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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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The P. aeruginosa cidAB and lrgAB genes control cell death and lysis, as well as the timing of seeding dispersal.(A) A sequence alignment between P. aeruginosa CidA (putative holin) and LrgA (putative anti-holin), the predicted CidA protein structure, and a diagram of P. aeruginosa cid/lrg genetic organization. Identical residues between the two sequences and residues absent in CidA are shaded. The black rectangle represents a sequence predicted to form a trans-membrane helix. _, residues predicted to be cytoplasmic; -, residues predicted to be periplasmic. (B) A growth comparison of P. aeruginosa PAO1, ΔcidAB, and ΔlrgAB (left graph) and the corresponding complemented strains (right graph). (C) The Psl matrix (green, HHA-FITC staining) cavity in the 2-day-old biofilm of ΔlrgAB, PAO1, and the ΔcidAB mutant. A horizontal sectioned image (square) and a vertical sectioned image (rectangle) are shown. The white bar marked by letter “a” represents the height of the Psl matrix cavity and the “b” bar shows the height of the corresponding microcolony (MC).
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ppat-1000354-g007: The P. aeruginosa cidAB and lrgAB genes control cell death and lysis, as well as the timing of seeding dispersal.(A) A sequence alignment between P. aeruginosa CidA (putative holin) and LrgA (putative anti-holin), the predicted CidA protein structure, and a diagram of P. aeruginosa cid/lrg genetic organization. Identical residues between the two sequences and residues absent in CidA are shaded. The black rectangle represents a sequence predicted to form a trans-membrane helix. _, residues predicted to be cytoplasmic; -, residues predicted to be periplasmic. (B) A growth comparison of P. aeruginosa PAO1, ΔcidAB, and ΔlrgAB (left graph) and the corresponding complemented strains (right graph). (C) The Psl matrix (green, HHA-FITC staining) cavity in the 2-day-old biofilm of ΔlrgAB, PAO1, and the ΔcidAB mutant. A horizontal sectioned image (square) and a vertical sectioned image (rectangle) are shown. The white bar marked by letter “a” represents the height of the Psl matrix cavity and the “b” bar shows the height of the corresponding microcolony (MC).

Mentions: Our results (Figure 6) as well as other reports [10],[20],[37], indicate that death and lysis of cells occurs as a function of their spatial orientation within the biofilm, like that recently reported in S. aureus [38]–[40]. This suggests that P. aeruginosa is capable of undergoing a form of programmed cell death similar to apoptosis in higher organisms. Previous studies with S. aureus show that the CidA and LrgA proteins function as a holin and anti-holin, respectively, to control cell death and the timing of cell lysis [40]. The holins are phage-encoded small integral membrane proteins that control the activity of murein hydrolases and timing of host cell lysis during bacteriophage infection, whereas the anti-holin molecule antagonizes holin activity [40]. Holins are the gatekeeper of the lysis process and at a precise time point, can form large holes in the cytoplasmic membrane of phage-infected bacteria [41]. cid/lrg orthologues are present in a variety of bacteria including P. aeruginosa [38]. Based on structural analysis, sequence homology, and gene organization, we have identified a putative cidAB (PA3432-3431) and lrgAB (PA4014-4013) locus in P. aeruginosa (Figure 7A). The PA3432 encoded protein was tentatively identified as CidA, since it had all the structural features of a holin. These include a small trans-membrane protein (129 amino acids, four predicted trans-membrane helices as shown by black rectangles in Figure 7A), a hydrophobic N-terminus, and a highly polar, charged C-terminal domain. PA4014 was defined to encode LrgA, which has characteristics of an anti-holin protein (Figure 7A). As with S. aureus, LrgA shares sequence similarity with CidA, but LrgA has an extended N-terminus including two positively charged residues (arginine), which are important for anti-holin function [42]. Similar to the organization in S. aureus, the P. aeruginosa cidA/lrgA orthologues had cidB/lrgB, respectively, immediately downstream, and upstream of cidA is a putative transcriptional regulator (cidR).


