Identity and effects of quorum-sensing inhibitors produced by Penicillium species

Thomas Bovbjerg Rasmussen1, Mette E. Skindersoe1, Thomas Bjarnsholt1, Richard K. Phipps2, Kathrine Bisgaard Christensen2, Peter Ostrup Jensen3, Jens Bo Andersen1, Birgit Koch1, Thomas Ostenfeld Larsen2, Morten Hentzer4, Leo Eberl5, Niels Hoiby3,6 and Michael Givskov1

1 Center for Biomedical Microbiology, BioCentrum-DTU, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark
2 Center for Microbial Biotechnology, BioCentrum-DTU, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark
3 Department of Clinical Microbiology, Rigshospitalet, DK-2100, Copenhagen Ø, Denmark
4 Carlsberg Research Center, Biosector, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark
5 Department of Microbiology, University of Zürich, CH-8008 Zürich, Switzerland
6 Department of Bacteriology, Institute of Medical Microbiology and Immunology, University of Copenhagen, Denmark

Correspondence
Michael Givskov
immg{at}pop.dtu.dk


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Quorum sensing (QS) communication systems are thought to afford bacteria with a mechanism to strategically cause disease. One example is Pseudomonas aeruginosa, which infects immunocompromised individuals such as cystic fibrosis patients. The authors have previously documented that blockage of the QS systems not only attenuates Ps. aeruginosa but also renders biofilms highly susceptible to treatment with conventional antibiotics. Filamentous fungi produce a battery of secondary metabolites, some of which are already in clinical use as antimicrobial drugs. Fungi coexist with bacteria but lack active immune systems, so instead rely on chemical defence mechanisms. It was speculated that some of these secondary metabolites could interfere with bacterial QS communication. During a screening of 100 extracts from 50 Penicillium species, 33 were found to produce QS inhibitory (QSI) compounds. In two cases, patulin and penicillic acid were identified as being biologically active QSI compounds. Their effect on QS-controlled gene expression in Ps. aeruginosa was verified by DNA microarray transcriptomics. Similar to previously investigated QSI compounds, patulin was found to enhance biofilm susceptibility to tobramycin treatment. Ps. aeruginosa has developed QS-dependent mechanisms that block development of the oxidative burst in PMN neutrophils. Accordingly, when the bacteria were treated with either patulin or penicillic acid, the neutrophils became activated. In a mouse pulmonary infection model, Ps. aeruginosa was more rapidly cleared from the mice that were treated with patulin compared with the placebo group.


Abbreviations: AHL, N-acylated homoserine lactone; 123-DHR, dihydrorhodamine 123; GFP, green fluorescent protein; PMN, polymorphonuclear leukocyte; QS, quorum sensing; QSI, QS inhibitor(y)


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The 20th century initially offered the promising prospect of penicillin and other antibiotics to fight bacterial infections, but ended with the gloomy scenario of emerging multi-resistant bacteria. The efficiency of conventional antibiotics in preventing bacterial proliferation is the source of their success, but at the same time is also the cause of their failure. In many cases, selective pressure imposed by the use of conventional antibiotics leads to increased expression of degrading enzymes and development of drug-efflux systems which operate with increased efficiency and therefore actively reduce the internal concentration of the antibiotics. Furthermore, research over the last two decades has revealed that bacteria in the biofilm mode exhibit a higher tolerance to antimicrobial treatments (Anwar et al., 1990). The biofilm mode of growth also protects opportunistic pathogens, such as Pseudomonas aeruginosa, against the action of the host immune system, which in turn facilitates establishment of chronic infections (Costerton et al., 1999).

The prospect of the 21st century as a post-antibiotic era highlights the importance of novel strategies to control bacterial diseases (Camara et al., 2002). Many Gram-negative bacteria utilize N-acylated homoserine lactones (AHLs) to coordinate expression of virulence in response to the density of the surrounding bacterial population in a process termed quorum sensing (QS). The AHL molecules are produced by LuxI homologues and constitute, in complex with LuxR homologues, transcriptional regulators. AHLs consist of a conserved homoserine lactone ring with a variable N-acyl chain. The predominant AHL variations include presence or absence of a keto or hydroxy group on the C-3 carbon atom, as well as the length and saturation of this chain (Fuqua et al., 1994; Salmond et al., 1995). A number of recent publications have pointed to QS as a new drug target. Fighting bacteria by interfering with their command language and thereby disrupting virulence expression instead of inhibiting growth could serve as an alternative to the conventional ways of combating bacterial infections (Finch et al., 1998; Habeck, 2003; Smith & Iglewski, 2003; Hentzer & Givskov, 2003). The strategy would be based on small molecules with variations in their chemical composition that would allow them to block the AHL receptor site of the LuxR homologues or alternatively block the formation of active dimers that are required for binding to and expression of target genes. A number of studies have identified several molecules that function as QS inhibitors (QSI) (Smith et al., 2003a, b; Reverchon et al., 2002; Olsen et al., 2002; Hentzer & Givskov, 2003). Much effort has been spent on synthesis of AHL analogues which antagonize the cognate signal molecules. Varying the length of the acyl side chain was found to be important; for example AHLs with extended side chains generally caused inhibition of the LuxR homologues (Chhabra et al., 1993; Passador et al., 1996; Schaefer et al., 1996; Zhu et al., 1998). Other modifications to the AHLs included alteration of the acyl chain by introducing ramified alkyl, cycloalkyl or aryl/phenyl substituents at the C-4 position, resulting in both inducers (analogues with non-aromatic substitutions) and antagonists (analogues with phenyl substitutions) (Reverchon et al., 2002). Modification of the lactone ring of AHLs by adding substituents to C-3 or C-4 did not give rise to strong QSI activity (Olsen et al., 2002). However, exchanging the homoserine ring with a five- or six-membered alchohol or ketone ring, Smith et al. (2003a, b) generated a number of activators and inhibitors, some of which blocked of Ps. aeruginosa QS in vitro. Their target specificity for the QS regulon was not verified by transcriptomics.

