1 Center for Biomedical Microbiology, BioCentrum-DTU, Building 301, Technical University of Denmark, DK-2800 Lyngby, Denmark
2 Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark
3 Department of Natural Sciences, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
4 School of Biotechnology and Biomolecular Science and Centre for Marine Biofouling and Bio-innovation, Biological Science Building, University of New South Wales, Randwick, Sydney, NSW 2052, Australia
Correspondence
Michael Givskov
immg{at}pop.dtu.dk
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ABSTRACT |
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A table with the sequences of the mutagenic oligonucleotides is available as supplementary material with the online version of this paper.
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INTRODUCTION |
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A number of studies have been carried out with mutated forms of LuxR-type proteins (Chai & Winans, 2004; Choi & Greenberg, 1991
; Hanzelka & Greenberg, 1995
; Kiratisin et al., 2002
; Lamb et al., 2003
; Luo et al., 2003a
; Shadel et al., 1990
; Slock et al., 1990
), and the crystal structure of the LuxR homologue TraR of Agrobacterium tumefaciens in complex with N-3-oxooctanoyl-L-homoserine lactone (OOHL) and DNA has been solved (Vannini et al., 2002
; Zhang et al., 2002
). This has led to the general assumption that the N-terminal domain of LuxR-type proteins binds the signal molecule (Hanzelka & Greenberg, 1995
; Kiratisin et al., 2002
; Lamb et al., 2003
; Vannini et al., 2002
; Zhang et al., 2002
), while the C-terminal domain is involved in DNA binding (Choi & Greenberg, 1991
; Kiratisin et al., 2002
; Lamb et al., 2003
; Vannini et al., 2002
; Zhang et al., 2002
). Despite sequence similarity in the signal-binding region, biochemical evidence suggests that the various LuxR homologues exhibit differences in autoinducer accessibility. In TraR, the signal molecule is completely embedded into a narrow cavity devoid of solvent contact (Vannini et al., 2002
; Zhang et al., 2002
). This is in accordance with the observations that removal of OOHL from the TraR-OOHL complex requires extensive dialysis in the presence of detergent (Zhu & Winans, 1999
), and that TraR activated by OOHL or N-3-oxohexanoyl-L-homoserine lactone (OHHL) remains active for as long as 8 h after removal of exogenous signal (Luo et al., 2003b
). In contrast, experiments with the purified LuxR protein show that the LuxR-OHHL complex can be reversibly inactivated by dilution, suggesting a weaker binding of the signal molecule (Urbanowski et al., 2004
). The LuxR homologue EsaR of Pantoea stewartii functions as a repressor, and the addition of signal triggers the release of the protein from DNA (Minogue et al., 2002
). The LuxR homologue RhlR of Pseudomonas aeruginosa has recently been reported to bind to the rhlAB las box both in the absence and in the presence of the AHL signal serving as a repressor and an activator, respectively (Medina et al., 2003
). The receptivity of these apo-proteins to their signal molecules indicates that their signal binding site must be highly accessible.
In most cases, quorum-sensing systems control the expression of virulence genes and biofilm development, traits that are important for the interaction with and colonization of eukaryotic hosts (Smith & Iglewski, 2003). This includes the regulation of virulence factors in human pathogens such as P. aeruginosa (Hentzer et al., 2003
; Wagner et al., 2003
; Schuster et al., 2003
) and Yersinia pseudotuberculosis (Atkinson et al., 1999
), factors involved in food spoiling mediated by serratia strains (Christensen et al., 2003
) and virulence factors of plant-pathogenic bacteria such as Erwinia carotovora (Whitehead et al., 2002
).
As interference with AHL-mediated gene expression offers an opportunity to control unwanted microbial activity without the use of growth-inhibitory agents, which select for resistant organisms, much work has been carried out to identify synthetic analogues of AHLs that function as AHL antagonists (Smith et al., 2003; Olsen et al., 2002
). In nature, algae and higher plants have been found to produce substances that inhibit AHL-regulated behaviour (Givskov et al., 1996
; Bauer & Robinson, 2002
). While the compounds responsible for this activity in higher plants have not yet been identified (Bauer & Robinson, 2002
), several halogenated furanones that inhibit AHL-controlled processes have been isolated from the marine red alga Delisea pulchra (de Nys et al., 1993
). Regarding the mechanism of action, several data sets show that halogenated furanones interact specifically with LuxR from Vibrio fischeri (Manefield et al., 1999
, 2002
).
