Discovery of antagonists of PqsR, a key player in 2-alkyl-4-quinolone- dependent quorum sensing in Pseudomonas aeruginosa

Article abstract of DOI:10.1016/j.chembiol.2012.01.015

The pqs quorum sensing communication system of Pseudomonas aeruginosa controls virulence factor production and is involved in biofilm formation, therefore playing an important role for pathogenicity. In order to attenuate P. aeruginosa pathogenicity, we followed a ligand-based drug design approach and synthesized a series of compounds targeting PqsR, the receptor of the pqs system. In vitro evaluation using a reporter gene assay in Escherichia coli led to the discovery of the first competitive PqsR antagonists, which are highly potent (K_d,app of compound 20: 7 nM). These antagonists are able to reduce the production of the virulence factor pyocyanin in P. aeruginosa. Our finding offers insights into the ligand-receptor interaction of PqsR and provides a promising starting point for further drug design.

Full text of DOI:10.1016/j.chembiol.2012.01.015

Chemistry & Biology
Article
Discovery of Antagonists of PqsR, a Key Player
in 2-Alkyl-4-quinolone-Dependent Quorum Sensing
in Pseudomonas aeruginosa
Cenbin Lu,1 Benjamin Kirsch,1 Christina Zimmer,1 Johannes C. de Jong,1 Claudia Henn,1 Christine K. Maurer,1
2
1,4,
Mathias Mu¨ sken, Susanne Haussler,2,3 Anke Steinbach,1, and Rolf W. Hartmann
*
*
¨
1Helmholtz Institute for Pharmaceutical Research Saarland, Campus C2.3, 66123 Saarbru¨ cken, Germany
2Twincore, Department of Pathophysiology of Bacterial Biofilms, Feodor-Lynen-Strasse 7, 30625 Hannover, Germany
3Helmholtz Centre for Infection Research, Chronic Pseudomonas Infection Research Group, Inhoffenstrasse 7, 38124 Braunschweig,
Germany
4Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, 66123 Saarbru¨ cken, Germany
*Correspondence: anke.steinbach@helmholtz-hzi.de (A.S.), rolf.hartmann@helmholtz-hzi.de (R.W.H.)
DOI 10.1016/j.chembiol.2012.01.015
SUMMARY
tion system known as quorum sensing (QS; Swift et al., 2001).
QS functions by secreting and sensing of signaling molecules
The pqs quorum sensing communication system of called autoinducers (AIs). Once at a high cell density, the AIs
reach a threshold concentration in the culture and interact with
specific receptors to regulate their target gene expression.
P. aeruginosa uses two lux-type QS systems known as las
Pseudomonas aeruginosa controls virulence factor
production and is involved in biofilm formation,
therefore playing an important role for pathogenicity.
(Gambello and Iglewski, 1991; Passador et al., 1993) and rhl
In order to attenuate P. aeruginosa pathogenicity, we
(Ochsner et al., 1994; Ochsner and Reiser, 1995). The synthase
LasI produces the molecule 3-oxo-C12-HSL (N-3-oxododeca-
followed a ligand-based drug design approach and
synthesized a series of compounds targeting PqsR,
the receptor of the pqs system. In vitro evaluation
using a reporter gene assay in Escherichia coli led
to the discovery of the first competitive PqsR antag-
onists, which are highly potent (Kd,app of compound
noyl-L-homoserine lactone), which activates the receptor
LasR. Similarly, RhlI produces C4-HSL (N-butanoyl-L-homoser-
ine lactone), which stimulates RhlR. While both homoserine-
mediated QS systems are broadly applied by various bacteria,
2-alkyl-4-quinolone-dependent QS occurs in Pseudomonas
20: 7 nM). These antagonists are able to reduce the (Pesci et al., 1999) and in Burkholderia species (Diggle et al.,
2006), whereas only P. aeruginosa produces Pseudomonas qui-
production of the virulence factor pyocyanin in
nolone signal (PQS). PQS and, to a lesser extent, its precursor
2-heptyl-4-hydroxyquinoline (HHQ; Figure 1)—the two most
predominant members of the 2-alkyl-4-hydroxyquinoline (HAQ)
P. aeruginosa. Our finding offers insights into the
ligand-receptor interaction of PqsR and provides
a promising starting point for further drug design.
´
family (Deziel et al., 2004)—activate PqsR (synonym MvfR:
multiple virulence factor regulator; Cao et al., 2001; Xiao et al.,
2006), a LysR-type transcriptional regulator (Maddocks and
Oyston, 2008) that drives the coordinated expression of nearly
INTRODUCTION
Pseudomonas aeruginosa is an opportunistic pathogen that 200 genes. Many of these genes are related to virulence factors,
causes life-threatening nosocomial infections and is a major such as phzA1-G1, which are involved in the biosynthesis of pyo-
problem for cystic fibrosis (CF) patients that leads to inflamma- cyanin; hcnAB, which is responsible for production of hydrogen
tion and chronic persistent lung infections. cyanide; lasB, which encodes elastase B; rhlAB, which is
It is responsible for 90% of chronic lung infections of CF involved in biosynthesis of rhamnolipids; and lecA, which codes
´
patients (Koch and Høiby, 1993) and is considered as the major for Lectin A (Cao et al., 2001; Deziel et al., 2005). Besides viru-
cause of mortality (Govan and Deretic, 1996). Its eradication is lence factors, the biofilm formation is also controlled by the
very difficult because it forms microcolonies encased with exo- pqs system (Diggle et al., 2003). Moreover, other QS-regulated
polysaccharides forming a biofilm community that defends the activities, for instance, membrane vesicle formation (Mash-
bacterial cells against adverse conditions, counterworks the burn-Warren et al., 2008, 2009), are under the control of pqs
human immune response, and decreases the susceptibility to signaling. Furthermore, PqsR drives the biosynthesis of the
antibiotics dramatically (Costerton et al., 1999). PQS precursor HHQ through activation of pqsABCD and phnAB
Moreover, this microorganism possesses an exceptional operons, which is further converted to PQS by the LasR-depen-
´
adaptability to the fluctuating environments and expresses an dent monooxygenase PqsH (Gallagher et al., 2002; Deziel et al.,
arsenal of virulence factors, including exotoxins, hemolysins, 2004; Schertzer et al., 2010). Thus, a positive autoinducing loop
and exoproteases. The coordinated production and secretion is triggered by activation of PqsR by either PQS or HHQ
of these virulence factors, as well as the biofilm formation, are (McGrath et al., 2004), which allows an initial rapid increase of
controlled by a cell-density-dependent cell-to-cell communica- extracellular PQS levels during an exponential growth phase.
