Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors
2003; Springer Nature; Volume: 22; Issue: 15 Linguagem: Inglês
10.1093/emboj/cdg366
ISSN1460-2075
Autores Tópico(s)Antimicrobial Resistance in Staphylococcus
ResumoArticle1 August 2003free access Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors Morten Hentzer Morten Hentzer BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark Search for more papers by this author Hong Wu Hong Wu Department of Clinical Microbiology, Rigshospitalet, DK-2100 Copenhagen Ø, Denmark Search for more papers by this author Jens Bo Andersen Jens Bo Andersen BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark Search for more papers by this author Kathrin Riedel Kathrin Riedel Lehrstuhl für Mikrobiologie, Technische Universität München, D-85350 Freising, Germany Search for more papers by this author Thomas B. Rasmussen Thomas B. Rasmussen BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark Search for more papers by this author Niels Bagge Niels Bagge Department of Bacteriology, Institute of Medical Microbiology and Immunology, University of Copenhagen, Denmark Search for more papers by this author Naresh Kumar Naresh Kumar School of Chemical Sciences, University of New South Wales, NSW 2052 Australia Search for more papers by this author Mark A. Schembri Mark A. Schembri BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark Search for more papers by this author Zhijun Song Zhijun Song Department of Clinical Microbiology, Rigshospitalet, DK-2100 Copenhagen Ø, Denmark Search for more papers by this author Peter Kristoffersen Peter Kristoffersen BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark Search for more papers by this author Mike Manefield Mike Manefield Centre for Ecology and Hydrology Oxford, Mansfield Road, Oxford, OX1 3SR UK Search for more papers by this author John W. Costerton John W. Costerton Center for Biofilm Engineering, Montana State University, Bozeman, MT, 59717 USA Search for more papers by this author Søren Molin Søren Molin BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark Search for more papers by this author Leo Eberl Leo Eberl Lehrstuhl für Mikrobiologie, Technische Universität München, D-85350 Freising, Germany Search for more papers by this author Peter Steinberg Peter Steinberg Center for Marine Biofouling and Bioinnovation, University of New South Wales, NSW 2052 Australia Search for more papers by this author Staffan Kjelleberg Staffan Kjelleberg Center for Marine Biofouling and Bioinnovation, University of New South Wales, NSW 2052 Australia Search for more papers by this author Niels Høiby Niels Høiby Department of Clinical Microbiology, Rigshospitalet, DK-2100 Copenhagen Ø, Denmark Department of Bacteriology, Institute of Medical Microbiology and Immunology, University of Copenhagen, Denmark Search for more papers by this author Michael Givskov Corresponding Author Michael Givskov BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark Search for more papers by this author Morten Hentzer Morten Hentzer BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark Search for more papers by this author Hong Wu Hong Wu Department of Clinical Microbiology, Rigshospitalet, DK-2100 Copenhagen Ø, Denmark Search for more papers by this author Jens Bo Andersen Jens Bo Andersen BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark Search for more papers by this author Kathrin Riedel Kathrin Riedel Lehrstuhl für Mikrobiologie, Technische Universität München, D-85350 Freising, Germany Search for more papers by this author Thomas B. Rasmussen Thomas B. Rasmussen BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark Search for more papers by this author Niels Bagge Niels Bagge Department of Bacteriology, Institute of Medical Microbiology and Immunology, University of Copenhagen, Denmark Search for more papers by this author Naresh Kumar Naresh Kumar School of Chemical Sciences, University of New South Wales, NSW 2052 Australia Search for more papers by this author Mark A. Schembri Mark A. Schembri BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark Search for more papers by this author Zhijun Song Zhijun Song Department of Clinical Microbiology, Rigshospitalet, DK-2100 Copenhagen Ø, Denmark Search for more papers by this author Peter Kristoffersen Peter Kristoffersen BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark Search for more papers by this author Mike Manefield Mike Manefield Centre for Ecology and Hydrology Oxford, Mansfield Road, Oxford, OX1 3SR UK Search for more papers by