Assembly and development of the Pseudomonas aeruginosa biofilm matrix.

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

The P. aeruginosa cidAB and lrgAB genes control cell death and lysis, as well as the timing of seeding dispersal.(A) A sequence alignment between P. aeruginosa CidA (putative holin) and LrgA (putative anti-holin), the predicted CidA protein structure, and a diagram of P. aeruginosa cid/lrg genetic organization. Identical residues between the two sequences and residues absent in CidA are shaded. The black rectangle represents a sequence predicted to form a trans-membrane helix. _, residues predicted to be cytoplasmic; -, residues predicted to be periplasmic. (B) A growth comparison of P. aeruginosa PAO1, ΔcidAB, and ΔlrgAB (left graph) and the corresponding complemented strains (right graph). (C) The Psl matrix (green, HHA-FITC staining) cavity in the 2-day-old biofilm of ΔlrgAB, PAO1, and the ΔcidAB mutant. A horizontal sectioned image (square) and a vertical sectioned image (rectangle) are shown. The white bar marked by letter “a” represents the height of the Psl matrix cavity and the “b” bar shows the height of the corresponding microcolony (MC).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000354-g007: The P. aeruginosa cidAB and lrgAB genes control cell death and lysis, as well as the timing of seeding dispersal.(A) A sequence alignment between P. aeruginosa CidA (putative holin) and LrgA (putative anti-holin), the predicted CidA protein structure, and a diagram of P. aeruginosa cid/lrg genetic organization. Identical residues between the two sequences and residues absent in CidA are shaded. The black rectangle represents a sequence predicted to form a trans-membrane helix. _, residues predicted to be cytoplasmic; -, residues predicted to be periplasmic. (B) A growth comparison of P. aeruginosa PAO1, ΔcidAB, and ΔlrgAB (left graph) and the corresponding complemented strains (right graph). (C) The Psl matrix (green, HHA-FITC staining) cavity in the 2-day-old biofilm of ΔlrgAB, PAO1, and the ΔcidAB mutant. A horizontal sectioned image (square) and a vertical sectioned image (rectangle) are shown. The white bar marked by letter “a” represents the height of the Psl matrix cavity and the “b” bar shows the height of the corresponding microcolony (MC).
Mentions: Our results (Figure 6) as well as other reports [10],[20],[37], indicate that death and lysis of cells occurs as a function of their spatial orientation within the biofilm, like that recently reported in S. aureus [38]–[40]. This suggests that P. aeruginosa is capable of undergoing a form of programmed cell death similar to apoptosis in higher organisms. Previous studies with S. aureus show that the CidA and LrgA proteins function as a holin and anti-holin, respectively, to control cell death and the timing of cell lysis [40]. The holins are phage-encoded small integral membrane proteins that control the activity of murein hydrolases and timing of host cell lysis during bacteriophage infection, whereas the anti-holin molecule antagonizes holin activity [40]. Holins are the gatekeeper of the lysis process and at a precise time point, can form large holes in the cytoplasmic membrane of phage-infected bacteria [41]. cid/lrg orthologues are present in a variety of bacteria including P. aeruginosa [38]. Based on structural analysis, sequence homology, and gene organization, we have identified a putative cidAB (PA3432-3431) and lrgAB (PA4014-4013) locus in P. aeruginosa (Figure 7A). The PA3432 encoded protein was tentatively identified as CidA, since it had all the structural features of a holin. These include a small trans-membrane protein (129 amino acids, four predicted trans-membrane helices as shown by black rectangles in Figure 7A), a hydrophobic N-terminus, and a highly polar, charged C-terminal domain. PA4014 was defined to encode LrgA, which has characteristics of an anti-holin protein (Figure 7A). As with S. aureus, LrgA shares sequence similarity with CidA, but LrgA has an extended N-terminus including two positively charged residues (arginine), which are important for anti-holin function [42]. Similar to the organization in S. aureus, the P. aeruginosa cidA/lrgA orthologues had cidB/lrgB, respectively, immediately downstream, and upstream of cidA is a putative transcriptional regulator (cidR).

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