Pseudomonas aeruginosa virulence analyzed in a Dictyostelium discoideum host system

Abstract

Pseudomonas aeruginosa is an important opportunistic pathogen that produces a variety of cell-associated and secreted virulence factors. P. aeruginosa infections are difficult to treat effectively because of the rapid emergence of antibiotic-resistant strains. In this study, we analyzed whether the amoeba Dictyostelium discoideum can be used as a simple model system to analyze the virulence of P. aeruginosa strains. The virulent wild-type strain PAO1 was shown to inhibit growth of D. discoideum. Isogenic mutants deficient in the las quorum-sensing system were almost as inhibitory as the wild type, while rhl quorum-sensing mutants permitted growth of Dictyostelium cells. Therefore, in this model system, factors controlled by the rhl quorum-sensing system were found to play a central role. Among these, rhamnolipids secreted by the wild-type strain PAO1 could induce fast lysis of D. discoideum cells. By using this simple model system, we predicted that certain antibiotic-resistant mutants of P. aeruginosa should show reduced virulence. This result was confirmed in a rat model of acute pneumonia. Thus, D. discoideum could be used as a simple nonmammalian host system to assess pathogenicity of P. aeruginosa.

FIG. 1.

Quorum sensing in P. aeruginosa. In P. aeruginosa, secreted components such as proteases (LasB elastase), rhamnolipids, and pyocyanin are under the transcriptional control of two quorum-sensing systems, las and rhl. Both systems involve a transcriptional regulator (LasR and RhlR, respectively) and an autoinducer synthase (LasI and RhlI, respectively). When the bacterial density reaches a threshold, the accumulation in the medium of signaling autoinducer molecules (• and ▪) induces the las and rhl pathways, leading to transcription of virulence genes. The las quorum-sensing system can also induce, to some extent, the rhl quorum-sensing system. SOD, superoxide dismutase.

FIG. 2.

Growth of D. discoideum clones in the presence of Klebsiella and P. aeruginosa. Approximately 200 D. discoideum cells were plated with a lawn of K. pneumoniae bacteria alone (A) or supplemented with P. aeruginosa strain PT5 (wild type [wt]) (B), PT462 (rhlR [C]), PT498 (lasR [D]), PT531 (lasR-rhlR [E]), or PT712 (rhlA [F]). Growth of D. discoideum created plaques in the bacterial lawn after 5 days of incubation at 25°C.

FIG. 3.

Quantitative assessment of D. discoideum growth in the presence of P. aeruginosa. Dictyostelium cells were applied as droplets onto a lawn of pure P. aeruginosa bacteria, as represented in the upper left panel. The numbers of Dictyostelium cells applied were 50,000 in drop number 1, 10,000 in 2, 2000 in 3, etc., as described in Materials and Methods. After 5 days at 25°C, the ability of Dictyostelium cells to create plaques in the bacterial lawn was recorded. The bacteria used here were PT5 (wild type [wt]), PT498 (lasR), PT462 (rhlR), and PT531 (lasR-rhlR). In a scale measuring growth of Dictyostelium cells, the results obtained were scored 0 for wild-type PT5, 1 for PT498, 5 for PT462, and 8 for PT531.

FIG. 4.

Lysis of Dictyostelium cells exposed to Pseudomonas supernatants. Dictyostelium cells were exposed to culture supernatants of wild-type (wt) strain PT5 (A, upper panels) and the double mutant PT531 (A, lower panels) and observed with a phase-contrast microscope. Arrows indicate individual Dictyostelium cells being lysed by wild-type supernatant during the indicated time frame. The kinetics of cell lysis induced by supernatants of the wild type (PT5), the lasR-rhlR double mutant (PT531) and the rhlA mutant (PT712) was determined by counting Dictoystelium cells under the microscope in a field containing approximately 100 cells at time zero (B).

FIG. 4.

Lysis of Dictyostelium cells exposed to Pseudomonas supernatants. Dictyostelium cells were exposed to culture supernatants of wild-type (wt) strain PT5 (A, upper panels) and the double mutant PT531 (A, lower panels) and observed with a phase-contrast microscope. Arrows indicate individual Dictyostelium cells being lysed by wild-type supernatant during the indicated time frame. The kinetics of cell lysis induced by supernatants of the wild type (PT5), the lasR-rhlR double mutant (PT531) and the rhlA mutant (PT712) was determined by counting Dictoystelium cells under the microscope in a field containing approximately 100 cells at time zero (B).