In nature, eukaryotes live closely associated with virulent prokaryotes. This has forced mammals to evolve different defence systems. Plants and fungi, however, do not possess active immune systems; instead they have to rely on physical and chemical defences. A well-studied example of this is the production of halogenated furanone compounds by the Australian alga Delisea pulchra (Givskov et al., 1996). This species produces the compounds in the central vesicle of gland cells, from which they are released to the surface of the plant (Dworjanyn & Steinberg, 1999), where they prevent extensive surface growth by bacteria and higher fouling organisms (Steinberg et al., 1997; Maximilien et al., 1998). The halogenated furanones have been shown to inhibit several QS-controlled phenotypes, including swarming motility of Serratia liquefaciens, toxin production of Vibrio harveyi and bioluminescence of Vibrio fischeri (Givskov et al., 1996; Kjelleberg et al., 1997; Manefield et al., 2000; Rasmussen et al., 2000). In a more clinical context, a synthetic derivative of the furanones (C-30) was found to downregulate expression of more than 80 % of the QS-regulated genes found in Ps. aeruginosa, many of which encode known virulence factors. This effect is not limited to planktonic bacteria: it also applies to biofilm-dwelling Ps. aeruginosa. Biofilms developed in the presence of furanone compounds become more susceptible to treatments with antibiotics and disinfectants (Hentzer et al., 2002, 2003). This is highly interesting given that Ps. aeruginosa is an opportunistic pathogen often found in people with compromised immune systems, such as cystic fibrosis patients, where it is responsible for persistent, chronic infections probably caused by biofilm formation within the host (Hoiby & Koch, 2000; Hoiby, 2000; Van Delden & Iglewski, 1998). Attenuating this bacterium with respect to virulence and persistence is undoubtedly desirable; the first proof of this concept was delivered by Hentzer et al. (2003), who were able to attenuate and eradicate bacteria colonizing mouse lungs.

In the present study, selected members of the filamentous fungal genus Penicillium were investigated for production of QSI activity. Microfungi produce a huge variety of secondary metabolites, including complex molecules such as alkaloids and polyketides. Some of these are mycotoxins, compounds which can cause disease and sickness in vertebrates at fairly low concentrations. The {beta}-lactam antibiotics, which include penicillin G and cephalosporin, were originally isolated from Penicillium species. The immunosuppressants cyclosporin and tacrolimus used in connection with organ transplants also originate as fungal metabolites. The ability to produce a wide variety of bioactive compounds makes the microfungi obvious candidates to screen for QSI products. Using a recently published screening system consisting of bacteria that will grow only when the growth medium is supplemented with a QSI compound (Rasmussen et al., 2005), extracts of 50 members of the genus Penicillium have been screened. Several fungi were found to produce QSI activities and two of the compounds were identified and examined for their specificity for the Ps. aeruginosa QS regulon, and their effects on both biofilm tolerance to antimicrobial treatments and pulmonary infections.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bacterial strains.
The two QSI selector strains, QSIS1 (Apr, luxI–phlA) and QSIS2 (Apr Gmr, lasB–sacB) were described by Rasmussen et al. (2005). We also used a quorum-sensing reporter strain such as Ps. aeruginosa PAO1 harbouring a lasB–gfp fusion (Hentzer et al., 2002). The Ps. aeruginosa PAO1 used for the in vitro experiments was obtained from the Pseudomonas Genetic Stock Center (www.pseudomonas.med.ecu.edu, strain PAO0001). This PAO1 isolate has served as DNA source for the Pseudomonas Genome Project (www.pseudomonas.com) and, subsequently, as template for design of the Ps. aeruginosa GeneChip (Affymetrix, Inc.). The {Delta}lasI rhlI mutant and the {Delta}lasR rhlR mutant were constructed using previously described knock-out systems (Beatson et al., 2002a, b). The knock-out mutants were verified by Southern blot analysis and by screening for absence of AHL production. When green fluorescent protein (GFP) was employed as a bacterial tag, we used a constitutively expressed stable GFP version encoded by a gene present on the plasmid pMRP9 (Davies et al., 1998).

The Ps. aeruginosa used for in vivo experiments was obtained from B. Iglewski, University of Rochester, Medical Center, Rochester, NY, USA. The {Delta}lasR rhlR mutant of the Iglewski PAO1 was constructed using the same knock-out systems as for PAO0001 (Beatson et al., 2002a, b). The knock-out mutants were verified by Southern blot analysis and by screening for absence of AHL production.

Growth medium and conditions.
ABT minimal medium (AB medium of Clark & Maaløe, 1967, plus 2·5 mg thiamine l–1) supplemented with 0·5 % glucose and 0·5 % Casamino acids was used. Media were supplemented with antibiotics where appropriate, and unless stated otherwise, all strains were incubated at 30 °C.

Fungal strains.
Fungal isolates (Table 1) were obtained from the IBT Culture Collection at BioCentrum-DTU, Technical University of Denmark. The cultures were inoculated in triplicate on Czapek yeast (autolysate) agar (CYA) (Pritt, 1979) and yeast extract sucrose agar (YES) (Frisvad & Filtenborg, 1983) and incubated at 25 °C for 7 days in the dark.


View this table:
[in this window]
[in a new window]
 
Table 1. Fungal extracts tested for QSI content

+, QSI activity, –, no activity. Extracts were prepared from fungi cultured on CYA and YES media (see Methods).

 
Preparation of fungal extracts.
Procedures for micro-extraction of fungal secondary metabolites were similar to those given by Smedsgaard (1997) with the following modifications. Plug extracts were prepared from nine plugs and extracted with 2 ml ethyl acetate/methylene chloride/methanol (3 : 2 : 1, by vol.) containing 50 p.p.m. trifluoroacetic acid.

Screening for QSIs.
The screening assays using QSIS1 and QSIS2 were performed as described by Rasmussen et al. (2005). QSIS1 is an E. coli lac+ strain harbouring pTBR2iB, which encodes an AHL-induced killing system. QSIS2 is a Ps. aeruginosa lasI rhlI double mutant harbouring pLasB-SacB1, encoding an AHL-induced killing system; this strain also harbours pSU2007, encoding constitutively expressed luxCDABE, giving rise to bioluminescence (Rasmussen et al., 2005).

Microfractionation of extracts.
An Agilent 1100 series HPLC with fraction collector coupled to a G2250A Micro plate sampler was used for microfractionation. Samples were fractionated on a Phenomenex Luna II C18 column (100 mmx4·6 mm, 5 µm) at 40 °C with a flow rate of 1 ml min–1 and the following H2O/CH3CN solvent gradient: 2 min at 10 % CH3CN/H2O; a linear gradient to 70 % CH3CN/H2O for 12 min; isocratic at 70 % for another 10 min; a linear gradient for 2 min to 100 % CH3CN, followed by isocratic at 100 % CH3CN for 4 min then returned to 10 % CH3CN/H2O in 2 min and re-equilibrated for 8 min. Solvents were HPLC-grade acetonitrile and MilliQ water, both with 50 p.p.m. trifluoroacetic acid added. HPLC eluate was collected into Nunculon polystyrene microtitre plates (Nunc) for HPLC samples containing less than 70 % acetonitrile. Solvent-resistant U96 PP 0·5 ml polypropylene plates (Nunc) were used for collection of HPLC eluate containing more than 70 % acetonitrile.

Identification of active compounds by LC-DAD-MS.
Fungal metabolites were analysed using an Agilent HP 1100 liquid chromatograph with a DAD system (Waldbronn) coupled to an LCT oaTOF mass spectrometer (Micromass) using a Z-spray ESI source and a Lock Spray probe. A Phenomenex Luna II C18 column (100 mmx2 mm, 3 µm) at 40 °C and a flow rate of 0·3 ml min–1 was used for separation. Solvents were HPLC-grade acetonitrile and MilliQ water. The water was buffered with 10 mM ammonium formate (analytical grade) and 20 mM formic acid, and the acetonitrile with 20 mM formic acid.

