Bacterial screen for QS inhibition.

Strain QSIS1 has previously been described in detail (73). The QS element is derived from the Vibrio fischeri LuxR-regulated QS system and was cloned in Escherichia coli, where it responds to a broad range of AHLs and QS inhibitors (2, 73, 74). QSIS1 harbors plasmid pUC18Not-luxR-P_luxI-RBSII-phlA T₀-T₁ Ap^r maintained in CSH37 (59), an E. coli strain that constitutively expresses β-galactosidase. The general concept of the selector system implies the presence of a QS regulator (luxR or a homologue) gene and a QS-controlled promoter fused to a gene encoding a toxic gene product. In the presence of signal molecules, the gene under QS control is expressed, causing cell death. However, in the presence of a QSI compound, production of the toxic gene product is switched off and the bacteria are rescued and able to grow normally. The toxic gene product of the QSIS1 selector system is cytolytic phospholipase A, which originates from Serratia liquefaciens MG1 (33, 34). Because signal molecules are added to the assay from an external source, QSIs targeting the AHL synthetase are not identified, whereas compounds that enhance the degradation of AHL, impede AHL uptake, or block the interaction of AHL with the luxR protein are found.

Briefly, the preparation of QSIS1 screens was performed as follows: BT medium (B medium [16] plus 2.5 mg thiamine per liter) containing 2% agar (wt/vol) was melted, 10% A10 buffer (16) was added, and the mixture was cooled to 45°C. To the resulting ABT agar were then added N-3-oxo-hexanoyl-l-homoserine lactone (Sigma-Aldrich, Germany), ampicillin (VepiDan, Denmark), 5-bromo-4-chloro-3-indoxyl-β-d-galactopyranoside (X-Gal; Apollo Scientific, United Kingdom), and 1-methyl-2-pyrrelidon (Merck, Germany) to final concentrations of 100 nM, 100 μg/ml, 40 μg/ml, and 0.2% (vol/vol), respectively; and the medium was supplemented with 0.5% (wt/vol) glucose and 0.5% (wt/vol) Casamino Acids. An overnight culture of QSIS1 (grown in ABT medium supplemented with 0.5% [wt/vol] glucose, 0.5% [wt/vol] Casamino Acids, and 100 μg/ml ampicillin) was added to a final concentration of 0.4%.

Plates for the screening assay were subsequently made by pouring the mixture into a box to give an agar thickness of 5 mm. Wells with diameters of 5 mm were made in the agar plates. Upon solidification, 50 μl of a 1- to 10-μg/ml solution of the antibiotic to be tested for QSI activity was added to the wells. The plate was then incubated at 30°C for 16 h. The appearance of a circular zone of growth (which appeared blue due to the presence of hydrolyzed X-Gal) indicated the presence of compounds with QSI activity in the sample tested.

Sample preparation for P. aeruginosa GeneChip analysis.

ABT medium supplemented with 0.5% Casamino Acids was inoculated with exponentially growing P. aeruginosa PAO1 cells to an optical density at 600 nm (OD₆₀₀) of 0.05, and the cells were grown at 37°C and 200 rpm in five 500-ml flasks containing 100 ml each. When the OD₆₀₀ was 0.3, AZM (8 and 2 μg/ml; Zithromax; Pfizer), CPR (0.04 μg/ml; Fluka), or CFT (0.25 μg/ml; Fortum; GlaxoSmithKline) was added. These concentrations of antibiotics did not affect the growth of P. aeruginosa. Samples were retrieved when the OD₆₀₀ reached 2.0, immediately transferred to 2 volumes of RNAlater (Ambion), and stored at −80°C. RNA was isolated with an RNeasy mini purification kit (Qiagen), according to the protocol provided by the manufacturer, including the on-column DNase treatment. The synthesis of cDNA was performed with 12 μg RNA, 300 ng/μl random primers (Invitrogen), 1,500 U SuperScript III reverse transcriptase (Invitrogen), and 30 U SUPERase·In RNase inhibitor (Ambion), according to the Affymetrix expression analysis protocol. The cDNA synthesized was purified with a QIAquick PCR purification kit (Qiagen), and 3 to 4 μg cDNA was fragmented with 0.2 U FPLC pure DNase I (Amersham Bioscience) per μg cDNA. The fragmented cDNA was labeled at the terminus with biotin-ddUTP (Enzo Bioarray terminal labeling kit) and hybridized for 18 h at 55°C to a P. aeruginosa genome microarray GeneChip (Affymetrix). The chips were washed and stained according to the Affymetrix protocol. The microarray hybridization signal intensity was scaled to an overall signal average of 500; and the microarray was evaluated for the presence, marginal presence, or absence of probe sets (representing different genes or open reading frames) by the use of Affymetrix GeneChip operating system (version 1.4) software. The expression values only for probe sets estimated as being “present” were included in the further analyses. The microarray experiments were conducted twice for the antibiotic-treated cultures (two arrays for 2 μg/ml AZM, two arrays for 8 μg/ml AZM, two arrays for 0.04 μg/ml CPR, and two arrays for 0.25 μg/ml CFT), while the untreated controls (references) were tested in four replicates. The data presented here are based on the average of each set.

