Identification of Pseudomonas aeruginosa phenazines that kill Caenorhabditis elegans

Abstract

Pathogenic microbes employ a variety of methods to overcome host defenses, including the production and dispersal of molecules that are toxic to their hosts. Pseudomonas aeruginosa, a Gram-negative bacterium, is a pathogen of a diverse variety of hosts including mammals and the nematode Caenorhabditis elegans. In this study, we identify three small molecules in the phenazine class that are produced by P. aeruginosa strain PA14 that are toxic to C. elegans. We demonstrate that 1-hydroxyphenazine, phenazine-1-carboxylic acid, and pyocyanin are capable of killing nematodes in a matter of hours. 1-hydroxyphenazine is toxic over a wide pH range, whereas the toxicities of phenazine-1-carboxylic acid and pyocyanin are pH-dependent at non-overlapping pH ranges. We found that acidification of the growth medium by PA14 activates the toxicity of phenazine-1-carboxylic acid, which is the primary toxic agent towards C. elegans in our assay. Pyocyanin is not toxic under acidic conditions and 1-hydroxyphenazine is produced at concentrations too low to kill C. elegans. These results suggest a role for phenazine-1-carboxylic acid in mammalian pathogenesis because PA14 mutants deficient in phenazine production have been shown to be defective in pathogenesis in mice. More generally, these data demonstrate how diversity within a class of metabolites could affect bacterial toxicity in different environmental niches.

Figure 1. Phenazine synthesis by P. aeruginosa is essential for killing C. elegans.

(A) Phenazine synthesis pathway of P. aeruginosa. (B) Killing of wild-type P. aeruginosa PA14 and Δphz mutant and partial complementation of Δphz killing with plasmids containing either the phzA1-G1 or phzA2-G2 operon. (C) Complementation of killing in the Δphz mutant by addition of synthetic phenazine-1-carboxylic acid (100 µg/mL) to the agar medium prior to plating and growth of the bacteria.

Figure 2. phenazine-1-carboxylic acid and 1-hydroxyphenazine are toxic to C. elegans.

(A) Killing of C. elegans after four hours of exposure to synthetic phenazines (4, 8, 16, 32, 64, and 128 µg/mL final concentrations) added to naive PGS agar plates. Data points for phenazine-1-carboxylic acid, pyocyanin, and phenazine-1-carboxamide are overlapping. (B) Killing of C. elegans after four hours of exposure to synthetic phenazines added to PGS agar plates after growth of Δphz bacteria. (C) Killing of C. elegans by P. aeruginosa PA14 mutants in the phenazine synthesis pathway.

Figure 3. Toxicity of phenazine-1-carboxylic acid is pH dependent.

Nematode death after exposure to 100 µg/mL of phenazine-1-carboxylic acid or 1-hydroxyphenazine in PGS agar plates buffered at pH 4 (50 mM sodium acetate), 5 (50 mM sodium citrate), 6 (50 mM potassium phosphate), 7 (50 mM potassium phosphate), or 8 (50 mM potassium phosphate). There is no observable killing by phenazine-1-carboxylic acid at pH 6, 7, or 8, or by DMSO without phenazines under any of the buffer conditions.

Figure 4. Toxicity of pyocyanin is pH dependent.

(A, B, C) Nematode death on PGS agar with wild-type PA14, phzM, phzH, phzS, and Δphz. After bacterial growth, agar was melted and potassium phosphate pH 7 (100 mM final concentration) or equal volume of water was added. Worms were added after the agar had cooled and solidified. (D) Toxicity of exogenously added pyocyanin (10 µg/mL) in Δphz agar raised to pH 6, 7, or 8 with 100 mM potassium phosphate. Pyocyanin and buffer were added after bacterial growth.