Dose response.
To establish a dose–response relationship of patulin and penicillic acid, dilution rows were made with growth medium (ABT with 0·5 % Casamino acids) in a microtitre dish. Each well contained 100 µl diluted QSI and 200 µl of a 1 : 100 diluted overnight culture of PAO1 lasB-gfp(ASV) (Hentzer et al., 2002), grown in ABT with 0·5 % Casamino acids, was added. Growth was monitored as OD450 over a time-course of 14 h, and GFP expression was measured at 515 nm.

DNA array analysis.
ABT minimal medium supplemented with 0·5 % Casamino acids (200 ml) was inoculated with exponentially growing Ps. aeruginosa PAO1 cells (OD600<0·5) at an OD600 of 0·05. At an OD600 of 0·7, the culture was split into two 100 ml cultures, which were grown on in 500 ml conical flasks on an orbital air shaker operating at 200 r.p.m. at 37 °C. To one culture QSI compound was added (at a non-growth-inhibitory concentration); the second culture served as an untreated control. Patulin was added to 8 µM and penicillic acid was added to 147 µM, with both treated and untreated cultures showing similar growth rates. Samples were retrieved at OD600 2·0, mixed with 2 vols RNA Later (Ambion) and stored at –80 °C until RNA extraction. RNA extraction was performed with the Qiagen RNeasy Purification kit, using the bacterial protocol. To remove all DNA, the purified RNA was treated for 1 h with 11 U DNase I; RNA was then retrieved by using the Qiagen RNeasy Purification kit. cDNA was synthesized by mixing 10 µg RNA with 250 ng random primers (Invitrogen Life Technologies) in a total volume of 30 µl. The rest of the assay was performed according to the protocol supplied by Affymetrix.

Preparation of polymorphonuclear leukocytes (PMNs).
Human blood samples were obtained from normal healthy volunteers by venous puncture, and collected in BD Vacutainers coated with heparin and lithium (Becton-Dickinson, 388330). The blood was mixed with dextran (T-500), 1 : 5, and the erythrocytes were sedimented for 40 min. The supernatant was applied to Lymphoprep (Axis-Shield Poc.) and centrifuged at 800 g for 15 min at 5 °C. The supernatant was discarded and the PMN neutrophils were treated with 2 ml 0·2 % NaCl in order to lyse remaining erythrocytes. Lysis was terminated by adding 2 ml 1·6 % NaCl and 6 ml Eagle-MEM (Bie & Berntsen). The cells were centrifuged at 350 g for 10 min at 5 °C, the supernatant was discarded and the PMNs were resuspended in Eagle-MEM.

Biofilm assays.
Biofilms were grown in continuous culture, once-through flow chambers, perfused with sterile ABtrace minimal medium containing 0·3 mM glucose. Patulin (1·2 µg ml–1) or penicillic acid (1·2 µg ml–1) was added to the medium when appropriate. Biofilm development was examined by confocal scanning laser microscopy (CSLM) using a Zeiss LSM 510 system (Carl Zeiss) equipped with an argon laser and a helium-neon laser for excitation of fluorophores. Bacterial viability in biofilm cultures was assessed by using the BacLight live/dead staining kit (Molecular Probes) as described elsewhere (Huber et al., 2001).

PMN treatment of biofilms.
In order to inoculate PMNs into the biofilm chambers, the flow was stopped and the flow cells were clamped off. PMNs in the order of 1·5x106 were inoculated in each flow channel. The flow cells were incubated top down in a 37 °C water bath with shaking, until microscopic inspection.

Monitoring the oxidative burst of PMNs.
Isolated PMNs were incubated for approximately 30 min in Eagle-MEM (3x107 cells ml–1) with 10 % normal human AB serum, 5 µM SYTO 62 (Molecular Probes) to stain the nuclei (dsDNA). For detecting the oxidative burst of the PMNs 0·1 mg dihydrorhodamine 123 (123-DHR) ml–1 (D-1054, Sigma) was added to the PMNs in order to stain the H2O2 in the phagosomes (Bassoe et al., 2003). One hundred microlitres of the PMN mixture was added to each biofilm.

Experimental animals.
Female BALB/c mice were purchased from M&B Laboratory Animals at 10–11 weeks of age. The mice were of equal size and were maintained on standard mouse chow and water ad libitum for 1 week before challenge. All animal experiments were authorized by the National Animal Ethics Committee.

Immobilization of Ps. aeruginosa in seaweed alginate beads.
Ps. aeruginosa was immobilized in seaweed alginate beads as described by Pedersen et al. (1990). The suspension was adjusted to 2·5x108 c.f.u. ml–1 and confirmed by colony counts; 0·04 ml of the suspension was inoculated into the left lung of each mouse.

Challenge procedure.
The mice were anaesthetized by subcutaneous injection of 0·2 ml Hyp/Mid [2·5 mg Hypnorm ml–1 (Janssen) and 1·25 mg midazolam ml–1 (Roche) in sterile water]. Sedated mice were immobilized and the trachea was exposed and penetrated with an 18G needle. The inoculum was introduced into the left lung approximately 11 mm from the tracheal penetration site with a bead-tipped needle (Moser et al., 1997). The incision was sutured with silk and healed without any complications. Pentobarbital (DAK), 2·0 ml per kg body weight, was used to kill the animals, 5 days after infection.

Bacteriology.
Lungs from mice were prepared for bacteriological examination as described by Moser et al. (1997). Isolated lungs were homogenized on ice. A serial dilution of the lung homogenate was performed and plated on blue agar plates (Statens Serum Institut, Copenhagen, Denmark), selective for Gram-negative bacilli, for colony counting (Hoiby, 1974).


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Screening of Penicillium extracts
Extracts of the fungi (Table 1) were prepared as described in Methods. As an initial, promiscuous screen, we employed the QSIS1 selector, which identifies inhibitors of the lux QS system. The selector bacterium harbours a QS-controlled gene that causes cell death when expressed. In the assay, the QSIS bacteria are cast into agar along with signal molecules that activate the killing gene. A well is made in the agar in which the test sample is placed. The sample diffuses into the agar from the well, creating a concentration gradient allowing for screening of several concentrations of the sample. Presence of a QSI is signalled by growth of the selector bacteria; therefore only non-toxic compounds are identified. The QSIS2 selector was employed as a second screen for inhibitors of the Ps. aeruginosa QS systems. It is based on a QS-controlled sacB gene which leads to cell death when expressed in the presence of sucrose (Rasmussen et al., 2005). By means of these two screens, a number of QSI-producing Penicillium species were identified (Fig. 1, Table 1).



