Article Figures & Data
Figures
- 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).
Tables
- TABLE 1.
P. aeruginosa strains used in this study
Strain Phenotype or genotype Source or reference 35 PT5 PAO1 wild type Laboratory collection PT149 PT5NfxC (previously called PAO-7H) 18 PT454 PT5ΔrhlI::Tn501 Hgr 17 PT462 PT5ΔrhlR::Tn501 Hgr 17 PT466 PT5ΔlasI Tcr 17 PT498 PT5ΔlasR Tcr 17 PT502 PT5ΔrhlI::Tn501 ΔlasI Hgr Tcr This study PT531 PT5rhlR::Tn501 ΔlasR Hgr Tcr 17 PT625 PT5NalC 19 PT637 PT149mexE::ΩHg 20 PT648 PT5nfxB 19 PT712 PT5rhlA::Gm 17 - TABLE 2.
Growth of D. discoideum in the presence of various P. aeruginosa mutants
Strain D. discoideum growth on substrate(s)a K. pneumoniae + P. aeruginosa P. aeruginosab PT5 (wild type) − 0 PT462 (rhlR) + 5 PT498 (lasR) − 1 PT531 (rhlR-lasR) + 8 PT454 (rhlI) + 4 PT466 (lasI) − 1 PT502 (rhlI-lasI) + 8 PT712 (rhlA) + 2 PT149 (NfxC) + 4 PT637 (NfxC, mexE) − 0 PT625 (NalC) − 0 PT648 (nfxB) − 1 ↵a Clonal growth of D. discoideum was tested on a mixture of K. pneumoniae and P. aeruginosa cells, as described in Fig. 2. +, growth of D. discoideum on the given substrate; −, no growth of D. discoideum.
↵b The ability of D. discoideum cells to form plaques was tested on a lawn of pure P. aeruginosa cells as described in the legend to Fig. 3.
- TABLE 3.
Virulence of P. aeruginosa efflux mutants in a rat model of acute pneumonia
Strain Inoculum (106 CFU) n % Mortalitya P Time to death (days) Lung wt (g)b Bacterial counts in lungs (log CFU/g) PT5 (wild type) 3.0 22 72 1.6 ± 0.63 3.98 ± 0.50 8.30 ± 0.54 PT625 (NalC) 1.7 18 50 0.19 2.1 ± 1.0 3.84 ± 0.9 8.53 ± 0.38 PT648 (nfxB) 2.1 11 45 0.15 1.6 ± 0.89 4.83 ± 1.35 8.85 ± 0.28 PT149 (NfxC) 2.1 18 0 <0.001 NRc NDd ND PT637 (NfxC, mexE) 0.9 12 42 0.006 2.8 ± 1 4 ± 1.21 8.44 ± 0.48
