Drosophila are protected from Pseudomonas aeruginosa lethality by transgenic expression of paraoxonase-1

Pseudomonas aeruginosa uses quorum sensing, an interbacterial communication system, to regulate gene expression. The signaling molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) is thought to play a central role in quorum sensing. Since 3OC12-HSL can be degraded by paraoxonase (PON) family members, we hypothesized that PONs regulate P. aeruginosa virulence in vivo. We chose Drosophila melanogaster as our model organism because it has been shown to be a tractable model for investigating host-pathogen interactions and lacks PONs. By using quorum-sensing–deficient P. aeruginosa, synthetic acyl-HSLs, and transgenic expression of human PON1, we investigated the role of 3OC12-HSL and PON1 on P. aeruginosa virulence. We found that P. aeruginosa virulence in flies was dependent upon 3OC12-HSL. PON1 transgenic flies expressed enzymatically active PON1 and thereby exhibited arylesterase activity and resistance to organophosphate toxicity. Moreover, PON1 flies were protected from P. aeruginosa lethality, and protection was dependent on the lactonase activity of PON1. Our findings show that PON1 can interfere with quorum sensing in vivo and provide insight into what we believe is a novel role for PON1 in the innate immune response to quorum-sensing–dependent pathogens. These results raise intriguing possibilities about human-pathogen interactions, including potential roles for PON1 as a modifier gene and for PON1 protein as a regulator of normal bacterial florae, a link between infection/inflammation and cardiovascular disease, and a potential therapeutic modality.

The paraoxonase (PON) family in mammals is made up of 3 members: PON1, PON2, and PON3. PON-like proteins are evolutionarily conserved, including in Caenorhabditis elegans, but interestingly are absent in Drosophila melanogaster. PON1 is an approximately 39- to 45-kDa protein and was the first family member studied. It was originally described as an enzyme capable of degrading paraoxon and other organophosphates (1–3). Later, it was found that PONs also play an important role in lipid oxidation and atherosclerosis (4, 5). More recently, all 3 members of the PON family have been shown to also possess lactonase activity (6–10), and structure-reactivity studies suggest that PON’s native enzyme activity is as a lactonase (11). Importantly, lactones are key quorum-sensing signaling molecules for a number of Gram-negative bacteria, including Pseudomonas aeruginosa.

P. aeruginosa is a major cause of clinically relevant infections in hospitalized patients, immunocompromised people, burn wound patients, and those with chronic lung disease, such as cystic fibrosis (CF). Quorum sensing is an interbacterial mode of communication accomplished through the coordinated production, secretion, and detection of chemical signals (quorum-sensing signals) that trigger expression of specific bacterial genes. The quorum-sensing signals self produced by P. aeruginosa are in the form of small molecules, or autoinducers, termed acyl-homoserine lactones (acyl-HSLs) (12). P. aeruginosa uses acyl-HSL quorum-sensing molecules to regulate the expression of genes implicated in virulence and biofilm formation (13–15). Two hierarchically regulated acyl-HSL systems are present in P. aeruginosa: the las and rhl systems. LasI synthesizes N-3-oxododecanoyl HSL (3OC12-HSL), and LasR is the transcription factor that responds to 3OC12-HSL. RhlI synthesizes N-butanoyl HSL (C4-HSL), and RhlR is the transcription factor that responds to C4-HSL (12). Quorum-sensing–deficient P. aeruginosa is less virulent in animal models of burn wound infection, neonatal pneumonia, and acute/chronic pulmonary infections, suggesting an important role for quorum sensing in P. aeruginosa virulence (16–21). We (6, 9) and others (7, 8) have shown that all of the PONs can inactivate 3OC12-HSL. In addition, PON1 inhibits P. aeruginosa biofilm growth in an in vitro biofilm model (6).

These observations suggest the hypothesis that quorum-sensing molecules are important for P. aeruginosa virulence and that lactonases, specifically PONs, can protect the host from lethal P. aeruginosa infection. In order to test this hypothesis, an in vivo model of infection is required. However, this has been difficult to study in mammals since all 3 PONs are present on the same locus on chromosome 7, each PON can degrade 3OC12-HSL, and deletion of individual PON family members leads to compensation by the remaining members (6, 9). Therefore, we turned to other organisms and found that D. melanogaster does not express PON or a PON homolog. The fruit fly immune response has been extensively studied, and findings from this model organism have led to exciting discoveries of mammalian counterparts of D. melanogaster’s innate immune response (22, 23). We took what we believe is a novel approach of expressing PON1 in D. melanogaster and testing for protection from P. aeruginosa–induced lethality. We chose to study PON1 as opposed to PON2 or PON3, since PON1 can degrade organophosphates and protection from organophosphate killing would confirm in vivo PON1 activity in transgenic D. melanogaster. By using quorum-sensing–deficient strains of P. aeruginosa, synthetic acyl-HSLs, and transgenic lactonase (PON1) expression in flies, we have found that 3OC12-HSL is required for P. aeruginosa virulence and PON1 protects flies from P. aeruginosa–induced lethality by disrupting quorum-sensing pathways.

P. aeruginosa lethality in D. melanogaster is quorum-sensing dependent. For these studies, we infected D. melanogaster by pricking the fly’s abdomen with a needle previously dipped in a P. aeruginosa suspension (24, 25). In this model, P. aeruginosa is lethal to flies. Figure 1, A and B, shows low- and high-magnification scanning electron micrographs of a whole fly and abdomen, respectively, at approximately 24 hours following inoculation. P. aeruginosa infection is evidenced by the presence of rod-shaped bacteria encased in an extracellular matrix.

Figure 1

Quorum-sensing–dependent P. aeruginosa infection of Drosophila. (A and B) Scanning electron micrographs at low and high magnification of Drosophila at 24 hours following infection with wild-type P. aeruginosa (PAO1). (A) Low-magnification image shows a whole fly with an opening in the distal abdomen. Scale bar: 0.5 mm. (B) High-magnification image of boxed area on whole fly. Arrow indicates bacteria and asterisk denotes extracellular matrix. Scale bar: 2.5 μm. (C and D) GFP signal (quorum-sensing activity) after infection of Drosophila with a quorum-sensing reporter strain of P. aeruginosa (under control of the lasB promoter). GFP signal at 0 hours (C) and 18 hours (D) following abdominal inoculation with PAO1. Scale bars: 0.5 mm. (E) Fly survival after infection with PAO1 and quorum-sensing–deficient strains of P. aeruginosa. da-GAL4/+ flies were infected with PAO1 (squares), ΔlasI/rhlI (circles), or ΔlasR/rhlR (triangles) strains. n = 30 flies per group for each experiment. *P < 0.001, comparing PAO1 versus ΔlasI/rhlI and PAO1 versus ΔlasR/rhlR fly survival; log-rank test. (F) P. aeruginosa bacterial counts after infection with PAO1 or ΔlasI/rhlI. At 6, 12, and 18 hours following infection, flies were anesthetized, surface sterilized, and homogenized for performance of quantitative bacterial counts. Data are displayed as log₁₀ CFU/fly and represent the mean ± SEM. n = 3–5 per time point with 10–20 flies per group per experiment.

