Controlling localization of Escherichia coli populations using a two-part synthetic motility circuit: An accelerator and brake.

Probiotics, whether taken as capsules or consumed in foods, have been regarded as safe for human use by regulatory agencies. Being living cells, they serve as "tunable" factories for the synthesis of a vast array of beneficial molecules. The idea of reprogramming probiotics to act as controllable factories, producing potential therapeutic molecules under user-specified conditions, represents a new and powerful concept in drug synthesis and delivery. Probiotics that serve as drug delivery vehicles pose several challenges, one being targeting (as seen with nanoparticle approaches). Here, we employ synthetic biology to control swimming directionality in a process referred to as "pseudotaxis." Escherichia coli, absent the motility regulator cheZ, swim sporadically, missing the traditional "run" in the run:tumble swimming paradigm. Upon introduction of cheZ in trans and its signal-generated upregulation, engineered bacteria can be "programmed" to swim toward the source of the chemical cue. Here, engineered cells that encounter sufficient levels of the small signal molecule pyocyanin, produce an engineered CheZ and swim with programmed directionality. By incorporating a degradation tag at the C-terminus of CheZ, the cells stop running when they exit spaces containing pyocyanin. That is, the engineered CheZ modified with a C-terminal extension derived from the putative DNA-binding transcriptional regulator YbaQ (RREERAAKKVA) is consumed by the ClpXP protease machine at a rate sufficient to "brake" the cells when pyocyanin levels are too low. Through this process, we demonstrate that over time, these engineered E. coli accumulate in pyocyanin-rich locales. We suggest that such approaches may find utility in engineering probiotics so that their beneficial functions can be focused in areas of principal benefit.

Pyocyanin as anti-tyrosinase and anti tinea corporis: A novel treatment study.

OBJECTIVE: The aim of this study was to evaluate the efficiency of pyocyanin pigment as a novel compound active against tyrosinase with its depigmentation efficiency for combating Trichophyton rubrum which could be a major causative agent of tinea corporis. METHODS: Fifty swabs of fungal tinea corporis infections were collected and identified. Five MDRPA isolates were tested for their levels of pyocyanin production. The purified extracted pyocyanin was characterized by UV spectrum and FT-IR analysis. Pyocyanin activity against tyrosinase was determined by dopachrome micro-plate. In addition, the antidermatophytic activity of pyocyanin against T. rubrum was detected by radial growth technique. In vivo novel trial was conducted to evaluate the efficiency and safety of pyocyanin as an alternative natural therapeutic compound against T. rubrum causing tinea corporis. RESULTS: Purified pyocyanin showed highly significant inhibitory activity against tyrosinase and T. rubrum. In vivo topical treatments with pyocyanin ointment revealed the efficiency of pyocyanin (MIC 2000 µg/ml) to cure tinea corporis compared to fluconazole, which showed a partial curing at a higher concentration (MIC 3500 µg/ml) after two weeks of treatment. In addition, the results revealed complete healing and disappear of hyperpigmentation by testing the safety of pyocyanin ointment and its histopathological efficiency in the skin treatment without any significant toxic effect. CONCLUSION: Pyocyanin pigment could be a promising anti-tyrosinase and a new active compound against T. rubrum, which could be a major causative agent of tinea corporis. In fact, if pyocyanin secondary metabolite is going to be used in practical medication, it will support the continuous demand of novel antimycotic natural agents against troublesome fungal infections.

Development of a simple and low cost microbioreactor for high-throughput bioprocessing.

A simple microbioreactor for high-throughput bioprocessing made from low cost polymer polytetrafluoroethylene (PTFE) tubes with a working volume of 1.5 ml is described. We have developed a microfluidic system that handles a small population of cells of a model microorganism, Pseudomonas aeruginosa DS10-129. Under the conditions of the microbioreactor, the organism produced extracellular secondary metabolites by using nutrient broth modified with glycerol. Pyocyanins were isolated from the fermented medium as a metabolite of interest. Antibiotic properties of pyocyanin were effective against a number of microorganisms such as Staphylococcus aureus, S. epidermis, Bacillus subtilis, Micrococcus luteus and Saccharomyces cerevisiae. Batch fermentation of the model organism in the microbioreactor was compared to shake-flask and conventional bench fermenter methods. Results obtained from the microbioreactor compared favourably with the conventional processes.

