Identification of Pseudomonas aeruginosa Phenazines that Kill Caenorhabditis elegans

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.Chemistry and Chemical Biolog

Metabolomics Strategies for Discovery of Biologically Active or Novel Metabolites

Along with genes and proteins, metabolites play important roles in sustaining life. There remains much to be learned about the in vivo roles of metabolites. Metabolomics is a comparative tool to study global metabolite levels in samples under various conditions. This dissertation describes the development and application of metabolomics strategies for discovery of biologically active or novel metabolites with priori knowledge about genes, proteins, or phenotypes. The power of metabolomics for discovery of novel metabolites from genes is demonstrated through the work with the pyochelin (pch) gene cluster. Comparison of the extracellular metabolomes of pch gene cluster mutants to the wild-type Pseudomonas aeruginosa (strain PA14) identified 198 ions regulated by the pch genes. In addition to known metabolites, a pair of novel metabolites were characterized as 2-alkyl-4,5-dihydrothiazole-4-carboxylates (ATCs). Subsequent assays revealed that ATCs bind iron and that their production is regulated by iron levels and dependent on pchE gene in the pch gene cluster. Metabolomics can also facilitate discovery of active metabolites from proteins, as shown in the work with orphan nuclear receptor Nur77. We applied a metabolomics platform for detected protein-metabolite interactions to identify lipids that bind to Nur77. Using this approach, we discovered that the Nur77 ligand-binding domain (Nur77LBD) enriched unsaturated fatty acids (UFAs) in tissue lipid mixtures. Subsequent biophysical and biochemical assays indicate that UFAs bind to Nur77LBD to cause changes in the conformation and oligomerization of the receptor. Last, analogous to classic fractionation experiments, metabolomics can also be applied to discover active metabolites from phenotypes. Using combination of genetics, biochemistry, and metabolomics, we identified three phenazine compounds produced by Pseudomonas aeruginosa that are toxic to the nematode Caenorhabditis elegans. 1-hydroxyphenazine, phenazine-1-carboxylic acid (PCA), 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 PCA and pyocyanin are strictly pH-dependent at non-overlapping pH ranges. The diversity within a class of metabolites can be used to modulate bacterial toxicity in different environmental niches.Chemistry and Chemical Biolog

Phenazine synthesis by <i>P. aeruginosa</i> is essential for killing <i>C. elegans</i>.

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

phenazine-1-carboxylic acid and 1-hydroxyphenazine are toxic to <i>C. elegans</i>.

<p>(A) Killing of <i>C. elegans</i> 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 <i>C. elegans</i> after four hours of exposure to synthetic phenazines added to PGS agar plates after growth of Δ<i>phz</i> bacteria. (C) Killing of <i>C. elegans</i> by <i>P. aeruginosa</i> PA14 mutants in the phenazine synthesis pathway.</p

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

<p>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.</p

Levels of phenazines in agar plated with PA14 mutants (µg/mL).

<p>nd = not detected.</p>*<p>phenazine-1-carboxamide was detected in all strains except Δ<i>phz</i>. However, levels were below the quantifiable detection limit of 1 µg/mL for wild-type, <i>phzH</i>, and <i>phzS</i>.</p

Toxicity of pyocyanin is pH dependent.

<p>(A, B, C) Nematode death on PGS agar with wild-type PA14, <i>phzM</i>, <i>phzH</i>, <i>phzS</i>, and Δ<i>phz</i>. 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 Δ<i>phz</i> agar raised to pH 6, 7, or 8 with 100 mM potassium phosphate. Pyocyanin and buffer were added after bacterial growth.</p