this author John W. Costerton John W. Costerton Center for Biofilm Engineering, Montana State University, Bozeman, MT, 59717 USA Search for more papers by this author Søren Molin Søren Molin BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark Search for more papers by this author Leo Eberl Leo Eberl Lehrstuhl für Mikrobiologie, Technische Universität München, D-85350 Freising, Germany Search for more papers by this author Peter Steinberg Peter Steinberg Center for Marine Biofouling and Bioinnovation, University of New South Wales, NSW 2052 Australia Search for more papers by this author Staffan Kjelleberg Staffan Kjelleberg Center for Marine Biofouling and Bioinnovation, University of New South Wales, NSW 2052 Australia Search for more papers by this author Niels Høiby Niels Høiby Department of Clinical Microbiology, Rigshospitalet, DK-2100 Copenhagen Ø, Denmark Department of Bacteriology, Institute of Medical Microbiology and Immunology, University of Copenhagen, Denmark Search for more papers by this author Michael Givskov Corresponding Author Michael Givskov BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark Search for more papers by this author Author Information Morten Hentzer1, Hong Wu2, Jens Bo Andersen1, Kathrin Riedel3, Thomas B. Rasmussen1, Niels Bagge4, Naresh Kumar5, Mark A. Schembri1, Zhijun Song2, Peter Kristoffersen1, Mike Manefield6, John W. Costerton7, Søren Molin1, Leo Eberl3, Peter Steinberg8, Staffan Kjelleberg8, Niels Høiby2,4 and Michael Givskov 1 1BioCentrum-DTU, Technical University of Denmark, DK-2800 Lyngby, Denmark 2Department of Clinical Microbiology, Rigshospitalet, DK-2100 Copenhagen Ø, Denmark 3Lehrstuhl für Mikrobiologie, Technische Universität München, D-85350 Freising, Germany 4Department of Bacteriology, Institute of Medical Microbiology and Immunology, University of Copenhagen, Denmark 5School of Chemical Sciences, University of New South Wales, NSW 2052 Australia 6Centre for Ecology and Hydrology Oxford, Mansfield Road, Oxford, OX1 3SR UK 7Center for Biofilm Engineering, Montana State University, Bozeman, MT, 59717 USA 8Center for Marine Biofouling and Bioinnovation, University of New South Wales, NSW 2052 Australia *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:3803-3815https://doi.org/10.1093/emboj/cdg366 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Traditional treatment of infectious diseases is based on compounds that kill or inhibit growth of bacteria. A major concern with this approach is the frequent development of resistance to antibiotics. The discovery of communication systems (quorum sensing systems) regulating bacterial virulence has afforded a novel opportunity to control infectious bacteria without interfering with growth. Compounds that can override communication signals have been found in the marine environment. Using Pseudomonas aeruginosa PAO1 as an example of an opportunistic human pathogen, we show that a synthetic derivate of natural furanone compounds can act as a potent antagonist of bacterial quorum sensing. We employed GeneChip® microarray technology to identify furanone target genes and to map the quorum sensing regulon. The transcriptome analysis showed that the furanone drug specifically targeted quorum sensing systems and inhibited virulence factor expression. Application of the drug to P.aeruginosa biofilms increased bacterial susceptibility to tobramycin and SDS. In a mouse pulmonary infection model, the drug inhibited quorum sensing of the infecting bacteria and promoted their clearance by the mouse immune response. Introduction As the 21st century commences, it is becoming increasingly apparent that the 20th century, which opened with the promise of the eradication of most infectious diseases, closed with the specter of re-emergence of many deadly infectious diseases. It has become clear that bacteria can adapt by mutation to the selective pressure imposed by antibiotics. Today, a global concern has emerged that we are entering a post-antibiotic era with a reduced capability to combat microbes (Geddes, 2000). A large number of opportunistic pathogenic Gram-negative bacteria employ N-acylhomoserine lactone (AHL) as their command language to coordinate population behavior during invasion and colonization of higher organisms (Hentzer et al., 2002a). The communication systems share common regulatory features: AHL signal molecules are synthesized from precursors by a synthase protein I and interact with transcriptional activator proteins R to regulate expression of target genes. AHL signaling is