Stationary phase-specific virulence factor overproduction by a lasR mutant of Pseudomonas aeruginosa

Abstract

Secreted virulence factors of the human pathogen Pseudomonas aeruginosa are often under quorum sensing control. Cells lacking the quorum-sensing regulator LasR show reduced virulence factor production under typical laboratory conditions and are hypo-virulent in short-term animal infection models, yet lasR mutants are frequently associated with long-term infection in cystic fibrosis patients. Here, I show that in stationary-phase or slow-growth conditions, lasR cells continuously and strongly produce the important virulence factor pyocyanin while wild-type cells do not. Pyocyanin overproduction by lasR cells is permitted by loss of repression by RsaL, a LasR-dependent negative regulator. lasR cells also contribute pyocyanin in mixed cultures, even under "cheating" conditions where they depend on their wild-type neighbors for nutrients. Finally, some clinical P. aeruginosa isolates with lasR mutations can overproduce pyocyanin in the laboratory. These results imply that slow-growing clinical populations of lasR cells in chronic infections may contribute to virulence by producing pyocyanin under conditions where lasR⁺ cells do not.

Figure 1. A lasR mutant overproduces pyocyanin under stationary-phase culture conditions.

A. A simplified diagram showing some of the regulatory pathways linking the three quorum-sensing systems of P. aeruginosa and exoproduct synthesis. Each system and its regulatory pathways is color coded. B. Plot of the number of live cells (colony-forming units) in static cultures of strains with wild-type (WT) PA14 (MTC772), PA14 lasR (MTC774), and PA14 lasR rhlR (MTC797) backgrounds in LB at 25°C. C. Pyocyanin quantification in static cultures of wild-type (WT) PA14 (MTC1), PA14 lasR (MTC390), and PA14 lasR rhlR (MTC626) in LB at 25°C. The inset image shows the appearance of the cultures in a 12-well plate after 7 d. All plots show the mean ± standard deviation of 3 biological replicates.

Figure 2. Wild-type and lasR mutant cells display distinct virulence gene expression patterns in stationary-phase culture.

Gene expression was measured using chromosomally integrated lux reporters in the listed strain backgrounds. Plots show the mean ± standard deviation of 3 biological replicates each composed of 3 technical replicates.

Figure 3. Pyocyanin production by stationary-phase lasR mutant cells requires Rhl and quinolone signaling.

A. Pyocyanin quantification in the listed strains with or without the listed signaling molecules after 6 days of LB static culture at 25°C. The images to the right of the bar graph show the appearance of representative cultures. *, p<0.005; **, p<0.001. The differences between lasR and the 100 µM-complemented autoinducer-negative samples were not significant (0.04<p<0.33). B. Pyocyanin quantification in static LB cultures of the listed strains and conditions at 25°C. C. Pyocyanin quantification in static LB cultures of the listed strains at 25°C. All plots show the mean ± standard deviation of 3 biological replicates.

Figure 4. RsaL prevents pyocyanin production by stationary-phase lasR ⁺ cells.

A. Transcription from the rsaL promoter in wild-type (PA14) and lasR mutant cells in static 25°C LB culture. B. Pyocyanin quantification in static LB cultures of the listed strains and conditions at 25°C. All plots show the mean ± standard deviation of 3 biological replicates.

Figure 5. A lasR mutant contributes pyocyanin in a cheating mixture.

Starting mixtures were 1:4 lasR mutant to PA14 or PA14 phz. All plots show the mean ± standard deviation of 3 biological replicates. A. Pyocyanin quantification in shaking M9-casein medium at 25°C. Inset shows appearance of cultures after 7 days of incubation. B. Relative proportions of lasR mutant cells in the indicated mixtures after 7 days of incubation. The difference between the two mixtures was not significant (p = 0.12).

Figure 6. Clinical lasR isolates can overproduce pyocyanin.

A. Locations of mutations in the LasR protein detected by sequencing. The mutations are in clinical isolates from Ref. 17. B. Appearance of static LB cultures of PA14 and lasR laboratory strains and of clinical isolates (CF1-6) with and without lasR mutations. The cultures were grown for 12 days at 25°C to allow weaker phenotypes to develop.