Research Article

Host monitoring of quorum sensing during Pseudomonas aeruginosa infection

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Science  20 Dec 2019:
Vol. 366, Issue 6472, eaaw1629
DOI: 10.1126/science.aaw1629

Spying on bacterial signals

Many bacteria produce small molecules for monitoring population density and thus regulating their collective behavior, a process termed quorum sensing. Pathogens like Pseudomonas aeruginosa, which complicates cystic fibrosis disease, produce different quorum-sensing ligands at different stages of infection. Moura-Alves et al. used experiments in human cells, zebrafish, and mice to show that a host organism can eavesdrop on these bacterial conversations. A host sensor responds differentially to bacterial quorum-sensing molecules to activate or repress different response pathways. The ability to “listen in” on bacterial signaling provides the host with the capacity to fine-tune physiologically costly immune responses.

Science, this issue p. eaaw1629

Structured Abstract

INTRODUCTION

The interaction between a bacterial pathogen and its host can be viewed as an “arms race” in which each participant continuously responds to the evolving strategies of the other partner. A mechanism allowing bacteria to rapidly adapt to such changing circumstances is provided by density-dependent cell-to-cell communication known as quorum sensing (QS). QS involves a hierarchy of signaling molecules, which in pathogenic bacteria is associated with biofilm formation and virulence regulation. Notably, some QS molecules are detected by the host, and these can provoke specific immune responses. However, the receptors and their signaling pathways that the host uses to eavesdrop on bacteria remain poorly understood.

RATIONALE

We hypothesized that if a host sensor can detect and differentiate between bacterial QS molecules and their expression patterns, it will allow hosts to customize their immune responses according to the stage and state of infection. We recently showed that the aryl hydrocarbon receptor (AhR) directly recognizes pigmented bacterial virulence factors, such as the phenazines produced by Pseudomonas aeruginosa, which are downstream products of QS. Upon binding phenazines, the AhR elicits diverse immune responses that coordinate host resistance to infection. As a result of its capacity to sense a broad array of ligands, we postulated that the host AhR is well positioned to spy on bacterial communications, continuously monitor bacterial infection dynamics, and thereby signal to the host to tune immune responses according to the state of infection.

RESULTS

Our results demonstrated that infected hosts show differential modulation of host AhR signaling over the course of P. aeruginosa infection in zebrafish, mice, and human cells. AhR signaling depended on the relative abundances of several classes of P. aeruginosa QS molecules, including homoserine lactones (e.g., N-3-oxo-dodecanoyl-homoserine lactone), quinolones (e.g., 4-hydroxy-2-heptylquinoline), and phenazines (e.g., pyocyanin). In vitro and in vivo studies showed that the AhR not only detects P. aeruginosa QS molecules in a qualitative way but also quantifies their relative abundances. Quantitative assessment enables the host to sense bacterial community densities that may have distinct gene expression programs and infection dynamics, and thereby to regulate the scale and intensity of host defense mechanisms, which can range from induction of inflammatory mediators to immune cell recruitment and bacterial clearance.

CONCLUSION

Our findings emphasize a crucial role for host AhR as master regulator of host defense responses, capable of tuning immunity according to the stage of infection and disease. By inhibiting profuse and inessential immune responses, the host can counteract some of the detrimental effects of infection and avoid collateral damage. We propose that host surveillance of bacterial communication allows not only a trade-off between energy expenditure and efficient defense in the host, but also a trade-off between energy expenditure and virulence in the pathogen.

QS is not restricted to P. aeruginosa, and we postulate that monitoring of bacterial QS by hosts may be a widespread phenomenon. Different therapeutic strategies to manipulate P. aeruginosa QS have been attempted, including adaptive treatment regimens for cystic fibrosis patients, who suffer severely from this pathogen. A better understanding of the cross-talk between host AhR and bacterial QS could pave the way to specific host-directed therapies to treat infectious diseases, tailored not only to the type of infection but also to the specific stage of disease.

Bacterial communication under the radar of the host aryl hydrocarbon receptor (AhR).

The AhR spies on bacterial communication and translates the bacterial signaling vocabulary into the most appropriate host defenses. The expression of bacterial quorum-sensing molecules, such as homoserine lactones, quinolones, and phenazines, varies according to community density and state of infection. The AhR can detect the type and quantity of quorum-sensing molecules and hence the state of infection, and thus tunes host defenses.

