A new assay for rhamnolipid detection—important virulence factors of Pseudomonas aeruginosa
- 1.1k Downloads
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.
- Alhede M, Bjarnsholt T, Jensen PO, Phipps RK, Moser C, Christophersen L, Christensen LD, van Gennip M, Parsek M, Hoiby N, Rasmussen TB, Givskov M (2009) Pseudomonas aeruginosa recognizes and responds aggressively to the presence of polymorphonuclear leukocytes. Microbiology 155(Pt 11):3500–8. doi: 10.1099/mic.0.031443-0 PubMedCrossRefGoogle Scholar
- Deziel E, Lepine F, Milot S, Villemur R (2003) rhlA is required for the production of a novel biosurfactant promoting swarming motility in Pseudomonas aeruginosa: 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs), the precursors of rhamnolipids. Microbiology 149(Pt 8):2005–13Google Scholar
- Diggle SP, Winzer K, Chhabra SR, Worrall KE, Camara M, Williams P (2003) The Pseudomonas aeruginosa quinolone signal molecule overcomes the cell density-dependency of the quorum sensing hierarchy, regulates rhl-dependent genes at the onset of stationary phase and can be produced in the absence of LasR. Mol Microbiol 50(1):29–43PubMedCrossRefGoogle Scholar
- Diggle SP, Matthijs S, Wright VJ, Fletcher MP, Chhabra SR, Lamont IL, Kong X, Hider RC, Cornelis P, Camara M, Williams P (2007) The Pseudomonas aeruginosa 4-quinolone signal molecules HHQ and PQS play multifunctional roles in quorum sensing and iron entrapment. Chem Biol 14(1):87–96. doi: 10.1016/j.chembiol.2006.11.014 PubMedCrossRefGoogle Scholar
- Hoffman LR, Kulasekara HD, Emerson J, Houston LS, Burns JL, Ramsey BW, Miller SI (2009) Pseudomonas aeruginosa lasR mutants are associated with cystic fibrosis lung disease progression. J Cyst Fibros 8(1):66–70. doi: 10.1016/j.jcf.2008.09.006
- Jensen PO, Bjarnsholt T, Phipps R, Rasmussen TB, Calum H, Christoffersen L, Moser C, Williams P, Pressler T, Givskov M, Hoiby N (2007) Rapid necrotic killing of polymorphonuclear leukocytes is caused by quorum-sensing-controlled production of rhamnolipid by Pseudomonas aeruginosa. Microbiology 153(Pt 5):1329–38. doi: 10.1099/mic.0.2006/003863-0 PubMedCrossRefGoogle Scholar
- Marshall SE, Hong SH, Thet NT, Jenkins AT (2013) Effect of lipid and fatty acid composition of phospholipid vesicles on long-term stability and their response to Staphylococcus aureus and Pseudomonas aeruginosa supernatants. Langmuir 29(23):6989–95. doi: 10.1021/la401679u PubMedCrossRefGoogle Scholar
- Sanchez M, Aranda FJ, Teruel JA, Espuny MJ, Marques A, Manresa A, Ortiz A (2010) Permeabilization of biological and artificial membranes by a bacterial dirhamnolipid produced by Pseudomonas aeruginosa. J Colloid Interface Sci 341(2):240–7. doi: 10.1016/j.jcis.2009.09.042 PubMedCrossRefGoogle Scholar
- Schertzer JW, Whiteley M (2012) A bilayer-couple model of bacterial outer membrane vesicle biogenesis. MBio 3(2) doi:10.1128/mBio.00297-11Google Scholar
- Smith EE, Buckley DG, Wu Z, Saenphimmachak C, Hoffman LR, D'Argenio DA, Miller SI, Ramsey BW, Speert DP, Moskowitz SM, Burns JL, Kaul R, Olson MV (2006) Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc Natl Acad Sci U S A 103(22):8487–92. doi: 10.1073/pnas.0602138103 PubMedCentralPubMedCrossRefGoogle Scholar