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
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ABSTRACT |
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INTRODUCTION |
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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 -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.
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METHODS |
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The Ps. aeruginosa used for in vivo experiments was obtained from B. Iglewski, University of Rochester, Medical Center, Rochester, NY, USA. The 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 l1) 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.
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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 min1 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 min1 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 doseresponse 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 ml1) or penicillic acid (1·2 µg ml1) 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 ml1) 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) ml1 (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 1011 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. ml1 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 ml1 (Janssen) and 1·25 mg midazolam ml1 (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
).
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RESULTS AND DISCUSSION |
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Doseresponse 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 lasBgfp(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.
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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 35 % 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 (phzCG, PA1901PA1905), the rhamnosyl transferase AB operon (rhlAB, PA3478PA3479), 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 PA3326PA3336) 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 (PA1430PA1432, PA3437 and PA3476), and genes involved in the synthesis of PQS (pqsAE, pqsH, PA0996PA1000, 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 luxIgfp 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.
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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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
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, 2129.[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, 35983604.
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, 36053613.
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, 373383.[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, 667676.[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, 441454.[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, 27522757.
Clark, D. J. & Maaløe, O. (1967). DNA replication and the division cycle Escherichia coli. J Mol Biol 23, 99112.[CrossRef]
Costerton, J. W., Stewart, P. S. & Greenberg, E. P. (1999). Bacterial biofilms: a common cause of persistent infections. Science 284, 13181322.
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, 295298.
Dworjanyn, S. A. & Steinberg, P. (1999). Localisation and surface quantification of secondary metabolites in the red alga Delisea pulchra. Mar Biol 133, 727736.[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, 569571.
Frisvad, J. C. & Filtenborg, O. (1983). Classification of terverticillate penicillia based on profiles of mycotoxins and other secondary metabolites. Appl Environ Microbiol 46, 13011310.[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, 1174.
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, 269275.[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, 66186622.
Habeck, M. (2003). Stop talking at the back. Drug Discov Today 8, 279280.[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, 13001307.
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, 87102.[Medline]
Hentzer, M., Wu, H., Andersen, J. B. & 15 other authors (2003). Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors. EMBO J 22, 38033815.
Hoiby, N. (1974). Epidemiological investigations of the respiratory tract bacteriology in patients with cystic fibrosis. Acta Pathol Microbiol Scand [B] Microbiol Immunol 82, 541550.[Medline]
Hoiby, N. (2000). Prospects for the prevention and control of pseudomonal infection in children with cystic fibrosis. Paediatr Drugs 2, 451463.[Medline]
Hoiby, N. & Koch, C. (2000). Maintenance treatment of chronic Pseudomonas aeruginosa infection in cystic fibrosis. Thorax 55, 349350.
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, 25172528.[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, 831841.[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, 8593.
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, 199210.[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, 20792084.
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, 11191127.[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, 233246.
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, 838842.[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, 10931100.[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, 329335.[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, 13111322.[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, 325328.[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, 59956000.
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, 203211.[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, 32373244.[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, 17991814.
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, 11531157.[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, 615624.[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, 28972901.
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, 20662079.
Smedsgaard, J. (1997). Micro-scale extraction procedure for standardized screening of fungal metabolite production in cultures. J Chromatogr A 760, 264270.[CrossRef][Medline]
Smith, R. S. & Iglewski, B. H. (2003). Pseudomonas aeruginosa quorum sensing as a potential antimicrobial target. J Clin Invest 112, 14601465.
Smith, K. M., Bu, Y. & Suga, H. (2003a). Induction and inhibition of Pseudomonas aeruginosa quorum sensing by synthetic autoinducer analogs. Chem Biol 10, 8189.[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, 563571.[CrossRef][Medline]
Steinberg, P. D., Schneider, R. & Kjelleberg, S. (1997). Chemical defences of seaweeds against microbial colonization. Biodegradation 8, 211220.[CrossRef]
Van Delden, C. & Iglewski, B. H. (1998). Cell-to-cell signalling and Pseudomonas aeruginosa infections. Emerg Infect Dis 4, 551560.[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, 20802095.
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, 1390413909.
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, 53855394.
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, 10541061.
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, 53985405.
Received 19 October 2004;
revised 21 January 2005;
accepted 25 January 2005.
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