Chemistry & Biology 19, 381–390, March 23, 2012 ª2012 Elsevier Ltd All rights reserved 381

Chemistry & Biology
Discovery of the Antagonists of PqsR
assay in Escherichia coli. Agonistic and antagonistic properties
were determined, and competition experiments were conducted
to investigate the binding site of the antagonists. For examina-
tion of the effect in P. aeruginosa PA14 cells, we determined
the extracellular levels of the virulence factor pyocyanin and
PQS. The antibacterial effect of the compounds on an E. coli
tolC strain was tested.
Figure 1. Structures of HHQ and PQS
PQS and, to a lesser extent, its precursor HHQ activate PqsR to drive target
gene transcription.
RESULTS
Design of PqsR Antagonists
To our knowledge, the protein structure of PqsR has not been
published and antagonists of this receptor are not known. The
natural ligands are all agonists. Nevertheless, we used them
for the design of potential antagonists (ligand-based approach)
since it has been known for a long time that antagonists can
be obtained by structural modification of agonists (Hartmann
et al., 1980; Klebe, 2009).
QS inhibitors (QSIs) that specifically interfere with the bacterial
cell-to-cell communication are discussed as an alternative
approach to conventional antibacterial therapy (Rasmussen
and Givskov, 2006; Bjarnsholt and Givskov, 2007). The selective
intervention in pathogenicity without effect on bacterial growth
may reduce natural selection pressure and therefore delay or
avoid the development of resistance.
Although PQS is the most potent natural ligand, we used the
less potent HHQ (Xiao et al., 2006) for the following reasons:
(1) The 3-hydroxy group of PQS has been proved to be respon-
sible for interaction with lipid A of outer membrane lipopolysac-
charides (Mashburn-Warren et al., 2008). Thus, HHQ lacking this
group should exhibit a lower tendency to membrane association.
(2) HHQ does not display iron chelating (Bredenbruch et al.,
2006; Diggle et al., 2007) or pro-oxidant properties in contrast
Since the pqs system plays a critical role for the pathogenicity,
we consider its receptor PqsR as an attractive target protein
for the development of QSIs to disrupt the pqs-dependent
gene expression. This concept has been supported by the fact
that P. aeruginosa pqsRꢀ mutant strains display a reduced
mortality rate in mice (Xiao et al., 2006). The mutation of pqs-
controlled virulence genes results in decreased pathogenicity
in plants, nematodes, and insects (Mahajan-Miklos et al., 1999;
Jander et al., 2000). Moreover, based on the fact that PQS is
produced in high amounts in the sputum of CF patients (Collier
et al., 2002), it can be assumed that blocking the pqs QS system
should make the P. aeruginosa lung infection in CF patients
better treatable.
¨
to PQS (Haussler and Becker, 2008). Therefore, modification of
HHQ should avoid these unwanted interactions. In this work,
we modified the stereo-electronic configuration of HHQ by
changing the length of the alkyl side chain and by the introduc-
tion of electron-donating groups (EDGs) and -withdrawing
groups (EWGs) into the benzene moiety of the quinolone struc-
ture. Besides, several corresponding PQS analogs were pre-
pared for comparison.
Our primary goal is to develop PqsR antagonists as QSIs of the
pqs system. Although, to our knowledge, no PqsR antagonist
has been published to date, initial investigations of ligand-
receptor interaction of this target have been conducted by other Synthesis
research groups. In a recent study, Hodgkinson and co-workers HHQ and its analogs were prepared in 2-3 steps according to
presented structure-activity relationships (SARs) of PQS a literature procedure (Woschek et al., 2007, Figure 2). The
analogs. It was found that various substituents in the quinolone condensation of b-ketoesters with aniline or substituted anilines
core, like hydroxy and methoxy, influence the agonistic activity followed by cyclization of the resulting enamine in refluxing di-
(Hodgkinson et al., 2010) and that the alkyl side chain plays an phenyl ether yielded HHQ and its derivatives 1-7, 9-12, 14-21,
important role for activation of PqsR (Fletcher et al., 2007; Hodg- 24, and 26-30. Compounds 22, 23, and 25 were commercially
kinson et al., 2010).
available. Hydroxy-substituted products 8 and 13 were obtained
In this article, we present the first PqsR antagonists to our by demethylation of the methoxy-substituted intermediates 9
knowledge. Following a ligand-based drug design approach, and 14 (Konieczny et al., 2005).
a set of HHQ and PQS analogs were synthesized, the side chain
Compounds 7, 8, 10-21, and 26-30 are described for the first
was varied and substituents were introduced into the carbocy- time, to our knowledge. The synthesis of PQS and its congeners
clic moiety of the quinolone molecule. The biological evaluation (31-42) is provided in the Supplemental Information available
was performed in vitro with a b-galactosidase reporter gene online.
Figure 2. Synthesis Route of HHQ and PQS
Analogs
Reagents and conditions: (i) p-TsOH, n-hexane, reflux; (ii)
Ph2O, reflux; (iii) BF3
d SMe2, dichlormethane, room
temperature, then CH3OH.
See also Figure S3.
382 Chemistry & Biology 19, 381–390, March 23, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology
Discovery of the Antagonists of PqsR
Table 1. Agonistic and Antagonistic Activities of HHQ Analogs
PqsR Stimulation Induced
Inhibition of PqsR Stimulation Induced
by 1 mM HHQ in the Presence of 10 mM
Test Compd (Full Inhibition = 1.00)
by 10 mM Test Compd Compared
Compd
R
R00
to 50 nM PQS (= 1.00)
Variation of Side Chain
1
2
3
4
5
6
7
CH3
H
H
H
H
H
H
H
0.03
0.18
0.33
0.19
—
n-C5H11
n-C6H13
n-C7H15
n-C8H17
n-C9H19
n-C3H6Ph
0.03
0.49*
0.67a,*
0.69a,*
0.35*
0.29*
0.09
0.26*
0.32*
Introduction of Substituents in the Carbocyclic Ring
8
n-C7H15
n-C7H15
n-C7H15
n-C7H15
n-C7H15
n-C7H15
n-C7H15
n-C7H15
n-C7H15
n-C7H15
n-C7H15
n-C7H15
n-C7H15
n-C7H15
7-OH
0.46*
0.74*
0.02
0.05
0.67*
0.66*
0.18
0.54*
0.65*
0.29*
0.05
0.00
0.00
0.00
0.02
ꢀ0.24
0.21*
0.17
9
7-OCH3
8-OCH3
8-C2H5
8-F
10
11
12
13
14
15
16
17
18
19
20
21
ꢀ0.04
ꢀ0.09
0.23*
0.12
6-OH
6-OCH3
6-CH3
6-F
0.21*
0.49*
1.00*
0.95*
1.00*
0.61*
6-Cl
6-CN
6-CF3
6-NO2
6-CF3, 8-OCH3
Variation of Side Chainb and CF3 Position of Compd 19
22
23
24
25
26
27
28
19b
29
30
H
6-CF3
6-CF3
6-CF3
6-CF3
6-CF3
6-CF3
6-CF3
6-CF3
7-CF3
8-CF3
0.00
0.02
0.00
0.00
0.02
0.02
0.01
0.00
0.50*
0.10
0.05
0.12
0.12
0.38
0.64*
0.93*
0.97*
1.00*
0.00
0.25
CH3
C2H5
n-C3H7
n-C4H9
n-C5H11
n-C6H13
n-C7H15
n-C7H15
n-C7H15
b-Galactosidase reporter gene assay was performed in E. coli transformed with the plasmid pEAL08-2 encoding PqsR and the reporter gene lacZ
controlled by the pqsA promoter. For the agonist test, the compounds were measured at 10 mM and 1mM (data not shown); for the antagonist test,
the compounds were measured at 10 mM and 1 mM (data not shown) in the presence of 1 mM HHQ. Mean value of at least two independent experiments
with n = 4, SD < 25%. Significance: For the agonist test, induction compared to the basal value; for the antagonist test, decrease of the HHQ or PQS-
induced induction. Cmpd, compound. *p < 0.05.