Interaction of the Pseudomonas aeruginosa secretory products pyocyanin and pyochelin generates hydroxyl radical and causes synergistic damage to endothelial cells. Implications for Pseudomonas-associated tissue injury.

Pyocyanin, a secretory product of Pseudomonas aeruginosa, has the capacity to undergo redox cycling under aerobic conditions with resulting generation of superoxide and hydrogen peroxide. By using spin trapping techniques in conjunction with electron paramagnetic resonance spectrometry (EPR), superoxide was detected during the aerobic reduction of pyocyanin by NADH or porcine endothelial cells. No evidence of hydroxyl radical formation was detected. Chromium oxalate eliminated the EPR spectrum of the superoxide-derived spin adduct resulting from endothelial cell exposure to pyocyanin, suggesting superoxide formation close to the endothelial cell plasma membrane. We have previously reported that iron bound to the P. aeruginosa siderophore pyochelin (ferripyochelin) catalyzes the formation of hydroxyl free radical from superoxide and hydrogen peroxide via the Haber-Weiss reaction. In the present study, spin trap evidence of hydroxyl radical formation was detected when NADH and pyocyanin were allowed to react in the presence of ferripyochelin. Similarly, endothelial cell exposure to pyocyanin and ferripyochelin also resulted in hydroxyl radical production which appeared to occur in close proximity to the cell surface. As assessed by 51Cr release, endothelial cells which were treated with pyocyanin or ferripyochelin alone demonstrated minimal injury. However, endothelial cell exposure to the combination of pyochelin and pyocyanin resulted in 55% specific 51Cr release. Injury was not observed with the substitution of iron-free pyochelin and was diminished by the presence of catalase or dimethyl thiourea. These data suggest the possibility that the P. aeruginosa secretory products pyocyanin and pyochelin may act synergistically via the generation of hydroxyl radical to damage local tissues at sites of pseudomonas infection.

Impairment of airway mucociliary transport by Pseudomonas aeruginosa products. Role of oxygen radicals.

We previously showed that the supernatant of a Pseudomonas aeruginosa (PA) culture and its constituents pyocyanin and 1-hydroxyphenazine inhibit ciliary activity of dispersed tracheal epithelial cells in vitro via the generation of oxygen radicals by phagocytes. In the present study, we wished to determine if tracheal mucus velocity (TMV) is also impaired by PA supernatant and if oxygen radicals have a mediating role. In conscious sheep, TMV (measured with a radiographic method) was determined before and serially following aerosol challenge with the cell-free supernatant of a PA culture or unconditioned culture medium (control). TMV decreased from a mean (+/- SEM) baseline of 6.7 +/- 1.1 mm/min (n = 6) by 29, 35, and 25% at 0.5, 3, and 24 h after challenge, respectively (p < 0.05), and returned to baseline 1 wk later (-6%, p = NS). Control medium had no effect on TMV (maximum decrease by 15% at 0.5 h). Aerosolized catalase blunted the effect of PA supernatant on TMV. To determine if the impairment of TMV involved ciliary inhibition, tissues were mounted in a chamber and ciliary beat frequency (CBF) and surface liquid velocity (SLV) were measured with a microscopic method. PA supernatant decreased both CBF (maximum mean decrease 12%; n = 5, p < 0.05) and SLV (maximum mean decrease 78%; n = 5, p < 0.05) in a dose-dependent fashion, with a correlation between the two parameters; these effects were blocked by catalase.(ABSTRACT TRUNCATED AT 250 WORDS)