View larger version (108K):
[in this window]
[in a new window]
 
Fig. 1. QSIS1 (top rows) and QSIS2 (bottom rows) assays on extracts from four Penicillium spp.: (a) P. olsonii, (b) P. roqueforti, (c) P. hordei and (d) P. carneum. The fungi were grown on two media, CYA (left columns) and YES (right columns). For QSIS1, a blue ring indicates presence of QSI compound(s). QSIS2 indicates presence of QSI by emitting light visualized by a photosensitive CCD camera.

 
Fractionation of extracts and identification of active compounds
In order to identify the individual fungal metabolites inhibiting the QS systems of Ps. aeruginosa, Penicillium radicicola (IBT 10696) and Penicillium coprobium (IBT 6895) were selected for further studies using microfractionation and LC-DAD-MS. QSIS1 screening of IBT 6895 showed a single active well in the microtitre plate assay of the collected fractions (Fig. 2), whereas IBT 10696 extract resulted in activity in five wells (data not shown).



View larger version (54K):
[in this window]
[in a new window]
 
Fig. 2. Screening of 96 fractions of P. coprobium (IBT6895) extract. The wells marked C contain a positive control (furanone C-30).

 
The corresponding UV data from the microfractionation samples suggested that the QSI compounds from P. radicicola and P. coprobium were penicillic acid and patulin respectively (Fig. 3). This was confirmed by LC-DAD-MS analysis of the remaining contents of the active fractions and by comparison with authentic samples. This is believed to be the first report of these two well-known fungal metabolites (Frisvad et al., 2004) acting as QSI compounds.



View larger version (5K):
[in this window]
[in a new window]
 
Fig. 3. Structures of penicillic acid (left), patulin (middle) and a general Delisea pulchra furanone structure (right).

 
The identification and presence of these two compounds probably explains why several of the other species also showed QSI activity. Recently the following terverticillate Penicillium species were reported as penicillic acid producers: P. aurantiogriseum, P. carneum, P. cyclopium, P. freii, P. melanoconidium, P. neoechinulatum, P. polonicum, P. radicicola, P. tulipae, P. viridicatum. The following species were reported as patulin producers: P. carneum, P. clavigerum, P. concentricum, P. coprobium, P. dipodomyicola, P. expansum, P. glandicola, P. gladioli, P. griseofulvum, P. marinum, P. paneum, P. sclerotigenum, P. vulpinum (Frisvad et al., 2004). However, extracts prepared from some of these fungi did not appear active in the QSI screens used in the present study. One likely explanation is that some of the fungal strains produce only trace amounts of either patulin or penicillic acid, which is therefore below the detection limit of the QSI screen. The search for compounds other than patulin and penicillic acid is in progress on other fungal species known as non- producers of patulin and penicillic acid.

Dose–response analysis of penicillic acid and patulin
Pure penicillic acid and patulin were tested in the QSIS1 and QSIS2 systems and found to be positive (data not shown). To examine the effects of these two fungal metabolites more closely, we employed a lasB–gfp(ASV) fusion harboured by Ps. aeruginosa PAO1 as a QS monitor (Hentzer et al., 2002). Being a type IV QS gene, lasB is induced in late exponential/early stationary phase (Whiteley et al., 1999). Induction by the addition of C4 HSL and 3-oxo-C12 HSL caused a burst of GFP production. Conversely, if a QSI is present the burst of GFP expression will be reduced (Hentzer et al., 2003). As the QSIS systems only provide a crude estimation of the optimal concentration of the QSI, a dilution series of the compounds was incubated with the QS monitor. Growth and green fluorescence were recorded over a period of 14 h. Upon induction, the untreated cultures increased GFP production approximately 10-fold. Addition of either penicillic acid or patulin reduced the induction of fluorescence in a concentration-dependent manner. At the highest concentrations tested (40 µM and 80 µM for patulin and penicillic acid, respectively) the induction of the QS-controlled lasB promoter was abolished. These concentrations did not affect growth rate of the cultures (Fig. 4). This suggests that the two compounds specifically block bacterial QS.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4. Dose–response curves of patulin (a) and penicillic acid (b). Dilution series were incubated with the QS monitor PAO1 (lasB–gfp) (Hentzer et al., 2002). Growth (top panels) and specific fluorescence (bottom panels, expressed as relative fluorescence units per OD450 unit) were followed over 14 h. Patulin was tested at 40 µM ({blacklozenge}), 10 µM ({blacktriangleup}), 3 µM ({blacksquare}), 0·6 µM ({bullet}) and 0 µM (+). Penicillic acid was tested at 80 µM ({blacklozenge}), 20 µM ({blacktriangleup}), 5 µM ({blacksquare}), 1 µM ({bullet}) and 0 µM (+).

 
Definition of the QS regulon
The QS regulon was previously identified by means of DNA microarray analysis of gene expression (Hentzer et al., 2003). Briefly, the QS regulon was defined as genes induced by the addition of C4 HSL and 3-oxo-C12 HSL to a lasI rhlI double mutant. It is possible, however, that the presence of high, exogenous AHL concentrations during the entire growth cycle could influence the expression profile of genes in a non-natural manner, which would then affect mapping of the QS regulon. To pursue this particular problem we chose to define the QS regulon as consistently downregulated (>5-fold) genes in both a lasI rhlI and a lasR rhlR mutant as compared to their parent wild-type (Table 2). In addition, the expression profiles of a lasR and a rhlR mutant were determined (Table 2). The 172 genes of this alternative QS regulon can be divided into four groups on the basis of their expression pattern within the four mutants. Group A contains 34 genes which require lasR for expression, i.e. they are downregulated in both the lasR and the lasR rhlR mutants. The 23 genes responsive to rhlR are located in group B. Group C consists of 92 genes which are downregulated in all three mutants. Finally, group D comprises the 23 genes that are downregulated only in the lasR rhlR double mutant; if only one of lasR or rhlR was mutated, no effect was observed. Remarkably, the genes in group D generally exhibited a lower degree of regulation (less average fold change) than those in group C. We speculate that the group D genes are only partially regulated by QS. Of the 172 genes in the alternative QS regulon, 99 are in common with the previous QS regulon defined by Hentzer et al. (2003). It must be emphasized that the previous QS regulon was based on several sample points throughout the growth cycle whereas the present QS regulon was based on a single sample point at OD600 2, where the majority of QS genes are being expressed (Hentzer et al., 2003).


View this table:
[in this window]
[in a new window]
 
Table 2. Genes downregulated in the four QS mutants and by treatment with patulin (Pat) and penicillic acid (PA)

 
Target specificity
Next, DNA microarray analysis was performed to determine the target specificity of the two QSI compounds towards the QS-regulated genes in Ps. aeruginosa. Cultures of Ps. aeruginosa PAO1 were grown to exponential phase, then split into subcultures at OD600 0·5. These cultures were either not treated or treated with 8 µM patulin and 147 µM penicillic acid, respectively (concentrations at which growth was not affected). Samples for DNA microarray analysis were taken at OD600 2·0. Absolute expression values from treated cultures or QS mutant cultures were compared with values from untreated cultures; changes in expression are reported as simple fold changes (Table 2). Genes found ‘not present’ by the Affymetrix Micro Array Suite software were excluded from the calculations. According to Hentzer et al. (2003) and Wagner et al. (2003) changes in expression below 5-fold are disregarded.