aThe stimulations induced by 4 and 5 at 1 mM were 0.75 (p < 0.05) and 0.62 (p < 0.05), respectively.
bCompounds 22-28 and 19 here were tested at 5 mM, for antagonist test, in the presence of 50 nM PQS.
See also Tables S1 and S2.
Biological Evaluation
the b-galactosidase reporter gene lacZ controlled by the pqsA
promoter (Cugini et al., 2007).
Evaluation of the Agonistic and Antagonistic Activities
in Reporter Gene Assay
For the variation of the side chain, we found that, in the agonist
The PqsR-mediated transcriptional effect of the compounds was test, HHQ with n-heptyl revealed a very high activity (compound
evaluated as previously described in a HTS b-galactosidase 4, Table 1). Compound 3 with an n-hexyl side chain still showed
reporter gene assay in E. coli containing the plasmid pEAL08-2, moderate activity, but further shortening of the chain length
which encodes PqsR under the control of the tac promoter and (compound 1 and 2) resulted in complete loss of potency.
Chemistry & Biology 19, 381–390, March 23, 2012 ª2012 Elsevier Ltd All rights reserved 383

Chemistry & Biology
Discovery of the Antagonists of PqsR
kinson et al., 2010). In order to improve this physicochemical
property of compound 19 and to clarify the effect of the side
chain on the antagonistic activity, short chain compounds 22-
28 were synthesized. Indeed, shortening of the side chain
improved the aqueous solubility (Table S2) but also resulted in
a decrease of activity. At 5 mM, compounds 27 and 28 were as
potent as compound 19. However, at a lower concentration of
50 nM (data not shown), only compound 19 revealed antago-
nistic activity. This result shows that the antagonistic activity of
the analogs of compound 19 also strongly depends on the alkyl
side chain as described for the agonistic activity of HHQ (Table 1)
and PQS (Table S1).
For the purpose of obtaining a more active antagonist,
we combined the structures of the very potent compound 19
with the antagonist 10 to prepare compound 21. It showed
higher antagonistic potency than 10 but was not as effective
as 19.
Figure 3. Determination of IC50 Values
IC50 values were determined in the reporter gene assay. Compounds 18 (-),
19 (B) and 20 (;) were tested at five concentrations in competition with 50 nM
PQS. IC50 values: compound 18, 259 114 nM; 19, 54 23 nM; and 20, 51
19 nM. Mean value of two experiments with n = 4.
The corresponding PQS analogs of compounds 9, 12, 14, 15,
and 16 either displayed high agonistic activity (compounds 37
and 39; Table S1) or showed no antagonistic potency
(compound 36, 38, and 40).
Elongation of the side chain by one carbon (compound 5) did
To further characterize the potency of the antagonists, we
not reduce the PqsR stimulation at 10 mM (whereas, at 1 mM, determined IC50 values using the same reporter gene assay
a slight decrease was observed). The HHQ analog with n-nonyl (Figure 3).
(compound 6) showed a diminished activity. Introduction of
HHQ analogs with trifluoromethyl and nitro functional groups
a 3-phenylpropyl side chain (compound 7) also led to a decrease in 6-position were found to be equipotent (compound 19:
in activity. These results indicate that the ability of HHQs to acti- IC50 = 54 23 nM; compound 20, IC50 = 51 19 nM) and were
vate PqsR is dependent on the side chain with n-heptyl being the more active than compound 18 with a nitrile group (IC50
=
optimal. This phenomenon was also found for PQS side-chain 259 114 nM).
analogs in this study (compounds 31-35; Table S1), which is in
The fitting of dose-response curves of PQS with increasing
agreement with previous observations (Fletcher et al., 2007; concentrations of the antagonists 18-20 (0-200 nM) by nonlinear
Hodgkinson et al., 2010). However, PQS analog 35 with a longer Schild regression analysis allowed the calculation of the
side chain (n-nonyl) was better tolerated by PqsR than the corre- apparent dissociation constant Kd,app (Figure 4).
sponding HHQ analog (compound 6). In the antagonist test, none
For compounds 18-20, Kd,app values were determined and
of the HHQ side-chain analogs at 10 mM was able to strongly high affinities were observed (Kd,app = 556 nM, 17 nM, and
inhibit the PqsR stimulation.
To continue our search for PqsR antagonists, we then focused
7 nM, respectively).
Furthermore, direct evidence for binding of a selected antag-
on the introduction of EDGs and EWGs into the benzene part of onist (compound 18) to PqsR was provided by surface plasmon
HHQ. Introduction of an EDG such as hydroxy or methoxy into 7- resonance (SPR) biosensor experiments. For this purpose,
position resulted only in agonists (compounds 8 and 9), and the a truncated soluble form of PqsR, PqsRC87 (Xiao et al., 2006),
agonistic potency of compound 8 was significantly reduced was cloned, heterologeously expressed in E. coli, purified as
compared to HHQ (p < 0.05). Contrariwise, their congeners SUMO-tagged fusion protein (His6SUMO-PqsRC87), biotinylated
with methoxy or ethyl group in 8-position (compounds 10 and (Klein et al., 2011), and immobilized on a streptavidin-coated
11) showed no agonistic activity, while compound 12 with an sensorchip. A high affinity to the truncated target was deter-
electron-withdrawing fluoro substituent in the same position re- mined (Kd = 57 nM; Figure S1).
vealed agonistic effect. Regarding the 6-position, the introduc-
To gain further information about the binding site of the three
tion of hydroxy, methyl, or fluoro led to agonists (compounds antagonists 18-20 to PqsR, we performed competitive experi-
13, 15, and 16). Substitution with methoxy or chloro (compounds ments in the presence of PQS.