Quantification of mRNA by real-time PCR. (i) Primer design.

The primers used for mRNA amplification (Table ​(Table1)1) were designed by the use of Integrated DNA Technologies Primer Quest software (http://www.idtdna.com). Briefly, the sizes of the amplicon designed ranged from 80 to 200 bp, and the primer melting temperatures were designed for 60°C, with a melting temperature difference of less than 2°C for each primer pair. The primer sequences were also subjected to a BLAST analysis against the P. aeruginosa PAO1 genome sequence to eliminate the possibility of nonspecific binding.

(ii) Real-time PCR.

Each real-time PCR mixture (final volume, 20 μl) contained 9 μl of cDNA, 10 μl of 2×SYBR green quantitative PCR master mix (Fermentas), and 0.5 μM of each forward and reverse primer. Real-time PCR was performed with a Chromo4 real-time detector (Bio-Rad [formerly MJ Research]) by using the following cycling parameters: an initial denaturation at 95°C for 10 min before 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. The data were analyzed by the efficiency-corrected relative quantification method (71). Expression of the target genes was normalized to the expression of reference gene rpoD, as the DNA microarray analysis showed that the expression of this gene was stable under all conditions. Furthermore, Savli et al. (77) have found that among the housekeeping genes, this gene has the most stable expression. To control for variations between runs, the housekeeping gene and the various target genes for each individual treatment were amplified at the same time on a 96-well plate. The changes in the levels of expression of the target genes in the treated samples were calculated relative to the average level of expression of the respective gene in the nontreated sample. The efficiency of the reverse transcription-PCR (RT-PCR) for the gene pairs (under treated versus untreated conditions) varied less than 5%.

Accordingly, the average efficiencies of each gene in this study were very similar for the conditions compared, allowing accurate analysis.

Supernatants for virulence factor assays.

P. aeruginosa PAO1 cultures were grown either for 16 to 20 h (for hemolysis assay) or to an OD₆₀₀ of 2.0 (for the protease, chitinase, and elastase assays), as described above, with 8 or 2 μg/ml AZM, 0.04 μg/ml CPR, 0.25 μg/ml CFT, or no antibiotic (control). The P. aeruginosa PAO1 QS mutant ΔlasI-ΔrhlI was also grown along with the treated and untreated P. aeruginosa PAO1 cultures. Cells were harvested by centrifugation, and the supernatants were filtered sterilized (TPP syringe filter; pore size, 0.22 μm). The supernatants were either used immediately or stored at −20°C.

Hemolysis assay.

The filter-sterilized supernatants were autoclaved to destroy enzymatic activity and mixed with venous blood from a healthy individual at a volumetric ratio of 30:1 (supernatant to blood), observed for hemolysis after 20 min, and left at room temperature for 4 h to allow the sedimentation of intact erythrocytes. Hemolytic activity was observed as a clearing and a nonprecipitating red coloring of the blood solution, while a lack of hemolytic activity was characterized by the precipitation of intact erythrocytes. The experiment was performed four times with supernatants from four individual growth experiments.

Protease assay.

The filter-sterilized supernatants (300 μl each) were added to the wells of ABT agar plates (ABT medium supplemented with 2% agar) containing 5% skim milk, and the plates were incubated overnight at 37°C. The zones of clearance (radii) were measured on photographs of the plates by the use of a ruler. The experiment was performed three times as four replicates with supernatants from three individual growth experiments. The values from the four replicates were averaged and used as one value to represent each of the three experiments.

Elastase assay.

