Interbacterial Antagonism Mediated by a Released Polysaccharide

Abstract

Pseudomonas aeruginosa and Staphylococcus aureus are two common pathogens causing chronic infections in the lungs of people with cystic fibrosis (CF) and in wounds, suggesting that these two organisms coexist in vivo. However, P. aeruginosa utilizes various mechanisms to antagonize S. aureus when these organisms are grown together in vitro. Here, we suggest a novel role for Psl in antagonizing S. aureus growth. Psl is an exopolysaccharide that exists in both cell-associated and cell-free forms and is important for biofilm formation in P. aeruginosa. When grown in planktonic coculture with a P. aeruginosa psl mutant, S. aureus had increased survival compared to when it was grown with wild-type P. aeruginosa. We found that cell-free Psl was critical for the killing, as purified cell-free Psl was sufficient to kill S. aureus. Transmission electron microscopy of S. aureus treated with Psl revealed disrupted cell envelopes, suggesting that Psl causes S. aureus cell lysis. This was independent of known mechanisms used by P. aeruginosa to antagonize S. aureus. Cell-free Psl could also promote S. aureus killing during growth in in vivo-like conditions. We also found that Psl production in P. aeruginosa CF clinical isolates positively correlated with the ability to kill S. aureus. This could be a result of P. aeruginosa coevolution with S. aureus in CF lungs. In conclusion, this study defines a novel role for P. aeruginosa Psl in killing S. aureus, potentially impacting the coexistence of these two opportunistic pathogens in vivo. IMPORTANCE Pseudomonas aeruginosa and Staphylococcus aureus are two important opportunistic human pathogens commonly coisolated from clinical samples. However, P. aeruginosa can utilize various mechanisms to antagonize S. aureus in vitro. Here, we investigated the interactions between these two organisms and report a novel role for P. aeruginosa exopolysaccharide Psl in killing S. aureus. We found that cell-free Psl could kill S. aureus in vitro, possibly by inducing cell lysis. This was also observed in conditions reflective of in vivo scenarios. In accord with this, Psl production in P. aeruginosa clinical isolates positively correlated with their ability to kill S. aureus. Together, our data suggest a role for Psl in affecting the coexistence of P. aeruginosa and S. aureus in vivo.

Keywords: Pseudomonas aeruginosa; Psl; Staphylococcus aureus; cell lysis; cystic fibrosis; exopolysaccharide; polymicrobial; wound.

FIG 1

P. aeruginosa Psl production antagonizes growth of S. aureus. (A) S. aureus USA300 was grown in coculture with either P. aeruginosa PAO1 or ΔP_psl on solidified medium using a cross-streak assay. Arrows point to the inhibition zones of S. aureus growth that were observed after overnight incubation. (B) S. aureus survival over the course of 8 h when grown alone or cocultured with PAO1 or ΔP_psl. Data are means and standard deviations (SD); individual points indicate the means for biological replicates (N = 5; n = 3). Significance was determined using Student’s t test. *, P < 0.05 compared to PAO1. (C) S. aureus survival when cocultured for 7 h with PAO1, ΔpslD, or the complemented ΔpslD/pslD⁺ strain. N = 4; n = 3. Data are means and SD; individual points indicate biological replicates. S. aureus survival (B and C) is presented as CFU normalized to the starting CFU at 0 h. Significance was determined using a one-way ANOVA. *, P < 0.05 compared to PAO1.