In total, patulin and penicillic acid targeted 157 and 300 genes, respectively. According to the above QS definition, 49 % of the 157 genes targeted by patulin and 34 % of the 300 targeted by penicillic acid were QS controlled. Since the number of chromosomal genes is approximately 5600, patulin and penicillic acid affected the expression of 3–5 % of all genes. QS-controlled genes are overrepresented among the genes downregulated by the compounds, indicating that they have specificity for QS-regulated genes. Of the QS regulated genes, 45 % and 60 % were affected by patulin and penicillic acid respectively. Interestingly, patulin downregulated 43 % of the group B genes, 58 % of the group C genes and 26 % of the group D genes, but only 15 % of the group A genes. This suggests that RhlR-controlled genes are preferentially targeted by patulin. Similarly, penicillic acid downregulated 65 %, 80 % and 30 % of groups B, C and D, respectively, whereas only 21 % of group A genes were downregulated. Once again this suggests that RhlR-controlled genes are preferentially targeted by penicillic acid.

Hentzer et al. (2003) found an 80 % target specificity of furanone C-30 to QS-regulated genes. However, when the same analysis was performed according to the QS regulon described above, it was found that C-30 exhibits a target specificity of 64 %. Thus, the QS target specificity of the QSIs identified in the present study is comparable to that of C-30.

Among the genes repressed by patulin and penicillic acid treatment, many encode virulence factors which had previously been reported to be controlled by QS (Hentzer et al., 2003; Schuster et al., 2003; Wagner et al., 2003; Whiteley et al., 1999). These include the genes encoding LasA protease (lasA, PA1871), LasB elastase (lasB, PA3724), chitinase (chiC, PA2300) and chitin-binding protein (chiD, PA0852), and the genes encoding phenazine biosynthesis proteins (phzC–G, PA1901–PA1905), the rhamnosyl transferase AB operon (rhlAB, PA3478–PA3479), fucose-binding lectin (lecB, PA3361) and PA-I galactophilic lectin (pa1L, PA2570). Pyoverdine production seems to be unaffected by patulin and penicillic acid, and the expression level of pvd genes was unaltered in the QS mutants; however, the PvdS-regulated endoprotease gene encoding PrpL (PA4175) was downregulated by both patulin and penicillic acid as well as in the QS mutants. PrpL is able to cleave lactoferrin, transferrin, elastin and casein, and may potentially play a role in virulence (Wilderman et al., 2001). Nouwens et al. (2003) found reduced levels of PrpL in culture supernatants of Ps. aeruginosa strains containing a las mutation, but not in rhl mutants (Nouwens et al., 2003). The expression of prpL in a lasI rhlI mutant is restored by the addition of 3-oxo-C12 HSL, which further supports the idea that expression of prpL is primarily LasR regulated (Hentzer et al., 2003; Schuster et al., 2003). It is not known if this is by direct regulation by the las QS system or via an intermediary regulator such as PvdS. The significant downregulation of chiC by patulin and penicillic acid was not unexpected considering that the major structural component of fungal cells is chitin. This suggests that the QSI properties of some mycotoxins are not just coincidental, but are instead a strategy used by some fungi to minimize damage caused by bacteria producing chitinase. A number of other genes, including osmC (PA0052, osmotically inducible protein) coxA and coxB (PA0105 and PA106, cytochrome c oxidase subunit I and II) and several genes encoding hypothetical proteins previously identified as being QS regulated (such as the operon at PA3326–PA3336) were also found to be downregulated in the presence of patulin and penicillic acid. One of these genes, fabH2, encodes a 3-oxoacyl-ACP synthase, probably involved in synthesis of the 3-oxo-C12 HSL signal molecule. This suggests that one of the modes of action by which penicillic acid inhibits QS is by preventing signal molecule synthesis. It should be noted that the QSIS1 and 2 screens select molecules which interfere with the LuxR homologue proteins; thus inhibition of AHL synthesis is just an additional feature of penicillic acid as QSI rather than its primary activity.

Genes encoding central parts of the QS circuit, such as lasR, rsaL, lasI, rhlR and rhlI (PA1430–PA1432, PA3437 and PA3476), and genes involved in the synthesis of PQS (pqsA–E, pqsH, PA0996–PA1000, PA2587) were almost unaffected by treatment with the two QSI compounds. This suggests that patulin and penicillic acid do not directly interfere with the regulatory systems controlling transcription of the lasRI and rhlRI genes, but instead act on these QS regulators at the post-transcriptional level. This suggests that the two QSI compounds interfere with the RhlR and LasR proteins.

Patulin accelerates LuxR turnover
Patulin was selected for more thorough analysis of its effect on bacterial phenotypes related to pathogenicity since it was found to actively inhibit Ps. aeruginosa QS at a concentration substantially lower than the LD50 (Hayes, 1981). Manefield et al. (2002) demonstrated the ability of furanone compounds to cause instability of the LuxR QS regulatory protein.

LuxR was overexpressed from an IPTG-inducible promoter in the presence of high concentrations of GroESL in accordance with Manefield et al. (2002). After cells were washed free of the inducer, they were treated with 3·5 µM patulin (this concentration does not affect growth). The amount of LuxR protein present in the samples was visualized after 1 h of treatment by Western blotting with a chemiluminescent LuxR antibody. Digital images of the gel reported chemoluminescence with increasingly lighter colours indicating higher band intensity. Treatment with patulin markedly decreased the amount of LuxR present in the samples (Fig. 5). As a reference, furanone C-2 was included as a control (Manefield et al., 2002). The reduced amount of LuxR correlated with the strong signal obtained in the QSIS systems and with downregulation of the fluorescent signal from a LuxR-controlled luxI–gfp fusion (data not shown). This is in accordance with a model where patulin interacts directly with QS regulators. Interestingly, a third LuxR homologue, designated QscR, has been identified in the Ps. aeruginosa chromosome (Chugani et al., 2001). This protein is a negative regulator of several QS-controlled virulence factors and is able to form multimers and heterodimers with both LasR and RhlR (Ledgham et al., 2003). No effect of the QSIs on QscR transcription was observed in our DNA array experiments (Table 2). Whether the inhibitors interact directly with QscR, as is the case for LuxR, is presently unknown. Another member of the LuxR family of transcriptional regulators, VqsR, has also been found in Ps. aeruginosa. This protein is a major virulence factor regulator and has been found to positively affect expression of QS-controlled genes (Juhas et al., 2004). Again, we have not investigated whether the QSIs interact directly with this regulator, but there is no effect on transcription of vqsR when Ps. aeruginosa is treated with penicillic acid or patulin. Interestingly, vqsR is significantly downregulated in both of the double mutants (Table 2). As we speculate that the inhibitors mainly target the RhlR protein, the lack of effect on vqsR transcription from the QSIs is not a surprising result.



