Cyclic voltammetric, fluorescence and biological analysis of purified aeruginosin A, a secreted red pigment of Pseudomonas aeruginosa PAO1

Abstract

The opportunistic pathogen Pseudomomas aeruginosa produces multiple pigments during in vitro culture and in vivo during colonization of burn wounds and in the airways of cystic fibrosis (CF) patients. One pigment is a deep 'merlot'-coloured compound known as aeruginosin A (AA). However, the red pigment(s) of P. aeruginosa are often collectively called pyorubrin, of which there is no known chemical composition. Here, we purified and confirmed by MS and assessed the physicochemical properties of AA (2-amino-6-carboxy-10-methylphenazinium betaine) by first focusing on its ability to redox-cycle using cyclic voltammetry and its spectroscopic (as well as fluorescent) properties, experiments that were conducted at physiological pH. AA exhibited reversible electrochemistry at a glassy carbon electrode within a potential range of -500 to -200 mV. Electrochemical anodic and cathodic peak currents were observed at -327 and -360 mV, respectively, with a low formal reduction potential of -343.5 mV versus Ag/AgCl. AA absorbed at 516 nm and fluoresced at 606 nm. Results from the spectro-electrochemistry of pyorubrin revealed that its strongest fluorescence was in its parent or oxidized form. Production of AA by P. aeruginosa was found to be controlled by the rhl component of the intercellular signalling system known as quorum sensing and was produced maximally during the stationary growth phase. However, unlike its downstream blue redox-active toxin, pyocyanin, AA had no adverse effects on methicillin-resistant Staphylococcus aureus USA300, Escherichia coli DH5-α or human keratinocytes. We close with some thoughts on the potential commercial use(s) of AA.