often referred to as quorum sensing (QS) because the system enables a given bacterial species to sense when a critical (i.e. quorate) population density has been reached in the host and in response activate expression of target genes required for succession (Fuqua et al., 1994). QS-controlled genes often encode virulence factors and gene products required for bacteria–host interactions (Pirhonen et al., 1993; Parsek and Greenberg, 2000; Pearson et al., 2000). More importantly, there is growing evidence that QS influences more complex behavioral processes such as the ability to form surface-associated, structured and cooperative consortia referred to as biofilms (Davies et al., 1998; Eberl et al., 1999; Huber et al., 2001). Biofilm formation plays an important role in bacterial pathogenesis and is a common cause of persistent infections (Costerton et al., 1999; Høiby et al., 2001; Middleton et al., 2002; Singh et al., 2002). Biofilm bacteria are resistant to disinfectants, antibiotics and the action of host immune defenses (Koch and Høiby, 1993; Costerton et al., 1999). Pseudomonas aeruginosa is an increasingly prevalent opportunistic human pathogen and is the most common Gram-negative bacterium found in nosocomial and life-threatening infections of immunocompromised patients (Van Delden and Iglewski, 1998). Patients with cystic fibrosis are especially disposed to P.aeruginosa infections, and for these persons the bacterium is responsible for high rates of morbidity and mortality (Høiby and Frederiksen, 2000; Lyczak et al., 2002). Pseudomonas aeruginosa possesses two QS systems: the LasR–LasI and the RhlR–RhlI, with the cognate signal molecules N-(3-oxo-dodecanoyl)-L-homoserine lactone (OdDHL) and N-buturyl-L-homoserine lactone (BHL), respectively. The two QS circuits orchestrate a symphony of virulence factors such as exoproteases, siderophores, exotoxins and several secondary metabolites (Passador et al., 1993; Winson et al., 1995). In vitro immunoassays on human leukocytes have shown that OdDHL possesses immunomodulatory properties, for example, inhibition of lymphocyte proliferation and downregulation of tumor necrosis factor-α production and IL-12 production (Telford et al., 1998). In addition, OdDHL has been demonstrated to activate T cells in vivo to produce inflammatory cytokine γ-interferon (Smith et al., 2001) and thereby potentially promote a Th2-dominated response leading to increased tissue damage and inflammation. We have attempted to attenuate bacterial pathogenesis by interfering with bacterial QS systems. Our approach is based on natural signal antagonists isolated from a marine environment. Seaweeds are devoid of advanced immune systems but some have evolved to rely, at least in part, on secondary metabolite chemistry for protection against colonizing organisms. In particular, the Australian red macro-alga (seaweed) Delisea pulchra is largely unfouled in nature due to the production of biologically active halogenated furanones (de Nys et al., 1993). These secondary metabolites are released at the surface of the plant at concentrations that inhibit colonization by both prokaryotes and eukaryotes (de Nys et al., 1995, 2002; Maximilien et al., 1998; Dworjanyn et al., 1999). We subsequently discovered that these compounds are QS inhibitors (QSIs), resulting in inhibition of colonization traits in a number of bacteria (Givskov et al., 1996; Gram et al., 1996; Maximilien et al., 1998; Hentzer et al., 2002b). The present article demonstrates that the P.aeruginosa communication systems can be blocked by a novel halogenated furanone compound. This is a highly specific and effective approach to attenuating bacterial virulence and controlling bacterial infections. Results Development of furanone compounds Our laboratories have previously reported on the generation of synthetic furanone compounds and their QSI activities (Manefield et al., 2002). In this work we have applied a novel substance, termed furanone C-30 (Figure 1). This compound displays an enhanced antagonistic activity against P.aeruginosa QS systems. Figure 1.From algal metabolite to Pseudomonas drug. (A) Compound 2, a natural furanone compound isolated from (C) D.pulchra. (B) compound C-30, a synthetic furanone with enhanced QSI activity. Download figure Download PowerPoint Inhibition of virulence factor production To test the efficacy of furanone C-30 to inhibit P.aeruginosa QS-controlled phenotypes, we investigated the effect on production of some QS-controlled extracellular virulence factors, namely protease, pyoverdin and