Evolution of the Pseudomonas aeruginosa quorum-sensing hierarchy

Abstract

The bacterial pathogen Pseudomonas aeruginosa activates expression of many virulence genes in a cell density-dependent manner by using an intricate quorum-sensing (QS) network. QS in P. aeruginosa involves two acyl-homoserine-lactone circuits, LasI-LasR and RhlI-RhlR. LasI-LasR is required to activate many genes including those coding for RhlI-RhlR. P. aeruginosa causes chronic infections in the lungs of people with cystic fibrosis (CF). In these infections, LasR mutants are common, but rhlR-rhlI expression has escaped LasR regulation in many CF isolates. To better understand the evolutionary trajectory of P. aeruginosa QS in chronic infections, we grew LasR mutants of the well-studied P. aeruginosa strain, PAO1, in conditions that recapitulate an environment where QS signal synthesis by other bacteria might still occur. When QS is required for growth, addition of the RhlI product butyryl-homoserine lactone (C4-HSL), or bacteria that produce C4-HSL, to LasR mutants results in the rapid emergence of a population with a LasR-independent RhlI-RhlR QS system. These evolved populations exhibit subsequent growth without added C4-HSL. The variants that emerge have mutations in mexT, which codes for a transcription factor that controls expression of multiple genes. LasR-MexT mutants have a competitive advantage over both the parent LasR mutant and a LasR-MexT-RhlR mutant. Our findings suggest a plausible evolutionary trajectory for QS in P. aeruginosa CF infections where LasR mutants arise during infection, but because these mutants are surrounded by C4-HSL-producing P. aeruginosa, variants rewired to have a LasR-independent RhlIR system quickly emerge.

Keywords: LasR; RhlR; acyl-homoserine lactone; cystic fibrosis; sociomicrobiology.

Fig. 1.

Characteristics of three protease-producing LasR mutant variants (LasR-1, LasR-2, and LasR-3) derived from the LasR mutant (LasR-0). (A) RhlR activity of each variant and their nonprotease producing parent LasR-0, measured as GFP fluorescence and reported as relative fluorescence units (RFU). All strains contained the P_rhlA-gfp fusion plasmid pProbe-GT-P_rhlAgfp. (B) Relative levels of C4-HSL in culture fluid after 8 or 18 h of cell growth. Values are relative to those produced by P. aeruginosa PAO1 (WT) at 18 h. (C) Pyocyanin production by P. aeruginosa PAO1, the parent LasR mutant, and LasR mutant variants. (D) Cyanide levels of cultures. Data in A are means of triplicate experiments, and data in B–D are means of duplicate experiments. In all panels, error bars represent the SEM.

Fig. 2.

The influence of mexT, pqsA, and pqsE mutations on RhlR activity in the P. aeruginosa LasR-PsdR mutant. (A) Growth of P. aeruginosa mutants in casein broth. Bacteria were tagged with mCherry, and fluorescence (RFU) was used as a proxy for growth, as described in Materials and Methods. (B) RhlR activity in the LasR-PsdR mutant and mutants derived from this strain, measured as GFP fluorescence (RFU). All strains contained the P_rhlA-gfp fusion plasmid pProbe-GT-P_rhlAgfp. (C) RhlR activity in strain PAO1 compared with RhlR activity in the LasR-PsdR-MexT mutant, measured as in B. (D) Relative concentrations of C4-HSL produced by the WT and mutants after 18 h. (E) Image of culture tubes after 2 d of growth of the indicated strains in casein broth. The LasR-PsdR-MexT mutant culture is blue green as a result of pyocyanin production. Data in A and D are means of duplicate experiments, and data in B and C are means of triplicate experiments. In all panels, error bars represent the SEM.

Fig. 3.

Competition between the LasR-PsdR-MexT mutant and QS mutants that cannot grow in casein broth by themselves. (A) Competition experiments with the LasR-PsdR parent. The competitions were initiated with either a low (circles) or high (squares) frequency of LasR-PsdR-MexT. (B) Competition with the LasR-PsdR-MexT-RhlR mutant. Arrows indicate a transfer to fresh casein broth (inoculum 2% vol/vol). Data are the means of duplicate experiments, and error bars represent the SEM.

Fig. 4.

Emergence of PAO1 LasR mutant variants from coinoculation of PAO1 LasR mutant with clinical P. aeruginosa isolates in casein broth. PAO1 LasR mutant was tagged with mCherry and its growth was measured by monitoring mCherry fluorescence (RFU), as described in Materials and Methods. The clinical isolates were E92 (triangles), E130 (circles), and E131 (squares). Cocultures with E130 and E131 were transferred on days 4 and 8; those with E92 were transferred on days 6 and 9 (inoculum 2% vol/vol). Data are means of duplicate experiments, and error bars represent the SEM.