Abstract
In order to study N-acyl homoserine lactone (AHL)-based quorum sensing in vivo, we present a protocol using an Escherichia coli strain equipped with a luxR-based monitor system, which in the presence of exogenous AHL molecules expresses a green fluorescent protein (GFP). Lungs from mice challenged intratracheally with alginate beads containing both a P. aeruginosa strain together with the E. coli monitor strain can be investigated at different time points postinfection. Epifluorescent or confocal scanning laser microscopy (CSLM) is used to detect the GFP-expressing E. coli monitor strain in the lung tissues, indicating production and excretion of AHLs in vivo by the infecting P. aeruginosa.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Costerton, J. W., Stewart, P. S. and Greenberg, E. P. (1999) Bacterial biofilms: a common cause of persistent infections, Science. 284, 1318–1322.
Høiby, N. (1974) Pseudomonas aeruginosa infection in cystic fibrosis. Relationship between mucoid strains of Pseudomonas aeruginosa and the humoral immune response, Acta Pathol Microbiol Scand B Microbiol Immunol. 82, 551–558.
Singh, P. K., Parsek, M. R., Greenberg, E. P. and Welsh, M. J. (2002) A component of innate immunity prevents bacterial biofilm development, Nature. 417, 552–555.
Bjarnsholt, T., Jensen, P. O., Burmølle, M., Hentzer, M., Haagensen, J. A., Hougen, H. P., et al. (2005) Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent, Microbiology. 151, 373–383.
Høiby, N., Krogh Johansen, H., Moser, C., Song, Z., Ciofu, O. and Kharazmi, A. (2001) Pseudomonas aeruginosa and the in vitro and in vivo biofilm mode of growth, Microbes Infect. 3, 23–35.
Pamp, S. J., Gjermansen, M., Johansen, H. K. and Tolker-Nielsen, T. (2008) Tolerance to the antimicrobial peptide colistin in Pseudomonas aeruginosa biofilms is linked to metabolically active cells, and depends on the pmr and mexAB-oprM genes, Mol Microbiol. 68, 223–240.
Passador, L., Cook, J. M., Gambello, M. J., Rust, L. and Iglewski, B. H. (1993) Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication, Science. 260, 1127–1130.
Pearson, J. P., Passador, L., Iglewski, B. H. and Greenberg, E. P. (1995) A second N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa, Proc Natl Acad Sci U S A. 92, 1490–1494.
Diggle, S. P., Winzer, K., Chhabra, S. R., Worrall, K. E., Cámara, M. and 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, 29–43.
Hentzer, M. and Givskov, M. (2003) Pharmacological inhibition of quorum sensing for the treatment of chronic bacterial infections, J Clin Invest. 112, 1300–1307.
Hentzer, M., Riedel, K., Rasmussen, T. B., Heydorn, A., Andersen, J. B., Parsek, M. R., et al. (2002) Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound, Microbiology. 148, 87–102.
Wu, H., Song, Z., Hentzer, M., Andersen, J. B., Heydorn, A., Mathee, K., et al. (2000) Detection of N-acylhomoserine lactones in lung tissues of mice infected with Pseudomonas aeruginosa, Microbiology. 146 (Pt 10), 2481–2493.
Wu, H., Song, Z., Hentzer, M., Andersen, J. B., Molin, S., Givskov, M., et al. (2004) Synthetic furanones inhibit quorum-sensing and enhance bacterial clearance in Pseudomonas aeruginosa lung infection in mice, J Antimicrob Chemother. 53, 1054–1061.
Andersen, J. B., Heydorn, A., Hentzer, M., Eberl, L., Geisenberger, O., Christensen, B. B., et al. (2001) gfp-based N-acyl homoserine-lactone sensor systems for detection of bacterial communication, Appl Environ Microbiol. 67, 575–585.
Andersen, J. B., Sternberg, C., Poulsen, L. K., Bjorn, S. P., Givskov, M. and Molin, S. (1998) New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria, Appl Environ Microbiol. 64, 2240–2246.
Hentzer, M., Wu, H., Andersen, J. B., Riedel, K., Rasmussen, T. B., Bagge, N., et al. (2003) Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors, EMBO J. 22, 3803–3815.
Kohler, T., van Delden, C., Curty, L. K., Hamzehpour, M. M. and Pechere, J. C. (2001) Overexpression of the MexEF-OprN multidrug efflux system affects cell-to-cell signaling in Pseudomonas aeruginosa, J Bacteriol. 183, 5213–5222.
van Gennip, M., Christensen, L. D., Alhede, M., Phipps, R., Jensen, P. Ø., Christophersen, L., et al. (2009) Inactivation of the rhlA gene in Pseudomonas aeruginosa prevents rhamnolipid production, disabling the protection against polymorphonuclear leukocytes, APMIS. 117, 537–546.
Pedersen, S. S., Shand, G. H., Hansen, B. L. and Hansen, G. N. (1990) Induction of experimental chronic Pseudomonas aeruginosa lung infection with P. aeruginosa entrapped in alginate microspheres, APMIS. 98, 203–211.
Bjarnsholt, T., van Gennip, M., Jakobsen, T. H., Christensen, L. D., Jensen, P. Ø. and Givskov, M. (2010) In vitro screens for quorum sensing inhibitors and in vivo confirmation of their effect, Nat Protoc. 5, 282–293.
Moser, C., Johansen, H. K., Song, Z., Hougen, H. P., Rygaard, J. and Høiby, N. (1997) Chronic Pseudomonas aeruginosa lung infection is more severe in Th2 responding BALB/c mice compared to Th1 responding C3H/HeN mice, APMIS. 105, 838–842.
Høiby, N. (1974) Epidemiological investigations of the respiratory tract bacteriology in patients with cystic fibrosis, Acta Pathol Microbiol Scand B Microbiol Immunol. 82, 541–550.
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C. (1994) Green fluorescent protein as a marker for gene expression, Science. 263, 802–805.
Cormack, B. P., Valdivia, R. H. and Falkow, S. (1996) FACS-optimized mutants of the green fluorescent protein (GFP), Gene. 173, 33–38.
Acknowledgment
We acknowledge the contributions made by Drs. Morten Hentzer, Hong Wu, Thomas Bovbjerg Rasmussen, Jens Bo Andersen, and Allan Bech Christensen with regard to the initial developments of the model systems.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Christensen, L.D., van Gennip, M., Jakobsen, T.H., Givskov, M., Bjarnsholt, T. (2011). Imaging N-Acyl Homoserine Lactone Quorum Sensing In Vivo. In: Rumbaugh, K. (eds) Quorum Sensing. Methods in Molecular Biology, vol 692. Humana Press. https://doi.org/10.1007/978-1-60761-971-0_11
Download citation
DOI: https://doi.org/10.1007/978-1-60761-971-0_11
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60761-970-3
Online ISBN: 978-1-60761-971-0
eBook Packages: Springer Protocols