The filter-sterilized supernatants were mixed 2:1 with phosphate buffer (0.1 M, pH 6.3), and 2 mg/ml elastin Congo red (Sigma) was added. The mixture was incubated at 37°C with shaking (200 rpm) for 1 week. After centrifugation, the absorbance of the supernatant at 495 nm was measured with a spectrophotometer zeroed on an elastin Congo red sample incubated with medium alone. The procedure was modified from that described previously (63). The experiment was performed three times in triplicate with supernatants from three individual growth experiments. The values from the triplicate experiments were averaged and used as one value to represent each of the three experiments.

Chitinase assay.

Chitinase was measured by a modified chitin azure assay (30, 36). The filter-sterilized supernatants were mixed 2:1 with sodium citrate buffer (0.1 M, pH 4.8), and 0.5 mg/ml chitin azure (Sigma) was added. The supernatant-chitin azure mixtures were incubated at 37°C with shaking (200 rpm) for 1 week. The samples were then centrifuged at 15,000 × g for 10 min, and the absorbance at 570 nm was determined. The samples were compared to blanks incubated with medium only. The experiment was performed three times in triplicates with supernatants from three individual growth experiments. The values from the triplicate experiments were averaged and used as one value to represent each of the three experiments.

Quantification of rhamnolipid.

A standard curve for rhamnolipid B (concentration versus total ionization current) was calculated from liquid chromatography-electrospray ionization-mass spectrometry (MS) data. To minimize potential differences in the ionization levels of rhamnolipid between the samples, the rhamnolipid standards used to calculate the concentration curve were analyzed immediately prior to as well as after the samples were analyzed. The total ionization current was determined on the [M + NH₄]⁺ ion at 668.4 over the 7 s over which rhamnolipid B was eluted. High-pressure liquid chromatography (LC)-MS analysis was performed with an Agilent 1100 series high-pressure liquid chromatograph connected to a Micromass LCT time-of-flight mass spectrometer. The concentration of rhamnolipid in the untreated culture was set equal to an index value of 100.

Docking of AZM, CFT, and CPR against the LBD of the LasR protein.

The X-ray crystallographic structure of the ligand binding domain (LBD) of the LasR protein (Protein Data Bank accession no. 2UV0) was used for the docking of AZM, CFT, and CPR to the LasR receptor site by using the program Molegro Virtual Docker (86) and the template docking model with default settings. OdDHL was set as the template ligand in the LBD of the LasR protein. The ligands were ranked by using the Molegro Virtual Docker rerank scoring function.

Data analysis and statistics.

One-way analysis of variance followed by Dunnett's test was used to evaluate the effects of AZM, CFT, and CPR treatment and the effect of the lasI-rhlI mutation on the expression of elastases, chitinase, and protease compared to their expression in untreated wild-type strain P. aeruginosa PAO1. Tests were performed with the InStat (version 3) program from GraphPad Software (San Diego, CA). Differences were considered significant if the P value was <0.05 or less.

Heat-stable hemolysin.

We investigated the effects of AZM, CPR, and CFT on the production of heat-stable hemolysin. P. aeruginosa produces at least two hemolysins, the heat-labile phospholipase C (phlC, PA0844) and the heat-stable rhamnolipid (rhlA, rhlB, rhlC, and rhlG; PA3479, PA3478, PA1130, and PA3387, respectively) (9, 46, 83). Rhamnolipid synthesis is turned on in early stationary phase of P. aeruginosa PAO1 cultures (62, 89). After 14 to 20 h of growth (under the conditions outlined in Materials and Methods), the concentration of rhamnolipid in the supernatant exceeds 50 μg/ml, which is sufficient to lyse erythrocytes within 20 min (unpublished observations). The lysis of erythrocytes by rhamnolipid can be used as a simple assay for the detection of rhamnolipid by mixing autoclaved supernatant with blood and incubating the mixture at room temperature for 20 min. The autoclaving eliminates the activity of the heat-labile enzyme phospholipase. A clearing and a nonprecipitating red coloring of the blood-supernatant solution indicates that rhamnolipid is present at a concentration well above 50 μg/ml, while a lack of hemolytic activity (characterized by the precipitation of intact blood cells) indicates that little (less than 50 μg/ml) or no rhamnolipid is present in the supernatant. Investigating autoclaved supernatants from P. aeruginosa grown in the presence or the absence of AZM, CPR, and CFT and supernatants from the QS mutant ΔlasI-ΔrhlI, we found that only the untreated wild type produced enough rhamnolipid to cause the rapid hemolysis of red blood cells. However, treatment with CFT did not fully abolish the hemolytic activity (Fig. ​(Fig.33).

