(seeFig. 1). During growth of the bacteria the AHLs, produced by
the I synthetase, accumulate in the medium until a critical threshold
concentration is reached. At this concentration the R protein forms
a multimeric complex with the AHLs. This complex exhibits DNA
binding properties and is believed to interact with specific recogni-
tion sequences positioned in the promoter regions of QS regulated
target genes. Binding can either (dependent on the genes) upregu-
late or downregulate transcription initiation (seereview [10]).
The ability to monitor the production of AHLs by pathogenic
Gram-negative bacteria during infection can contribute to the
understanding of the host–pathogen interaction. In principle, the
presence of exogenous AHLs can be detected by a reporter gene
fused to any QS target gene maintained in a bacterial background
devoid of the signal generator (I). We have previously designed a
sensitive green fluorescent protein (GFP) reporter, which enables
visualization of cell–cell communication at the single-cell level and
it has been used to detect in vitro and in vivo production of AHLs
byP. aeruginosa[11–16].2 Materials
2.1 Bacterial Strains
and Growth Media
- Any AHL producingP. aeruginosastrain can be used, we useP.
aeruginosa(PAO1) originally obtained from Professor Barbara
Iglewski (University of Rochester Medical Center, NY, USA).
This strain is well tolerated by the mice, it is QS-proficient,
however it features a reduced production of C 4 -HSL [17],
resulting in lowered rhamnolipid production, which we have
shown to be extremely toxic for the mice [18]. After choosing a
bacterial strain, a dose–response experiment must be con-
ducted to optimize the inoculum since the virulence of bacte-
rial strains significantly affects the load of bacteria that can get
instilled in the lungs without leading to mortality. It must be
emphasized that our end point of the mouse model is not death
but visualization of QS or gradual eradication of the bacteria.
Fig. 1The basics of AHL regulation. The AHL regulatory components are
encoded by anRgene and anIgene. The gene products of these two genes
are the regulator (R protein) and the signal synthetase (I protein), respectively204 Louise Dahl Hultqvist et al.