Our initial experiments were designed to test the hypothesis that quorum sensing occurs following P. aeruginosa infection of flies and that killing is dependent upon an intact quorum-sensing system. To test this hypothesis, we first inoculated flies with a quorum-sensing reporter strain of P. aeruginosa that expresses GFP under control of the lasB promoter (a quorum-sensing–regulated gene). Figure 1C shows that immediately following inoculation (time = 0 hours), there was no quorum-sensing activity. However, at 18 hours following infection, the fly abdomen was intensely green, confirming increased quorum-sensing activity (Figure 1D). To directly test whether quorum sensing was required for P. aeruginosa virulence in flies, we inoculated flies with (a) wild-type P. aeruginosa (PAO1); (b) strains deficient in 3OC12-HSL and C4-HSL production (ΔlasI/rhlI); or (c) strains deficient in the receptors required to respond to 3OC12-HSL and C4-HSL (ΔlasR/rhlR). Flies inoculated with medium alone had less than 10% mortality (data not shown). When flies were infected with PAO1, greater than 80% mortality occurred by 24 hours following infection (Figure 1E). In contrast, flies infected with the ΔlasI/rhlI or ΔlasR/rhlR mutants showed increased survival. These results are consistent with our hypothesis and previous reports from others showing that quorum sensing is important for P. aeruginosa virulence in D. melanogaster (26–29). When flies were inoculated with a very high dose of ΔlasI/rhlI (10¹¹ CFU/ml), enhanced fly killing was observed, suggesting that this high bacterial load overwhelmed the fly’s host defense response (Supplemental Figure 1B; supplemental material available online with this article; doi: 10.1172/JCI35147DS1).

One potential explanation for decreased killing by mutant strains could be that bacterial dissemination or growth is altered. We compared bacterial counts in flies at 6, 12, and 18 hours following infection with either PAO1 or ΔlasI/rhlI. Bacterial counts were similar at all time points examined in flies infected with either PAO1 or the quorum-sensing–deficient strain ΔlasI/rhlI (Figure 1F), suggesting that differences in bacterial replication are not responsible for the less virulent phenotype.

We next hypothesized that C4- and 3OC12-HSL feeding would restore virulence to ΔlasI/rhlI -infected flies but not to ΔlasR/rhlR-infected flies. Flies were fed 5% sucrose alone or containing C4- and 3OC12-HSL for 48 hours (Figure 2A), inoculated with the ΔlasI/rhlI or ΔlasR/rhlR mutants, and then maintained in the feeding assay for the remainder of the experiment. Acyl-HSLs did not alter food consumption rates in D. melanogaster (Figure 2, B and C). We found that, compared with flies fed 5% sucrose alone, flies fed C4- and 3OC12-HSL had decreased survival following ΔlasI/rhlI infection (Figure 2D). In contrast, C4- and 3OC12-HSL feeding had no effect on fly survival following ΔlasR/rhlR infection (Figure 2E). These data show that P. aeruginosa is virulent in D. melanogaster, virulence is dependent upon an intact quorum-sensing system, and exogenous acyl-HSLs can restore virulence to P. aeruginosa strains deficient in C4- and 3OC12-HSL production.

Figure 2

acyl-HSL feeding enhances virulence of quorum-sensing–deficient P. aeruginosa mutants. (A) Photomicrograph of fly-feeding vials with filter paper at the base containing either 5% sucrose alone (tubes 1 and 3) or 5% sucrose with C4- and 3OC12-HSL (tube 2). Filter paper in tubes 2 and 3 contained green food dye to confirm similar rates of food consumption between feeding groups and also consumption of acyl-HSLs. Scale bar: 2.5 cm. (B and C) Representative low-power images of Drosophila after 24 hours feeding on either 5% sucrose alone (B) or 5% sucrose containing C4- and 3OC12-HSL (containing green dye) (C). Note similar levels of green dye in the abdomen of flies consuming sucrose alone or sucrose containing acyl-HSLs. Scale bars: 0.25 mm. (D and E) Flies were fed 5% sucrose (squares) or 5% sucrose containing C4-HSL (5 μM) and 3OC12-HSL (60 μM) (circles). 48 hours later, flies were infected with ΔlasI/rhlI (D) or ΔlasR/rhlR (E) strains of P. aeruginosa. Fly survival was monitored over time. n = 30 flies per group for each experiment. *P < 0.01, comparing survival between ΔlasI/rhlI versus ΔlasI/rhlI + C4- and 3OC12-HSL groups; log-rank test.

PON1 expression and enzyme activity in PON1 transgenic flies. To test whether the lactonase function of PON1 can protect flies from P. aeruginosa infection, PON1 transgenic D. melanogaster were constructed using the GAL4–upstream activation sequence (GAL4-UAS) system, as previously described (30). Five transformed PON1-expressing fly lines were created, and PON1 protein levels were determined by immunoblotting. While da-GAL4/+ (da, daughterless) flies (control flies) did not express PON1, UAS-PON1/da-GAL4 flies (PON1 transgenic flies) expressed PON1, with variable levels of PON1 protein expression observed (PON1R9c > PON1R31 > PON1R1 ≈ PON1R4 > PON1R19) (Figure 3A). Multiple PON1 bands with differing electrophoretic mobility were observed and are consistent with prior reports of PON1 undergoing N-linked core glycosylation (8, 31).

Figure 3

PON1 expression and activity in PON1 transgenic Drosophila. (A) Western blot analysis for PON1 in da-GAL4/+ (control) and UAS-PON1/da-GAL4 flies. PON1 flies were constructed using the GAL4-UAS binary system under control of the da promoter driving ubiquitous PON1 expression. Fly heads were obtained from 1- to 3-day-old flies, homogenized, and used for immunoblotting with PON1 antibody. Lysates from CHO cells infected with an adenovirus-expressing human PON1 served as the positive control. All PON1 fly lines obtained were tested for PON1 protein. β-tubulin was used as a loading control. (B) Arylesterase activity was tested in fly homogenates from da-GAL4/+ and UAS-PON1/da-GAL4 transgenic fly lines by measuring phenylacetate degradation. mOD, optical density × 10^–3. (C) Correlation of arylesterase activity and PON1 protein expression in control and PON1 transgenic flies. Densitometry analysis of PON1 and β-tubulin immunoreactive bands was performed, and data are expressed in relative units. (D) Lactonase activity in control and PON1 transgenic flies. Whole-fly lysates were combined with the synthetic lactone TBBL (0.25 mM), and lactonase activity (thiol moiety release) was monitored with DTNB (0.5 mM) at an absorbance of 412 nm. Data represent the mean ± SEM with n = 3–4 per group. *P < 0.01, lactonase activity between da-GAL4/+ and UAS-PON1/da-GAL4 flies; Student’s t test.

