1-hydroxyphenazine and Pseudomonas-Infections

A novel immunochemical approach to diagnose Pseudomonas aeruginosa infections is reported, which is based on the quantification of relevant and specific virulence factors secreted by this microorganism. Specific antibodies have been raised using hapten PC1 (a 1:1 mixture of 9-hydroxy- and 6-hydroxy-phenazine-2-carobxylic acids), designed to recognize 1-hydroxyphenazine (1-OHphz), which is the main metabolite of pyocyanin (PYO). PYO is one of the most important virulence factors produced by nearly all P. aeruginosa strains, and other species do not produce this factor. With these antibodies, an immunochemical analytical procedure able to quantify both 1-OHphz and PYO in complex clinical samples has been developed. 1-OHphz can be directly measured in solubilized sputum samples diluted 20 times with the assay buffer. Quantification of PYO is accomplished after conversion to 1-OHphz in just 20 min under basic conditions. A LOD of 0.60 ± 0.01 nM (4.80 ± 0.08 nmol kg(-1) sputum) is reached for both biomarker targets under the conditions established, a value that is much below the reported concentrations on sputum samples obtained from infected patients (up to 100 μM). The assay is robust, reproducible, accurate, can be run in about 2 h, and many samples can be measured simultaneously. The present reported assay could represent a significant improvement in the diagnosis of infectious diseases caused by this pathogen.

Topics: Animals; Antibodies; Biomarkers; Female; Immunochemistry; Models, Molecular; Molecular Structure; Phenazines; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Rabbits

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.

Topics: Animals; Bacterial Toxins; Caenorhabditis elegans; Phenazines; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine

Pseudomonas aeruginosa colonizes and infects human tissues, although the mechanisms by which the organism evades the normal, predominantly neutrophilic, host defenses are unclear. Phenazine products of P. aeruginosa can induce death in Caenorhabditis elegans. We hypothesized that phenazines induce death of human neutrophils, and thus impair neutrophil-mediated bacterial killing. We investigated the effects of two phenazines, pyocyanin and 1-hydroxyphenazine, upon apoptosis of neutrophils in vitro. Pyocyanin induced a concentration- and time-dependent acceleration of neutrophil apoptosis, with 50 microM pyocyanin causing a 10-fold induction of apoptosis at 5 h (p < 0.001), a concentration that has been documented in sputum from patients colonized with P. aeruginosa. 1-hydroxyphenazine was without effect. In contrast to its rapid induction of neutrophil apoptosis, pyocyanin did not induce significant apoptosis of monocyte-derived macrophages or airway epithelial cells at time points up to 24 h. Comparison of wild-type and phenazine-deleted strains of P. aeruginosa showed a highly significant reduction in neutrophil killing by the phenazine-deleted strain. In clinical isolates of P. aeruginosa pyocyanin production was associated with a proapoptotic effect upon neutrophils in culture. Pyocyanin-induced neutrophil apoptosis was not delayed either by treatment with LPS, a powerfully antiapoptotic bacterial product, or in neutrophils from cystic fibrosis patients. Pyocyanin-induced apoptosis was associated with rapid and sustained generation of reactive oxygen intermediates and subsequent reduction of intracellular cAMP. Treatment of neutrophils with either antioxidants or synthetic cAMP analogues significantly abrogated pyocyanin-induced apoptosis. We conclude that pyocyanin-induced neutrophil apoptosis may be a clinically important mechanism of persistence of P. aeruginosa in human tissue.

Topics: Apoptosis; Bucladesine; Cell Nucleus; Cells, Cultured; Culture Media, Conditioned; Cyclic AMP; Dose-Response Relationship, Drug; Exotoxins; Humans; Kinetics; Lipopolysaccharides; Neutrophils; Phenazines; Pseudomonas aeruginosa; Pseudomonas Infections; Pyocyanine; Reactive Oxygen Species

The airways of cystic fibrosis patients colonised by Pseudomonas aeruginosa contain the redox active phenazine derivative, 1-hydroxyphenazine (OHP). As the presence of reactive oxygen species is of importance to tissue damage in cystic fibrosis, OHP was investigated for its ability to reduce molecular oxygen to superoxide. In the presence of NADPH, OHP reduced cytochrome c in a dose-dependent manner. This effect was not inhibited by superoxide dismutase and demonstrates an electron transport role for OHP. The OHP/NADPH system was unable to reduce molecular oxygen to superoxide as judged by an inability to oxidase epinephrine to adrenochrome. However, using lucigenin-enhanced chemiluminescence to detect superoxide, it was found that pathophysiologically relevant concentrations of OHP (5-25 microM) effectively scavenged superoxide from a xanthine/xanthine oxidase system. Similarly, in the presence of OHP, superoxide availability from contact-activated neutrophils was substantially reduced. It is concluded that OHP is an efficient scavenger of superoxide and that electron transfer from superoxide to OHP represents a major mechanism for reduction of OHP in vivo. Reduced OHP has the potential to alter cellular function by participating in the reduction of iron-containing proteins and in this manner contribute to the pathogenesis of P. aeruginosa infection in cystic fibrosis.

Topics: Acridines; Cystic Fibrosis; Cytochrome c Group; Electron Transport; Epinephrine; Free Radical Scavengers; Humans; Luminescent Measurements; NADP; Neutrophils; Oxygen; Phenazines; Pseudomonas aeruginosa; Pseudomonas Infections; Reactive Oxygen Species; Superoxide Dismutase; Superoxides; Xanthine; Xanthine Oxidase; Xanthines