The mucoid switch in Pseudomonas aeruginosa represses quorum sensing systems and leads to complex changes to stationary phase virulence factor regulation

Abstract

The opportunistic pathogen Pseudomonas aeruginosa chronically infects the airways of Cystic Fibrosis (CF) patients during which it adapts and undergoes clonal expansion within the lung. It commonly acquires inactivating mutations of the anti-sigma factor MucA leading to a mucoid phenotype, caused by excessive production of the extracellular polysaccharide alginate that is associated with a decline in lung function. Alginate production is believed to be the key benefit of mucA mutations to the bacterium in the CF lung. A phenotypic and gene expression characterisation of the stationary phase physiology of mucA22 mutants demonstrated complex and subtle changes in virulence factor production, including cyanide and pyocyanin, that results in their down-regulation upon entry into stationary phase but, (and in contrast to wildtype strains) continued production in prolonged stationary phase. These findings may have consequences for chronic infection if mucoid P. aeruginosa were to continue to make virulence factors under non-growing conditions during infection. These changes resulted in part from a severe down-regulation of both AHL-and AQ (PQS)-dependent quorum sensing systems. In trans expression of the cAMP-dependent transcription factor Vfr restored both quorum sensing defects and virulence factor production in early stationary phase. Our findings have implications for understanding the evolution of P. aeruginosa during CF lung infection and it demonstrates that mucA22 mutation provides a second mechanism, in addition to the commonly occurring lasR mutations, of down-regulating quorum sensing during chronic infection this may provide a selection pressure for the mucoid switch in the CF lung.

Figure 1. mucA22, mucoid strains show differential regulation of virulence factor production throughout grow.

A, Change in cyanide concentration in liquid culture supernatant (primary y axis, square markers) throughout growth (secondary y axis, triangle markers) for PAO1 (wild type, non-mucoid) (black); M04 (mucA22, mucoid) (dark grey); and M04S (non-mucoid suppressor isolated from M04) (light grey). B and C respectively, pyocyanin and elastase levels in culture supernatant normalised to OD₆₀₀ of culture at 2 hours (mid-exponential phase), 6 hours (early stationary phase),15 hours (mid stationary phase) and 24 hours (late stationary phase). Values are means of 3 independent replicates with SE error bars. Culture conditions and cyanide, pyocyanin and elastase assays were as described in Figure 1.

Figure 2. mucA mutation alters the expression of cyanide, pyocyanin and elastase genes.

Expression profiles of hcnA (A), phzM (B), phzS (C) and lasB, (D) for PAO1 (wild type, non-mucoid) (black); M04 (mucA22, mucoid) (dark grey); and M04S (non-mucoid suppressor isolated from M04) (light grey). Values are averages of 3 biological replicates and the value for each replicate was the average of duplicate qRT-PCR technical replicates. Error bars are ±SEM. Data were analysed by ANOVA and post-hoc by Tukey's Multiple Comparison Test.

Figure 3. mucA mutation leads to suppression of quorum sensing signal molecule production throughout growth.

Activities of the LasIR (A) RhlIR (B) and PQS (C) systems was compared in PAO1 (wild type, non-mucoid) (black), M04 (mucA22, mucoid) (dark grey) and M04S (non-mucoid suppressor isolated from MucA04) (light grey) using bioluminescent reporter strains to assay 3-oxo-C₁₂ (lasIR) C₄-HSL (RhlIR) and PQS signal molecule levels in supernatant. Bioluminescence was measured using a luminometer. Culture conditions were as described in Figure 1. Values are means of 3 independent replicates and error bars are ±SEM.

Figure 4. mucA mutation leads to suppression of quorum sensing gene expression.

qRT-PCR expression profiles of quorum sensing regulatory genes for: PAO1 (wild type, non-mucoid) (black); M04 (mucA22, mucoid) (dark grey); and M04S (non-mucoid suppressor isolated from MucA04) (light grey). Values are averages of 3 biological replicates and the value for each replicate was the average of duplicate qRT-PCR technical replicates Error bars are ±SE and data were analysed by ANOVA and post-hoc by Tukey's Multiple Comparison Test.

Figure 5. Plasmid expression of the cAMP-dependent transcription factor Vfr restores the early stationary phase cyanide production and the quorum sensing defect in the mucA22 mutant.

Cyanide concentration and QS molecule levels were measured in culture supernatants of wildtype (PAO1), muc22 mutant (M04) and its suppressor strain (M04S) with or without the plasmid pPa-vfr. Cyanide levels after (A) 6 hours growth (early stationary phase) and (B) 24 hours growth (late stationary phase). (C) Relative 3-oxo-C12 AHL levels; (D) relative C4-AHL levels; (E) relative PQS levels after 8 hours growth (early stationary phase). Values are relative to the PAO1 8 hour value. All experiments are averages of three independent replicates error bars are ±SEM and data were analysed by ANOVA and post-hoc by Tukey's Multiple Comparison Test. **** p<0.001; *** p<0.01.

Figure 6. Model to explain early stationary phase down regulation of cyanide, pyocyanin and elastase production by mucA22 mutation of P. aeruginosa.

This figure is an adaptation and extension of Figure 7 from . Solid arrows indicate positive regulation and T-bar negative regulation. MucA22 mutation results in release of AlgU, which is then free to associate with core RNA polymerase and direct expression from its target genes, which include the gene for the transcriptional regulator AlgR. AlgR is a repressor of the cAMP-dependent transcriptional regulator, VfR, which is a positive regulator of the LasRI quorum sensing system. AlgR inhibition Vfr transcription will result in down regulation of the LasRI quorum sensing system, which since it is the dominant QS system will act to down regulate both the RhlRI and AQ (PQS) QS systems, resulting in suppression of the entire QS network and decreased expression of virulence factors in early stationary phase.