A multitask biosensor for micro-volumetric detection of N-3-oxo-dodecanoyl-homoserine lactone quorum sensing signal
Introduction
The Gram negative bacterium Pseudomonas aeruginosa is an important cause of infection, particularly in hospitals where it is responsible for about 10% of all nosocomial infections. Furthermore, P. aeruginosa chronic lung infection remains the major cause of morbidity and mortality among patients suffering from cystic fibrosis (CF), a genetic disease that affects about 1/2500 newborns in USA and Europe. P. aeruginosa infections are often recalcitrant to conventional antimicrobial chemotherapy since this bacterium is endowed with both innate and acquired resistance to many antibiotics, and adopts a biofilm mode of growth in vivo (Driscoll et al., 2007).
An appealing approach for identifying new drugs against P. aeruginosa is to search for inhibitors of virulence-related traits, as opposed to growth inhibitors per se. Selective targeting of the pathogenic potential of the bacterium by anti-virulence drugs would prevent or inhibit the establishment of the infection process, providing the host immune system with a better chance of clearing the infection (Cegelski et al., 2008, Rasko and Sperandio, 2010).
P. aeruginosa possesses a network of quorum sensing (QS) systems based on the production, secretion and perception of different signal molecules. The N-3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) is required for optimal production of other QS signals, namely N-butanoyl-homoserine lactone (C4-HSL) and 2-heptyl-3-hydroxy-4-quinolone (PQS) (Williams and Cámara, 2009).
The QS cascade functions in a way that when 3OC12-HSL signal molecule reaches a certain concentration in the P. aeruginosa growth environment, it binds and activates the LasR signal receptor protein, a transcriptional regulator that triggers the expression of hundreds of genes. These include, among others, the lasI gene encoding the 3OC12-HSL-producing synthase, the genes required for the synthesis of the other P. aeruginosa QS signals, and the genes for production of some virulence factors and biofilm formation (Williams and Cámara, 2009). A number of studies support the role of QS in P. aeruginosa infection and highlight its importance as a candidate for novel virulence-targeted antimicrobials (reviewed in Winstanley and Fothergill, 2009, Bjarnsholt and Givskov, 2007, Rasko and Sperandio, 2010). Therefore, search for QS inhibitors (QSI) to be used as lead compounds for the development of “non-antibiotic” drugs against P. aeruginosa has become an intensive field of industrial and academic research (Kjelleberg et al., 2008, Wang et al., 2008, Pan and Ren, 2009 and references therein).
Several reporter strains have been constructed, endowed with different specificity and sensitivity for QS signal molecules produced by different bacterial species. These biosensors consist of a reporter gene expressed under the control of a QS-dependent promoter, introduced into a strain unable to produce QS signal molecules (e.g., Escherichia coli or a P. aeruginosa QS mutant) but expressing the signal receptor specific for that promoter (e.g., LasR or its homologs). The presence of the specific QS signal molecule is recognized by the cognate signal receptor which triggers the expression of the reporter gene. Biosensors of this kind have been constructed primarily for the non-quantitative detection of different QS signal molecules produced by bacteria, but some of them have occasionally been employed for the quantification of QS signals in laboratory cultures and/or clinical samples (reviewed in Steindler and Venturi, 2007), or for the screening of QSI. A QSI screening procedure is generally carried out by detecting a reduction of the biosensor response to exogenously added synthetic signal molecule in the presence of a putative inhibitory compound. These QSI screening procedures have proven to be useful for the identification of inhibitors of signal receptor activity. However, since the signal molecule is exogenously provided, this approach is not suited to identify inhibitors of the synthesis and export of the signal molecule or of other key processes related to QS (reviewed in Wang et al., 2008, Kjelleberg et al., 2008).
