Photodynamic oxidation of Escherichia coli membrane phospholipids: new insights based on lipidomics

RATIONALE: The irreversible oxidation of biological molecules, such as lipids, can be achieved with a photosensitizing agent and subsequent exposure to light, in the presence of molecular oxygen. Although lipid peroxidation is an important toxicity mechanism in bacteria, the alterations caused by the photodynamic therapy on bacterial phospholipids are still unknown. In this work, we studied the photodynamic oxidation of Escherichia coli membrane phospholipids using a lipidomic approach.\n\nMETHODS: E. coli ATCC 25922 were irradiated for 90 min with white light (4 mW cm(-2), 21.6 J cm(-2)) in the presence of a tricationic porphyrin [(5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin triiodide, Tri-Py(+)-Me-PF]. Lipids were extracted and separated by thin-layer chromatography. Phospholipid classes were quantified by phosphorus assay and analyzed by electrospray ionization tandem mass spectrometry. Fatty acids were analyzed by gas chromatography. Quantification of lipid hydroperoxides was performed by FOX2 assay. Analysis of the photodynamic oxidation of a phospholipid standard was also performed.\n\nRESULTS: Our approach allowed us to see that the photodynamic treatment induced the formation of a high amount of lipid hydroperoxides in the E. coli lipid extract. Quantification of fatty acids revealed a decrease in the unsaturated C16:1 and C18:1 species suggesting that oxidative modifications were responsible for their variation. It was also observed that photosensitization induced the oxidation of phosphatidylethanolamines with C16:1, C18:1 and C18:2 fatty acyl chains, with formation of hydroxy and hydroperoxy derivatives.\n\nCONCLUSIONS: Membrane phospholipids of E. coli are molecular targets of the photodynamic effect induced by Tri-Py(+) -Me-PF. The overall change in the relative amount of unsaturated fatty acids and the formation of PE hydroxides and hydroperoxides evidence the damages in bacterial phospholipids caused by this lethal treatment.publishe

Characterization by electrospray ionization and tandem mass spectrometry of rhamnolipids produced by two Pseudomonas aeruginosa strains isolated from Brazilian crude oil

In this work, biosurfactants produced by two Pseudomonas aeruginosa strains isolated from Brazilian crude oils were identified by proton nuclear magnetic resonance ((1)H NMR) and further characterized by mass spectrometry (MS) coupled with electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) analysis in positive mode and their surface activities evaluated. Mono-rhamnolipids and di-rhamnolipids were identified for both isolates, but the most abundant were found to be mono-rhamnolipids. The similarity of rhamnolipids produced by the two strains was in good agreement with their surface activities. Both biosurfactants exhibited similar aqueous solution surface tensions, high emulsification indexes and critical micelle concentration values. The results obtained show that ESI-MS and MS/MS analysis alone provide a fast and highly specific characterization of biosurfactants produced by microbial strains.This work was supported by PARTEX OIL AND GAS. The authors acknowledge the financial support from Fundacao para a Ciencia e a Tecnologia for the Pest-C/CTM/LA0011/2011 and doctoral research grant of Jorge F.B. Pereira SFRH/BD/60228/2009