14 and 17) led to a drop of agonistic activity. The agonistic
As depicted in Figure 5, compounds 18-20 were displaced in
potency of three HHQ analogs with strong EWGs in 6-position, a dose-dependent manner by the native ligand PQS. This result
nitrile, trifluoromethyl, or nitro (compounds 18-20), was indicates that these compounds are efficient competitors to the
completely eliminated. What was most interesting was that these receptor. For compound 18, a higher concentration (250 nM) is
compounds exhibited strong antagonistic properties by needed to reach the similar antagonistic effect in comparison
completely or almost completely inhibiting the PqsR stimulation. with the other two compounds.
The antagonistic potency of two position isomers of 19, Effect on Virulence Factors Production in P. aeruginosa
compounds 29 and 30 with trifluoromethyl in 7- or 8-position, PA14
was strongly reduced compared to the 6-position analog.
To examine the biological effect of the intervention in the pqs
We assumed that the alkyl side chain of HHQ contributes to system, we investigated the impact of the PqsR antagonists 18
the poor water solubility as reported for the PQS analogs (Hodg- and 19 on the production of the PQS-regulated virulence factor
384 Chemistry & Biology 19, 381–390, March 23, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology
Discovery of the Antagonists of PqsR
Figure 4. Determination of the Apparent Kd Values
Compounds 18 (A), 19 (B) and 20 (C) at four concentrations, 0 nM (C), 50 nM (,), 100 nM (:) and 200 nM (B) were competed with PQS (2.5-5,000 nM) in the
reporter gene assay. The Schild slope was constrained equal to 1.0. Apparent Kd values: compound 18, 556 nM; 19, 17 nM; and 20, 7 nM.
See also Figure S1.
pyocyanin in P. aeruginosa PA14 with agonists 15 and 17 for Determination of Antibacterial Effect
comparison.
We evaluated the antibacterial effect of all compounds using
As shown in Figure 6, the antagonist compound 19 is able a filter disc diffusion technique on E. coli tolC strain (Table S3).
to reduce the pyocyanin production in P. aeruginosa PA14 It was found that the PqsR antagonists 18, 19, and 20 did not
supernatants by 74% at a concentration of 3 mM. In a comparison exhibit any antibacterial properties against E. coli tolC (25 mg in
of the effects of both antagonists, compound 19 is more filter disc). Furthermore, growth kinetics of P. aeruginosa PA14
active than 18. This observation is consistent with the higher in the presence of two antagonists 18 and 19 were determined
activity of compound 19 observed in the reporter gene assay. and are shown to be unaffected at a concentration of 5 mM (Fig-
As expected, the agonists 15 and 17 did not display significant ure S2). These results are in line with our approach of selectively
reduction of pyocyanin production at the concentrations of targeting bacterial QS-controlled virulence without any impact
0.5–5 mM.
The production of elastase as well as rhamnolipids, in which
on bacterial viability.
pqs system is involved, was also examined in P. aeruginosa DISCUSSION
PA14. Unlike the result from the pyocyanin assay, the production
of elastase and rhamnolipids was unaffected in the presence of The usage and misusage of antibiotics in the traditional treat-
compound 18 or 19 at 5 mM (data not shown).
ment of infections result in natural selection pressure that leads
to the widespread emergence of antibiotic resistance, which is
Effect on Extracellular PQS Levels
In a further experiment in P. aeruginosa PA14, we examined a serious and urgent medical problem. QSIs are compounds
whether the extracellular PQS levels were also affected by that are able to interfere with bacterial QS signaling pathways
the PqsR antagonists. Therefore, PQS in PA14 culture superna- thereby impairing QS-mediated group behaviors such as viru-
tants was quantified using liquid chromatography with tandem lence factor production and biofilm formation without inhibition
mass spectrometry (LC-MS/MS). The pqsAꢀ mutant, deficient of the bacterial growth. We regard PqsR, the receptor of the
in PQS and pyocyanin production, was used as the reference P. aeruginosa-specific pqs QS system, as an attractive target
strain. However, first results indicate that the extracellular to develop QSIs and expect that PqsR antagonists should limit
PQS levels were not significantly reduced in the presence of pqs-related pathogenicity.
the PqsR antagonist, compound 19 at concentrations up to
5 mM.
Following a ligand-based approach, we modified the structure
of HHQ. Considering that some variation of the side chain of PQS
Chemistry & Biology 19, 381–390, March 23, 2012 ª2012 Elsevier Ltd All rights reserved 385

Chemistry & Biology
Discovery of the Antagonists of PqsR
Figure 5. Dose-Dependent Displacement of
Antagonists by PQS
Competitive binding studies of compounds 18-20 were
performed in the reporter gene assay using increasing
concentrations of PQS (50-500 nM). Mean value of one
experiment with n = 4, SD < 10%. Black bars, control;
black and white mosaic bars, compound 18; black and
white dotted bars, compound 19; hatched bars,
compound 20.
ligands in P. aeruginosa (Hodgkinson et al.,
2010; Heeb et al., 2011).
It is most interesting that all HHQ analogs with
strong EWGs in 6-position revealed antago-
nistic potency. The introduction of nitrile, tri-
or introduction of certain functional groups into the benzene part fluoromethyl, and nitro led to the discovery of the first PqsR
of the quinolone moiety resulted in decrease of agonistic activity antagonists (compounds 18, 19, and 20). Investigation of the
of PQS (Fletcher et al., 2007; Hodgkinson et al., 2010), we binding mode of these compounds identified them as competi-
assumed that such modifications on HHQ may also remove tive antagonists. From analysis of the IC50 and Kd, app values,
the agonistic potency and provide a starting point to discover the following correlation between activity and electronegativity
PqsR antagonists. Indeed, three highly potent PqsR antagonists can be drawn: The increase of the electronegativity (nitro z tri-
were identified from HHQ core-substituted analogs.
fluoromethyl > nitrile; Sanderson, 1983; Bratsch, 1985) of the
substituent in 6-position results in a rise in potency. In order to
examine whether the antagonistic effects are singly attributable
to the nature of the substituents, their position was varied in
compound 19. It is interesting that the position isomers with tri-
fluoromethyl in 7- or 8-position (compounds 29 and 30) were
unable to antagonize the PqsR stimulation induced by HHQ.
This implies that not only the electron-withdrawing effects but
also the position of the EWG at the benzene core are
responsible.
SARs and Biological Implications
The data from the reporter gene assay reveal that the HHQ side
chain plays an important role for the activities of the compounds,
whereas the substituents at the benzene core are decisive for
agonistic and antagonistic properties. It is interesting that
agonists, compounds with agonistic and antagonistic properties
as well as pure antagonists, were obtained.
