Fig 1. Alkyl-quinolone production is necessary and sufficient for surface-induced killing of D. discoideum by P. aeruginosa.
(A) Representative images of D. discoideum infected with surface-attached wild type and ΔpqsA P. aeruginosa after 1 h co-culture (scale bars = 30 μm). Fluorescent calcein-AM staining indicates cell death. (B) Schematic of the PQS pathway depicting the functions of relevant genes. Solid and dotted arrows represent biosynthetic reactions and gene regulation, respectively. Genes above arrows indicate genetic requirements for pathways. (C) Quantification of D. discoideum killing by PQS pathway mutants. Expression of pqsABCDE is driven by the endogenous pqsA promoter or a strong constitutive promoter inserted upstream of the pqsA gene (Pconst−pqsABCDE). Values are mean ± SEM from 3 biological replicates, and approximately 200–300 cells were analyzed for each measurement. (D) Cytotoxicity of purified HHQ and PQS to D. discoideum in axenic cultures grown for 3 days at 22°C. Values are mean ± SD of five biological replicates (n = 5). E) MTT cell viability assay of TIB-67 mouse monocytes after 48 h of treatment with various concentrations of the alkyl-quinolones HHQ, PQS, or HQNO in a 96-well format. Percent survival is relative to an untreated control. Values are mean ± SEM of three biological replicates (n = 3). (F) Quantification of survival of TIB-67 monocytes after 18 h co-cultured with surface-attached P. aeruginosa. Values are mean ± SEM of 4–5 biological replicates (n = 4–5). Approximately 2500–3000 cells were analyzed for each measurement. For statistical analysis, mutants were compared to wild type unless shown otherwise. Statistical analyses are Student’s t-test (two-tailed, * = p<0.05, ** = p<0.01, *** = p<0.001).