In the present report, we characterize the influence of selected amino acid substitutions on the interaction between LuxR, signal molecules, signal antagonists and quorum-sensing inhibitors. Based on this, we propose a model for the binding modes of the molecules in the LuxR receptor site.
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METHODS |
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Mutagenesis and plasmid construction.
Recent data indicate that the translation of LuxR is initiated at an ATG codon situated 6 bp upstream of the originally proposed start codon (Urbanowski et al., 2004). To facilitate the comparison with previously published LuxR mutants we have decided to keep the previous numbering of the residues. Site-directed mutations were introduced into the luxR gene from V. fischeri encoded by the plasmid pJBA89 (Andersen et al., 2001
). All mutations were created by overlap extension PCR (Ho et al., 1989
). The sequences of the mutagenic oligonucleotides are shown in the Supplementary Table available with the online journal. PCR was performed with the Expand High Fidelity PCR system (Roche Diagnostics). Except for the PCR products used to create the mutants pJBA89L42A and pJBA89L42S, the overlap extension products were digested with EcoRI and XbaI. The resulting 585 bp fragments were cloned into pJBA89 digested with EcoRI and XbaI, using standard cloning techniques (Sambrook et al., 1989
). The overlap extension products used to create pJBA89L42A and pJBA89L42S were digested with EcoRI and KpnI. The resulting 996 bp fragments were cloned into pJBA89 digested with EcoRI and KpnI. All mutations were verified by sequencing, which was carried out by GATC Biotech. Primers for sequencing and PCR were obtained from TAG Copenhagen.
Determination of green fluorescent protein (GFP) expression.
The LuxR-controlled green-fluorescent-AHL monitor strain E. coli XL1-Blue(pJBA89), which carries luxR+ and a PluxI-gfp(ASV) fusion (Andersen et al., 2001), or the strain containing a selection of pJBA89 derivative plasmids with mutations in luxR, was cultured in ABT minimal medium. To obtain exponentially growing cultures minimal ABT medium was inoculated from overnight cultures to an initial OD600 of 0·05 and grown for 45 h at 30 °C.
For GFP measurements, aliquots of exponential phase E. coli XL1-Blue cultures carrying pJBA89 with or without mutations in luxR were distributed to the wells of microtitre dishes in which different AHLs, AHL analogues or OHHL in combination with quorum-sensing inhibitors at the required concentrations were already present. Every 15 min for 2 h, the relative units of fluorescence (RFU) of each sample were measured with a Wallac Victor2, 1420 Multilabel Counter using a 485 nm excitation filter and a 535 nm emission filter. The OD450 was also measured for each sample in order to determine cell-specific fluorescence. This cell-specific fluorescence was plotted versus time, and the linear part of the curve was used for linear regression to obtain the change in cell-specific GFP expression versus time. All measurements were repeated at least twice.
Antagonist preparation.
The halogenated furanones C-2 and C-56 were synthesized as previously described (Manny et al., 1997). The halogenated furanone C-4 was extracted from D. pulchra and purified by HPLC according to protocols established by de Nys et al. (1993)
. The halogenated furanone C-30 was synthesized according to protocols reported by Wells (1963)
. N-(propylsulfanylacetyl)-L-homoserine lactone (pros-ahl) and n-(pentylsulfanylacetyl)-L-homoserine lactone (PenS-AHL) were synthesized as described previously by Persson et al. (2005)
.
Western blotting.
In order to measure GFP expression and perform Western blotting under identical conditions, we originally grew our E. coli XL1-Blue(pJBA89) cultures in ABT minimal medium at 30 °C. Unfortunately very low levels of LuxR were produced in this medium compared to growth in LB broth, as judged by Western blotting (data not shown). Use of LB caused a higher background than use of ABT minimal medium in our GFP measurements (data not shown). There was significantly more LuxR protein present in cells grown at 37 °C compared to cells grown at 30 °C. For Western blotting of the LuxR proteins we therefore used either the E. coli XL1-Blue cells harbouring pJBA89 with or without luxR mutations grown in LB at 37 °C to OD600 11·5, or E. coli XL1-Blue cells carrying the Ptac-luxR expression plasmid pHK724 (Kaplan & Greenberg, 1987) and pGroESL (Goloubinoff et al., 1989
) grown in LB at 37 °C to OD600 0·30·4 in the presence of 100 µM of the inducer IPTG. Based on four Western blots of proteins from cells with pJBA89 with or without luxR mutations, and numerous Western blots with E. coli XL1-Blue(pHK724)(pGroESL), we concluded that the highest reproducibility was obtained with E. coli XL1-Blue(pHK724)(pGroESL). This strain was therefore selected for quantitative measurements.