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FIG. 3.

Hemolysis of autoclaved supernatants from P. aeruginosa PAO1 cultures grown for 20 h. PAO1 was grown without antibiotics (a) and in the presence of 2 μg/ml AZM (b), 8 μg/ml AZM (c), 0.25 μg/ml CFT (d), or 0.04 μg/ml CPR (e). The results for the ΔlasI-ΔrhlI mutant of PAO1 are also shown (f).

AZM at sub-MICs has previously been reported to affect an rhlAB::lacZ gene fusion (84). We decided to directly quantify the effect of AZM as well as the effects of CFT and CPR on rhamnolipid production. We thus determined the concentration of rhamnolipid in the supernatants of treated and untreated P. aeruginosa cells by LC-MS. When the cells were grown in the presence of 8 μg/ml AZM, the concentration of rhamnolipid was reduced by 85% compared to that in the untreated cultures, and 2 μg/ml AZM reduced the rhamnolipid content of the supernatants by 80% compared to that in the untreated cultures, whereas CPR (0.04 μg/ml) and CFT (0.25 μg/ml) reduced the concentration of rhamnolipid to 55 to 65% of that in the untreated cultures. The QS mutant ΔlasI-ΔrhlI did not produce any measurable amount of rhamnolipid (Fig. ​(Fig.44).

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FIG. 4.

Rhamnolipid contents, in relative units (RU), in supernatants from P. aeruginosa cultures grown either without antibiotics (black bar) or in the presence of 2 μg/ml AZM (bar with horizontal lines), 8 μg/ml AZM (bar with vertical lines), 0.25 μg/ml CFT (dark gray bar), or 0.04 μg/ml CPR (light gray bar). The ΔlasI-ΔrhlI mutant of PAO1 was also included in the experiment but did not produce a measurable concentration of rhamnolipid (last slot [no column visible]). The concentration of rhamnolipid in the untreated culture was set equal to an index value of 100. The graph is based on the average of the indexes of three independent experiments. *, P < 0.05; **, P < 0.01 (Dunnett's post test).

Protease.

The production of extracellular protease in P. aeruginosa PAO1 cultures grown in the absence or the presence of AZM, CPR, or CFT and in ΔlasI-ΔrhlI mutant cultures was assayed by adding filter-sterilized supernatants from overnight cultures to wells made in agar plates containing 5% skim milk. The protease activity was lowered by AZM, CPR, and CFT (Fig. ​(Fig.55).

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FIG. 5.

Protease activities of supernatants from P. aeruginosa cultures grown either without antibiotics (black bar) or in the presence of 2 μg/ml AZM (bar with horizontal lines), 8 μg/ml AZM (bar with vertical lines), 0.25 μg/ml CFT (dark gray bar), or 0.04 μg/ml CPR (light gray bar). The ΔlasI-ΔrhlI mutant of PAO1 was also included in the experiment but did not produce a measurable concentration of protease (last slot [no column visible]). The graph is based on the average results of three independent experiments. *, P < 0.05; **, P < 0.01 (Dunnett's test).

Elastase.

The extracellular elastase activities in the supernatants of P. aeruginosa PAO1 cultures (untreated or grown with AZM, CPR, or CFT) were quantified by use of a previously described procedure (63). The QS mutant ΔlasI-ΔrhlI was assayed by the same procedure. AZM treatment reduced the elastase activity of PAO1 almost to the level of that for the ΔlasI-ΔrhlI mutant. CPR and CFT also reduced the elastase activity (Fig. ​(Fig.66).

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FIG. 6.

Elastolytic activities of supernatants from PAO1 cultures grown to an OD₆₀₀ of 2.0 either without antibiotics (black bar) or in the presence of: 2 μg/ml AZM (bar with horizontal lines), 8 μg/ml AZM (bar with vertical lines), 0.25 μg/ml CFT (dark gray bar), or 0.04 μg/ml CPR (light gray bar). The elastolytic activity of the ΔlasI-ΔrhlI mutant of PAO1 is also shown (white bar). The graph is based on the average results of three independent experiments. *, P < 0.05; **, P < 0.01 (Dunnett's post test).

Chitinase.