View larger version (109K):
[in this window]
[in a new window]
 
Fig. 5. Western blot analysis of LuxR content using a chemiluminescent LuxA antibody. LuxR-overproducing E. coli cells were either untreated (A), or treated with 20 µM furanone C-2 (B), 100 µM furanone C-2 (C) or 3·5 µM patulin (D). Lighter colours indicate higher band density.

 
QSI effect on biofilm tolerance to tobramycin
It has previously been shown that Ps. aeruginosa biofilm cells are highly tolerant to antibiotic treatments (Anwar et al., 1990). Davies et al. (1998) demonstrated that a QS mutant of PAO1 is more susceptible to antimicrobial treatments than the wild-type counterpart. Furthermore, Hentzer et al. (2003) showed that biofilms treated with the QSI compound furanone C-30 became susceptible to both SDS and tobramycin treatment. We tested whether patulin exhibited a similar effect. Two sets of PAO1 biofilms were allowed to form in flow chambers in which the medium contained either no patulin or 8 µM patulin for 3 days. The biofilms were then challenged with tobramycin (340 µg ml–1) for 24 h. The effect of the antibiotic treatment was assessed by means of live/dead staining (Fig. 6). In the biofilm treated with tobramycin only a few cells, mainly localized in the top layer, were dead, whereas almost all the cells in the biofilm treated with both tobramycin and patulin were dead. Treatment with patulin alone did not affect development of the biofilm (data not shown).



View larger version (58K):
[in this window]
[in a new window]
 
Fig. 6. Biofilms formed by Ps. aeruginosa in the absence (left) or presence (right) of 1·2 µg patulin ml–1 and treated with tobramycin for 24 h. The cells are stained with the Baclight live/dead kit, which stains live cells green and dead cells red.

 
PMN activation is affected by a QS-controlled mechanism
In recent work by Bjarnsholt et al. (2005) it was found that the activation of neutrophile PMNs by Ps. aeruginosa is controlled by QS. A lasR rhlR mutant activated PMNs whereas the wild-type Ps. aeruginosa PAO1 failed to do so. In addition, when the wild-type was treated with furanone C-30, activation was observed. We wanted to investigate if the QS inhibitory effect observed by patulin and penicillic acid created a similar increase in activation of PMNs. Biofilms of wild-type Ps. aeruginosa PAO1 were grown for 3 days with and without 1·2 µg ml–1 patulin and penicillic acid. PMNs were injected into the biofilm flow chambers and development of oxidative burst was recorded after 2 h by 123-DHR staining (Fig. 7). A marked difference in green fluorescence signal intensity was observed for the PMNs incubated on the wild-type biofilms compared to the QSI-treated biofilms. These experiments suggest that a QSI-treated biofilm, in contrast to its untreated counterpart, fully activates the PMNs to produce H2O2.



View larger version (77K):
[in this window]
[in a new window]
 
Fig. 7. Biofilms formed by Ps. aeruginosa PAO1 in the absence of QSI (a), and in the presence of 1·2 µg patulin ml–1 (b) or 1·2 µg penicillic acid ml–1 (c). Freshly isolated PMNs were exposed to biofilm bacteria in the biofilm flow chambers and stained with 123-DHR. PMNs developing oxidative burst appear green fluorescent. Bars, 50 µm.

 
Ps. aeruginosa in mouse lungs is cleared rapidly when treated with patulin
We have shown that a functional QS system plays a major role in the persistence of a pulmonary infection in mice with respect to both clearance of the bacteria and the onset of the innate immune defence (Bjarnsholt et al., 2005). We investigated whether patulin could promote a faster clearing of bacteria in mice, as seen with other QSIs (Hentzer et al., 2003; Wu et al., 2004; Rasmussen et al., 2005). BALB/c mice were used to establish a pulmonary model of chronic lung infection. The immune response of the BALB/c mice is Th-2 dominated (Moser et al., 1999), resembling the immune response of cystic fibrosis patients to Ps. aeruginosa pulmonary infection (Moser et al., 2000). In order to study the effect of patulin in a pulmonary infection model two groups of 72 mice were treated with either patulin or placebo (saline). The treatments were given as a subcutaneous injection every 24 h for 7 days, administering 2·5 µg per g body weight (~16 µM; the average mouse volume is taken as 20 ml). After 1 day of prophylactic treatment, the mice were intratracheally challenged with alginate beads containing Ps. aeruginosa (Pedersen et al., 1990). After the challenge, a higher mortality was observed for the placebo-treated group on day 1: 12 died compared to 5 in the patulin-treated group; however, the difference was not found to be statistically significant.

Clearance of bacteria from the lungs was assessed on day 1 and day 3 after bacterial challenge. On day 1 there was only a slight difference between the two groups, with the patulin-treated group showing faster clearance. On day 3, a 20-fold lower bacterial content was observed with the patulin-treated group, suggesting that the treatment promoted clearing compared to the placebo (P<0·04) This is in accordance with the results of Rasmussen et al. (2005), Hentzer et al. (2003) and Wu et al. (2004) using other QSI compounds.

Conclusions
A recently published screening system, the QSI selector, was used to screen extracts prepared from 50 Penicillium species. Approximately 33 % of the extracts contained potential QSIs. The extracts from P. radicicola and P. coprobium were subjected to further analysis, which showed that the QSI produced by P. radicicola was penicillic acid and the QSI produced by P. coprobium was patulin. The ability to inhibit QS was verified by DNA array analysis, which suggested that penicillic acid and patulin targeted the RhlR and the LasR proteins. Ps. aeruginosa biofilms formed in the presence of patulin were susceptible to tobramycin treatment, whereas control biofilms were tolerant. Furthermore, the wild-type Ps. aeruginosa biofilms formed in the presence of patulin induced oxidative burst in PMNs that settled on top of the biofilm bacteria. The reduction in virulence factor expression and activation of PMN oxidative burst may explain why patulin accelerated the clearance of Ps. aeruginosa from the lungs of infected mice. Even though the two mycotoxins are not currently drug lead compounds, this work demonstrates that fungi produce QSIs. Ongoing work in our laboratories is aimed at identifying non-toxin QSIs produced by fungi.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Danish Technical Research Council, the Villum Kann-Rasmussen Foundation to M. G., and from the Cystic Fibrosis Foundation, Therapeutics Inc. and the German Mukoviszidose e.V. to M. G. and L. E. The technical assistance of Linda Stabell was greatly appreciated.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Anwar, H., Dasgupta, M. K. & Costerton, J. W. (1990). Testing the susceptibility of bacteria in biofilms to antibacterial agents. Antimicrob Agents Chemother 34, 2043–2046.[Medline]

Bassoe, C. F., Li, N., Ragheb, K., Lawler, G., Sturgis, J. & Robinson, J. P. (2003). Investigations of phagosomes, mitochondria, and acidic granules in human neutrophils using fluorescent probes. Cytometry 51, 21–29.[CrossRef][Medline]