In order to confirm that PON1 was enzymatically active in transgenic flies, we measured arylesterase activity in da-GAL4/+ and UAS-PON1/da-GAL4 transgenic fly lysates. Consistent with presence of enzymatically active PON1, UAS-PON1/da-GAL4 flies degraded more phenylacetate than da-GAL4/+ flies (Figure 3B). Of interest, control flies showed low levels of arylesterase activity independent of PON1 expression. Others have reported carboxylesterase and acetylesterase activity in D. melanogaster but did not observe arylesterase activity (32). These contradictory findings may be due to differing approaches utilized to assay for arylesterase activity. We next compared arylesterase activity to PON1 protein expression and, as expected, found a strong correlation (Figure 3C). Finally, we also found increased arylesterase activity in hemolymph from PON1 flies compared with that in control flies (data not shown). For all subsequent in vivo experiments, we chose to use the PON1R9c line based upon its high level of PON1 protein expression and enzyme activity. This fly line will be referred to as UAS-PON1/da-GAL4 in the remainder of experiments described.

We hypothesized that PON1 transgenic flies would degrade lactone molecules and, in subsequent in vivo experiments, disrupt P. aeruginosa quorum-sensing signaling pathways. Lactonase activity was quantified using a colorimetric assay that measures degradation of the synthetic lactone 5-thiobutyl butyrolactone (TBBL) (33). PON1R9c flies had significantly greater lactonase activity (0.203 ± 0.088 U/ml) compared with da-GAL4/+ flies (0.001 ± 0.001 U/ml) (Figure 3D). Similar to what was shown in the arylesterase data, PON1 protein levels (in the 5 PON1 lines tested) correlated with lactonase activity (R² (coefficient of determination) = 0.684, data not shown). These in vitro arylesterase and lactonase studies demonstrate that PON1 is enzymatically active in transgenic flies.

PON1 flies are protected from organophosphate toxicity. To test for PON1 function in vivo, we next took advantage of PON1’s promiscuous ability to hydrolyze organophosphate insecticides (1). Chlorpyrifos is a toxic organophosphate insecticide whose mechanism of action is acetylcholinesterase inhibition. Flies fed chlorpyrifos died in a dose-dependent manner (Figure 4A). At a dose of 50 parts per million (ppm), 100% of da-GAL4/+ flies died by 4 hours after initiation of chlorpyrifos feeding. In contrast and as expected from the biochemical data, UAS-PON1/da-GAL4 flies were markedly protected from chlorpyrifos-induced toxicity (Figure 4B). PON1 has a number of enzymatic activities, and protection from organophosphate toxicity could represent a nonspecific effect. Therefore, we asked whether PON1 transgenic flies would be protected from demeton-S-methyl (DSM) exposure. DSM is another toxic organophosphate that is not metabolized by PON1 (34). Both da-GAL4/+ and UAS-PON1/da-GAL4 flies died at a similar rate following DSM treatment (Figure 4C). These data show that PON1 protects D. melanogaster from chlorpyrifos lethality and that this protection does not represent a general resistance to toxic compounds conferred by PON1.

Figure 4

PON1 flies are protected from chlorpyrifos toxicity. (A) da-GAL4/+ flies were fed 0.5, 5, 50, 500, or 5000 ppm chlorpyrifos in 5% sucrose. Fly survival was monitored over time. (B and C) da-GAL4/+ (squares) or UAS-PON1/da-GAL4 (circles) flies were fed chlorpyrifos (50 ppm in 5% sucrose) (B) or DSM (150 ppm in 5% sucrose) (C), and survival was followed over time. n = 30 flies per group for each experiment. *P < 0.001, comparing survival between da-GAL4/+ and UAS-PON1/da-GAL4 flies; log-rank test.

PON1 flies are protected from P. aeruginosa–induced lethality. Based upon the important role of 3OC12-HSL for P. aeruginosa virulence and the lactonase activity of PON1-expressing flies, we hypothesized that PON1 transgenic D. melanogaster would be protected from P. aeruginosa–induced lethality. As shown before (Figure 1D), wild-type P. aeruginosa (PAO1) killed the majority of control or da-GAL4/+ flies. However, Figure 5A shows that UAS-PON1/da-GAL4 flies were protected from PAO1 killing. Importantly, we also noted that this protection occurred to a degree similar to that seen when control flies were infected with ΔlasI/rhlI. By 18 hours following infection, 53% ± 13% of da-GAL4/+ flies were still alive compared with 87% ± 4% of UAS-PON1/da-GAL4 flies (n = 5; P < 0.05). Comparable levels of bacteria were cultured from da-GAL4/+ and UAS-PON1/da-GAL4 flies at 6, 12, and 18 hours following infection (Figure 5B). Finally, UAS-PON1/da-GAL4 flies were also protected from high-dose (10¹¹ CFU/ml) PAO1 challenge (Supplemental Figure 2A). These results indicate that PON1 expression protects flies from P. aeruginosa independent of bacterial growth, suggesting modulation of virulence factors. To test whether PON1 expression would select for quorum-sensing mutants, we cocultured wild-type PAO1 and ΔlasI/rhlI in the presence or absence of PON1 (Supplemental Methods). Over 18 hours, there was no enrichment for either P. aeruginosa strain (Supplemental Figure 3, A and B).