The chitinase activities in the supernatants of P. aeruginosa PAO1 cultures (untreated or grown with AZM, CPR, or CFT) and QS mutant ΔlasI-ΔrhlI cultures were quantified by measuring the degradation of chitin azure. AZM and CPR reduced the chitinolytic activity of PAO1 almost to the level of that for the ΔlasI-ΔrhlI mutant. CFT did not decrease chitinase activity as much as AZM and CPR did, although it still had an effect (Fig. ​(Fig.77).

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FIG. 7.

Chitinase activities of supernatants from PAO1 cultures grown to an OD₆₀₀ of 2.0 either without antibiotics (black bar) or in the presence of: 2 μg/ml AZM (bar with horizontal lines), 8 μg/ml AZM (bar with vertical lines), 0.25 μg/ml CFT (dark gray bar), or 0.04 μg/ml CPR (light gray bar). The chitinase activity of the ΔlasI-ΔrhlI mutant of PAO1 is also shown (white bar). The graph is based on the average results of three independent experiments. *, P < 0.05; **, P < 0.01 (Dunnett's post test).

DNA microarray analysis of gene expression in the presence of AZM, CFT, and CPR.

In order to obtain knowledge of the mechanism by which virulence factor production was altered by the presence of the three antibiotics, we performed DNA microarray analyses with P. aeruginosa cultures treated with AZM, CPR, and CFT at concentrations that did not affect growth. The medium with exponentially growing P. aeruginosa cultures (OD₆₀₀, 0.1) was supplemented with either AZM (2 and 8 μg/ml), CFT (0.25 μg/ml), or CPR (0.04 μg/ml) or the medium was left untreated as a control. By applying the growth conditions described in Materials and Methods, an OD₆₀₀ of 2.0 corresponds to the transition from exponential phase into stationary phase, and at this point a substantial fraction of QS-regulated genes are maximally regulated (induced or repressed) (80). An OD₆₀₀ of 2.0 was therefore chosen as the sample point for the transcriptomic analysis. Total RNA was extracted from the samples and subjected to cDNA synthesis. The absolute expression values from cultures grown in the presence of antibiotics were compared with those from untreated cultures, and the changes in expression are reported. As described previously (38, 89), changes in expression of less than fivefold were disregarded. Only probe sets that were present, as validated with Affymetrix GeneChip operating system (version 1.4) software, were included in the analyses. According to these criteria, non-growth-inhibitory concentrations of AZM altered the expression of 477 (8 μg/ml) and 323 (2 μg/ml) genes, with most genes (419 and 277, respectively) being downregulated. Non-growth-inhibitory concentrations of CFT (0.25 μg/ml) and CPR (0.04 μg/ml) affected the expression of 122 (114 repressed) and 271 genes (215 repressed), respectively (Table ​(Table22).

AZM, CFT, and CPR affect expression of QS-regulated genes.

The QS regulon has been defined by (among others) Rasmussen et al. (74). That particular study (74) considered genes to be regulated by QS if their levels of expression in both ΔlasI-ΔrhlI and ΔlasR-ΔrhlR mutants were consistently altered more than fivefold compared to the levels of expression in their wild-type counterpart. The majority of all QS-regulated genes defined in this way are inducible with exogenous AHLs in a ΔlasI-ΔrhlI background. Only one gene (PA1556, a probable cytochrome c oxidase subunit) is consistently upregulated in both the ΔlasI-ΔrhlI and the ΔlasR-ΔrhlR mutants and is thus QS repressible. Using this QS regulon, we analyzed the fraction of QS-regulated genes among the genes repressed by the three antibiotics. These analyses showed that AZM (8 μg/ml) repressed 71% of the 174 QS-regulated genes in P. aeruginosa PAO1 and that 29% of all genes being repressed by AZM were members of the QS regulon. Lowering of the concentration of AZM to 2 μg/ml caused a relief in the inhibition of QS-regulated genes (47% of all QS genes repressed), and the total number of genes downregulated fell to 277, while the specificity toward the QS regulon increased slightly to 30%. CPR had the highest specificity for the QS regulon (48%) and targeted 49% of all QS genes in P. aeruginosa, whereas the specificity for CFT was 39% and CFT repressed only 25% of all QS genes. For comparison, the specificity of C30 was 48%, and it downregulated 72% of all QS genes in P. aeruginosa (raw data for C30 are from a previous report [38] and have been subjected to the same data treatment and criteria as the raw data for AZM, CFT, and CPR). The effects of AZM, CFT, and CPR on the expression of genes belonging to the QS regulon are shown in Table ​Table33.

