Identification of Pseudomonas aeruginosa Phenazines that Kill Caenorhabditis elegans

Stanford University, United States of America
PLoS Pathogens (Impact Factor: 7.56). 01/2013; 9(1):e1003101. DOI: 10.1371/journal.ppat.1003101
Source: PubMed


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.

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Available from: Grace Yuen, Mar 08, 2014
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    • "Although toxic proteins have been described (Wei et al., 2003), the toxins are typically secondary metabolites. For example, P. aeruginosa PA14 is capable of killing C. elegans through oxidative stress by using the phenazine compound pyocyanin as a virulence factor (Mahajan-Miklos et al., 1999; Cezairliyan et al., 2013). "
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    ABSTRACT: Soil bacteria can be prolific producers of secondary metabolites and other biologically active compounds of economic and clinical importance. These natural products are often synthesized by large multi-enzyme complexes such as polyketide synthases (PKSs) or non-ribosomal peptide synthases (NRPSs). The plant-associated Gram-negative bacterium, Serratia plymuthica A153, produces several secondary metabolites and is capable of killing the nematode worm Caenorhabditis elegans; a commonly used model for the study of bacterial virulence. In this study, we show that disruption of the hybrid PKS/NRPS zeamine (zmn) gene cluster results in the attenuation of "fast-killing" of C. elegans, indicating that zeamine has nematicidal activity. C. elegans also exhibits age-dependent susceptibility to zeamine, with younger worms being most sensitive to the bioactive molecule. The zmn gene cluster is widely distributed within Serratia and phytopathogenic Dickeya species and investigation of strains harboring the zmn gene cluster showed that several of them are highly virulent in C. elegans. Zeamine was described previously as a phytotoxin and broad-spectrum antibacterial compound. In addition to its nematicidal properties, we show here that zeamine can also kill Saccharomyces cerevisiae and Schizosaccharomyces pombe. The expression of the zmn gene cluster and regulation of zeamine production were also investigated. Transcription of the cluster was growth phase-dependent, and was modulated by the post-transcriptional RNA chaperone, Hfq. The results of this study show that zeamine is a highly toxic molecule with little, or no, apparent host specificity in very diverse biological systems. In its current form, zeamine(s) may be useful as a lead compound suitable for chemical modification and structure-activity assays. However, because of widespread non-selective toxicity in multiple bioassays, unmodified zeamine(s) is unlikely to be suitable as a therapeutic antibiotic.
    Frontiers in Microbiology 02/2015; 6:137. DOI:10.3389/fmicb.2015.00137 · 3.99 Impact Factor
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    • "The diversity of toxic molecules produced and released by P. aeruginosa facilitates its pathogenicity and contributes to impair lung function in CF. Cezairliyan et al. (2013) Zebrafish embryos The T3SS, biofilm formation and quorum-sensing systems are involved in virulence, and these systems correlate with increased P. aeruginosa virulence in murine models and in humans. Clatworthy et al. (2009) Zebrafish embryos Helped to support a connection between the cystic fibrosis transmembrane conductance regulator (CFTR) and the innate immune response. "
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    • "In contrast, the fast killing assay results in C. elegans death within a period of hours. While the fast killing assay does not require live bacteria and is due to the production of secreted toxins including phenazines (Cezairliyan et al., 2013), the slow killing pathway requires ingestion of live bacteria and many different virulence genes (Feinbaum et al., 2012). P. aeruginosa has been shown to survive ingestion, colonize within an extracellular matrix in the nematode lumen, and cause much less damage to the intestinal epithelial barrier than S. aureus (Sifri, Begun & Ausubel, 2005; Irazoqui et al., 2010). "
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    ABSTRACT: Academic editor Siouxsie Wiles Additional Information and Declarations can be found on page 16 DOI 10.7717/peerj.521 ABSTRACT Caenorhabditis elegans is commonly used as an infection model for pathogenesis studies in Pseudomonas aeruginosa. The standard virulence assays rely on the slow and fast killing or paralysis of nematodes but here we developed a behaviour assay to monitor the preferred bacterial food sources of C. elegans. We monitored the food preferences of nematodes fed the wild type PAO1 and mutants in the type III secre-tion (T3S) system, which is a conserved mechanism to inject secreted effectors into the host cell cytosol. A ΔexsEΔpscD mutant defective for type III secretion served as a preferred food source, while an ΔexsE mutant that overexpresses the T3S effectors was avoided. Both food sources were ingested and observed in the gastrointestinal tract. Using the slow killing assay, we showed that the ΔexsEΔpscD had reduced vir-ulence and thus confirmed that preferred food sources are less virulent than the wild type. Next we developed a high throughput feeding behaviour assay with 48 possible food colonies in order to screen a transposon mutant library and identify potential virulence genes. C. elegans identified and consumed preferred food colonies from a grid of 48 choices. The mutants identified as preferred food sources included known virulence genes, as well as novel genes not identified in previous C. elegans infection studies. Slow killing assays were performed and confirmed that several preferred food sources also showed reduced virulence. We propose that C. elegans feeding behaviour can be used as a sensitive indicator of virulence for P. aeruginosa PAO1.
    PeerJ 08/2014; DOI:10.7717/peerj.521 · 2.11 Impact Factor
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