The side chain must consist of at least six C atoms to result in
biologically active compounds. It can be longer than the one of
the natural ligands HHQ and PQS (7 C atoms). In this regard, it
is worth noting that HHQ and PQS analogs with a n-nonyl side
chain (compound 6, Table 1; compound 35, Table S1), which
are natural occurring HAQs, obviously also function as PqsR
Combining the substituents of a weak antagonist (compound
10) and a strong antagonist (compound 19), bisubstituted HHQ
compound 21 was identified as a moderate antagonist. We
suppose that, for compound 21, the electronic effects of the
electron-donating methoxy group in 8-position and the elec-
tron-withdrawing trifluoromethyl group in 6-position may coun-
teract each other (for example, their effect on the NH group of
the quinolone core).
Intricate Interplay between Pyocyanin and the pqs
System
Pyocyanin is an important virulence factor in P. aeruginosa that is
required for full pathogenicity and is associated with morbidity
and mortality in CF patients (Courtney et al., 2007). Its production
has been described to be controlled by PqsR, which activates
´
the transcription of the pqsABCDE operon (Cao et al., 2001; De-
ziel et al., 2004) in which pqsABCD is responsible for HHQ
biosynthesis (Bredenbruch et al., 2005; Pistorius et al., 2011).
HAQ-deficient mutants (pqsAꢀ and pqsRꢀ) are unable to
´
produce pyocyanin (Cao et al., 2001; Deziel et al., 2004), while
PQS-deficient mutant pqsHꢀ still produces 25% pyocyanin
compared with wild-type (Xiao et al., 2006). Moreover, pyocya-
nin formation is also controlled in a HAQ-independent manner
by the action of PqsE, which is coregulated in the pqsABCDE
operon; it is able to restore pyocyanin levels in HAQ-deficient
pqsAꢀ or pqsRꢀ mutants (Farrow et al., 2008; Rampioni et al.,
2010). Therefore, PqsR antagonists that block the transcription
Figure 6. Effect on Pyocyanin Production in P. aeruginosa PA14
The pyocyanin levels in P. aeruginosa PA14 were spectrophotometrically
determined at A520 nm in the presence of compounds (cmpds.) 15, 17, 18, and
19 at 0.5 mM (black bars), 1.5 mM (hatched bars), 3 mM (small-checkered bars),
5 mM (large-checkered bars). Mean value of one experiment with n = 4, SD <
15%. Significance: reduction of pyocyanin production compared to the
control. *p < 0.003. For antibacterial activity, see also Table S3 and Figure S2.
386 Chemistry & Biology 19, 381–390, March 23, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology
Discovery of the Antagonists of PqsR
EXPERIMENTAL PROCEDURES
of the pqsABCDE operon—and, therefore, the production of
HAQs and PqsE—should disrupt pyocyanin formation. Treat-
ment of P. aeruginosa PA14 with compounds 18 and 19 revealed
that PqsR ligands with antagonistic activity are capable of dimin-
ishing extracellular pyocyanin levels relative to control in the low
micromolar range while PqsR ligands with agonistic activity
(compounds 15 and 17) did not exhibit a significant effect.
What is most interesting is that the extracellular PQS levels
that are abolished in a pqsRꢀ mutant were not affected in the
presence of compounds 18 or 19 at 5 mM. Differences in genetic
backgrounds of E. coli and P. aeruginosa like efflux pumps (e.g.,
Synthesis of the Title Compounds 18–20
Chemical and Analytical Methods
1H and 13C NMR spectra were recorded on a Bruker DRX-500 instrument.
Chemical shifts are given in parts per million (ppm), and tetramethylsilane
was used as internal standard for spectra obtained in CDCl3, MeOH-d4, and
DMSO-d6. All coupling constants (J) are given in hertz. LC/MS was performed
on an MSQ electro spray mass spectrometer (Thermo Fisher). The system was
operated by the standard software Xcalibur. An RP C18 NUCLEODUR 100-5
(125 3 3 mm) column (Macherey-Nagel GmbH) was used as stationary phase
with water/acetonitrile mixtures as eluents. All solvents were high-pressure
liquid chromatography grade. Reagents were used as obtained from commer-
cial suppliers without further purification. Flash chromatography was per-
formed on silica gel 60, 70-230 mesh (Fluka), and the reaction progress was
determined by thin-layer chromatography analyses on silica gel 60, F254
(Merck). Visualization was accomplished with UV light. All microwave irradia-
tion experiments were carried out in a multiSYNTH all-in-one microwave
(MLS GmbH). We measured the melting points using melting point apparatus
SMP3 (Stuart Scientific). The apparatus is uncorrected.
´
Lamarche and Deziel, 2011) might play a role, as discussed by
Hodgkinson and co-workers, where PQS is about 1,000 times
more sensitive to transcriptional activation of the pqsA-lacZ
fusion in the heterologous E. coli than in P. aeruginosa (Fletcher
et al., 2007; Hodgkinson et al., 2010). Besides, we speculate that
there may be some unknown effects (e.g., inhibition of enzymes
involved in biosynthesis of pyocyanin by PqsR antagonists.
Mavrodi et al., 2001) in QS circuitry. Further studies to investigate
these unexpected phenomena are currently underway. Anyway,
we have clearly demonstrated that the tight binding to PqsR
(compound 18 by SPR) leads to strong antagonistic effects
(reporter gene assay). However, only pyocyanin production is
reduced; no effects are observed regarding PQS and rhamnoli-
pid formation and elastase activity.
General Procedure
A solution of b-ketoester (9.24 mmol, 1 equiv), aniline (9.24 mmol, 1 equiv), and
p-TsOH $ H2O (50 mg, 0.29 mmol) in n-hexane (20 ml) was heated at reflux
using a Dean-Stark separator for 5 hr. After cooling, the solution was concen-
trated in vacuo, and the residue was added dropwise to refluxing (260ꢁC)
diphenyl ether (5 ml). Refluxing was continued for 30 min. After cooling to
room temperature, Et2O (15 ml) and 2 M HCl (20 ml) were added and the
mixture was left overnight at 5ꢁC. If a crystalline solid had formed, it was
collected and washed with Et2O. If no solid had formed, ammonia was added
to basify the mixture. HHQ analogs were purified by crystallization from ethyl
acetate or column chromatography on silica gel (Woschek et al., 2007).