Beatson, S. A., Whitchurch, C. B., Semmler, A. B. & Mattick, J. S. (2002a). Quorum sensing is not required for twitching motility in Pseudomonas aeruginosa. J Bacteriol 184, 3598–3604.[Abstract/Free Full Text]

Beatson, S. A., Whitchurch, C. B., Sargent, J. L., Levesque, R. C. & Mattick, J. S. (2002b). Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa. J Bacteriol 184, 3605–3613.[Abstract/Free Full Text]

Bjarnsholt, T., Jensen, P. O., Burmolle, M., Hentzer, M., Haagensen, J. A., Hougen, H. P., Calum, H., Madsen, K. G., Moser, C., Molin, S., Holby, N. & Givskov, M. (2005). Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent. Microbiology 151, 373–383.[CrossRef][Medline]

Camara, M., Williams, P. & Hardman, A. (2002). Controlling infection by tuning in and turning down the volume of bacterial small-talk. Lancet Infect Dis 2, 667–676.[CrossRef][Medline]

Chhabra, S. R., Stead, P., Bainton, N. J., Salmond, G. P., Stewart, G. S., Williams, P. & Bycroft, B. W. (1993). Autoregulation of carbapenem biosynthesis in Erwinia carotovora by analogues of N-(3-oxohexanoyl)-L-homoserine lactone. J Antibiot 46, 441–454.[Medline]

Chugani, S. A., Whiteley, M., Lee, K. M., D'Argenio, D., Manoil, C. & Greenberg, E. P. (2001). QscR, a modulator of quorum-sensing signal synthesis and virulence in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 98, 2752–2757.[Abstract/Free Full Text]

Clark, D. J. & Maaløe, O. (1967). DNA replication and the division cycle Escherichia coli. J Mol Biol 23, 99–112.[CrossRef]

Costerton, J. W., Stewart, P. S. & Greenberg, E. P. (1999). Bacterial biofilms: a common cause of persistent infections. Science 284, 1318–1322.[Abstract/Free Full Text]

Davies, D. G., Parsek, M. R., Pearson, J. P., Iglewski, B. H., Costerton, J. W. & Greenberg, E. P. (1998). The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280, 295–298.[Abstract/Free Full Text]

Dworjanyn, S. A. & Steinberg, P. (1999). Localisation and surface quantification of secondary metabolites in the red alga Delisea pulchra. Mar Biol 133, 727–736.[CrossRef]

Finch, R. G., Pritchard, D. I., Bycroft, B. W., Williams, P. & Stewart, G. S. (1998). Quorum sensing: a novel target for anti-infective therapy. J Antimicrob Chemother 42, 569–571.[Free Full Text]

Frisvad, J. C. & Filtenborg, O. (1983). Classification of terverticillate penicillia based on profiles of mycotoxins and other secondary metabolites. Appl Environ Microbiol 46, 1301–1310.[Medline]

Frisvad, J. C., Smedsgaard, J., Larsen, T. O. & Samson, R. A. (2004). Mycotoxins, drugs and other extrolites produced by species in Penicillium subgenus Penicillium. Stud Mycol 49, 1–174.

Fuqua, W. C., Winans, S. C. & Greenberg, E. P. (1994). Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. J Bacteriol 176, 269–275.[Medline]

Givskov, M., de Nys, R., Manefield, M., Gram, L., Maximilien, R., Eberl, L., Molin, S., Steinberg, P. D. & Kjelleberg, S. (1996). Eukaryotic interference with homoserine lactone-mediated prokaryotic signalling. J Bacteriol 178, 6618–6622.[Abstract/Free Full Text]

Habeck, M. (2003). Stop talking at the back. Drug Discov Today 8, 279–280.[CrossRef][Medline]

Hayes, A. W. (1981). Mycotoxin Teratogenicity and Mutagenicity. New York: CRC Press.

Hentzer, M. & Givskov, M. (2003). Pharmacological inhibition of quorum sensing for the treatment of chronic bacterial infections. J Clin Invest 112, 1300–1307.[Abstract/Free Full Text]

Hentzer, M., Riedel, K., Rasmussen, T. B. & 9 other authors (2002). Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound. Microbiology 148, 87–102.[Medline]

Hentzer, M., Wu, H., Andersen, J. B. & 15 other authors (2003). Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors. EMBO J 22, 3803–3815.[Abstract/Free Full Text]

Hoiby, N. (1974). Epidemiological investigations of the respiratory tract bacteriology in patients with cystic fibrosis. Acta Pathol Microbiol Scand [B] Microbiol Immunol 82, 541–550.[Medline]

Hoiby, N. (2000). Prospects for the prevention and control of pseudomonal infection in children with cystic fibrosis. Paediatr Drugs 2, 451–463.[Medline]

Hoiby, N. & Koch, C. (2000). Maintenance treatment of chronic Pseudomonas aeruginosa infection in cystic fibrosis. Thorax 55, 349–350.[Free Full Text]

Huber, B., Riedel, K., Hentzer, M., Heydorn, A., Gotschlich, A., Givskov, M., Molin, S. & Eberl, L. (2001). The cep quorum-sensing system of Burkholderia cepacia H111 controls biofilm formation and swarming motility. Microbiology 147, 2517–2528.[Medline]

Juhas, M., Wiehlmann, L., Huber, B. & 8 other authors (2004). Global regulation of quorum sensing and virulence by VqsR in Pseudomonas aeruginosa. Microbiology 150, 831–841.[CrossRef][Medline]

Kjelleberg, S., Steinberg, P., Givskov, M., Gram, L., Manefield, M. & de Nys, R. (1997). Do marine natural products interfere with prokaryotic AHL regulatory systems? Aquat Microb Ecol 13, 85–93.

Ledgham, F., Ventre, I., Soscia, C., Foglino, M., Sturgis, J. N. & Lazdunski, A. (2003). Interactions of the quorum sensing regulator QscR: interaction with itself and the other regulators of Pseudomonas aeruginosa LasR and RhlR. Mol Microbiol 48, 199–210.[CrossRef][Medline]

Manefield, M., Harris, L., Rice, S. A., de Nys, R. & Kjelleberg, S. (2000). Inhibition of luminescence and virulence in the black tiger prawn (Penaeus monodon) pathogen Vibrio harveyi by intercellular signal antagonists. Appl Environ Microbiol 66, 2079–2084.[Abstract/Free Full Text]

Manefield, M., Rasmussen, T. B., Henzter, M., Andersen, J. B., Steinberg, P., Kjelleberg, S. & Givskov, M. (2002). Halogenated furanones inhibit quorum sensing through accelerated LuxR turnover. Microbiology 148, 1119–1127.[Medline]

Maximilien, R., de Nys, R., Holmstrom, C., Gram, L., Givskov, M., Kjelleberg, S. & Steinberg, P. (1998). Chemical mediation of bacterial surface colonisation by secondary metabolites from the red alga Delisea pulchra. Aquat Microb Ecol 15, 233–246.