FIG 2

Cell-free Psl derived from P. aeruginosa spent medium antagonizes S. aureus growth. (A) Cell-associated (associated) and cell-free (free) Psl from P. aeruginosa was quantified by immunoblotting. The cell pellets and spent media of centrifuged overnight cultures were used as the source of cell-associated and cell-free Psl, respectively. Psl was quantified by densitometry of the blot and normalized to PAO1 cell-associated Psl. Data are means and SD; individual points indicate biological replicates (N = 3; n = 3). Significance was determined with one-way ANOVA. *, P < 0.05 compared to PAO1. (B) S. aureus survival when cocultured for 7 h with P. aeruginosa PAO1, ΔP_psl, pagP::Tn, or pagP::TnΔpsl strains. S. aureus survival is presented as CFU normalized to the starting CFU at 0 h. Data are means and SD; individual points indicate biological replicates (N = 3; n = 3). Significance was determined using Student’s t test compared to PAO1. *, P < 0.05; ns, not significant. (C) S. aureus survival when grown for 5 h in spent medium of PAO1 or the ΔP_psl or pagP::Tn strain that was diluted 1:1 in fresh medium. Data are means and SD; individual points indicate means for biological replicates (N = 4; n = 3). Significance was determined using Student’s t test. *, P < 0.05 compared to PAO1. (D) S. aureus was grown for 4 h in spent medium from PAO1, ΔP_psl, or pagP::Tn strains that had been pretreated with (+) or without (−) PslG. Data are means and SD; individual points indicate biological replicates (N = 4; n = 3). S. aureus survival (B and C) is presented as CFU normalized to the starting CFU at 0 h. Significance was determined using Student’s t test, compared to the no-PslG pretreated group. *, P < 0.05; ns, not significant.

FIG 3

Cell-free Psl kills S. aureus by disrupting the cell envelope. (A) S. aureus USA300 was incubated with spent medium from P. aeruginosa PAO1, ΔP_psl, or pagP::Tn for 2 h. Changes in cell morphology were visualized by TEM. Arrows indicate cells with disrupted cell envelopes. (B) Total and disrupted cell counts for each group were enumerated, and the percentage of disrupted cells within each group was calculated. Data are means and SD; individual points indicate biological replicates (N = 4; n = 3). Significance was determined using a one-way ANOVA compared to PAO1. *, P < 0.05.

FIG 4

Cell-free Psl promotes S. aureus killing under in vivo-like conditions. (A) Twenty-four-hour S. aureus USA300 biofilms were incubated with or without P. aeruginosa PAO1, ΔP_psl, or pagP::Tn spent medium for 5 h. Biofilm biomass was quantified by crystal violet staining. Data are means and SD; individual points indicate biological replicates (N = 5; n = 3). Significance was determined using one-way ANOVA. *, P < 0.05, and ns, not significant, compared to the medium-only control. (B) S. aureus survival when cocultured with P. aeruginosa PAO1, ΔP_psl, or pagP::Tn in SCFM2 for 7 h. Data are means and SD; individual points indicate biological replicates (N = 3; n = 3). Significance was determined using Student’s t test. *, P < 0.05 compared to PAO1.

FIG 5

Psl production in P. aeruginosa clinical isolates positively correlates with the ability to kill S. aureus. S. aureus USA300 was cocultured with each of the 16 P. aeruginosa CF isolates, PAO1, and the ΔP_psl and pagP::Tn strains. The number of CFU at 7 h was divided by that at 0 h to quantify survival. (A) Linear regression analysis was performed to determine any correlation between the two parameters indicated. Cell-free Psl production by the designated strains was measured by immunoblot assay using Psl antibody and then normalized to PAO1. Individual points indicate biological replicates (N = 3; n = 3). (B) P. aeruginosa clinical isolates with low Psl production showed reduced S. aureus killing activity. Six of the CF isolates produced no cell-free Psl (−), while the other 10 produced variable amounts of cell-free Psl (+; the arrow indicates the increasing production of Psl), compared with that produced by PAO1. The number of CFU of S. aureus USA300 cocultured with each of the isolates, PAO1, and the ΔP_psl and pagP::Tn strains for 7 h was divided by that at 0 h to quantify S. aureus survival. Data are means and SD (N = 3; n = 3).

FIG 6

Purified cell-free Psl is sufficient to kill S. aureus. Survival of S. aureus grown in media supplemented with (A) increasing concentrations of purified cell-free Psl from PAO1 or the pagP::Tn mutant or cell-free products from the ΔP_psl mutant or (B) 60 μg/mL of d-mannose (Man), 20 μg/mL of d-glucose (Glu), 20 μg/mL of l-rhamnose (Rha), or a combination of the three monosaccharides (Combined). S. aureus grown without cell-free Psl or monosaccharides (untreated) was used as a control. OD₆₀₀ was measured after 16 h. S. aureus survival is presented as OD₆₀₀ normalized to that of the control. (A) The x axis is a log scale. Data are means and SD; individual points indicate the mean of biological replicates (N = 3; n = 3). Significance was determined using Student’s t test compared to the ΔP_psl mutant. *, P < 0.05.