Defining the Functional Properties of Cyclopropane Fatty Acid Synthase from Pseudomonas aeruginosa PAO1.

Cyclopropane fatty acid synthases (CFAS) catalyze the conversion of unsaturated fatty acids to cyclopropane fatty acids (CFAs) within bacterial membranes. This modification alters the biophysical properties of membranes and has been correlated with virulence in several human pathogens. Despite the central role played by CFAS enzymes in regulating bacterial stress responses, the mechanistic properties of the CFAS enzyme family and the consequences of CFA biosynthesis remain largely uncharacterized in most bacteria. We report the first characterization of the CFAS enzyme from Pseudomonas aeruginosa (PA) - an opportunistic human pathogen with complex membrane biology that is frequently associated with antimicrobial resistance and high tolerance to various external stressors. We demonstrate that CFAs are produced by a single enzyme in PA and that cfas gene expression is upregulated during the transition to stationary phase and in response to oxidative stress. Analysis of PA lipid extracts reveal a massive increase in CFA production as PA cells enter stationary phase and help define the optimal membrane composition for in vitro assays. The purified PA-CFAS enzyme forms a stable homodimer and preferentially modifies phosphatidylglycerol lipid substrates and membranes with a higher content of unsaturated acyl chains. Bioinformatic analysis across bacterial phyla shows highly divergent amino acid sequences within the lipid binding domain of CFAS enzymes, perhaps suggesting distinct membrane binding properties among different orthologues. This work lays an important foundation for further characterization of CFAS in Pseudomonas aeruginosa and for examining the functional differences between CFAS enzymes from different bacteria.

Evaluation of Quatsome Morphology, Composition, and Stability for Pseudomonas aeruginosa Biofilm Eradication.

Biofilm infections are a major cause of food poisoning and hospital-acquired infections. Quaternary ammonium compounds are a group of effective disinfectants widely used in industry and households, yet their efficacy is lessened when used as antibiofilm agents compared to that against planktonic bacteria. It is therefore necessary to identify alternative formulations of quaternary ammonium compounds to achieve an effective biofilm dispersal. Quaternary ammonium amphiphiles can form vesicular structures termed "quatsomes" in the presence of cholesterol. In addition to their intrinsic antimicrobial properties, quatsomes can also be used for the delivery of other types of antibiotics or biomarkers. In this study, quatsomes were prepared from binary mixtures of cholesterol and mono- or dialkyl-quaternary ammonium compounds; then, the integrity and stability of their vesicular structure were assessed and related to monomer chain number and chain length. The quatsomes were used to treat Pseudomonas aeruginosa biofilms, showing effective antibiofilm abilities comparable to those of their monomers. A systematic liquid chromatography-mass spectrometry method for quantifying quatsome vesicle components was also developed and used to establish the significance of cholesterol in the quatsome self-assembly processes.