Figure 5

PON1 flies are protected from P. aeruginosa lethality. (A) da-GAL4/+ (squares) and UAS-PON1/da-GAL4 (circles) flies were infected with P. aeruginosa, and fly survival was monitored over time. n = 30 flies per group for each experiment. *P < 0.01, comparing da-GAL4/+ and UAS-PON1/da-GAL4 survival; log-rank test. (B) P. aeruginosa bacterial counts following infection in da-GAL4/+ and UAS-PON1/da-GAL4 flies. Bacterial quantification was performed as described for Figure 1. Data are displayed as log 10 CFU/fly and represent the mean ± SEM. n = 3–5 per time point with 10–20 flies per group per experiment. (C) PON1 transgenic flies are protected from lethality following infection with clinical isolates of P. aeruginosa. Light-shaded symbols, PA-7JJA2; and dark-shaded symbols, PA-7DVM2. *P < 0.01, comparing da-GAL4/+ and UAS-PON1/da-GAL4 fly survival following either PA-7JJA2 or PA-7DVM2 infection; log-rank test. (D) acyl-HSL quorum-sensing activity by P. aeruginosa isolates. Supernatants from overnight bacterial cultures were incubated with the acyl-HSL detector strain A. tumefaciens NTL4, and β-galactosidase activity was determined. Data are mean ± SEM and n = 3 per bacterial strain. ^†P < 0.001, compared with PAO1. (E–F) da-GAL4/+ and UAS-PON1/da-GAL4 flies were infected with either (E) S. marcescens or (F) S. aureus (SH1000), and survival was followed over time. n = 30 flies per group for each experiment. ^†P < 0.001, comparing survival between da-GAL4/+ and UAS-PON1/da-GAL4 flies following S. marcescens infection; log-rank test.

When flies were infected with 2 clinical isolates of P. aeruginosa (PA-7JJA2 and PA-7DVM2) from CF patients, we also observed improved survival in UAS-PON1/da-GAL4 flies (Figure 5C), demonstrating that protection is not only limited to infection with PAO1. In order to determine whether these clinical isolates were producing levels of acyl-HSLs comparable to those seen with PAO1, we used the acyl-HSL detector strain Agrobacterium tumefaciens NTL4. Figure 5D shows equivalent levels of acyl-HSL activity in PAO1, PA-7JJ2, and PA-7DVM2 strains, with minimal activity observed in the ΔlasI/rhlI isolate. To determine whether PON1 could protect flies from another bacterial species that also uses acyl-HSL–dependent quorum sensing, we next infected da-GAL4/+ and UAS-PON1/da-GAL4 flies with Serratia marcescens. Similar to our results following P. aeruginosa infection, we also observed that PON1 transgenic flies were protected from S. marcescens lethality (Figure 5E). Finally, we infected flies with the SH1000 strain of Staphylococcus aureus (no acyl-HSL production) and found similar fly death in da-GAL4/+ and UAS-PON1/da-GAL4 flies (Figure 5F). These results suggest that PON1 protection is specific and may occur against bacteria that utilize acyl-HSLs to regulate virulence factor production.

PON1 protection from P. aeruginosa–induced lethality is dependent upon 3OC12-HSL inactivation. As the las and rhl systems are hierarchically regulated, with increasing concentrations of 3OC12-HSL leading to virulence gene activation and C4-HSL production, we hypothesized that 3OC12-HSL alone would be sufficient for P. aeruginosa–induced lethality. To directly test this hypothesis, da-GAL4/+ flies were infected with a strain of P. aeruginosa unable to produce 3OC12-HSL (ΔlasI mutant). Similar to our findings with the ΔlasI/rhlI and ΔlasR/rhlR mutants, ΔlasI killed fewer control flies than wild-type PAO1 (Figure 6A). Similarly, the ΔlasI strain also failed to kill PON1-expressing flies (Figure 6B). Having established the importance of 3OC12-HSL in P. aeruginosa virulence, we next hypothesized that exogenous 3OC12-HSL would enhance ΔlasI virulence in da-GAL4/+ flies but degradation of exogenous 3OC12-HSL by UAS-PON1/da-GAL4 flies would prevent restoration of ΔlasI virulence. As predicted, feeding 3OC12-HSL to da-GAL4/+ flies but not UAS-PON1/da-GAL4 flies augmented ΔlasI -induced lethality (Figure 6, A and B). These results further emphasize the importance of 3OC12-HSL in quorum-sensing control of P. aeruginosa’s virulence. Moreover, PON1 degradation of either endogenous or exogenous 3OC12-HSL can decrease mortality from P. aeruginosa infection.

Figure 6

3OC12-HSL fails to enhance P. aeruginosa virulence in PON1 flies. (A) da-GAL4/+ or (B) UAS-PON1/da-GAL4 flies were fed 5% sucrose (squares) or 5% sucrose containing 3OC12-HSL (60 μM) (circles). 48 hours later, flies were infected with the ΔlasI mutant strain of P. aeruginosa (deficient in 3OC12-HSL production). n = 30 flies per group for each experiment. Fly survival was monitored over time. *P < 0.05, comparing ΔlasI and 3OC12-HSL + ΔlasI survival in da-GAL4/+ flies; log-rank test.

PON1 flies disrupt P. aeruginosa quorum-sensing signaling. Finally, we began to investigate a possible mechanism or mechanisms for PON1’s protection by studying the effects of PON1 expression on regulation of quorum-sensing–controlled genes. For these studies, we used 2 complementary approaches. First, we infected flies with a quorum-sensing reporter strain of P. aeruginosa (PAO1-qsc102-lacZ) that expresses β-galactosidase under control of qsc102, which responds primarily to 3OC12-HSL (13). Following infection, da-GAL4/+ flies had significant X-gal staining in their abdomen, but this signal was less pronounced in UAS-PON1/da-GAL4 flies (Figure 7A). UAS-PON1/da-GAL4–infected flies scored 3.8 ± 0.8 compared with 6.2 ± 0.7 in da-GAL4/+-infected flies (P < 0.05), according to a visual analog scale for X-gal staining, and lacZ expression (quantified by RT-PCR) was decreased following P. aeruginosa infection in UAS-PON1/da-GAL4 compared with da-GAL4/+ flies (P < 0.05) (Figure 7B). Second, we measured the mRNA levels of several quorum-sensing regulated genes that are important for P. aeruginosa virulence (lasA, lasB, toxA, and aprA). Compared with da-GAL4/+ flies, UAS-PON1/da-GAL4 flies had lower expression levels for 3 of the 4 genes tested (lasA, lasB, and aprA; P < 0.05 for lasB and aprA), with greater than 50% reduction observed for aprA after PAO1 infection (Figure 7B). As a control, we found that the level of a non–quorum-sensing controlled gene (polA) was similar between da-GAL4/+ and UAS-PON1/da-GAL4 flies after infection (Figure 7B). Finally, since P. aeruginosa has other non–acyl-HSL–dependent quorum-sensing systems, PON1 could cause upregulation of these systems. As expected, we found no difference in pqsA expression, an upstream regulator of Pseudomonas quinolone signal (PQS) production, between da-GAL4/+ and UAS-PON1/da-GAL4 flies following P. aeruginosa infection (data not shown). In summary, these findings show that PON1 protection from P. aeruginosa infection is dependent upon 3OC12-HSL and suggest disruption of quorum-sensing–controlled signals (i.e., virulence factors) as a possible mechanism.

