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Identification of biofilm-associated cluster (bac) in Pseudomonas aeruginosa involved in biofilm formation and virulence.

Macé C, Seyer D, Chemani C, Cosette P, Di-Martino P, Guery B, Filloux A, Fontaine M, Molle V, Junter GA, Jouenne T - PLoS ONE (2008)

Bottom Line: The pA3731 gene is found to control positively the ability to swarm and to produce extracellular rhamnolipids, and belongs to a cluster of 4 genes (pA3729-pA3732) not previously described in P. aeruginosa.Though the protein PA3731 has a predicted secondary structure similar to that of the Phage Shock Protein, some obvious differences are observed compared to already described psp systems, e.g., this unknown cluster is monocistronic and no homology is found between the other proteins constituting this locus and psp proteins.We consequently named this locus bac for biofilm-associated cluster.

View Article: PubMed Central - PubMed

Affiliation: Université de Rouen, Laboratoire Polymères, Biopolymères, Surfaces, CNRS FRE 3101, Plate-forme protéomique de l'IFRMP 23, Mont-Saint-Aignan, France.

ABSTRACT
Biofilms are prevalent in diseases caused by Pseudomonas aeruginosa, an opportunistic and nosocomial pathogen. By a proteomic approach, we previously identified a hypothetical protein of P. aeruginosa (coded by the gene pA3731) that was accumulated by biofilm cells. We report here that a Delta pA3731 mutant is highly biofilm-defective as compared with the wild-type strain. Using a mouse model of lung infection, we show that the mutation also induces a defect in bacterial growth during the acute phase of infection and an attenuation of the virulence. The pA3731 gene is found to control positively the ability to swarm and to produce extracellular rhamnolipids, and belongs to a cluster of 4 genes (pA3729-pA3732) not previously described in P. aeruginosa. Though the protein PA3731 has a predicted secondary structure similar to that of the Phage Shock Protein, some obvious differences are observed compared to already described psp systems, e.g., this unknown cluster is monocistronic and no homology is found between the other proteins constituting this locus and psp proteins. As E. coli PspA, the amount of the protein PA3731 is enlarged by an osmotic shock, however, not affected by a heat shock. We consequently named this locus bac for biofilm-associated cluster.

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Quantification of biofilm formation by the PAO1, ΔpA3731 and ΔpA3731comp strains using (A) the crystal violet test; (B) the Biofilm Ring Test®, control without bacteria; (C), adherence to human A549 pneumocyte cells (cells and bacteria were stained with 20% Giemsa); A549, control without bacteria; Magnification, ×1000).PAO1 (Grey); ΔpA3731 (Horizontal line); ΔpA3731comp (Oblique line). Bars: SEM (n = 10); *, P≤0.05.
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pone-0003897-g002: Quantification of biofilm formation by the PAO1, ΔpA3731 and ΔpA3731comp strains using (A) the crystal violet test; (B) the Biofilm Ring Test®, control without bacteria; (C), adherence to human A549 pneumocyte cells (cells and bacteria were stained with 20% Giemsa); A549, control without bacteria; Magnification, ×1000).PAO1 (Grey); ΔpA3731 (Horizontal line); ΔpA3731comp (Oblique line). Bars: SEM (n = 10); *, P≤0.05.

Mentions: Growth kinetics of planktonic cells in LB broth (Figure 1) showed that the pA3731 mutation did not alter bacterial growth (generation time, 30 min for PAO1 and ΔpA3731). The complemented strain (ΔpA3731comp) grew a little bit more slowly (generation time 35 min). To determine whether the pA3731gene is a true biofilm specific factor, we first investigated the influence of the pA3731 mutation on the ability of P. aeruginosa to adhere on polystyrene plastic surfaces. Determination by crystal violet staining (Fig. 2A) revealed a significant (P≤0.05, n = 10) decrease of about 77% of the ring at the air-liquid interface as compared with the wild-type, after incubation for 24 h at 37°C in LB broth, and the mutant was consequently considered as non adherent. This phenotype was then confirmed by using the BioFilm Ring Test ®, i.e., an innovative biofilm assay recently developed [7]. Images obtained with the parent strain and the mutant confirmed the alteration in the biofilm-forming ability of the ΔpA3731 strain (Fig. 2B). Whereas a biofilm was already formed by the wild-type after 2 h of incubation at 37°C (BioFilm Index (BFI) of 2.4±0.1), a BFI value of 4.0±0.2 was obtained with the mutant. After 5 h of incubation, a significant difference (P≤0.05, n = 10) was still observed between the two strains, BFI values of 1.8±0.1 and 2.7±0.2 being obtained with PAO1 and ΔpA3731 strains, respectively. The parental strain PAO1 and its ΔpA3731mutant were then compared for their ability to adhere on A549 human pneumocyte cells. Microscopic examinations showed that the wild-type strain could adhere diffusely to the cell line, bacteria scattering over the eukaryotic cell surface (Fig. 2C). An adhesion index of 9.5±3.1 was measured. The mutation on the pA3731gene made the bacterium unable to adhere on pneumocytes, which is highlighted by the adhesion index of 1.1±0.3 obtained for the mutant. Due to the difficulty to distinguish in the “biofilm formation” what is due to adhesion, growth, production of exopolymères, we evaluated the number of bacteria adhering on polystyrene wells after incubation for 0.5 h in Phosphate Buffered Saline (PBS). Results (Fig. 3) showed a significant (P≤0.05, n = 10) decrease of the number of adherent cells with the mutant as compared with the wild-type. Complementation of the ΔpA3731 mutant with pMMB67-HE14 expressing pA3731 restored the wild-type phenotypes


Identification of biofilm-associated cluster (bac) in Pseudomonas aeruginosa involved in biofilm formation and virulence.