chitinase. The production of these virulence factors was partially or completely suppressed in P.aeruginosa cultures grown in the presence of 1 or 10 μM (∼2.5 μg/ml) furanone C-30 (Figure 2). Importantly, the furanone did not affect growth of the planktonic cultures (Figure 2A). QS-deficient mutants of P.aeruginosa PAO1 show similar growth rates to the parental wild-type strain (Glessner et al., 1999). Figure 2.Influence of furanone C-30 on growth and expression of virulence factors of P.aeruginosa PAO1. Cultures were grown in the absence (circles) or presence of 1 μM (squares) and 10 μM (triangles) furanone C-30. (A) Growth rate; (B) exoprotease activity; (C) pyoverdin activity; (D) chitinase activity. The data represent mean values of three independent experiments. Error bars represent the standard errors of the means. Download figure Download PowerPoint Identification of target genes of furanone C-30 action QSI-screening assays and repression of P.aeruginosa virulence factor production suggest that QS circuits are targeted by the furanones. However, these observations do not exclude other targets of the furanone. DNA microarray technology offers the ability to overview the bacterial transcriptome and hence to reveal furanone target specificity by monitoring changes in transcript accumulation. We have used an Affymetrix GeneChip® P.aeruginosa Genome Array covering all the 5570 predicted P.aeruginosa PAO1 genes to study the effect of furanone C-30 on global gene expression. Planktonic cultures of P.aeruginosa PAO1 were grown with or without addition of furanone C-30. Analysis of microarray hybridization signals showed that 93 genes (1.7% of all genes) were >5-fold affected by the furanone compound (Figure 3). In all, 85 genes (1.5%) were repressed and eight genes (0.1%) were activated in response to C-30. About 43% of the C-30-regulated genes encode hypothetical proteins of unknown function. Currently, 44% of the P.aeruginosa genes are classified as encoding such hypothetical proteins (Whiteley et al., 2001). Among the 85 furanone-repressed genes, 30% have previously been reported as QS-controlled major P.aeruginosa virulence factors. These genes include the lasB gene encoding elastase, lasA encoding LasA protease, the rhlAB operon for rhamnolipid production, the phzA-G operon encoding phenazine biosynthesis, the hcnABC operon for hydrogen cyanide production and the chiC gene encoding chitinase activity. Figure 3.Effect of furanone C-30 on genome-wide gene expression profile of P.aeruginosa. Differential gene expression in planktonic cultures of P.aeruginosa PAO1 in response to 10 μM C-30 analyzed by microarrays. Positive values represent C-30-induced genes. Negative values indicate C-30-repressed genes. The dashed lines indicate 5-fold induction or repression. Genes significantly affected by C-30 are indicated. The color coding of the individual genes indicates which of the two QS systems are required for induction: blue, LasR-controlled genes (activated by addition of only OdDHL); gray, both LasR and RhlR are required for full expression (activated by OdDHL, and further induced by addition of BHL); yellow, RhlR-controlled genes (genes induced only by simultaneous addition of OdDHL and BHL). C-30-regulated genes recorded as non-QS-controlled are not colored. Data represent samples retrieved at an OD600 of 2.0. Download figure Download PowerPoint Transcription of the lasRI and rlhRI genes was not significantly affected by C-30 (<2-fold repression). However, we observed that expression of two QS-associated genes, fabH1 and fabH2, was drastically repressed by C-30. These genes encode 3-oxo-acyl carrier protein (ACP) synthase III. Furthermore, two ACP-encoding genes (PA3334 and PA1869) were significantly downregulated by C-30. Acyl-ACPs have been proposed to be the acyl donors for synthesis of AHLs (Schaefer et al., 1996; Parsek et al., 1999). Transcription of the phnAB operon encoding anthranilate synthase was repressed by C-30. Recently, the phnAB operon was suggested to encode the biosynthetic function for the Pseudomonas quinolone signal PQS (Gallagher et al., 2002). PQS signaling is, in concert with the AHL-based QS systems, involved in regulation of virulence factor production, in particular phenazine, pyocyanin and hydrogen cyanide, and in autolysis of P.aeruginosa colonies (D'Argenio et al., 2002), and hence potentially also in biofilms. Among the C-30-activated genes we observed the mexEF genes encoding a multidrug efflux transporter and the mexR gene encoding the multidrug resistance