The Pseudomonas aeruginosa PSL Polysaccharide Is a Social but Noncheatable Trait in Biofilms

Abstract

Extracellular polysaccharides are compounds secreted by microorganisms into the surrounding environment, and they are important for surface attachment and maintaining structural integrity within biofilms. The social nature of many extracellular polysaccharides remains unclear, and it has been suggested that they could function as either cooperative public goods or as traits that provide a competitive advantage. Here, we empirically tested the cooperative nature of the PSL polysaccharide, which is crucial for the formation of biofilms in Pseudomonas aeruginosa We show that (i) PSL is not metabolically costly to produce; (ii) PSL provides population-level benefits in biofilms, for both growth and antibiotic tolerance; (iii) the benefits of PSL production are social and are shared with other cells; (iv) the benefits of PSL production appear to be preferentially directed toward cells which produce PSL; (v) cells which do not produce PSL are unable to successfully exploit cells which produce PSL. Taken together, this suggests that PSL is a social but relatively nonexploitable trait and that growth within biofilms selects for PSL-producing strains, even when multiple strains are on a patch (low relatedness at the patch level).IMPORTANCE Many studies have shown that bacterial traits, such as siderophores and quorum sensing, are social in nature. This has led to an impression that secreted traits act as public goods, which are costly to produce but benefit both the producing cell and its surrounding neighbors. Theories and subsequent experiments have shown that such traits are exploitable by asocial cheats, but we show here that this does not always hold true. We demonstrate that the Pseudomonas aeruginosa exopolysaccharide PSL provides social benefits to populations but that it is nonexploitable, because most of the fitness benefits accrue to PSL-producing cells. Our work builds on an increasing body of work showing that secreted traits can have both private and public benefits to cells.

Keywords: Pseudomonas aeruginosa; biofilms; social evolution.

FIG 1

PSL production provides community benefits to cells grown in biofilms. (A) The PSL⁻ strain was significantly defective in biofilm formation on beads compared to the PSL⁺ strain (F_1,15 = 11.26, P = 0.0043; n = 3), but no major differences were seen between either strain’s growth in the unattached fractions (F_1,16 = 0.67, P = 0.4251; n = 3). (B) PSL⁻ cells cocultured with PSL⁺ cells at a 1:1 ratio increased their proportional numbers in a biofilm. *, P < 0.0001; **, P > 0.08; n = 4. (C) Confocal micrograph images of surface-attached populations of PSL⁻/PSL⁺ cocultures at a 1:1 ratio. PSL⁻ cells (red) coaggregated with PSL⁺ cells (green).

FIG 2

PSL mutants are not social cheats within biofilms. (A) The relative fitness of PSL⁻ cells was equal or lower than that of the PSL⁺ strain across all starting frequencies of the mutant in mixed cultures in biofilms (t¹³ = −3.242, P = 0.0064). PSL⁻ strain fitness was equal to that of the PSL⁺ strain across all starting frequencies in unattached populations (t¹³ = −0.741, P = 0.4716). (B) The PSL⁻/PSL⁺ cell coculture biomass remained similar across all starting ratios in both biofilm (t¹³ = −1.769, P = 0.1004) and unattached populations (t¹³ = 1.881, P = 0.0825).

FIG 3

The PSL⁺ strain smothers and outcompetes the PSL⁻ strain during biofilm growth. Confocal micrograph images of PSL⁻/PSL⁺ (1:1 cell ratio) mixed population biofilms at 0 h (A and B) and 48 h (C and D). PSL⁻ cells were only attached to the surface as coaggregates with PSL⁺ cells, and they were eventually outcompeted by PSL⁺ cells when the biofilm matured. Panels A and C show three-dimensional rendered images, and panel B and D represent corresponding open box top-down views. Red, PSL⁺ cells; green, PSL⁻ cells.

FIG 4

PSL production results in increased tolerance toward antibiotics. (A) PSL⁻/PSL⁺ cell mixed biofilms yielded consistent biomasses regardless of the starting ratio of the two strains (t¹³ = −0.732, P = 0.4769; n = 3). Treatment of the mixed biofilm with a high concentration of gentamicin eliminated the entire biofilm population when the starting proportion of PSL⁻ cells increased above that of PSL⁺. cells. (B) In the intermediate PSL⁻:PSL⁺ coculture, where the mixed biofilm was partially eradicated, there were fewer PSL⁻ cells in the surviving biofilm in the presence of gentamicin (t⁴ = 4.07, P < 0.02; n = 3).

FIG 5

PSL production is favored in high- and low-relatedness treatments in biofilms. PSL⁺ cells had a selective advantage under conditions promoting biofilm formation (black) (F_1,20 =542.7, P < 0.0001; n = 6). However, this observation was not reciprocated under unattached conditions (gray). The concurrent loss of PSL⁻ cells from both low-related and high-related biofilm populations through subsequent selection rounds suggested there is a strong selection toward PSL⁺ strains (under biofilm-promoting conditions only), as PSL⁻ strains were unable to cheat on PSL⁺ strains.