aeruginosa biofilm matrix.

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

Lectin staining results of surface attached bacteria; Psl is anchored on the bacteria cell surface in a helical pattern.(A) MOA-FITC (green) and membrane stain FM4-64 (red) double staining of WFPA801 cells. The green image depicts the staining of helical structures around the cell surface as indicated in the inset. The white arrow points out the division site. (B) An optical section of HHA-FITC stained WFPA801 cells without (1) or with (1') deconvolution. (C) HHA-FITC and MOA-TRITC (white) double staining of WFPA801 cells. (D) HHA-FITC stained PAO1 cells. In all panels, green and red signals are merged images of the FITC-lectins and FM4-64 stained cells. The gray signal represents DIC images while green with gray are merged images of the FITC-lectins with DIC images. Scale bars, 1 µm.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000354-g001: Lectin staining results of surface attached bacteria; Psl is anchored on the bacteria cell surface in a helical pattern.(A) MOA-FITC (green) and membrane stain FM4-64 (red) double staining of WFPA801 cells. The green image depicts the staining of helical structures around the cell surface as indicated in the inset. The white arrow points out the division site. (B) An optical section of HHA-FITC stained WFPA801 cells without (1) or with (1') deconvolution. (C) HHA-FITC and MOA-TRITC (white) double staining of WFPA801 cells. (D) HHA-FITC stained PAO1 cells. In all panels, green and red signals are merged images of the FITC-lectins and FM4-64 stained cells. The gray signal represents DIC images while green with gray are merged images of the FITC-lectins with DIC images. Scale bars, 1 µm.
Mentions: To visualize Psl on the bacterial cell surface and to detect Psl exopolysaccharide during the attachment stage of biofilm development, we used fluorescently labeled lectins MOA (from Marasmium oreades agglutinin) or HHA (from Hippeastrum hybrid) to stain individual P. aeruginosa cells. The lectins MOA and HHA detect the galactose and mannose structure in Psl, respectively, and we previously demonstrated that they specifically stained Psl [21]. The cells were allowed to attach to a glass surface keeping Psl in its native state on the bacterial surface. When surface-attached P. aeruginosa WFPA801 (Psl-overproducing strain) cells were stained by FITC-MOA, we found that Psl was associated with the bacterial cell surface. More interestingly, most cells had a helical-like patterned fluorescence signal (green in Figure 1A). Such patterns were only seen with the MOA stained Psl and not with FM4-64 (red in Figure 1A) that stained the cell membrane. These helical patterns were observed on dividing (Figure 1A) as well as non-dividing cells (Figure 1B and 1C). The helical pattern of Psl on the cell surface was also seen with HHA staining (green, Figure 1B). Moreover, FITC-HHA and TRITC-MOA double staining of WFPA801 cells showed a similar helical pattern (compare green with white images in Figure 1C).

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