BdlA, DipA and Induced Dispersion Contribute to Acute Virulence and Chronic Persistence of Pseudomonas aeruginosa

The human pathogen Pseudomonas aeruginosa is capable of causing both acute and chronic infections. Differences in virulence are attributable to the mode of growth: bacteria growing planktonically cause acute infections, while bacteria growing in matrix-enclosed aggregates known as biofilms are associated with chronic, persistent infections. While the contribution of the planktonic and biofilm modes of growth to virulence is now widely accepted, little is known about the role of dispersion in virulence, the active process by which biofilm bacteria switch back to the planktonic mode of growth. Here, we demonstrate that P. aeruginosa dispersed cells display a virulence phenotype distinct from those of planktonic and biofilm cells. While the highest activity of cytotoxic and degradative enzymes capable of breaking down polymeric matrix components was detected in supernatants of planktonic cells, the enzymatic activity of dispersed cell supernatants was similar to that of biofilm supernatants. Supernatants of non-dispersing Delta bdlA biofilms were characterized by a lack of many of the degradative activities. Expression of genes contributing to the virulence of P. aeruginosa was nearly 30-fold reduced in biofilm cells relative to planktonic cells. Gene expression analysis indicated dispersed cells, while dispersing from a biofilm and returning to the single cell lifestyle, to be distinct from both biofilm and planktonic cells, with virulence transcript levels being reduced up to 150-fold compared to planktonic cells. In contrast, virulence gene transcript levels were significantly increased in non-dispersing Delta bdlA and Delta dipA biofilms compared to wild-type planktonic cells. Despite this, bdlA and dipA inactivation, resulting in an inability to disperse in vitro, correlated with reduced pathogenicity and competitiveness in cross-phylum acute virulence models. In contrast, bdlA inactivation rendered P. aeruginosa more persistent upon chronic colonization of the murine lung, overall indicating that dispersion may contribute to both acute and chronic infections

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BdlA, DipA and Induced Dispersion Contribute to Acute Virulence and Chronic Persistence of <i>Pseudomonas aeruginosa</i>

<div><p>The human pathogen <i>Pseudomonas aeruginosa</i> is capable of causing both acute and chronic infections. Differences in virulence are attributable to the mode of growth: bacteria growing planktonically cause acute infections, while bacteria growing in matrix-enclosed aggregates known as biofilms are associated with chronic, persistent infections. While the contribution of the planktonic and biofilm modes of growth to virulence is now widely accepted, little is known about the role of dispersion in virulence, the active process by which biofilm bacteria switch back to the planktonic mode of growth. Here, we demonstrate that <i>P. aeruginosa</i> dispersed cells display a virulence phenotype distinct from those of planktonic and biofilm cells. While the highest activity of cytotoxic and degradative enzymes capable of breaking down polymeric matrix components was detected in supernatants of planktonic cells, the enzymatic activity of dispersed cell supernatants was similar to that of biofilm supernatants. Supernatants of non-dispersing Δ<i>bdlA</i> biofilms were characterized by a lack of many of the degradative activities. Expression of genes contributing to the virulence of <i>P. aeruginosa</i> was nearly 30-fold reduced in biofilm cells relative to planktonic cells. Gene expression analysis indicated dispersed cells, while dispersing from a biofilm and returning to the single cell lifestyle, to be distinct from both biofilm and planktonic cells, with virulence transcript levels being reduced up to 150-fold compared to planktonic cells. In contrast, virulence gene transcript levels were significantly increased in non-dispersing Δ<i>bdlA</i> and Δ<i>dipA</i> biofilms compared to wild-type planktonic cells. Despite this, <i>bdlA</i> and <i>dipA</i> inactivation, resulting in an inability to disperse <i>in vitro</i>, correlated with reduced pathogenicity and competitiveness in cross-phylum acute virulence models. In contrast, <i>bdlA</i> inactivation rendered <i>P. aeruginosa</i> more persistent upon chronic colonization of the murine lung, overall indicating that dispersion may contribute to both acute and chronic infections.</p></div

The non-dispersing Δ<i>bdlA</i> mutant and complemented Δ<i>bdlA</i> mutants impaired in biofilm dispersion are avirulent.