Figure 7

Quorum-sensing–dependent virulence gene activation is suppressed in PON1 flies. (A) da-GAL4/+ and UAS-PON1/da-GAL4 flies were infected with a quorum-sensing reporter strain of P. aeruginosa, PAO-1qsc102-lacZ. 18 hours later, X-gal staining was performed. Shown are representative images of da-GAL4/+ and UAS-PON1/da-GAL4 flies following infection. Scale bars: 0.5 mm. (B) Quorum-sensing–regulated (QS-regulated) and nonregulated genes. da-GAL4/+ and UAS-PON1/da-GAL4 flies were infected with P. aeruginosa, and 18 hours later, flies were sacrificed for mRNA analysis with RT-PCR. Data are expressed as percentage change (± SEM) in mRNA levels between daGAL4/+ and UAS-PON1/da-GAL4 flies. *P < 0.05 for mRNA expression between da-GAL4/+ and UAS-PON1/da-GAL4 flies following P. aeruginosa infection.

Our results from studies with quorum-sensing–deficient strains of P. aeruginosa and synthetic quorum-sensing molecules, specifically 3OC12-HSL, reveal that quorum sensing is required for P. aeruginosa virulence in vivo. Moreover, our work is the first, to our knowledge, to demonstrate that the in vivo expression of a lactonase, PON1, is able to disrupt quorum-sensing pathways and protect from P. aeruginosa–induced virulence. These findings are important because they allow us to better understand a possible role for PONs in the innate immune response to quorum-sensing–dependent bacterial pathogens.

Some of the original descriptions of quorum sensing and bacterial function focused on control of bioluminescence in the marine organism Vibrio fischeri (35, 36). Since then, many bacterial pathogens have been found to use quorum sensing, with the P. aeruginosalas-rhl system being one of the most widely studied. The earliest report of quorum sensing in P. aeruginosa focused on 3OC12-HSL’s (Pseudomonas autoinducer or PAI-1) control of elastase production (37). Subsequent studies have shown that quorum sensing regulates a large number of P. aeruginosa genes, specifically those involved in virulence, pathogenesis, and biofilm formation (12).

The most convincing data demonstrating a link between quorum sensing and P. aeruginosa virulence come from a number of animal models of infection, including burn wound, neonatal pneumonia, acute and chronic pneumonia, and ocular keratitis (16–21, 38). These studies have primarily been performed with P. aeruginosa laboratory strains deficient in 1 or more of the key components of the las and rhl systems, including lasI, lasR, rhlI, and/or rhlR. In general, these studies have found that, compared with wild-type P. aeruginosa, quorum-sensing–deficient strains result in minimal inflammation and tissue destruction, decreased bacterial dissemination, and less persistent infection. In some studies, mutant strains were complemented with plasmids expressing the absent quorum-sensing components and enhanced virulence was observed (17, 18, 38). Here, we report findings similar to those described above, but in D. melanogaster, through the use of quorum-sensing–deficient mutant strains of P. aeruginosa.

Our data show that C4- and 3OC12-HSL can complement the virulence of P. aeruginosa in vivo, in agreement with previous work. To the best of our knowledge, this is the first in vivo work to test the direct effect of 3OC12-HSL on bacterial virulence. Moreover, we found that the 3OC12-HSL–deficient strain of P. aeruginosa, ΔlasI, resulted in decreased lethality in flies and this was complemented by 3OC12-HSL feeding. This is consistent with hierarchal control of 3OC12-HSL and C4-HSL (39, 40). These findings are important because we and others have found that C4-HSL is a poor substrate for PON (6, 7). Taken together, these data convincingly show that 3OC12-HSL is required for P. aeruginosa virulence in vivo.

Certain naturally occurring lactonases can degrade or inactivate quorum-sensing signals. Expression of the lactonase aiiA, from Bacillus sp. bacteria, has been shown to decrease Erwinia carotovora–induced soft rot in plants, a quorum-sensing–dependent process (41–43). Several recent lines of evidence from in vitro studies have led to the hypothesis that PON’s lactonase activity may represent an important host defense mechanism against certain bacterial pathogens.

To test this hypothesis in vivo, we required an infection model in a whole organism and the ability to directly study PON’s role in host defense. Mice have all 3 PONs, making results from single PON-knockout models difficult to interpret and a triple-knockout mouse difficult to construct. Moreover, as explained before, acyl-HSL complementation is not easily possible in murine models. Because of the absence of PON in D. melanogaster, well-characterized models of infection in insects, and genetic tools available for D. melanogaster research, we took the approach of studying P. aeruginosa virulence in PON1 transgenic flies. In contrast with the typical paradigm in which immune mechanisms, such as Toll, Imd, and JAK/STAT pathways (23, 44) have been first identified in D. melanogaster and subsequently studied in humans, few if any studies have first used D. melanogaster to investigate putative components of the immune response in humans.

Our data show that PON1 transgenic flies are protected from lethality due to P. aeruginosa infection. In these studies, we began to investigate potential mechanisms for this effect. Although P. aeruginosa bacterial counts tended to be slightly lower following infection of PON1 flies compared with control flies, this small difference was neither statistically significant nor likely to be responsible for the dramatic increase in PON1 fly survival. Whether PON has a direct antibacterial effect on P. aeruginosa growth remains to be tested. Based upon the in vitro lactonase activity of PON1 flies and results from in vivo experiments with the ΔlasI mutant and exogenous 3OC12-HSL, PON1 flies appear to be protected secondary to degradation of 3OC12-HSL and disruption of downstream quorum-sensing pathways. To further address this possibility, we examined regulation of quorum-sensing–controlled genes following P. aeruginosa infection in control and PON1 flies. Of the many genes regulated by quorum sensing in P. aeruginosa (13–15), 4 of the most relevant virulence genes reported to be regulated by acyl-HSLs were tested. Expression of lasA, lasB, and aprA was lower in PON1 flies. Interestingly, aprA encodes an alkaline protease, which was recently shown to be required for P. entomophila virulence in flies (45). While we did not test to determine whether 3OC12-HSL or PON1 directly affect the fly’s innate immune response to P. aeruginosa, our data in which 3OC12-HSL feeding restored virulence to ΔlasI/rhlI but not ΔlasR/rhlR suggest this is not the case.