2-Heptyl-4-oxo-1,4-dihydroquinoline-6-carbonitrile; 18. Compound 18 was
obtained from 4-aminobenzonitrile (441 mg, 3.73 mmol) and ethyl 3-oxodeca-
noate (800 mg, 3.74 mmol) after crystallization as a white solid (164 mg,
0.61 mmol, 16%), melting point (mp) 196-199ꢁC. 1H-NMR (500 MHz, DMSO-
d6): d = 0.74 (t, J = 7.0 Hz, 3H), 1.13-1.21 (m, 8H), 1.56 (quint, J = 7.0 Hz,
2H), 2.50 (t, J = 7.5 Hz, 2H), 5.94 (s, 1H), 7.55 (d, J = 8.5 Hz, 1H), 7.84 (dd,
J = 2.0 Hz, 8.5 Hz, 1H), 8.27 (d, J = 2.0 Hz, 1H) 11.69 (brs, 1H). 13C-NMR
(125 MHz, DMSO-d6): d = 13.8, 21.9, 28.0, 28.3, 28.3, 31.0, 33.2, 104.9,
109.9, 118.7, 119.5, 124.1, 130.6, 133.4, 142.5, 154.9, 175.6. LC/MS: m/z
269.28 (MH+), 99.4%.
In conclusion, the PqsR antagonists provide an interesting
starting point for further drug design efforts on this target protein
to develop effective antivirulence drugs. In a next step, the
discovered antagonists will be further optimized concerning their
aqueous solubility.
SIGNIFICANCE
PqsR is the receptor of pqs quorum sensing cell-to-
cell communication system of the human pathogen
P. aeruginosa, which controls the expression of various
virulence factors and is involved in biofilm formation. This
receptor plays an important role for pathogenicity and
therefore appears to be an attractive target for antivirulence
drugs. This work describes, to our knowledge, the discovery
of the first antagonists of PqsR with IC50 and Kd,app values in
the low nanomolar range that have been determined
in heterologous E. coli reporter gene system. An activity
in vivo is demonstrated by the antagonist 19, which reduces
virulence factor pyocyanin production by 74% in
P. aeruginosa PA14 at a concentration of 3 mM. We examined
the binding of these active compounds in competition
experiments and identified them as competitive antago-
nists. As expected, the PqsR antagonists do not reduce
viability of P. aeruginosa; therefore, they should not induce
natural selection pressure. This property makes these
compounds important as they could overcome the short-
comings of traditional antibiotics. These PqsR antagonists
are highly valuable scientific tools for in-depth study of
the ligand-receptor interaction of PqsR and the function of
the pqs system. Our finding provides an important
step toward further drug design targeting PqsR and may
open new avenues for the combat against P. aeruginosa
infection.
2-Heptyl-6-(trifluoromethyl)quinolin-4(1H)-one; 19. Compound 19 was ob-
tained from 4-(trifluoromethyl)aniline (602 mg, 3.74 mmol) and ethyl 3-oxode-
canoate (800 mg, 3.74 mmol) after acidification with concentrated HCl and
crystallization as a white solid (94 mg, 0.30 mmol, 8%), mp 233-237ꢁC. 1H-
NMR (500 MHz, DMSO-d6): d = 0.83 (t, J = 6.5 Hz, 3H), 1.25-1.34 (m, 8H),
1.70 (quint, J = 7.5 Hz, 2H), 2.73 (t, J = 7.5 Hz, 2H), 6.34 (s, 1H), 7.92-7.97
(m, 2H), 8.36 (d, J = 0.5 Hz, 1H). 13C-NMR (125 MHz, DMSO-d6): d = 14.2,
22.4, 28.7, 28.9, 31.5, 33.8, 108.5, 120.6, 122.6 (q, JCF = 3.7 Hz), 123.1,
124.6 (q, JCF = 270.0 Hz), 124.7 (q, JCF = 32.0 Hz), 128.3 (q, JCF = 3.0 Hz),
142.5, 157.6, 174.9. LC/MS: m/z 312.31 (MH+), 97.0%.
2-Heptyl-6-nitroquinolin-4(1H)-one; 20. Compound 20 was obtained from
4-nitroaniline (515 mg, 3.73 mmol) and ethyl 3-oxodecanoate (800 mg,
3.74 mmol) after crystallization as a brown solid (12 mg, 0.04 mmol, 1%),
mp 185-186ꢁC. 1H-NMR (500 MHz, MeOH-d4): d = 0.91 (t, J = 7.0 Hz, 3H),
1.33-1.50 (m, 8H), 1.88 (quint, J = 7.5 Hz, 2H), 3.08 (t, J = 7.5 Hz, 2H), 7.08
(s, 1H), 8.15 (d, J = 9.5 Hz, 1H), 8.73 (dd, J = 2.0 Hz, 9.5 Hz, 1H), 9.18 (d, J =
2.0 Hz, 1H). 13C-NMR (125 MHz, MeOH-d4): d = 11.6, 20.9, 27.3, 27.5, 27.6,
30.0, 32.8, 105.1, 118.3, 118.9, 119.6, 126.2, 140.7, 144.3, 162.4, 170.6.
LC/MS: m/z 289.29 (MH+), 98.6%.
Synthesis of other HHQ analogs, PQS analogs, and b-ketoesters are
provided in the Supplemental Information.
Reporter Gene Assay
The ability of the compounds to either stimulate or antagonize the PqsR-
dependent transcription was evaluated as previously described using a
b-galactosidase reporter gene assay (Cugini et al., 2007) in E. coli expressing
PqsR, with some modifications to enable a higher throughput (Griffith and
Chemistry & Biology 19, 381–390, March 23, 2012 ª2012 Elsevier Ltd All rights reserved 387

Chemistry & Biology
Discovery of the Antagonists of PqsR
Wolf, 2002). PQS, HHQ, and its analogs were diluted in ethyl acetate and
added to the wells of a 96-deep-well plate, and the solvent was evaporated.
Overnight cultures of E. coli DH5a cells containing the plasmid pEAL08-2,
which encodes PqsR under the control of the tac promoter and the b-galacto-
sidase reporter gene lacZ controlled by the pqsA promoter, were diluted 1:100
in Luria-Bertani (LB) medium with ampicillin (50 mg/ml). The culture was incu-
bated at 37ꢁC with shaking until it reached an OD600 of 0.2. For the determina-
tion of agonistic activities, 1 ml aliquots were supplemented with either PQS
(50 nM) or the test compound: for HHQ analogs, 10 mM; for 6-trifluoromethyl
HHQs 22-28, 5 mM; PQS analogs 31-42, 50 nM. Ethyl acetate was used as
a control. Antagonistic effects of the compounds were evaluated in the pres-
ence of either 1 mM HHQ or 50 nM PQS. The b-galactosidase activity was
determined after a 2.5 hr incubation period at 37ꢁC with shaking (150 rpm).