The DNA array data show that members of the QS regulon are overrepresented among the genes repressed by sub-MICs of AZM, CFT, and CPR. This suggests that these antibiotics exhibit some specificity for QS-regulated gene expression through an interaction with or an effect on QS regulatory components. Investigating the expression profiles of genes regulated by LasR, RhlR, or LasR-RhlR, we further aimed to elucidate the potential QS target(s) of the antibiotics. The QS-regulated genes can be divided into four groups. Group A contains 44 LasR-dependent genes, i.e., genes downregulated in both a ΔlasR mutant and a ΔlasR-ΔrhlR mutant. Group B consists of RhlR-dependent genes (14 genes), i.e., genes downregulated in both a ΔrhlR mutant and a ΔlasR-ΔrhlR mutant. Group C comprises the main fraction of QS-regulated genes (102 genes in total) and consists of genes which are downregulated in all three types of mutants: ΔlasR, ΔrhlR, and ΔlasR-ΔrhlR. Finally, group D consists of 14 LasR-RhlR-dependent genes, which are downregulated only if both LasR and RhlR are knocked out, as in the ΔlasR-ΔrhlR mutant. Interestingly it seems that AZM, CFT, and CPR had less of a preference for the group B genes (the RhlR-specific genes) (Fig. ​(Fig.88).

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FIG. 8.

LasR, RhlR, and LasR-RhlR specificities of AZM (white bars), CFT (black bars), CPR (light gray bars), and C30 (dark gray bars). See the text for the definitions of groups (Gr.) A, B, C, and D.

Even though all three antibiotics exhibited a higher degree of specificity for the LasR-regulated genes than for the RhlR-dependent genes, none of these antibiotics downregulated the AHL synthetase genes. AZM, however, caused the downregulation of lasR sevenfold, which may also contribute to the QSI effect of AZM. This is in accordance with the observation that AZM (2 μg/ml) has been reported to decrease the level of expression of a lasR::lacZ gene fusion (84).

Among the genes affected by AZM, CFT, and CPR, many encode previously identified QS-regulated genes such as chitinase (chiC, PA2300) and chitin-binding protein (cbpD, PA0852) (30, 80, 89). Other known QS-induced genes, such as the genes encoding the osmotically inducible protein (osmC, PA0059) and cytochrome c oxidase (coxAB and coIII, PA0105 to PA0108), are downregulated by the presence of the three antibiotics (38, 80). The endoprotease gene piv (also known as prpL [PA4175]) was also downregulated by AZM, CFT, and CPR. This protease is capable of degrading elastin, decorin, and the transferrin family of proteins, including lactoferrin (92). PA5099 (a probable transporter), which belongs to the QS regulon, was downregulated more than 300 times by AZM and CPR and 50 times by CFT. The regulon contains only one gene, PA1556, which is repressed by QS. This gene, encoding cytochrome c oxidase, was upregulated by all three antibiotics, and so was the neighboring gene PA1557, a probable cytochrome oxidase subunit. Many genes from the region from PA2134 to PA2190 of the genome were downregulated by AZM, CFT, and CPR. This region has been found to be associated with stationary growth and to be under QS/rpoS control (79, 80, 89, 90). Of the 57 genes in this 60.69-kbp region, 46 were found to be present in all the arrays. Of the 46 genes, 35 genes were repressed more than fivefold by AZM and CPR. CPR and especially AZM seemed to exhibit higher target specificities for QS genes than CFT, which is in accordance with the findings of the virulence factor assays described earlier in this section. Many QS-regulated virulence factors, such as the alkaline metalloproteinase (aprA, PA1249), the PfpI protease (pfpI, PA0355), the LasA protease (lasA, PA1871), elastase B (lasB, PA3724), the cytotoxic galactophilic lectin (PA-IL; lecA, PA2570), and rhamnolipid (rhlA and rhlB; PA3478 and PA3479, respectively), were repressed more than fivefold by AZM and CPR but not by CFT. These results were confirmed by RT-PCR, which showed that AZM downregulated rhlA expression 45-fold, while CPR and CFT reduced the level of rhlA expression 10- and 3-fold, respectively.

AZM, CFT, and CPR alter the expression of a variety of genes.