There are limited and conflicting data on the role of quorum sensing in human diseases. Most studies have focused on chronic infections such as those in CF and burn wounds. Singh et al. suggested a role of quorum sensing in biofilm formation in CF patients and found a characteristic acyl-HSL signature from sputum samples (46). 3OC12-HSL activity in CF sputum samples has subsequently been measured by others (47, 48). In contrast, genomic analysis of CF P. aeruginosa strains has shown that over time, quorum-sensing genes are lost (49). Similarly, several groups have found quorum-sensing mutants in isolates from mechanically ventilated patients and from respiratory tract, urinary tract, and wound infections (50, 51). These studies have ignored the host factors that may regulate acyl-HSLs. We speculate that PON may play an important role in setting up these chronic infections by degrading 3OC12-HSL and decreasing virulence, thus avoiding catastrophic outcomes. Interestingly, a recent paper showed social cheating by P. aeruginosa quorum-sensing mutants. Isolates deficient in lasR emerged over time and took advantage of members of the bacterial population, thereby decreasing the metabolic burden imposed by quorum sensing (52).

The role of PONs in the host defense response to infection in humans remains to be fully determined. Our findings may be applicable to human P. aeruginosa infection, and the relative contribution of different PONs may be different in sepsis, burn wound infections, pulmonary infections, and other human P. aeruginosa infections. Since PON1 is primarily localized in the bloodstream, we postulate that PON1 would be most important for bloodstream infections. Alternatively, PON1 may reach tissue sites of infection, similar to other serum proteins, by leakage out of the vasculature (as might occur in burn wound infections). We have previously shown that PON2 is the predominant isoform of PON in human airway epithelial cells and that loss of PON2 (in murine airway epithelial cells from PON2-knockout mice) impairs 3OC12-HSL–inactivating activity (9). PON1-knockout mice demonstrated slightly improved survival following an intraperitoneal challenge with P. aeruginosa; however, absence of PON1 led to increased PON2 and PON3 levels, making these data difficult to interpret (6). Several polymorphisms have been described for PON family members, and natural variation in PON activity as a genetic modifier may be responsible for differing rates of pulmonary decline in CF patients with a similar CFTR genotype, development of ventilator-associated pneumonia or burn wound infections by P. aeruginosa, or chronic P. aeruginosa colonization in idiopathic bronchiectasis.

A limitation of this work is that our studies were performed using D. melanogaster and an injury model of infection. Whether PON will confer protection from P. aeruginosa infection in other model organisms, such as mice, or is important in humans for host defense against infection remains to be determined. However, in humans, decreased PON levels and PON single-nucleotide polymorphisms have been linked to numerous diseases, including cerebrovascular disease, atherosclerosis, coronary heart disease, inflammatory bowel disease, Behçet disease, sporadic amyotrophic lateral sclerosis, and Helicobacter pylori infection (53–57). The basis for these linkages is thought to be disruptions in the cellular oxidant-antioxidant balance. We speculate that some of these disease associations may be secondary to PON’s unique ability to disrupt quorum-sensing pathways. Yet-to-be-identified microbial pathogens or bacteria that are not culturable with traditional microbiology techniques may use lactone-based molecules for their disease pathogenesis. Some of these microorganisms might play an unrecognized role in these clinically important diseases. For example, infectious etiologies have been proposed for a number of illnesses not originally thought to be dependent upon microbial pathogens, including a proposed link between inflammation/infection (Chlamydia pneumoniae and H. pylori infection) and coronary heart disease (58, 59). Since acyl-HSL–dependent quorum-sensing pathways are very prevalent in Gram-negative bacteria (60), we speculate that PON may be important in the pathogenesis of a number of human illnesses with an underlying infectious etiology.

Taken together, our results using genetically modified strains of P. aeruginosa (deficient in key quorum-sensing components), acyl-HSL complementation, and PON1 transgenic D. melanogaster prove the importance of 3OC12-HSL for P. aeruginosa virulence. We speculate that genetic variability in PONs may be linked to the pathogenesis of P. aeruginosa infection. Novel therapeutic interventions aimed at directly regulating quorum sensing or PON activity may show promise for treatment. Finally, these results provide insight into what we believe is a newly described function of PON and suggest how this lactonase might be important for control of P. aeruginosa pathogenesis.

View Supplemental data

We thank Nick Gansemer, Yuhong Li, Thomas Recker, Michael Rector, Ashley Small, Sara Spielberger, Alex Thorne, and David Zabner for excellent assistance; Leonid Gaydukov and Dan Tawfik for lactonase activity measurements; and Kate Excoffon, Pete Greenberg, Bryant McAllister, and Michael Welsh for insightful discussions. These studies were supported in part by NHLBI 61234-09 and NIAID AI076671. David A. Stoltz is supported by a Parker B. Francis fellowship.

Address correspondence to: Joseph Zabner, Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, 440 EMRB, Iowa City, Iowa 52242, USA. Phone: (319) 335-7608; Fax: (319) 335-7623; E-mail: joseph-zabner@uiowa.edu.

Nonstandard abbreviations used: CF, cystic fibrosis; 3OC12-HSL, N-3-oxododecanoyl HSL; C4-HSL, N-butanoyl HSL; da, daughterless; DSM, demeton-S-methyl; DTNB, 5,5′-dithio-bis-2-nitrobenzoic acid; HSL, homoserine lactone; PON, paraoxonase; ppm, parts per million; TBBL, 5-thiobutyl butyrolactone; UAS, upstream activation sequence.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.118:3123–3131 (2008). doi:10.1172/JCI35147