<p>(A) Death of <i>Arabidopsis thaliana</i> 7 days post infection with <i>P. aeruginosa</i> PAO1, the isogenic Δ<i>bdlA</i> mutant and <i>ΔbdlA</i> mutants complemented with <i>bdlA</i>, a truncated BdlA variant (NoPAS-<i>bdlA</i>) and BdlA variants harboring alanine substitutions in various amino acids. #, indicates complemented Δ<i>bdlA</i> strains impaired in nutrient-induced dispersion, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004168#ppat.1004168-Petrova6" target="_blank">[58]</a>. All other complemented Δ<i>bdlA</i> strains were not impaired in nutrient-induced dispersion. (B) Death of <i>Arabidopsis thaliana</i> 7 days post-infection with <i>P. aeruginosa</i> PAO1 and selected isogenic mutants. Bars indicate average and median plant death rates while vertical lines indicate the highest and lowest plant death rates observed. (C) Death of <i>Arabidopsis thaliana</i> 7 days post infection with <i>P. aeruginosa</i> PA14, and the isogenic Δ<i>dipA</i> and <i>ΔrbdA</i> mutants. Control plants inoculated with ½ MS salts alone showed no symptoms over the course of the experiments. Experiments were carried out in triplicate using 8 plants per strain per replicate. *, significantly different from PAO1, P-value <0.05.</p

Identification of proteins present in supernatants.

<p>Identification of proteins present in supernatants.</p

Impaired dispersion correlates with increased persistence as determined using a chronic murine pneumonia infection model.

<p>CD1 mice were inoculated intratracheal through oral lavage using feeding needle with 1.2×10⁶ CFU of <i>Pseudomonas</i> strains. Lungs were harvested 14 days post-inoculation and CFU was determined. A total of 10 mice were used in per study. (A) Bacterial burden in the lung. Values presented are average, min, max, mean lung CFU/ml. (B) Fold change in lung CFU/ml 14 days post-infection compared to initial inoculum. Error bars indicate standard deviation. The values were tested by means of a Fisher test. **, significantly different from PAO1, P-value <0.001.</p

Detection of degradative activity in the extracellular proteome of <i>P. aeruginosa</i> PAO1 is growth mode dependent with <i>P. aeruginosa ΔbdlA</i> impaired in dispersion exhibiting lower degradative activity.

<p>Cytotoxic and degradative activities were determined using 10 µg of supernatant protein in 100 µl of sterile water, followed by measuring the zone of clearance 18 hr post-inoculation of the sterile protein solution into the wells of the respective agar plates. Degradative activity was determined using supernatants obtained from <i>P. aeruginosa</i> grown planktonically to exponential (A) and stationary phase (B). Supernatants of planktonic cells not treated with glutamate or nitric oxide are referred to as “control”. Additionally, supernatants of planktonic cells grown to exponential and stationary phase were exposed for 30 min to glutamate or SNP were used. (C–F) Degradative activities were furthermore determined in supernatants obtained from biofilms, and biofilms post-induction of dispersion with glutamate (remaining biofilm). Dispersed cells were obtained following dispersion in response to glutamate and SNP, which was used as a source of nitric oxide. (C) Proteolytic activity was detected using milk agar plates in supernatants obtained from biofilms, biofilms post-induction of dispersion, and dispersed cells. (D) Lipid hydrolysis was determined using tributyrin containing agar plates. (E) Hemolytic activity was detected using blood agar plates while (F) Psl degradation was detected on agar plates containing Psl extracted from a <i>P. aeruginosa</i> strain overexpressing Psl. Psl degradation was visualized as a zone of clearing following 24 hr incubation and staining the agar plate with iodine. Experiments were carried out at least in triplicate. Error bars indicate standard deviation.</p

Analysis of proteins present in supernatants of <i>P.</i> aeruginosa PAO1 and <i>ΔbdlA</i> biofilms.

<p>A total of 10 µg supernatant protein obtained from <i>P. aeruginosa</i> PAO1 and <i>ΔbdlA</i> biofilms was loaded per lane. Protein bands indicated by a letter and arrow were selected and subsequently identified by LC-MS/MS (see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004168#ppat-1004168-t001" target="_blank">Table 1</a>). Experiments were repeated in triplicate and a representative SDS-gel image is shown. Molecular masses are indicated on the right (in kDa).</p