OD600, OD420, and OD550 were measured, and the activity is expressed as ratio
of the ethyl acetate control relative to the cultures that received either PQS,
a test compound, or both. IC50 values of antagonists 18-20 were determined
by variation of the concentration of the test compounds in competition with
50 nM PQS. Binding affinities of antagonists 18-20 were determined by mutual
variations of concentrations of the test compounds and of PQS (50-500 nM).
The Gaddum/Schild IC50 shift model (GraphPad Prism, trial version 5.0) was
applied for nonlinear regression and determination of Kd values; the Schild
slope was constrained equal to 1.0 (Arunlakshana and Schild, 1959).
Binding Affinity for Compound 18
The binding experiment was performed at 12ꢁC at a constant flow rate of
50 ml/min in instrument running buffer (50 mM HEPES, pH 7.4, 150 mM
NaCl, 3 mM EDTA, 5% DMSO (v/v), 0.05% P20 (v/v). A 180 mM solution of
compound 18 in DMSO was directly diluted to a concentration of 9 mM
(50 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20 (v/v)) and
then diluted threefold from 9 mM down to 4.1 nM in the running buffer. Before
starting the experiments, 12 warm-up blank injections were performed. Zero-
buffer blank injections and DMSO calibrations were included for double refer-
encing. Individual concentrations were injected from lowest to highest
concentrations for 100 s association and 15 min dissociation time. Experi-
ments were performed twice with two independently immobilized SAD500
chips. Scrubber software was used for processing and analyzing data.
Pyocyanin Assay
P. aeruginosa PA14 cultures and a corresponding pqsAꢀ knockout strain as
a reference were grown at 37ꢁC under shaking conditions (180 rpm) in LB
medium in the absence and presence of test compounds (0.5 mM, 1.5 mM,
3 mM, and 5mM) overnight. After centrifugation (10 min, 2,600 3 g), culture
supernatants were extracted with equal volumes of chloroform. A total of
0.75 ml of the lower organic phase was supplemented with 0.25 ml of 0.2 N
HCl solution and shaken for 30 s. The upper reddish phase was collected,
and OD520 was measured.
Protein Expression and Purification
His6SUMO-PqsRC87 was expressed in E. coli and purified with a single
affinity chromatography step. Briefly, E. coli BL21 (DE3) cells containing the
pSUMO3_ck4_pqsRC87 plasmid were grown in LB medium (50 mg/ml
kanamycin) at 37ꢁC to an OD600 of approximately 0.8 units and induced with
0.2 mM IPTG for 16 hr at 16ꢁC. The cells were harvested by centrifugation
(5,000 rpm, 10 min, 4ꢁC), and the cell pellet was resuspended in 100 ml binding
buffer (50 mM Tris-HCl, pH 7.8, 150 mM NaCl, 20 mM imidazole, 10% glycerol
(v/v)) and lysed by sonication for a total process time of 2.5 min. Cell debris was
removed by centrifugation (13,000 rpm, 30 min), and the supernatant was
filtered through a syringe filter (0.2 mm). The clarified lysate was immediately
applied to a Ni-NTA column (GE Healthcare), washed with 50 mM Tris HCl,
pH 7.8, 150 mM NaCl, 20 mM imidazole, 10% glycerol (v/v), and eluted with
500 mM imidazole containing buffer. The protein-containing fractions were
buffer exchanged into 20 mM Tris, pH 7.4, 150 mM NaCl, and 10% glycerol
(v/v) using a PD10 column (GE Healthcare) and were judged pure by SDS-
PAGE. The His6SUMO-tagged proteins were used for biotinylation.
Elastase Assay
Elastase activity of P. aeruginosa PA14 cultures was measured with Elastine
Congo Red (ECR), which is cleaved upon elastolytic activity releasing the
soluble red pigment CR. In brief, 1 ml culture supernatants of overnight
cultures (180 rpm at 37ꢁC) were mixed with 1 ml of ECR buffer (0.1 M Tris-
HCl pH 7.2, 1 mM CaCl2,) containing 20 mg of ECR (Sigma-Aldrich) and incu-
bated for 3 hr at 37ꢁC with constant shaking. After incubation, insoluble ECR
was spinned down by centrifugation at 13,000 3 g for 5 min, and the absor-
bance of the supernatant was measured at 495 nm (measurement was per-
formed immediately after incubation since no stop reactant was added).
Rhamnolipid Assay
To determine rhamnolipid production of P. aeruginosa PA14 cultures, we per-
formed a modified orcinol assay for the detection of glycolipids. For that, 300 ml
aliquots of the supernatants of 24-hr-old cultures were extracted with 1,700 ml
diethylether. One milliliter of the ether phases was mixed with 600 ml 20 mM
HCl, and 500 ml of the resulting organic phases were transferred and dried
under the hood. After evaporation, 100 ml 1.6% orcinol and 900 ml 60%
H2SO4 were added, and the samples were incubated for 30 min at 80ꢁC. After
incubation, the absorbance was measured at 421 nm.
Minimal Biotinylation of His6SUMO-PqsRC87
Minimal biotinylation of the His6SUMO-PqsRC87 was achieved by mixing
56 nmol of His6SUMO-PqsRC87 with 28 nmol of EZ-link sulfoNHS LC-LC-biotin
(Thermofisher Scientific) that was freshly dissolved in water. Biotinylation reac-
tion mixture was incubated on ice for 2 hr. To remove unreacted biotin reagent,
we subjected the entire biotinylation mixture to size exclusion chromatography
on a Superdex200 HR (16/600) column equilibrated in storage buffer (13 PBS,
pH 7.4, 10% glycerol (v/v)). A protein peak containing biotinylated His6SUMO-
PqsRC87 protein was collected (0.3 mg/ml), stored at ꢀ80ꢁC, and used for SPR
studies.
Determination of Extracellular PQS Levels
Extracellular PQS produced by P. aeruginosa PA14 was determined in 250 ml
Erlenmeyer flasks containing 25 ml cultures in LB medium. Flasks were incu-
bated at 37ꢁC in an orbital shaker at 200 rpm. Cultures were inoculated with an
overnight culture to obtain a starting OD600 = 0.025. DMSO solutions of inhib-
itors were added to the cultures to a final DMSO concentration of 0.5%. For
PQS analysis, 500 ml of each culture (OD600 = 2.5) were mixed with 1 ml meth-
anol containing the internal standard. After centrifugation (9,000 3 g, 10 min),
160 ml of the supernatant were transferred to glass vials for LC-MS/MS anal-
ysis. For each sample, cultivation and extraction were performed in duplicates.
SPR Studies
We performed SPR binding studies using a Reichert SR7500DC instrument
optical biosensor (Reichert Technologie, Depew, NY, USA). SAD500 sensor
chips from Xantec (Xantec Analytics, Du¨ sseldorf) were used.
