O-Specific Antigen-Dependent Surface Hydrophobicity Mediates Aggregate Assembly Type in Pseudomonas aeruginosa

Abstract

Bacteria live in spatially organized aggregates during chronic infections, where they adapt to the host environment, evade immune responses, and resist therapeutic interventions. Although it is known that environmental factors such as polymers influence bacterial aggregation, it is not clear how bacterial adaptation during chronic infection impacts the formation and spatial organization of aggregates in the presence of polymers. Here, we show that in an in vitro model of cystic fibrosis (CF) containing the polymers extracellular DNA (eDNA) and mucin, O-specific antigen is a major factor determining the formation of two distinct aggregate assembly types of Pseudomonas aeruginosa due to alterations in cell surface hydrophobicity. Our findings suggest that during chronic infection, the interplay between cell surface properties and polymers in the environment may influence the formation and structure of bacterial aggregates, which would shed new light on the fitness costs and benefits of O-antigen production in environments such as CF lungs. IMPORTANCE During chronic infection, several factors contribute to the biogeography of microbial communities. Heterogeneous populations of Pseudomonas aeruginosa form aggregates in cystic fibrosis airways; however, the impact of this population heterogeneity on spatial organization and aggregate assembly is not well understood. In this study, we found that changes in O-specific antigen determine the spatial organization of P. aeruginosa cells by altering the relative cell surface hydrophobicity. This finding suggests a role for O-antigen in regulating P. aeruginosa aggregate size and shape in cystic fibrosis airways.

Keywords: O-antigen; Pseudomonas aeruginosa; cystic fibrosis; depletion aggregation; hydrophobicity; lipopolysaccharide.

FIG 1

The two types of aggregate assemblies formed by P. aeruginosa isolates in SCFM2 are due to ssg gene mutation. (A) In PAO1 and evolved isolates, aggregates assembled into either organized stacked structures (labeled S) or disorganized clumps (labeled C). (B) Stacked aggregates of PAO1 and A2 were significantly larger than aggregates formed by A9 and B9, and complementation with an intact ssg gene significantly increased the aggregate volume (P < 0.0001 by Kruskal-Wallis and Dunn’s multiple-comparison tests; error bars are median aggregate volumes with interquartile ranges, and each data point is representative of an aggregate). ns, not significant.

FIG 2

Loss of OSA leads to clumped aggregate assembly. (A) The loss of CPA (Δrmd) did not alter the type of aggregate assembly, and the loss of OSA (ΔwbpM) led to dispersed small aggregates. The loss of both CPA and OSA (ΔwbpL) changed the aggregate assembly type similarly to the ssg mutant. (B) There was a significant reduction in the aggregate volume in ssg, wbpL, and wbpM mutants, but only the loss of ssg and wbpL displayed large, clumped aggregates (P < 0.0001 by Kruskal-Wallis and Dunn’s multiple-comparison tests; error bars are median aggregate volumes with interquartile ranges, and each point is representative of an aggregate).

FIG 3

The aggregate assembly type is independent of exopolysaccharide production, lectins, and quorum sensing. (A) Loss of lectins (ΔlecA and ΔlecB), quorum sensing (ΔlasR), and exopolysaccharide components (Δpel Δpsl) did not change the aggregate assembly type, and aggregates were assembled in stacked forms similar to those seen in PAO1. (B) Stacked aggregates formed by cells lacking lectins (ΔlecA and ΔlecB), quorum sensing (ΔlasR), and exopolysaccharide components (Δpel Δpsl) were the same size as PAO1 aggregates (P = 0.1, P = 0.6, P > 0.999, and P > 0.999 by Kruskal-Wallis and Dunn’s multiple-comparison tests when aggregate volumes of ΔlecA, ΔlecB, ΔlasR, and Δpel Δpsl cells were compared to those of PAO1; error bars are median aggregate volumes with interquartile ranges).

FIG 4

Clumped aggregate assembly is not dependent on cell density. (A) The aggregate biovolume of PAO1 significantly increased after 180 min of growth (median biovolume of 0.34 to 0.75 over time). (B) In PAO1 ΔwbpL (lacking OSA), the biovolume remained the same over time (median biovolume of 0.33 to 0.27 over time).

FIG 5

Clumped aggregate assembly of P. aeruginosa is not serotype specific. (A) P. aeruginosa PA14, PAK, and STO1 formed stacked aggregates in SCFM2, and OSA⁻ mutant STO1 (STO1 ΔwbpM) led to a clumped assembly of aggregates. (B) The loss of OSA altered aggregate assembly from stacked to clumped in STO1 and significantly decreased the aggregate volume (P = 0.0048 by Kruskal-Wallis and Dunn’s multiple-comparison tests; error bars are median aggregate volumes with interquartile ranges, and each data point is representative of an aggregate).

FIG 6

Cell surface hydrophobicity determines the aggregate assembly type. (A) The relative cell surface hydrophobicity was dependent on OSA, and mutations in ssg, wbpL, and wbpM led to an increase in the relative hydrophobicity of PAO1 and STO1 (green bars). (B) There was heterogeneity in the relative hydrophobicity of cell surfaces of P. aeruginosa isolates collected from two CF expectorated sputum samples (CFP1 and CFP2).