Abstract

Pseudomonas aeruginosa rapidly adapts to altered conditions by quorum sensing (QS), a communication system that it uses to collectively modify its behavior through the production, release, and detection of signaling molecules. QS molecules can also be sensed by hosts, although the respective receptors and signaling pathways are poorly understood. We describe a pattern of regulation in the host by the aryl hydrocarbon receptor (AhR) that is critically dependent on qualitative and quantitative sensing of P. aeruginosa quorum. QS molecules bind to AhR and distinctly modulate its activity. This is mirrored upon infection with P. aeruginosa collected from diverse growth stages and with QS mutants. We propose that by spying on bacterial quorum, AhR acts as a major sensor of infection dynamics, capable of orchestrating host defense according to the status quo of infection.

Pseudomonas aeruginosa is a resourceful and ubiquitous Gram-negative bacterium that causes infectious diseases in a broad spectrum of organisms, including plants, animals, and humans (1). Its prevalence in burn victims, cystic fibrosis (CF) patients, and immunocompromised individuals (such as AIDS patients) is commonly associated with a poor, often fatal outcome (2). P. aeruginosa is also a major cause of nosocomial infections, such as bacterial pneumonia, urinary tract infection, and surgical-wound contamination (1). Because of its profound antibiotic resistance, therapy of P. aeruginosa is extremely difficult (1). Moreover, this pathogen possesses a wide range of mechanisms to adapt to different and sometimes harsh environments, further aggravating its eradication, even by antibiotic treatment (1).

One such important and unifying mechanism is the capacity of P. aeruginosa to perform quorum sensing (QS) (1, 3, 4). QS is a cell-to-cell signaling mechanism used by different bacteria to coordinate their activities in response to changes in community density. This coordination depends on chemical communication using different diffusible molecules, so-called autoinducers, and their receptors (Fig. 1A) (3, 4). In P. aeruginosa, QS regulates the production of a vast set of virulence factors, such as extracellular proteases and phenazines, and is crucial for colonization and infection, regulating diverse mechanisms such as biofilm formation and antimicrobial resistance (1, 35). Differences in P. aeruginosa virulence and transition from acute to chronic infection have been linked to changes in autoinducer levels and in the expression of QS-regulated genes (1, 3, 68). Consequently, QS constitutes an obvious target in the current search for novel treatment options for P. aeruginosa infections (3, 4, 9). Changes in the expression of autoinducers and QS-regulated genes may have an impact not only on bacterial community dynamics, but also on the host response during infection. It was previously reported that different QS-regulated molecules, such as homoserine lactones (HSLs), quinolones, and phenazines, can interact with host cells, thereby influencing a broad range of responses including immunomodulation (9). Thus far, the host receptors and signaling pathways, as well as the mechanisms involved in monitoring infection dynamics, are incompletely understood.

Fig. 1 AhR modulation by P. aeruginosa.

(A) Scheme of AhR sensing of P. aeruginosa QS molecules during infection. In this depiction of the P. aeruginosa signaling cascade during different bacterial growth stages. QS molecules are shown in black and proteins in colored circles, with different colors corresponding to each QS molecule. The black arrow with asterisk indicates a known interaction between P. aeruginosa phenazines and host AhR. (B) Luciferase activity of AhR reporter THP-1 (monocytic) and A549 (pneumocytic) cells upon 24 hours of infection with P. aeruginosa PA14 WT strain grown in lysogeny broth (LB) medium, at a multiplicity of infection (MOI) of 50 (pooled data from n = 3 independent experiments). (C) Luciferase activity of AhR reporter THP-1 and A549 cells upon 24 hours of stimulation with P. aeruginosa filtered supernatants (1:25 diluted), collected from different bacterial growth phases (pooled data from n = 4 independent experiments). (D) Expression of QS molecules in supernatants of PA14 WT, detected by HPLC. Data are from one representative experiment of two independent experiments. (E) Luciferase activity of AhR reporter THP-1 and A549 cells upon 4 hours of stimulation with different concentrations of P. aeruginosa homoserine lactones (3-o-C12-L-HSL or C4-L-HSL) and quinolones (HHQ or PQS) in the absence of P. aeruginosa 1-HP; pooled data from n = 6 (THP-1) or n = 4 (A549) independent experiments. (F and G) Same as (E) but in the presence of P. aeruginosa 1-HP; pooled data from n = 3 (THP-1) or n = 4 (A549) independent experiments (F); n = 3 (THP-1), n = 9 (A549, top), or n = 3 (A549, bottom) independent experiments (G). (H) CYP1A1 gene expression upon 24 hours of stimulation of A549 cells with QS molecules. Data are from one representative experiment of at least three independent experiments (n = 3 biological replicates). (I and J) CYP1A1 enzymatic activity after 24 hours of stimulation of Hepa-1c1c7 cells with 50 μM 1-HP alone (I) or in the presence or absence of other QS molecules (J). Data are pooled from n = 7 or n = 4 independent experiments, respectively. Pyo, pyocyanin; 1-HP, 1-hydroxyphenazine; PCA, phenazine-1-carboxylic acid; PCN, phenazine carboxamide, 3-o-C12-L-HSL, N-(3-oxodecanoyl)-l-homoserine lactone; C4-L-HSL, N-butyril-l-homoserine lactone; HHQ, 4-hydroxy-2-heptylquinoline; PQS, 2-heptyl-3,4-dihydroxyquinoline; IQS, 2-(2-hydroxylphenyl)-thiazole-4-carbaldehyde. Data are means ± SEM [(B), (C), (E), (F), (G), (I)] or means ± SD (H). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 [one-way analysis of variance (ANOVA) in (B), (C), (E), (F), (H), (J); two-tailed Student t test in (I)].

