The human pathogen Pseudomonas aeruginosa utilizes conserved virulence pathways to infect the social amoeba Dictyostelium discoideum

Abstract

Genetically accessible host models are useful for studying microbial pathogenesis because they offer the means to identify novel strategies that pathogens use to evade immune mechanisms, cause cellular injury, and induce disease. We have developed conditions under which the human pathogen Pseudomonas aeruginosa infects Dictyostelium discoideum, a genetically tractable eukaryotic organism. When D. discoideum is plated on nutrient agar plates with different P. aeruginosa strains, the bacteria form lawns on these plates with amoebae embedded in them. Virulent P. aeruginosa strains kill these amoebae and leave an intact bacterial lawn. A number of P. aeruginosa mutants have been identified that are avirulent in this assay. Amoebae feed on these bacteria and form plaques in their bacterial lawns. One avirulent mutant strain carries an insertional mutation in the lasR gene. LasR is a transcription factor that controls a number of virulence genes in a density-dependent fashion. Another class of avirulent P. aeruginosa mutants is defective in type III secretion. One mutant lacks the PscJ protein, a structural component of the secretion apparatus, suggesting that cytotoxins are injected into the D. discoideum cell. One of these cytotoxins is ExoU, and exoU mutants are avirulent toward D. discoideum. Complementation of the lasR and exoU mutations restores virulence. Therefore, P. aeruginosa uses conserved virulence pathways to kill D. discoideum.

Figure 1

D. discoideum cells do not form plaques in lawns of P. aeruginosa strain PA14. D. discoideum cells (AX3) were plated on SM/5 with K. aerogenes (Left) and P. aeruginosa strains PA14 (Right) at a density of ≈100 D. discoideum cells/plate. After 3 days, D. discoideum plaques appeared on plates with K. aerogenes. No plaques were formed on plates with the virulent P. aeruginosa strain.

Figure 2

Complementation of the lasR mutation restores LasR function and virulence but does not affect synthesis of the pigment pyocyanin. (Top) D. discoideum cells (AX3) were plated on nutrient agar (SM/5) with P. aeruginosa strain PA14, the isogenic lasR-mutant 12A1, complemented strains SUP16 (ΔlasR with pUCP18) and SUP17 (ΔlasR with plasR-PUH), and the pyocyanin-deficient strain ΔphenA/ΔphenB. Plates were incubated at 22°C for 5 days, and virulence was assessed by determining the number of Dictyostelium plaques. (Middle) Elastase activity of supernatants from overnight cultures were assayed by using elastin Congo red (ECR) as the substrate. Data represent the means of duplicate elastase assays, and activity is expressed as the activity relative to wild-type PA14 (=100%) +/−SDⁿ⁻¹. (Bottom) The ability of each strain to secrete the pigment pyocyanin was determined by assaying the amount of pyocyanin present in overnight cultures of each strain. Pyocyanin was extracted from supernatants and assayed as described in (20). Data represent the means of duplicate assays, and amounts of secreted pyocyanin are expressed as micrograms/milliliter culture medium +/− SDⁿ⁻¹.

Figure 3

Uptake of P. aeruginosa by D. discoideum. D. discoideum cells were placed in tissue culture wells at 10⁶ cells/ml. Cells were infected with bacteria at a multiplicity of infection of approximately 100:1. Cultures were incubated at 22°C for 30 min, at which time gentamicin was added to kill all extracellular bacteria. Amoebae were collected at indicated time points, lysed, and plated on nutrient agar plates to determine the colony-forming units (cfu) in these lysates. Data represent the means of duplicate experiments, and the number of internalized bacteria per well is expressed as cfu/ml +/− SDⁿ⁻¹.

Figure 4

D. discoideum forms plaques in thin lawns of P. aeruginosa PA14. D. discoideum cells (AX3) were plated with P. aeruginosa strains PA14 (wild type) and PA14 mutants 8C12 and 12A1 (ΔlasR) on nutrient agar plates with decreasing nutrient content. Agar plates contained 1/25 (SM/25), 1/50 (SM/50), and 1/100 (SM/100) of the nutrient content in SM plates. After 4 days at 22°C, D. discoideum formed plaques in lawns of 12A1 on SM/25 and plates with lower nutrient content, 8C12 and PA14 allowed plaque formation only on SM/100 plates.

Figure 5

Complementation of the exoU mutation restores virulence. D. discoideum cells (AX3) were plated with different bacterial associates on nutrient agar plates (SM/5) at a density of approximately 40 amoebae/plate. Emerging D. discoideum plaques on each plate were scored after 5 days. Virulence is expressed as the average number of emerging plaques per plate, and error bars represent the standard deviation of five replicates. The virulence of PA103, as well as isogenic mutants PA103∷pscJ (ΔpscJ), PA103∷exoU (ΔexoU), PA103∷exoU+pLAFRSKI (ΔexoU with plasmid control pLAFRSKI), and PA103∷exoU+pAH807 [ΔexoU with plasmid pAH807 (exoU+/spcU+)] was tested in this assay. As a reference for plating efficiency, AX3 was also cultured with K. aerogenes.

Figure 6

Expression of ExoU in P. aeruginosa PA103 prohibits plaque formation by D. discoideum. D. discoideum cells (AX3) were plated on SM/5 with P. aeruginosa strains PA103 (A), PA103∷exoU (ΔexoU) (B), and PA103∷exoU + pAH807 (ΔexoU with plasmid pAH807 [exoU⁺/spcU⁺]) (C) at a density of ≈100 D. discoideum cells/plate. Plaques were scored after 4 days of incubation at 22°C.