Cells carrying pHK724 and pGroESL were harvested by centrifugation at 10 000 g, at 4 °C for 5 min, washed once and then resuspended in an equal volume of fresh medium. Various concentrations of halogenated furanones and/or OHHL were added to aliquots of washed cells resuspended in fresh medium, and the samples were incubated at 37 °C for 0·5 or 1 h. After the treatment, samples were frozen immediately and kept at 20 °C. Samples of E. coli XL1-Blue cells carrying pJBA89, or pJBA89 derivatives with mutations in luxR, were taken directly from growing cultures and frozen. Prior to SDS-PAGE, the samples were adjusted to similar OD600 values. Western blots of the separated proteins were incubated with an anti-LuxR antibody from Quorum Sciences as the primary antibody, and an anti-rabbit horseradish-peroxidase-conjugated antibody from Amersham as the secondary antibody, according to the manufacturer's recommendations. Localization of the secondary antibody was visualized using chemiluminescent detection reagents from Amersham and a Hamamatsu C2400-47 double intensified CCD camera. Colour images were saved in 16-bit format (the scale has a resolution of 16 colours) using ARGUS-50 software (Hamamatsu).
Molecular modelling.
Our model of the AHL binding site in LuxR is based on the crystal structure of TraR from A. tumefaciens in complex with OOHL and DNA (PDB code 1L3L) (Zhang et al., 2002). Due to the low overall homology of TraR and LuxR, the modelling was restricted to the binding-site region defined by a sphere with a radius of 12 Å centred on the 3-carbonyl group of OOHL. The amino acid residues in TraR that are different from those of LuxR, according to the alignment by Whitehead et al. (2001)
, were replaced by the corresponding ones of LuxR using the program InsightII (Accelrys). The rotamer option in InsightII was then used to find the energetically most favourable conformations of the side chains. The calculations of the size, shape and the burial extent of the binding sites of TraR and LuxR were performed with the program PASS (Putative Active Sites with Spheres) developed by Brady & Stouten (2000)
.
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RESULTS AND DISCUSSION |
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In order to select residues for site-directed mutagenesis, furanone C-30 was superimposed on OOHL bound to TraR by molecular modelling, and amino residues situated close to C-30 and/or OOHL were identified (Fig. 2). The residues Y53, W57 and D70 of TraR form hydrogen bonds to the 1-carbonyl group, the ring carbonyl group and the NH group of OOHL, respectively (Zhang et al., 2002
). These three residues are highly conserved in LuxR-type proteins (Table 1
). According to this putative model of furanone binding, the TraR residue W57 would be expected to form hydrogen bonds to the ring carbonyl group of C-30, while the TraR residues D70 and Y53 cannot since halogenated furanones lack the NH group and the 1-carbonyl group present in AHLs (Fig. 1
). Since we used the TraRfuranone interaction as a model for the LuxRfuranone interaction, the residues in LuxR corresponding to the above-mentioned TraR residues were identified and subjected to site-directed mutagenesis. The LuxR Y62 residue (corresponding to Y53 in TraR) was changed to phenylalanine, which structurally resembles tyrosine but is unable to form hydrogen bonds. The W66 residue (corresponding to W57 in TraR) was changed to histidine, while D79 (corresponding to D70 in TraR) was changed to asparagine, changes which do not abolish the ability to form hydrogen bonds.
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These results are hard to explain by assuming the orientation of the OHHL signal in the binding pocket of LuxR resembles the OOHL orientation in TraR. F101 in TraR (corresponding to V109 in LuxR) and Y102 in TraR (corresponding to I110 in LuxR) are distantly situated from the acyl side chain of the signal molecule in the TraR/OOHL complex. Furthermore, a Y53F mutation in TraR resulted in an almost inactive protein (Chai & Winans, 2004), while the Y62F mutant of LuxR could be activated by all the tested signal molecules except BHL. The presence of the bulky side chain of L42 in LuxR (Fig. 6
) (corresponding to A38 in TraR) also argues against a similar positioning of the OHHL side chain in LuxR and the OOHL side chain in TraR, since the leucine residue at this position in LuxR would cause a reduction in the space available for the acyl side chain. The importance of a small amino acid residue at this position in TraR is highlighted by the observation that an A38V mutant of TraR was severely affected in signal-binding ability (Luo et al., 2003a
).