Macé C, Seyer D, Chemani C, Cosette P, Di-Martino P, Guery B, Filloux A, Fontaine M, Molle V, Junter GA, Jouenne T - PLoS ONE (2008)

Quantification of biofilm formation by the PAO1, ΔpA3731 and ΔpA3731comp strains using (A) the crystal violet test; (B) the Biofilm Ring Test®, control without bacteria; (C), adherence to human A549 pneumocyte cells (cells and bacteria were stained with 20% Giemsa); A549, control without bacteria; Magnification, ×1000).PAO1 (Grey); ΔpA3731 (Horizontal line); ΔpA3731comp (Oblique line). Bars: SEM (n = 10); *, P≤0.05.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2587700&req=5

pone-0003897-g002: Quantification of biofilm formation by the PAO1, ΔpA3731 and ΔpA3731comp strains using (A) the crystal violet test; (B) the Biofilm Ring Test®, control without bacteria; (C), adherence to human A549 pneumocyte cells (cells and bacteria were stained with 20% Giemsa); A549, control without bacteria; Magnification, ×1000).PAO1 (Grey); ΔpA3731 (Horizontal line); ΔpA3731comp (Oblique line). Bars: SEM (n = 10); *, P≤0.05.
Mentions: Growth kinetics of planktonic cells in LB broth (Figure 1) showed that the pA3731 mutation did not alter bacterial growth (generation time, 30 min for PAO1 and ΔpA3731). The complemented strain (ΔpA3731comp) grew a little bit more slowly (generation time 35 min). To determine whether the pA3731gene is a true biofilm specific factor, we first investigated the influence of the pA3731 mutation on the ability of P. aeruginosa to adhere on polystyrene plastic surfaces. Determination by crystal violet staining (Fig. 2A) revealed a significant (P≤0.05, n = 10) decrease of about 77% of the ring at the air-liquid interface as compared with the wild-type, after incubation for 24 h at 37°C in LB broth, and the mutant was consequently considered as non adherent. This phenotype was then confirmed by using the BioFilm Ring Test ®, i.e., an innovative biofilm assay recently developed [7]. Images obtained with the parent strain and the mutant confirmed the alteration in the biofilm-forming ability of the ΔpA3731 strain (Fig. 2B). Whereas a biofilm was already formed by the wild-type after 2 h of incubation at 37°C (BioFilm Index (BFI) of 2.4±0.1), a BFI value of 4.0±0.2 was obtained with the mutant. After 5 h of incubation, a significant difference (P≤0.05, n = 10) was still observed between the two strains, BFI values of 1.8±0.1 and 2.7±0.2 being obtained with PAO1 and ΔpA3731 strains, respectively. The parental strain PAO1 and its ΔpA3731mutant were then compared for their ability to adhere on A549 human pneumocyte cells. Microscopic examinations showed that the wild-type strain could adhere diffusely to the cell line, bacteria scattering over the eukaryotic cell surface (Fig. 2C). An adhesion index of 9.5±3.1 was measured. The mutation on the pA3731gene made the bacterium unable to adhere on pneumocytes, which is highlighted by the adhesion index of 1.1±0.3 obtained for the mutant. Due to the difficulty to distinguish in the “biofilm formation” what is due to adhesion, growth, production of exopolymères, we evaluated the number of bacteria adhering on polystyrene wells after incubation for 0.5 h in Phosphate Buffered Saline (PBS). Results (Fig. 3) showed a significant (P≤0.05, n = 10) decrease of the number of adherent cells with the mutant as compared with the wild-type. Complementation of the ΔpA3731 mutant with pMMB67-HE14 expressing pA3731 restored the wild-type phenotypes

Bottom Line: The pA3731 gene is found to control positively the ability to swarm and to produce extracellular rhamnolipids, and belongs to a cluster of 4 genes (pA3729-pA3732) not previously described in P. aeruginosa.Though the protein PA3731 has a predicted secondary structure similar to that of the Phage Shock Protein, some obvious differences are observed compared to already described psp systems, e.g., this unknown cluster is monocistronic and no homology is found between the other proteins constituting this locus and psp proteins.We consequently named this locus bac for biofilm-associated cluster.

View Article: PubMed Central - PubMed

Affiliation: Université de Rouen, Laboratoire Polymères, Biopolymères, Surfaces, CNRS FRE 3101, Plate-forme protéomique de l'IFRMP 23, Mont-Saint-Aignan, France.

ABSTRACT
Biofilms are prevalent in diseases caused by Pseudomonas aeruginosa, an opportunistic and nosocomial pathogen. By a proteomic approach, we previously identified a hypothetical protein of P. aeruginosa (coded by the gene pA3731) that was accumulated by biofilm cells. We report here that a Delta pA3731 mutant is highly biofilm-defective as compared with the wild-type strain. Using a mouse model of lung infection, we show that the mutation also induces a defect in bacterial growth during the acute phase of infection and an attenuation of the virulence. The pA3731 gene is found to control positively the ability to swarm and to produce extracellular rhamnolipids, and belongs to a cluster of 4 genes (pA3729-pA3732) not previously described in P. aeruginosa. Though the protein PA3731 has a predicted secondary structure similar to that of the Phage Shock Protein, some obvious differences are observed compared to already described psp systems, e.g., this unknown cluster is monocistronic and no homology is found between the other proteins constituting this locus and psp proteins. As E. coli PspA, the amount of the protein PA3731 is enlarged by an osmotic shock, however, not affected by a heat shock. We consequently named this locus bac for biofilm-associated cluster.

Show MeSH
Related in: MedlinePlus