Few biosensors based on the P. aeruginosa LasR/LasI system have been generated so far, all showing low sensitivity (i.e., high detection limit) and thus requiring solvent extraction to concentrate the signal molecule before testing (Pearson et al., 1994, Riedel et al., 2001, Lee et al., 2006). One exception is a biosensor generated in E. coli able to detect 3OC12-HSL levels in the pM range (Winson et al., 1998), which however cannot be employed to identify inhibitors of specific P. aeruginosa QS-related processes other than signal-mediated activation of LasR.
On this basis, we have undertaken a study aimed at the construction of a simple multi-task biosensor suitable for the direct detection of 3OC12-HSL levels in both laboratory and clinical samples, and for the high-throughput screening of molecules targeting the P. aeruginosa 3OC12-HSL-dependent QS. To our knowledge, the screening method based on this newly developed biosensor represents the first QSI screening system suitable for the identification of inhibitors of any process related to 3OC12-HSL-dependent QS, including LasR activity as well as 3OC12-HSL synthesis and export/import across the cell envelope.
Section snippets
Bacterial strains, growth media and reagents
Bacterial strains and plasmids used in this study are listed in Table S1. E. coli and P. aeruginosa cultures were grown in Luria–Bertani broth (LB; Sambrook et al., 1989), LB supplemented with 1.5% agar (LA) or Pseudomonas Isolation Agar (PIA). Where required, antibiotics were used at the following concentrations: ampicillin (Ap, 100 μg/ml), kanamycin (Km, 50 μg/ml), tetracycline (Tc, 12.5 μg/ml), nalidixic acid (Nal, 20 μg/ml), gentamycin (Gm, 10 μg/ml) for E. coli; Gm (200 μg/ml), carbenicillin
Construction of reporter strains for the detection of 3OC12-HSL
A transcriptional fusion between the LasR-dependent rsaL promoter (PrsaL) and the luxCDABE operon was constructed and evaluated as reporter system for 3OC12-HSL quantification. PrsaL was selected because this promoter is directly activated by LasR and several hundred-fold inducible in the presence of 3OC12-HSL (Rampioni et al., 2007, Whiteley and Greenberg, 2001, Schuster et al., 2003), whereas the luxCDABE operon is a convenient reporter system that has no background in P. aeruginosa and
Concluding remarks
The PA14-R3 biosensor provides direct quantification of 3OC12-HSL over a wide range of concentration (from pM to μM) in micro-volumes of laboratory or clinical samples. Since PA14-R3 can be used in microtiter plates and results are made available in approximately 4 h, it looks well suited for high-throughput studies. For instance, we showed that it can be successfully used for studying mutants with altered 3OC12-HSL production or for determining 3OC12-HSL concentration in sputum samples from CF
Acknowledgements
We thank Professor L.G. Rahme for the PA14 and PA14lasI strains, Professor M.G. Surette for the pMS402 and pDK201 plasmids, and Professors N. Kumar and M. Givskov for the FC30 compound. This work was supported by grants from the Ministry of University and Research of Italy (PRIN-2008) to P.V. and from the Italian Cystic Fibrosis Research Foundation to L.L. (Grant FFC#8/2008, with the contribution of FFC delegations from Latina, Catania, Ragusa-Vittoria, Paternò).
References (35)
- et al.
FEMS Microbiol. Lett.
(2005) - et al.
Plasmid
(2000) - et al.
J. Cystic Fibrosis
(2009) - et al.
FEMS Microbiol. Lett.
(2002) - et al.
Int. J. Med. Microbiol.
(2006) - et al.
Res. Microbiol.
(2004) - et al.
Curr. Opin. Microbiol.
(2009) - et al.
FEMS Microbiol. Lett.
(1998) - et al.
Philos. Trans. R. Soc. Lond. B: Biol. Sci.
(2007) - et al.
Nat. Rev. Microbiol.
(2008)
Proc. Natl. Acad. Sci. U. S. A.
Drugs
J. Bacteriol.
Mol. Microbiol.
Infect. Immun.
Br. Med. Bull.
Future Microbiol.
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Present address: Department of Biology and Biotechnology Charles Darwin, University of Rome Sapienza, Via dei Sardi 70, 00185 Rome, Italy.