Social cheating in a Pseudomonas aeruginosa quorum-sensing variant

Abstract

The opportunistic bacterial pathogen Pseudomonas aeruginosa has a layered acyl-homoserine lactone (AHL) quorum-sensing (QS) system, which controls production of a variety of extracellular metabolites and enzymes. The LasRI system activates genes including those coding for the extracellular protease elastase and for the second AHL QS system, RhlRI. Growth of P. aeruginosa on casein requires elastase production and LasR-mutant social cheats emerge in populations growing on casein. P. aeruginosa colonizes the lungs of individuals with the genetic disease cystic fibrosis (CF), and LasR mutants can be isolated from the colonized lungs; however, unlike laboratory-generated LasR mutants, many of these CF isolates have functioning RhlR-RhlI systems. We show that one such mutant can use the RhlR-RhlI system to activate expression of elastase and grow on casein. We carried out social-evolution experiments by growing this isolate on caseinate and, as with wild-type P. aeruginosa, elastase-negative mutants emerge as cheats, but these are not RhlR mutants; rather, they are mutants that do not produce the non-AHL Pseudomonas quinolone signal (PQS). Furthermore, we generated a RhlRI mutant and showed it had a fitness defect when growing together with the parent. Apparently, RhlR QS and PQS collude to support growth on caseinate in the absence of a functional LasR. Our findings provide a plausible explanation as to why P. aeruginosa LasR mutants, but not RhlR mutants, are common in CF lungs.

Keywords: Pseudomonas quinolone signal; acyl-homoserine lactone; chronic infection; cystic fibrosis; social evolution.

Fig. 1.

Production of C4-HSL, pyocyanin, and protease by the LasR⁻ mutant clinical isolate E80 and E80 RhlRI⁻ mutant. (A) Relative levels of C4-HSL in P. aeruginosa E80 (black squares), PAO1 (black circles), and a PAO1 LasR⁻ mutant (white circles) at different times during cell growth. Levels are normalized to the maximum amount of C4-HSL in PAO1 cultures. The data are means ± SD (bars) of four independent experiments. (B) Relative pyocyanin levels of the indicated strain. The pyocyanin levels produced by strain PAO1 are defined as 100%. Pyocyanin was measured after 24 h of growth in LB-Mops broth. Data represent means and SDs of six replicates. (C and D) Protease production on casein agar. High levels of extracellular proteases result in a zone of clearing around a colony, with a white zone of partial casein degradation at the periphery of the zone of clearing. The PAO1 LasR mutant and the E80 RhlRI mutant produce relatively little protease, as indicated by the lack of a zone of clearing. There is only a small zone of partial casein digestion. The images are of colonies after about 30 h of incubation.

Fig. 2.

Emergence of protease-deficient freeloaders during daily passage of strain E80 in casein broth. (A) An example of one serial transfer experiment (lineage B). Each photographic image was captured after 1 d of growth. The relative abundance of protease-deficient freeloaders (as a percentage of the total population) in culture samples just before transfer (50 μL) is shown above the indicated tubes, and the daily passage number is shown below the tubes. Cultures that only partially degrade casein have a milky white appearance due to protein precipitation. (B) Summary of results from 11 independent daily transfer experiments (A through K), including the transfer day (d) that no longer showed protein digestion and the percentage of freeloaders (protease-def) detected at the 2-d interval before the final day.

Fig. 3.

The relationship between PQS and protease production in strain E80. The top row shows an E80 colony, an E80 PqsR mutant, and the PqsR mutant complemented with a functional pqsR (PqsR⁻-C) in the att site. The middle and bottom rows show colonies of E80 freeloaders (1–6) and colonies of freeloaders 1 and 2 complemented with pqsR in the chromosomal att site (1-C and 2-C).

Fig. 4.

The E80 protease-deficient freeloaders show reduced levels of C4-HSL and 2-alkylquinolone production. (A) Relative C4-HSL levels in cultures of strain E80, E80 freeloaders (1–6), and E80 freeloaders 1 and 2 complemented with a chromosomal copy of pqsR (1-C and 2-C). Values are normalized to E80 levels. (B) Concentrations of PQS (black) and HHQ (white) produced in the indicated strains. For both A and B, data are the means of four replicates ± SD.

Fig. 5.

Growth and competition of the PqsR mutant, the RhlRI mutant, and the parent strain E80. (A) Growth of E80 (black), the RhlRI⁻ mutant (magenta), and the PqsR⁻ mutant (green) in LB-Mops broth. Cell density was measured as OD₆₀₀, and data are the means of three biological replicates ± SD. (B) Competition experiments showing the competitive indexes of competitor strains against the parent strain (E80 or E80-Gm) after 24 h in LB-Mops broth. Each symbol represents the outcome of an individual experiment; the solid lines represent means for each group. The starting percentage of the competitors was 10%. The RhlRI mutant was less fit than the parent, and the PqsR⁻ mutant was more fit than the parent. (C) The relative fitness of a PqsR mutant exhibits a negative frequency dependence. Data are from 24-h casein broth cultures. The outcomes above the dashed line indicate the competitor had a fitness advantage and below indicate the parent E80 had a fitness advantage.

Fig. 6.

Cyanide production by and sensitivity of a variety of P. aeruginosa strains and mutants. (A) Cyanide was monitored as described in Materials and Methods. The blue coloration of the filter paper reflects cyanide production. The top row is the parent strain E80 and various E80 mutants. The middle row is protease-deficient mutants 1–6, and the bottom row is strain PAO1, a PAO1 LasR mutant, and a PAO1 RhlR mutant shown for comparison. (B) Cyanide-killing curves for E80, the PqsR mutant, and the RhlRI mutant. Results are normalized to the number of cfu without cyanide. Data are the means of three replicates ± SD.