1. Gan, K.N., Smolen, A., Eckerson, H.W., La Du, B.N. 1991. Purification of human serum paraoxonase/arylesterase. Evidence for one esterase catalyzing both activities. Drug Metab. Dispos. 19:100-106.
   View this article via: PubMed Google Scholar
2. Furlong, C.E., et al. 1993. Human and rabbit paraoxonases: purification, cloning, sequencing, mapping and role of polymorphism in organophosphate detoxification. Chem. Biol. Interact. 87:35-48.
   View this article via: CrossRef PubMed Google Scholar
3. Furlong, C.E., Richter, R.J., Chapline, C., Crabb, J.W. 1991. Purification of rabbit and human serum paraoxonase. Biochemistry. 30:10133-10140.
   View this article via: CrossRef PubMed Google Scholar
4. Durrington, P.N., Mackness, B., Mackness, M.I. 2001. Paraoxonase and atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 21:473-480.
   View this article via: PubMed Google Scholar
5. Shih, D.M., et al. 1998. Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis. Nature. 394:284-287.
   View this article via: CrossRef PubMed Google Scholar
6. Ozer, E.A., et al. 2005. Human and murine paraoxonase 1 are host modulators of Pseudomonas aeruginosa quorum-sensing. FEMS Microbiol. Lett. 253:29-37.
   View this article via: PubMed Google Scholar
7. Yang, F., et al. 2005. Quorum quenching enzyme activity is widely conserved in the sera of mammalian species. FEBS Lett. 579:3713-3717.
   View this article via: CrossRef PubMed Google Scholar
8. Draganov, D.I., et al. 2005. Human paraoxonases (PON1, PON2, and PON3) are lactonases with overlapping and distinct substrate specificities. J. Lipid. Res. 46:1239-1247.
   View this article via: CrossRef PubMed Google Scholar
9. Stoltz, D.A., et al. 2007. Paraoxonase-2 deficiency enhances Pseudomonas aeruginosa quorum sensing in murine tracheal epithelia. Am. J. Physiol. Lung Cell Mol. Physiol. 292:L852-L860.
   View this article via: CrossRef PubMed Google Scholar
10. Gaidukov, L., Tawfik, D.S. 2005. High affinity, stability, and lactonase activity of serum paraoxonase PON1 anchored on HDL with ApoA-I. Biochemistry. 44:11843-11854.
    View this article via: CrossRef PubMed Google Scholar
11. Khersonsky, O., Tawfik, D.S. 2005. Structure-reactivity studies of serum paraoxonase PON1 suggest that its native activity is lactonase. Biochemistry. 44:6371-6382.
    View this article via: CrossRef PubMed Google Scholar
12. Parsek, M.R., Greenberg, E.P. 2000. Acyl-homoserine lactone quorum sensing in gram-negative bacteria: a signaling mechanism involved in associations with higher organisms. Proc. Natl. Acad. Sci. U. S. A. 97:8789-8793.
    View this article via: CrossRef PubMed Google Scholar
13. Whiteley, M., Lee, K.M., Greenberg, E.P. 1999. Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 96:13904-13909.
    View this article via: CrossRef PubMed Google Scholar
14. Wagner, V.E., Bushnell, D., Passador, L., Brooks, A.I., Iglewski, B.H. 2003. Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment. J. Bacteriol. 185:2080-2095.
    View this article via: CrossRef PubMed Google Scholar
15. Schuster, M., Lostroh, C.P., Ogi, T., Greenberg, E.P. 2003. Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J. Bacteriol. 185:2066-2079.
    View this article via: CrossRef PubMed Google Scholar
16. Tang, H.B., et al. 1996. Contribution of specific Pseudomonas aeruginosa virulence factors to pathogenesis of pneumonia in a neonatal mouse model of infection. Infect. Immun. 64:37-43.
    View this article via: PubMed Google Scholar
17. Rumbaugh, K.P., Griswold, J.A., Iglewski, B.H., Hamood, A.N. 1999. Contribution of quorum sensing to the virulence of Pseudomonas aeruginosa in burn wound infections. Infect. Immun. 67:5854-5862.
    View this article via: PubMed Google Scholar
18. Pearson, J.P., Feldman, M., Iglewski, B.H., Prince, A. 2000. Pseudomonas aeruginosa cell-to-cell signaling is required for virulence in a model of acute pulmonary infection. Infect. Immun. 68:4331-4334.
    View this article via: CrossRef PubMed Google Scholar
19. Wu, H., et al. 2001. Pseudomonas aeruginosa mutations in lasI and rhlI quorum sensing systems result in milder chronic lung infection. Microbiology. 147:1105-1113.
    View this article via: PubMed Google Scholar
20. Smith, R.S., Harris, S.G., Phipps, R., Iglewski, B. 2002. The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)homoserine lactone contributes to virulence and induces inflammation in vivo. J. Bacteriol. 184:1132-1139.
    View this article via: CrossRef PubMed Google Scholar
21. Imamura, Y., et al. 2005. Role of Pseudomonas aeruginosa quorum-sensing systems in a mouse model of chronic respiratory infection. J. Med. Microbiol. 54:515-518.
    View this article via: CrossRef PubMed Google Scholar
22. Lemaitre, B., et al. 1996. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell. 86:973-983.
    View this article via: CrossRef PubMed Google Scholar
23. Lemaitre, B., Hoffmann, J. 2007. The host defense of Drosophila melanogaster. Annu. Rev. Immunol. 25:697-743.
    View this article via: CrossRef PubMed Google Scholar
24. Boman, H.G., Nilsson, I., Rasmuson, B. 1972. Inducible antibacterial defence system in Drosophila. Nature. 237:232-235.
    View this article via: CrossRef PubMed Google Scholar
25. D’Argenio, D.A., Gallagher, L.A., Berg, C.A., Manoil, C. 2001. Drosophila as a model host for Pseudomonas aeruginosa infection. J. Bacteriol. 183:1466-1471.
    View this article via: CrossRef PubMed Google Scholar
26. Lau, G.W., et al. 2003. The Drosophila melanogaster toll pathway participates in resistance to infection by the gram-negative human pathogen Pseudomonas aeruginosa. Infect. Immun. 71:4059-4066.
    View this article via: CrossRef PubMed Google Scholar
27. Chugani, S.A., et al. 2001. QscR, a modulator of quorum-sensing signal synthesis and virulence in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 98:2752-2757.
    View this article via: CrossRef PubMed Google Scholar
28. Salunkhe, P., et al. 2005. A cystic fibrosis epidemic strain of Pseudomonas aeruginosa displays enhanced virulence and antimicrobial resistance. J. Bacteriol. 187:4908-4920.
    View this article via: CrossRef PubMed Google Scholar
29. Erickson, D.L., et al. 2004. Pseudomonas aeruginosa relA contributes to virulence in Drosophila melanogaster. Infect. Immun. 72:5638-5645.
    View this article via: CrossRef PubMed Google Scholar
30. Brand, A.H., Perrimon, N. 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 118:401-415.
    View this article via: PubMed Google Scholar
31. Josse, D., et al. 1999. Identification of residues essential for human paraoxonase (PON1) arylesterase/organophosphatase activities. Biochemistry. 38:2816-2825.