A few genes were consistently upregulated by AZM, CFT, and CPR. The QS repressible genes PA1556 (cytochrome c oxidase subunit) and PA1557 were induced more than five times. PA2260 (a hypothetical protein previously identified [89] to be a QS repressible gene) was induced by AZM and CFT but not by CPR.

Several genes were induced only by AZM. A high proportion of these genes have also previously been reported to be AZM inducible (60), although there were a number of differences in the experimental setup between the current study and the previous study (60). For example, the previous study (60) used the P. aeruginosa PAO1 strain DSM 1707 instead of the P. aeruginosa PAO1 strain from the Pseudomonas Genetic Stock Center used here. Moreover, the study by Nalca et al. (60) also applied the complex and rich brain heart infusion medium instead of minimal medium. Many of the genes induced by AZM encode ribosomal subunits, such as PA3742 (rplS) and PA4671 (probable ribosomal protein). The overexpression of ribosomal genes may be a mechanism that compensates for the effect of AZM, which targets the 50S ribosomal subunit. The initiation factor infA (PA2619) was also induced in the presence of AZM, in accordance with previous findings (60). In agreement with the findings of two recent studies (12, 53), we found that CPR induces the expression of the pyocin S genes (PA0985 for pyocin S5, PA1150 for pyocin S2, and PA3866 for the pyocin S3-like protein). The S-type pyocins are protease-sensitive bacteriocins and have colicin-like structures and modes of action.

The ribosome modulation factor (rmf, PA4296) was repressed by AZM but was not affected by treatment with CFT or CPR. Previous transcriptome analyses (60) also showed that rmf is repressed by AZM. The rmf of P. aeruginosa shows a high degree of homology to the rmf of E. coli (similarity, 66%). rmf mutants of E. coli have been shown to gradually lose their viability in stationary-phase cultures (61); however, a recent study concluded that rmf (in E. coli) may be required only under competitive growth conditions (13).

AZM induces expression of genes related to T3S.

In accordance with previous findings (60), we also found that AZM induced the expression of genes involved in type III secretion (T3S). P. aeruginosa uses its T3S system to deliver several cytotoxic products into the cytosol of eukaryotic cells. The expression of T3S increases the pathogenicity of P. aeruginosa and is associated with more severe infections and higher rates of mortality in animal models (1, 75, 78). QS has been shown to negatively modulate the expression of the T3S system in P. aeruginosa (10), which means that the T3S system is downregulated under high-cell-density conditions and thus presumably plays a role primarily in the early stages of the infection. Genes in the T3S system, such as PA1700 and PA1701 (conserved hypothetical proteins in T3S), pcrGV (PA1705 regulator in T3S and PA1706), exsB (PA1712, exoenzyme S synthesis protein B), exsD (PA1714, part of the pscB to pscL operon), and pscBCEO (PA1715, T3S export apparatus protein; PA1716, T3S outer membrane protein; PA1718, T3S export protein; PA1696, translocation protein in T3S), were upregulated by AZM. Neither CFT nor CPR, however, induced the genes in the T3S system gene cluster from PA1690 to PA1725.

AZM, CPR, CFT, and the two-component regulator system pprAB.

The QS-induced chemotaxis transducer PA4290 was downregulated by AZM, CFT, and CPR. AZM and CPR also repressed the main part of the genes in the region from PA4290 to PA4306. Many of these genes have been shown in previous studies to be regulated by QS. In those studies, QS genes were identified by mapping genes downregulated in ΔlasI-ΔrhlI mutants and restored to wild-type levels by addition of signal molecules (38, 80, 89). The two-component regulator system pprAB (PA4293, PA4296) was downregulated by AZM and CPR but not by CFT. pprA was downregulated 16 and 6 times by AZM and CPR, respectively, and pprB downregulated 10 and 5 times by AZM and CPR, respectively. These results obtained by DNA microarray analysis were confirmed by RT-PCR, which showed that AZM reduced the levels of pprA and pprB expression 19- and 15-fold, respectively, whereas CPR downregulated the level of pprA expression 11-fold and the level of pprB expression 18-fold. RT-PCR also confirmed that the effect of CFT on pprAB expression was insignificant.

This work was supported by grants from the German Mukoviszidose e.v., Deutsche Forschungsgemeinshaft, and the Danish Research Council (STF grant no. 2052-03-0013) to M.G.