Matrix Polysaccharides and SiaD Diguanylate Cyclase Alter Community Structure and Competitiveness of Pseudomonas aeruginosa during Dual-Species Biofilm Development with Staphylococcus aureus

Abstract

Mixed-species biofilms display a number of emergent properties, including enhanced antimicrobial tolerance and communal metabolism. These properties may depend on interspecies relationships and the structure of the biofilm. However, the contribution of specific matrix components to emergent properties of mixed-species biofilms remains poorly understood. Using a dual-species biofilm community formed by the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus, we found that whilst neither Pel nor Psl polysaccharides, produced by P. aeruginosa, affect relative species abundance in mature P. aeruginosa and S. aureus biofilms, Psl production is associated with increased P. aeruginosa abundance and reduced S. aureus aggregation in the early stages of biofilm formation. Our data suggest that the competitive effect of Psl is not associated with its structural role in cross-linking the matrix and adhering to P. aeruginosa cells but is instead mediated through the activation of the diguanylate cyclase SiaD. This regulatory control was also found to be independent of the siderophore pyoverdine and Pseudomonas quinolone signal, which have previously been proposed to reduce S. aureus viability by inducing lactic acid fermentation-based growth. In contrast to the effect mediated by Psl, Pel reduced the effective crosslinking of the biofilm matrix and facilitated superdiffusivity in microcolony regions. These changes in matrix cross-linking enhance biofilm surface spreading and expansion of microcolonies in the later stages of biofilm development, improving overall dual-species biofilm growth and increasing biovolume severalfold. Thus, the biofilm matrix and regulators associated with matrix production play essential roles in mixed-species biofilm interactions.IMPORTANCE Bacteria in natural and engineered environments form biofilms that include many different species. Microorganisms rely on a number of different strategies to manage social interactions with other species and to access resources, build biofilm consortia, and optimize growth. For example, Pseudomonasaeruginosa and Staphylococcus aureus are biofilm-forming bacteria that coinfect the lungs of cystic fibrosis patients and diabetic and chronic wounds. P. aeruginosa is known to antagonize S. aureus growth. However, many of the factors responsible for mixed-species interactions and outcomes such as infections are poorly understood. Biofilm bacteria are encased in a self-produced extracellular matrix that facilitates interspecies behavior and biofilm development. In this study, we examined the poorly understood roles of the major matrix biopolymers and their regulators in mixed-species biofilm interactions and development.

Keywords: Pseudomonas aeruginosa; SiaD; Staphylococcus aureus; biofilms; cyclic di-GMP; exopolysaccharide; microrheology.

FIG 1

The development of 19-h wild-type P. aeruginosa-S. aureus, P. aeruginosa ΔpelA-S. aureus, and P. aeruginosa ΔpslBCD-S. aureus dual-species biofilms imaged using confocal imaging every 6 h. The monospecies wild-type P. aeruginosa and S. aureus biofilms are shown for comparison. P. aeruginosa is mCherry tagged (red), and S. aureus is Gfp tagged (green). Images are representative of four biological replicates. The scale bar is 30 μm.

FIG 2

The development of 19-h biofilms formed by wild-type and matrix mutants of P. aeruginosa cocultured with S. aureus. Error bars represent standard errors of the means (SEM) (n ≥ 4). *, P < 0.05, α = 0.05 (unpaired two-sided t test with Welch’s correction). (A) Biovolumes per area of P. aeruginosa (solid lines) in the dual-species biofilms every 6 h. (B) Biovolumes per area of S. aureus (dashed lines) in the dual-species biofilms every 6 h. Note that the y axis scales for panels A and B are different. (C) Fitness of P. aeruginosa relative to S. aureus, where a selection constant of r_ij = 0 means that P. aeruginosa and S. aureus are equally competitive and a r_ij value of >0 means P. aeruginosa is more competitive than S. aureus.

FIG 3

Comparison of microcolony sizes in dual-species biofilms using the two-sample, two-sided Kolmogorov-Smirnov test. The red curve shows the difference between the two distributions (*, P < 0.05, **, P < 0.01, ***, P < 0.001; α = 0.05). (Top panel) Changes in size distribution as biofilms progresses from 13 to 19 h. (A to D) P. aeruginosa microcolonies in (A) wild-type P. aeruginosa-S. aureus, (B) mutant ΔpelA-S. aureus, and (C) mutant ΔpslBCD-S. aureus biofilms and S. aureus microcolonies in (D) mutant ΔpslBCD-S. aureus biofilms. (Bottom panel) Differences between P. aeruginosa microcolony size distributions formed by wild-type and matrix mutants of P. aeruginosa cocultured with S. aureus. (E and F) Wild-type P. aeruginosa-S. aureus compared to mutant ΔpelA-S. aureus at (E) 13 h and (F) 19 h. (G and H) Wild-type P. aeruginosa-S. aureus compared to mutant ΔpslBCD-S. aureus at (G) 13 h and (H) 19 h.

FIG 4

Microrheological measurements of wild-type and matrix mutants of P. aeruginosa cocultured with S. aureus. (A) MSD curves for wild-type P. aeruginosa-S. aureus, mutant ΔpelA-S. aureus, and mutant ΔpslBCD-S. aureus microcolonies. The MSD curve for TSB medium is shown for comparison. SA, S. aureus. The orange dotted lines indicate the line of best fit to the experimentally determined MSD using a power law function for the estimation of α. (B) Representative particle trajectories in wild-type P. aeruginosa-S. aureus, mutant ΔpelA-S. aureus, and mutant ΔpslBCD-S. aureus microcolonies. The red trajectory in the middle panel indicates a particle undergoing directed motion and superdiffusion, whereas the blue and yellow trajectories indicate subdiffusion. Error bars represent SEM.

FIG 5

Confocal images of 19-h biofilms of P. aeruginosa CdrA and diguanylate cyclase mutants cocultured with S. aureus. (A) P. aeruginosa ΔcdrA-S. aureus. (B) P. aeruginosa ΔsadC-S. aureus. (C) P. aeruginosa ΔsiaD-S. aureus. (D) P. aeruginosa ΔsiaD(siaD)-S. aureus. P. aeruginosa was mCherry tagged (red), and S. aureus was Gfp tagged (green). Images are representative of results from at least three biological replicates. The scale bar is 30 μm.

FIG 6

MSD curves for P. aeruginosa ΔsiaD-S. aureus, P. aeruginosa ΔcdrA-S. aureus, and P. aeruginosa ΔsadC-S. aureus microcolonies. SA, S. aureus. The orange dotted lines indicate the line of best fit to the experimentally determined MSD using a power law function for the estimation of α. Error bars represent SEM.

FIG 7

Relative levels of pyoverdine and PQS in 19-h P. aeruginosa-S. aureus biofilms. (A) Pyoverdine, as indicated by its fluorescence at an emission peak of 450 nm. (B). PQS, as indicated by the fluorescence emission of the green fluorescent protein from the PQS biosensor strain, mutant ΔpqsC(pqsA-gfp). Values are means (± standard deviations [SD]) of relative fluorescence units (RFU) determined from three biological replicates.

FIG 8

Schematic showing how matrix polysaccharides and SiaD contribute to P. aeruginosa predominance in dual-species P. aeruginosa-S. aureus communities. (A) Psl enhances P. aeruginosa competitiveness in early stages, possibly via SiaD activation, whereas Pel enables biofim expansion to increase P. aeruginosa predominance in the later stages. (B) Dominance of wild-type P. aeruginosa and SiaD and SiaD complement mutant over S. aureus, with their corresponding PQS/pyoverdine (PVD) levels.