LC-MS/MS Analysis
Immobilization of Biotinylated His6Sumo-PqsRC87
We performed the analyses using a TSQ Quantum mass spectrometer equip-
ped with an ESI source and a triple quadrupole mass detector (Thermo Finni-
gan, San Jose, CA). The MS detection was carried out at a spray voltage of
3.6 kV, a nitrogen sheath gas pressure of 4.0 3 105 Pa, an auxiliary gas pres-
sure of 1.0 3 105 Pa, a capillary temperature of 360ꢁC, a tube lens offset of
94 V, and source CID of 10 V.
Biotinylated His6SUMO-PqsRC87 was immobilized on a SAD500 (Streptavidin-
coated) sensor chip at 25ꢁC. HEPES (50 mM HEPES, pH 7.4, 150 mM NaCl,
3 mM EDTA) was used as the immobilization buffer. The streptavidin carbox-
ymethyl dextran surface was preconditioned for 30 min with running buffer
until the baseline was stable. Biotinylated His6SUMO-PqsRC87 was diluted
into running buffer to a concentration of 100 mg/ml and coupled to the surface
with a 4 min injection. Biotinylated His6SUMO-PqsRC87 (39494 Da) was immo-
bilized at densities of 2,556 RU (Chip (I)) and 4,463 RU (Chip (II)) for the binding
experiments of compound 18.
Observed ions were as follows (values are given, respectively, for mother ion
[m/z]; collision energy [V]; product ion [m/z]; scan time [s]; scan width [m/z]):
PQS: 260.160; 34; 175.053; 0.2; 3.000, internal standard (Amitriptyline):
278.000; 22; 232.970; 0.1; 3.000.
388 Chemistry & Biology 19, 381–390, March 23, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology
Discovery of the Antagonists of PqsR
Xcalibur software was used for data acquisition and quantification with the
use of a calibration curve relative to the area of the internal standard. All
samples were injected by autosampler (Surveyor, Thermo Finnigan) with
a volume of 20 ml. A Hypersil Gold 3 mm (150 3 2.1 mm) column (ThermoScien-
tific, Dreieich, Germany) was used as stationary phase under isocratic condi-
tions, with 55% of 10 mM ammonium acetate containing 0.1% trifluoroacetic
acid (TFA) (v/v) and 45% of acetonitrile containing 0.1% TFA (v/v) over 6.5 min
at a flow rate of 4,500 ml/min.
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Determination of Antibacterial Activity
Filter disc diffusion tests were performed on LB agar that was supplemented
with an overnight culture of E. coli tolC to a final OD600 of 0.01. Agar plates
were incubated at 30ꢁC with filter paper discs that were prepared to contain
25 mg test compounds. Chloramphenicol discs were included as reference.
De´ ziel, E., Le´ pine, F., Milot, S., He, J.X., Mindrinos, M.N., Tompkins, R.G., and
Rahme, L.G. (2004). Analysis of Pseudomonas aeruginosa 4-hydroxy-2-alkyl-
quinolines (HAQs) reveals a role for 4-hydroxy-2-heptylquinoline in cell-to-cell
communication. Proc. Natl. Acad. Sci. USA 101, 1339–1344.
Determination of the Growth Curves of P. aeruginosa PA14
Cultures of P. aeruginosa PA14 were inoculated with an overnight culture to
obtain a starting OD600 = 0.025 and grown in three replicates in 250 ml Erlen-
meyer flasks containing 25 ml LB medium at 37ꢁC and 200 rpm in an orbital
shaker. DMSO solutions of compounds 18 and 19 were added to the cultures
to a final DMSO concentration of 0.5%. We measured bacterial growth as
a function of OD600 using FLUOstar Omega (BMG LABTECH, Ortenberg,
Germany). P. aeruginosa PA14 cultures containing 0.5% DMSO were used
as a control.
De´ ziel, E., Gopalan, S., Tampakaki, A.P., Le´ pine, F., Padfield, K.E., Saucier,
M., Xiao, G., and Rahme, L.G. (2005). The contribution of MvfR to
Pseudomonas aeruginosa pathogenesis and quorum sensing circuitry regula-
tion: multiple quorum sensing-regulated genes are modulated without
affecting lasRI, rhlRI or the production of N-acyl-L-homoserine lactones.
Mol. Microbiol. 55, 998–1014.
Diggle, S.P., Lumjiaktase, P., Dipilato, F., Winzer, K., Kunakorn, M., Barrett,
´
D.A., Chhabra, S.R., Camara, M., and Williams, P. (2006). Functional genetic
analysis reveals a 2-Alkyl-4-quinolone signaling system in the human path-
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701–710.
SUPPLEMENTAL INFORMATION
´
Diggle, S.P., Winzer, K., Chhabra, S.R., Worrall, K.E., Camara, M., and
Supplemental Information includes three tables, three figures, and Supple-
mental Experimental Procedures and can be found with this article online at
doi:10.1016/j.chembiol.2012.01.015.
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.
ACKNOWLEDGMENTS
Diggle, S.P., Matthijs, S., Wright, V.J., Fletcher, M.P., Chhabra, S.R., Lamont,
I.L., Kong, X., Hider, R.C., Cornelis, P., Ca´ mara, M., and 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, 87–96.
We thank the DAAD (German Academic Exchange Service) for providing
a fellowship to C.L. We are very grateful to Dr. Cugini and Dr. Hogan
(Dartmouth Medical School, Hanover, NH, USA) for kindly supplying the
plasmid pEAL08-2. We thank Professor Rolf Mu¨ ller of the Helmholtz-Institute
for Pharmaceutical Research Saarland, Saarbru¨ cken, Germany, for providing
the E. coli tolC strain and the pSUMO3_ck4_pqsRC87 plasmid.
Farrow, J.M., 3rd, Sund, Z.M., Ellison, M.L., Wade, D.S., Coleman, J.P., and
Pesci, E.C. (2008). PqsE functions independently of PqsR-Pseudomonas
quinolone signal and enhances the rhl quorum-sensing system. J. Bacteriol.
190, 7043–7051.
Received: September 30, 2011
Revised: January 10, 2012
Accepted: January 13, 2012
Published: March 22, 2012
´
Fletcher, M.P., Diggle, S.P., Crusz, S.A., Chhabra, S.R., Camara, M., and
Williams, P. (2007). A dual biosensor for 2-alkyl-4-quinolone quorum-sensing
signal molecules. Environ. Microbiol. 9, 2683–2693.
Gallagher, L.A., McKnight, S.L., Kuznetsova, M.S., Pesci, E.C., and
Manoil, C. (2002). Functions required for extracellular quinolone signaling by
Pseudomonas aeruginosa. J. Bacteriol. 184, 6472–6480.
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390 Chemistry & Biology 19, 381–390, March 23, 2012 ª2012 Elsevier Ltd All rights reserved