operon repressor. Other C-30-activated genes included oxidoreductases, ABC transporters and MFS transporters. To obtain an independent validation of our microarray data, we have studied expression of reporter fusions using the green fluorescent protein (GFP) as the reporter. We observed that reporter gene expression correlated well with microarray data. For example, GFP expression from a lasB promoter fusion was 7-fold repressed in the presence of 10 μM C-30 (data not shown). In comparison, microarray analysis showed that a similar C-30 treatment caused lasB mRNA accumulation to fall ∼10-fold. Virulence factor measurements (Figure 2) showed that exoprotease was reduced 9-fold in the presence of 10 μM C-30. Mapping of the P.aeruginosa QS regulon Among the 1.7% of P.aeruginosa genes that were significantly affected by C-30, one-third have previously been reported as QS controlled, many of which encode major P.aeruginosa virulence factors. Until recently, QS target genes have only been identified by genetic analysis, not by transcriptome analysis. As a consequence, a larger fraction of our C-30-repressed genes might be as yet unidentified QS target genes. We have performed an in-depth mapping of genes responsive to exogenous OdDHL and BHL using a lasI rhlI double mutant constructed from the sequenced PAO1 strain (Stover et al., 2000). We identified 163 genes activated in response to addition of AHLs, corresponding to 2.9% of the PAO1 genome (Figure 4). The QS-induced genes are scattered throughout the genome, supporting the view that the QS systems function as global regulatory systems. Many of the identified QS-controlled genes are organized in putative operons. A simple estimate predicts 34 such operons. Sequence analysis showed that 20% of the QS-regulated genes and operons contained las box-like sequences in their corresponding promoter regions (Figure 4). Whiteley et al. (1999) categorized QS-controlled genes into four classes depending on early or late induction either by OdDHL or by OdDHL and BHL in combination. In our analysis we observed similar expression profiles notwithstanding different experimental conditions. Figure 4.Quorum-induced genes and C-30-repressed genes in P.aeruginosa. Quorum-induced genes identified in lasI rhlI mutant grown with or without added exogenous AHL signals. The transcription profile of the quorum-induced genes in P.aeruginosa PAO1 is shown together with the maximal repression caused by C-30. 1The gene number and name are from the Pseudomonas Genome Project (http://www.pseudomonas.com). Genes previously reported as QS regulated are shown in bold type]., genes organized in operon. The criteria for organization into putative operons were as follows: (i) genes are transcribed in the same orientation; (ii) intergenic regions are 5-fold repressed by C-30, and another 39% were 2- to 5-fold repressed (Figure 4). In general, there is a strong correlation between genes strongly induced and repressed by AHLs and C-30, respectively. Quorum-induced genes that are 1000-fold) expressed upon addition of AHLs to the lasI rhlI mutant culture, but rsaL transcription is not repressed by the addition of C-30. In order to investigate which cellular processes are controlled by QS, we have categorized the genes into functional groups according to the annotation by PseudoCAP (Supplementary figure S2, available at The EMBO Journal Online). This analysis shows that P.aeruginosa QS-controlled genes are slightly overrepresented in functional groups related to virulence and survival during infection, for example, adaptation/protection, antibiotic resistance and susceptibility, central intermediary metabolism, fatty acid and phospholipid metabolism, and protein secretion/export apparatus. In particular, many of the secreted factors (21%, corresponding to 18 genes) produced by P.aeruginosa are under QS control. All genes involved in quinolone signal response are under QS control; however, this functional group consists of only seven genes organized in three transcriptional units. The functional classification of C-30-repressed genes is strikingly similar to the grouping of QS-controlled genes, which indicates that the furanone interferes with QS-controlled gene expression in an unbiased fashion. We hypothesized that the constitution of the QS regulon might depend on experimental conditions. For instance, the biofilm environment might provide an ideal environment for QS signaling because bacteria are present in a very high local concentration. Additionally, the bacteria are believed to