Moser, C., Johansen, H. K., Song, Z., Hougen, H. P., Rygaard, J. & Hoiby, 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.[Medline]

Moser, C., Hougen, H. P., Song, Z., Rygaard, J., Kharazmi, A. & Hoiby, N. (1999). Early immune response in susceptible and resistant mice strains with chronic Pseudomonas aeruginosa lung infection determines the type of T-helper cell response. APMIS 107, 1093–1100.[Medline]

Moser, C., Kjaergaard, S., Pressler, T., Kharazmi, A., Koch, C. & Hoiby, N. (2000). The immune response to chronic Pseudomonas aeruginosa lung infection in cystic fibrosis patients is predominantly of the Th2 type. APMIS 108, 329–335.[CrossRef][Medline]

Nouwens, A. S., Beatson, S. A., Whitchurch, C. B., Walsh, B. J., Schweizer, H. P., Mattick, J. S. & Cordwell, S. J. (2003). Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1. Microbiology 149, 1311–1322.[CrossRef][Medline]

Olsen, J. A., Severinsen, R., Rasmussen, T. B., Hentzer, M., Givskov, M. & Nielsen, J. (2002). Synthesis of new 3- and 4-substituted analogues of acyl homoserine lactone quorum sensing autoinducers. Bioorg Med Chem Lett 12, 325–328.[CrossRef][Medline]

Passador, L., Tucker, K. D., Guertin, K. R., Journet, M. P., Kende, A. S. & Iglewski, B. H. (1996). Functional analysis of the Pseudomonas aeruginosa autoinducer PAI. J Bacteriol 178, 5995–6000.[Abstract/Free Full Text]

Pedersen, S. S., Shand, G. H., Hansen, B. L. & Hansen, G. N. (1990). Induction of experimental chronic Pseudomonas aeruginosa lung infection with P. aeruginosa entrapped in alginate microspheres. APMIS 98, 203–211.[Medline]

Pritt, J. I. (1979). The Genus Penicillium and its Teleomorphic States Eupenicillium and Taleromyces. London: Academic Press.

Rasmussen, T. B., Manefield, M., Andersen, J. B., Eberl, L., Anthoni, U., Christophersen, C., Steinberg, P., Kjelleberg, S. & Givskov, M. (2000). How Delisea pulchra furanones affect quorum sensing and swarming motility in Serratia liquefaciens MG1. Microbiology 146, 3237–3244.[Medline]

Rasmussen, T. B., Bjarnsholt, T., Skindersoe, M. E., Hentzer, M., Kristoffersen, P., Köte, M., Nielsen, J., Eberl, L. & Givskov, M. (2005). Screening for quorum sensing inhibitors using a novel genetic system – the QSI selector. J Bacteriol 187, 1799–1814.[Abstract/Free Full Text]

Reverchon, S., Chantegrel, B., Deshayes, C., Doutheau, A. & Cotte-Pattat, N. (2002). New synthetic analogues of N-acyl homoserine lactones as agonists or antagonists of transcriptional regulators involved in bacterial quorum sensing. Bioorg Med Chem Lett 12, 1153–1157.[CrossRef][Medline]

Salmond, G. P., Bycroft, B. W., Stewart, G. S. & Williams, P. (1995). The bacterial ‘enigma’: cracking the code of cell-cell communication. Mol Microbiol 16, 615–624.[CrossRef][Medline]

Schaefer, A. L., Hanzelka, B. L., Eberhard, A. & Greenberg, E. P. (1996). Quorum sensing in Vibrio fischeri: probing autoinducer-LuxR interactions with autoinducer analogs. J Bacteriol 178, 2897–2901.[Abstract/Free Full Text]

Schuster, M., Lostroh, C. P., Ogi, T. & Greenberg, E. P. (2003). Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J Bacteriol 185, 2066–2079.[Abstract/Free Full Text]

Smedsgaard, J. (1997). Micro-scale extraction procedure for standardized screening of fungal metabolite production in cultures. J Chromatogr A 760, 264–270.[CrossRef][Medline]

Smith, R. S. & Iglewski, B. H. (2003). Pseudomonas aeruginosa quorum sensing as a potential antimicrobial target. J Clin Invest 112, 1460–1465.[Abstract/Free Full Text]

Smith, K. M., Bu, Y. & Suga, H. (2003a). Induction and inhibition of Pseudomonas aeruginosa quorum sensing by synthetic autoinducer analogs. Chem Biol 10, 81–89.[CrossRef][Medline]

Smith, K. M., Bu, Y. & Suga, H. (2003b). Library screening for synthetic agonists and antagonists of a Pseudomonas aeruginosa autoinducer. Chem Biol 10, 563–571.[CrossRef][Medline]

Steinberg, P. D., Schneider, R. & Kjelleberg, S. (1997). Chemical defences of seaweeds against microbial colonization. Biodegradation 8, 211–220.[CrossRef]

Van Delden, C. & Iglewski, B. H. (1998). Cell-to-cell signalling and Pseudomonas aeruginosa infections. Emerg Infect Dis 4, 551–560.[Medline]

Wagner, V. E., Bushnell, D., Passador, L., Brooks, A. I. & Iglewski, B. H. (2003). Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment. J Bacteriol 185, 2080–2095.[Abstract/Free Full Text]

Whiteley, M., Lee, K. M. & Greenberg, E. P. (1999). Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 96, 13904–13909.[Abstract/Free Full Text]

Wilderman, P. J., Vasil, A. I., Johnson, Z., Wilson, M. J., Cunliffe, H. E., Lamont, I. L. & Vasil, M. L. (2001). Characterization of an endoprotease (PrpL) encoded by a PvdS-regulated gene in Pseudomonas aeruginosa. Infect Immun 69, 5385–5394.[Abstract/Free Full Text]

Wu, H., Song, Z., Hentzer, M., Andersen, J. B., Molin, S., Givskov, M. & Hoiby, N. (2004). Synthetic furanones inhibit quorum-sensing and enhance bacterial clearance in Pseudomonas aeruginosa lung infection in mice. J Antimicrob Chemother 53, 1054–1061.[Abstract/Free Full Text]

Zhu, J., Beaber, J. W., More, M. I., Fuqua, C., Eberhard, A. & Winans, S. C. (1998). Analogues of the autoinducer 3-oxooctanoyl-homoserine lactone strongly inhibit activity of the TraR protein of Agrobacterium tumefaciens. J Bacteriol 180, 5398–5405.[Abstract/Free Full Text]

Received 19 October 2004; revised 21 January 2005; accepted 25 January 2005.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Rasmussen, T. B.
Articles by Givskov, M.
Articles citing this Article
PubMed
PubMed Citation
Articles by Rasmussen, T. B.
Articles by Givskov, M.
Agricola
Articles by Rasmussen, T. B.
Articles by Givskov, M.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2005 Society for General Microbiology.