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This model can also explain the observed differences between the LuxR and TraR mutant proteins. Y62 forms a hydrogen bond to the ring carbonyl group, as does W57 of TraR (Zhang et al., 2002; Vannini et al., 2002
). The TraR W57Y mutant shows activation patterns similar to the wild-type protein, while the Y53F mutant remains inactive (Chai & Winans, 2004
). The corresponding LuxR mutants showed the opposite behaviour. The W66H mutant was inactive while the Y62F mutant could be fully activated although it required much higher signal concentrations than the wild-type. This difference may be caused by the significantly stronger hydrogen bond accepting capacity of the carbonyl oxygen present in the amide group, compared to a carbonyl oxygen of an ester group. Thus, the loss or change of hydrogen bond donation to an amide carbonyl oxygen, as in the LuxR mutant W66H and the TraR mutant Y53F, might be expected to affect the ligand binding more seriously and thereby cause inactivation of the protein.
A38 of TraR is situated near the acyl chain of the signal molecule (Zhang et al., 2002; Vannini et al., 2002
), while the corresponding LuxR residue L42 in our model is closer to the homoserine lactone moiety of the signal molecule (Fig. 6
). To further test the model, the mutants L42A and L42S were constructed. L42A was fully activated by a 15-fold higher OHHL concentration than the wild-type LuxR (Fig. 4
), while L42S required 1000-fold higher concentrations. Similar results were obtained for OOHL activation. For OHL and HHL, equal or slightly lower concentrations were required for activation of L42A, while L42S required 100500-fold higher concentrations than the wild-type (Fig. 4
). In comparison, the A38V mutation of TraR led to more pronounced effects: the mutant protein responded to OOHL at concentrations similar to the wild-type but could only attain half the activity of the wild-type protein, and was substantially less sensitive to OHL and OHHL indicating a role of the AHL side chain in the activity of this mutant (Luo et al., 2003a
). A remarkable difference between L42A and L42S mutants and the wild-type was observed with BHL (Fig. 4
), which activated L42A and L42S. The short hydrocarbon chain of BHL makes it less probable that this compound can bind to LuxR in the mode that we have proposed for OHHL and OOHL (Fig. 6
), due to lack of hydrophobic interaction between BHL and V109/I110. Thus, BHL most probably binds in a mode similar to that of OOHL in TraR (Fig. 6
). This makes BHL binding to the wild-type protein weaker due to steric repulsive interactions with L42. The L42A and L42S mutations should relieve these steric repulsions due to the significantly smaller size of alanine and serine compared to that of leucine (Fig. 7
). Our proposed binding mode for BHL is also supported by the observation that BHL, in contrast to all the other tested signal molecules, cannot activate the Y62F mutant (Fig. 4
). As mentioned above, the corresponding Y53F mutant in TraR is almost inactive.
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Interactions between signal analogues, LuxR and LuxR mutants
Much of the effort directed towards construction of quorum-sensing inhibitors has focused on the synthesis of compounds that resemble the signal molecules and can serve as competitive inhibitors (Smith et al., 2003; Olsen et al., 2002
; Persson et al., 2005
). The compounds ProS-AHL and PenS-AHL (Fig. 1
) differ from OHHL and OOHL, respectively, in the substitution of a carbonyl group with a sulfur atom, and differ from HHL and OHL by the replacement of a CH2 group with a sulfur atom. The significantly smaller size of a sulfur atom compared to a carbonyl group, the different geometries of the C-S-C and C-C-C moieties, and the higher degree of polarization of a sulfur atom compared to a CH2 group influence the steric properties and thereby the ability of the molecules to fit into the signal-binding cavity. This might explain the ability of the sulfur analogues to serve as quorum-sensing inhibitors.