    View this article via: CrossRef PubMed Google Scholar
32. Healy, M.J., Dumancic, M.M., Oakeshott, J.G. 1991. Biochemical and physiological studies of soluble esterases from Drosophila melanogaster. Biochem. Genet. 29:365-388.
    View this article via: CrossRef PubMed Google Scholar
33. Khersonsky, O., Tawfik, D. S. 2006. Chromogenic and fluorogenic assays for the lactonase activity of serum paraoxonases. Chembiochem. 7:49-53.
    View this article via: CrossRef PubMed Google Scholar
34. Geldmacher von Mallinckrodt, M., Diepgen, T.L. 1988. The human serum paraoxonase: polymorphism and specificity. Toxicol. Environ. Chem. 18:79-196.
35. Nealson, K.H., Platt, T., Hastings, J.W. 1970. Cellular control of the synthesis and activity of the bacterial luminescent system. J. Bacteriol. 104:313-322.
    View this article via: PubMed Google Scholar
36. Eberhard, A., et al. 1981. Structural identification of autoinducer of Photobacterium fischeri luciferase. Biochemistry. 20:2444-2449.
    View this article via: CrossRef PubMed Google Scholar
37. Passador, L., Cook, J.M., Gambello, M.J., Rust, L., Iglewski, B.H. 1993. Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication. Science. 260:1127-1130.
    View this article via: CrossRef PubMed Google Scholar
38. Zhu, H., et al. 2004. Pseudomonas aeruginosa with lasI quorum-sensing deficiency during corneal infection. Invest. Ophthalmol. Vis. Sci. 45:1897-1903.
    View this article via: CrossRef PubMed Google Scholar
39. Latifi, A., Foglino, M., Tanaka, K., Williams, P., Lazdunski, A. 1996. A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary-phase sigma factor RpoS. Mol. Microbiol. 21:1137-1146.
    View this article via: CrossRef PubMed Google Scholar
40. Pesci, E.C., Pearson, J.P., Seed, P.C., Iglewski, B.H. 1997. Regulation of las and rhl quorum sensing in Pseudomonas aeruginosa. J. Bacteriol. 179:3127-3132.
    View this article via: PubMed Google Scholar
41. Dong, Y.H., et al. 2001. Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase. Nature. 411:813-817.
    View this article via: CrossRef PubMed Google Scholar
42. Dong, Y.H., Xu, J.L., Li, X.Z., Zhang, L.H. 2000. AiiA, an enzyme that inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. Proc. Natl. Acad. Sci. U. S. A. 97:3526-3531.
    View this article via: CrossRef PubMed Google Scholar
43. Dong, Y.H., Gusti, A.R., Zhang, Q., Xu, J.L., Zhang, L.H. 2002. Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species. Appl. Environ. Microbiol. 68:1754-1759.
    View this article via: CrossRef PubMed Google Scholar
44. Pinheiro, V.B., Ellar, D.J. 2006. How to kill a mocking bug? Cell. Microbiol. 8:545-557.
    View this article via: CrossRef PubMed Google Scholar
45. Liehl, P., Blight, M., Vodovar, N., Boccard, F., Lemaitre, B. 2006. Prevalence of local immune response against oral infection in a Drosophila/Pseudomonas infection model. PLoS Pathog. 2:e56.
    View this article via: CrossRef PubMed Google Scholar
46. Singh, P.K., et al. 2000. Quorum-sensing signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature. 407:762-764.
    View this article via: CrossRef PubMed Google Scholar
47. Erickson, D.L., et al. 2002. Pseudomonas aeruginosa quorum-sensing systems may control virulence factor expression in the lungs of patients with cystic fibrosis. Infect. Immun. 70:1783-1790.
    View this article via: CrossRef PubMed Google Scholar
48. Middleton, B., et al. 2002. Direct detection of N-acylhomoserine lactones in cystic fibrosis sputum. FEMS Microbiol. Lett. 207:1-7.
    View this article via: CrossRef PubMed Google Scholar
49. Smith, E.E., et al. 2006. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc. Natl. Acad. Sci. U. S. A. 103:8487-8492.
    View this article via: CrossRef PubMed Google Scholar
50. Denervaud, V., et al. 2004. Characterization of cell-to-cell signaling-deficient Pseudomonas aeruginosa strains colonizing intubated patients. J. Clin. Microbiol. 42:554-562.
    View this article via: CrossRef PubMed Google Scholar
51. Schaber, J.A., et al. 2004. Analysis of quorum sensing-deficient clinical isolates of Pseudomonas aeruginosa. J. Med. Microbiol. 53:841-853.
    View this article via: CrossRef PubMed Google Scholar
52. Sandoz, K.M., Mitzimberg, S.M., Schuster, M. 2007. Social cheating in Pseudomonas aeruginosa quorum sensing. Proc. Natl. Acad. Sci. U. S. A. 104:15876-15881.
    View this article via: CrossRef PubMed Google Scholar
53. Ng, C.J., et al. 2005. The paraoxonase gene family and atherosclerosis. Free Radic. Biol. Med. 38:153-163.
    View this article via: CrossRef PubMed Google Scholar
54. Sanchez, R., et al. 2006. Paraoxonase 1, 2 and 3 DNA variants and susceptibility to childhood inflammatory bowel disease. Gut. 55:1820-1821.
    View this article via: CrossRef PubMed Google Scholar
55. Aslan, M., et al. 2006. Serum paraoxonase-1 activity in Helicobacter pylori infected subjects. Atherosclerosis. 196:270-274.
    View this article via: CrossRef PubMed Google Scholar
56. Karakucuk, S., et al. 2004. Serum paraoxonase activity is decreased in the active stage of Behcet’s disease. Br. J. Ophthalmol. 88:1256-1258.
    View this article via: CrossRef PubMed Google Scholar
57. Slowik, A., et al. 2007. Paraoxonase 2 gene C311S polymorphism is associated with a risk of large vessel disease stroke in a Polish population. Cerebrovasc. Dis. 23:395-400.
    View this article via: PubMed Google Scholar
58. Franceschi, F., et al. 2005. Helicobacter pylori infection and ischaemic heart disease: an overview of the general literature. Dig. Liver Dis. 37:301-308.
    View this article via: CrossRef PubMed Google Scholar
59. Kalayoglu, M.V., Libby, P., Byrne, G.I. 2002. Chlamydia pneumoniae as an emerging risk factor in cardiovascular disease. JAMA. 288:2724-2731.
    View this article via: CrossRef PubMed Google Scholar
60. Federle, M.J., Bassler, B.L. 2003. Interspecies communication in bacteria. J. Clin. Invest. 112:1291-1299.
    View this article via: JCI PubMed Google Scholar
61. Avet-Rochex, A., et al. 2005. Suppression of Drosophila cellular immunity by directed expression of the ExoS toxin GAP domain of Pseudomonas aeruginosa. Cell. Microbiol. 7:799-810.
    View this article via: CrossRef PubMed Google Scholar
62. Shaw, P.D., et al. 1997. Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography. Proc. Natl. Acad. Sci. U. S. A. 94:6036-6041.
    View this article via: CrossRef PubMed Google Scholar
63. Phillips, J.P., et al. 1990. Transfer and expression of an organophosphate insecticide-degrading gene from Pseudomonas in Drosophila melanogaster. Proc. Natl. Acad. Sci. U. S. A. 87:8155-8159.
    View this article via: CrossRef PubMed Google Scholar