exhibit a biofilm-specific physiology radically different from that of bacteria in a planktonic mode of growth (Sauer et al., 2002). To test our hypothesis, we grew biofilms of P.aeruginosa PAO1 and the lasI rhlI mutant in a silicone tube biofilm reactor as described in the Supplementary data. Our analysis showed that 254 genes were AHL induced in P.aeruginosa biofilms. Since biofilms are heterogeneous populations consisting of cells in many different growth stages, we compared gene induction ratios in biofilm samples with the planktonic data averaged over all five cell-density sample points. Among the planktonic QS genes showing an average induction ratio >5-fold, 86% of them were also >5-fold induced in the biofilm samples (Supplementary table S1). Effect on biofilm stress tolerance Many commonly used traditional antibiotics have been demonstrated to be ineffective on biofilm cells compared with planktonic cells (Anwar and Costerton, 1990). We wanted to test whether C-30 shows a similar inadequacy to cope with biofilm bacteria. For P.aeruginosa, it has been demonstrated that the ability to form the characteristic mushroom-structured and SDS-resistant biofilms is affected by QS (Davies et al., 1998). We observed that biofilms grown in the presence of C-30 were, in contrast to a non-furanone-treated control, efficiently dissolved by an overnight treatment with 0.1% SDS (see Supplementary figure S1). The sensitivity to tobramycin, an aminoglycoside antibiotic routinely used in cystic fibrosis clinics (Høiby et al., 2000), was also assessed. Bacterial viability staining showed that biofilms grown in the presence of C-30 were significantly more susceptible to this antibiotic (Figure 5). The antibiotics efficiently penetrated and killed the furanone-treated biofilm cells, leaving 5–10% of cells alive (mainly present at the substratum). In the non-furanone-treated control, only the cells at the surface of the biofilm were killed by the tobramycin treatment. C-30-treated planktonic P.aeruginosa cells were two to three orders of magnitude more sensitive to tobramycin (data not shown). Figure 5.Sensitivity of furanone C-30-treated P.aeruginosa biofilms to tobramycin. Scanning confocal laser microscopy (SCLM) photomicrographs of P.aeruginosa PAO1 biofilms grown in the absence (left panel) or presence (right panel) of 10 μM C-30. After 3 days, the biofilms were exposed to 100 μg/ml tobramycin for 24 h. Bacterial viability was assayed by staining using the LIVE/DEAD BacLight Bacterial Viability Kit: red areas are dead bacteria, and green areas are live bacteria. The biofilms were exposed to (A) no furanone and 100 μg/ml tobramycin, (B) 10 μM C-30 and 100 μg/ml tobramycin, (C) non-treated control and (D) 10 μM C-30 and no tobramycin. Download figure Download PowerPoint QS inhibition in vivo Cell-to-cell communication between infecting bacteria in the lungs of infected mice can be visualized by use of GFP reporter technology (Wu et al., 2000; Riedel et al., 2001). An Escherichia coli-based dual-labeled AHL sensor was introduced intratracheally to 4 × 107 colony-forming units (CFU) per lung. The sensor bacteria constitutively express the red fluorescent protein (RFP) for easy detection and localization in the lung tissue, and additionally express GFP in response to the presence of AHL signals. Introduction of OHHL into the mouse blood circulation caused activation of the LuxR-controlled PluxI-gfp(ASV) fusion (Figure 6A and B). This demonstrates that OHHL is transported by the blood, penetrates the lung tissue and induces QS-controlled gene expression in the infecting bacteria. Figure 6.Inhibition of QS in vivo. Photomicrographs of mouse lung tissue infected with an E.coli-based dual-labeled AHL sensor. The strain expresses GFP in response to the presence of exogenous AHL signals and carries a dsred expression cassette to provide a red fluorescent tag on the sensor bacteria for simple identification in tissue samples. Mice carrying the sensor bacteria in the lungs were administered OHHL and furanone C-30 via intravenous injection. (A) No injection; (B) 200 μM OHHL; (C) 200 μM OHHL + 2 μg/g BW C-30 (corresponds to ∼10 μM); (D) 400 μM OHHL + 2 μg/g BW C-30; (E) 800 μM OHHL + 2 μg/g BW C-30; (F) 1200 μM OHHL + 2 μg/g BW C-30. Download figure Download PowerPoint We used this model system to evaluate the efficacy of C-30 in vivo. Furanone C-30 [∼2 μg/g body weight (BW)] co-administered intravenously with OHHL caused repression of LuxR-controlled activation of
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