A new assay for rhamnolipid detection—important virulence factors of Pseudomonas aeruginosa
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Rhamnolipids (RLs) are heterogeneous glycolipid molecules that are composed of one or two l-rhamnose sugars and one or two β-hydroxy fatty acids, which can vary in their length and branch size. They are biosurfactants, predominantly produced by Pseudomonas aeruginosa and are important virulence factors, playing a major role in P. aeruginosa pathogenesis. Therefore, a fast, accurate and high-throughput method of detecting such molecules is of real importance. Here, we illustrate the ability to detect RL-producing P. aeruginosa strains with high sensitivity, based on an assay involving phospholipid vesicles encapsulated with a fluorescent dye. This vesicle-lysis assay is confirmed to be solely sensitive to RLs. We illustrate a half maximum concentration for vesicle lysis (EC50) of 40 μM (23.2 μg/mL) using pure commercial RLs and highlight the ability to semi-quantify RLs directly from the culture supernatant, requiring no extra extraction or processing steps or technical expertise. We show that this method is consistent with results from thin-layer chromatography detection and dry weight analysis of RLs but find that the widely used orcinol colorimetric test significantly underestimated RL quantity. Finally, we apply this methodology to compare RL production among strains isolated from either chronic or acute infections. We confirm a positive association between RL production and acute infection isolates (p = 0.0008), highlighting the role of RLs in certain infections.
KeywordsRhamnolipids Pseudomonas aeruginosa Lipid vesicles Detection
We would like to thank Prof. Mark C. Enright (University of Bath) and Southmead Hospital (Bristol, UK) for clinical bacterial strains, Dr. Stephan Heeb (University of Nottingham, UK) for the PAO1 quorum and rhlA mutant strains and Dr. Mathew J. Wargo (University of Vermont, USA) for the PAO1ΔplcH mutant strain. We also acknowledge the European Commission’s Seventh Framework Programme for funding via the EC-FP7 consortium project no.245500 Bacteriosafe and the Royal Society and NERC grant ref: NE/J007064/1.
Conflict of interest
The authors declare that they have no conflict of interest.
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