To determine the ability of ProS-AHL and PenS-AHL to inhibit LuxR-controlled GFP expression, cells carrying pJBA89 were incubated with a saturating amount of OHHL (i.e. 5 nM) and varying concentrations of ProS-AHL or PenS-AHL. Full inhibition of GFP expression was obtained with approximately 10 µM ProS-AHL (Fig. 8a), while approximately 100 µM PenS-AHL was needed to obtain full inhibition (data not shown). To examine the agonist activity at high concentrations, cells carrying pJBA89 were incubated with varying amounts of ProS-AHL. At high concentrations, addition of ProS-AHL stimulated a low level of GFP expression (Fig. 8b
). To determine the extent to which the introduced mutations in LuxR influenced the antagonist and agonist activity of ProS-AHL, a selection of LuxR mutants (which could be activated by OHHL) were incubated with ProS-AHL in the absence or presence of saturating amounts of OHHL (i.e. 5 nM for pJBA89, 75 nM for pJBA89L42A, 5 µM for pJBA89L42S and pJBA89M135A, 30 µM for pJBA89Y62F and 60 µM for pJBA89V109T and pJBA89I110T). Changes in GFP expression per unit time were recorded. In the antagonist assay (Fig. 8a
) expression of GFP in the presence of only OHHL was arbitrarily defined as 100 % activity. As shown in Fig. 8(a)
, all six mutations rendered the protein insensitive to inhibition by ProS-AHL, indicating that the mutations have a larger impact on the binding of ProS-AHL than on the binding of OHHL. For Y62F, V109T, I110T and M135A the agonist activity of ProS-AHL was less than for the wild-type (Fig. 8b
). In contrast, ProS-AHL served efficiently as an agonist for L42A and L42S (Fig. 8b
). Similarly to BHL, we propose that the L42A and L42S mutations enable ProS-AHL to bind to LuxR in an OOHL-TraR-like mode (Fig. 7
).
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Using the LuxR-based AHL monitor strain E. coli MT102(pSB403) it was shown that inhibition by 100 µM C-2 could be relieved by addition of 100 nM OHHL while the inhibition by 50 µM C-4 could not (Manefield et al., 1999). To re-examine the AHL-offered protection, we carried out a series of experiments with E. coli XL1-Blue(pJBA89) in which we investigated whether a surplus of OHHL would allow LuxR to activate PluxI in the presence of furanone compounds. In all cases, addition of OHHL had an effect. However, while all of the inhibition exerted by C-2 treatment and most of the inhibition exerted by C-4 treatment could be reversed by the addition of 50 nM OHHL, C-56 and C-30 required concentrations in the micromolar range (Fig. 10
). Furthermore, while full protection could be achieved against C-2 (50 nM OHHL against 250 µM C-2), only approximately 50 % protection could be obtained for C-30 (25 µM OHHL against 3 µM C-30).
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Perspective
The development of bacterial resistance to antibiotics is a serious side-effect of current anti-microbial treatments. The alternative to antibiotic-mediated killing or growth inhibition is to attenuate bacterial virulence such that the organism fails to establish a successful infection, and as a consequence is cleared by the host immune response. Specific targets for future anti-microbial drugs are likely to be key regulatory functions governing control over bacterial virulence, surface colonization and biofilm development. A major advantage of this strategy is that it most likely circumvents the problem of resistance, which is intimately connected to the use of conventional anti-bacterial agents. Examples of this are quorum-sensing inhibitors, which in contrast to the traditional anti-microbial agents can be used in low, sub-growth-inhibitory doses (Hentzer et al., 2003). In the present study we have performed site-directed mutagenesis of the signal binding site of LuxR to gain insight into the mechanism of inhibition and particularly the development of resistance towards quorum-sensing inhibitors. This analysis illustrates the lack of X-ray crystallographic data to support positioning of the OHHL signal molecule in the LuxR-binding cavity. Until that is achievable, we propose a model that flips the acyl side chain in the LuxR/signal molecule complex compared to the TraR/signal molecule complex.
Interestingly, point mutations in the signal binding site rendered LuxR insensitive to the activity of the synthetic inhibitor ProS-AHL. Are such mutant proteins also insensitive to OHHL activation and are cells harbouring such quorum sensors unable to signal each other? Not necessarily, one of these mutants could be activated in the presence of a 15-fold higher concentration of OHHL (compared to the concentration required to fully activate the wild-type protein). Although there is no selective pressure imposed by the inhibitors per se, it is conceivable that pathogenic bacteria in the long run might develop resistance to quorum-sensing inhibitors that are based on agonist structure. In contrast, our furanone analysis suggests that through time inhibitors have been selected in nature where single amino acid changes in a separated receptor site leading to resistance are less likely to occur. This highlights the importance of screening natural libraries for identification of quorum-sensing inhibitors, which might provide scaffolds for the development of powerful synthetic anti-microbial drugs.
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ACKNOWLEDGEMENTS |
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Received 9 February 2005;
revised 20 May 2005;
accepted 8 July 2005.
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