Recently, we demonstrated that the aryl hydrocarbon receptor (AhR), a highly conserved ligand-dependent transcription factor, directly recognizes P. aeruginosa phenazines and thereby plays an important role in infection control (10). AhR binds to phenazines, mediates their degradation, and regulates the expression of several host genes including detoxifying enzymes, chemokines, and cytokines. Accordingly, resistance of AhR-deficient (AhR–/–) mice to P. aeruginosa is diminished (10). Taking into consideration the vast set of ligands that AhR is able to detect and the numerous biological roles it can exert, we hypothesized that AhR monitors the course of bacterial infection and disease by sensing different bacterial QS molecules expressed at various stages of infection (Fig. 1A), and thereby orchestrates the most appropriate immune response against different stages of infection.

AhR senses bacterial QS molecules in vitro

Using luciferase AhR reporter cells (10), we infected THP-1 macrophages (THP-1 AhR reporter) and A549 alveolar type II pneumocytes (A549 AhR reporter) with P. aeruginosa laboratory wild-type UCBPP-PA14 (PA14 WT) and green fluorescent protein–labeled (PA14 WT-GFP) strains collected from distinct stages of bacterial growth (early log, OD600 < 0.3; mid-log, 0.5 < OD600 < 0.8; late log, OD600 > 1). AhR was more profoundly activated by bacteria from later growth phases (Fig. 1B and fig. S1A), whereas multiplicity of infection (MOI; fig. S1B) and percentage of infected cells remained comparable over the different growth stages (fig. S1, C and D). Similar results were obtained with filtered bacterial supernatants from PA14 WT strains (Fig. 1C and fig. S1E), pointing to different AhR signaling by distinct P. aeruginosa molecules. A comparable phenotype was observed using supernatants from PAO1, a different commonly used P. aeruginosa laboratory strain (fig. S1F). Among the obvious candidates are the P. aeruginosa phenazines, previously identified as AhR ligands (10). Consistently, increasing concentrations of the P. aeruginosa phenazine pyocyanin (Pyo) were detected in PA14 supernatants along bacterial growth stages (fig. S1, G and H), correlating with the observed AhR activation (Fig. 1, B and C, and fig. S1, A and E).

Phenazines are among the QS-regulated molecules expressed by P. aeruginosa, with Pyo providing a terminal signal of QS (3, 4, 11, 12). P. aeruginosa QS is regulated by four tightly controlled pathways, namely Las, Rhl, Pqs, and Iqs (Fig. 1A) (3, 4, 12). These pathways are tightly interconnected and their cognate autoinducer molecules are capable of activating a distinct downstream transcriptional pathway (Fig. 1A). In brief, N-3-oxo-dodecanoyl-homoserine lactone (3-o-C12-L-HSL) and N-butanoyl-homoserine lactone (C4-L-HSL) are produced in a sequential manner via Las and Rhl systems, and activate the receptors LasR and RhlR, respectively (3, 4, 12). A third pathway, Pqs, leads to the synthesis of the Pseudomonas quinolone signaling molecule 2-heptyl-3-hydroxy-4-quinolone (PQS) and its precursor 4-hydroxy-2-heptylquinoline (HHQ), which signal via the receptor PqsR (3, 4, 12). Recently, the Iqs pathway was discovered; however, the mechanisms by which 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde (IQS) and its receptor are produced are less well understood (1, 3).

Using high-performance liquid chromatography (HPLC), we confirmed a sequential autoinducer abundance in the supernatants of PA14 (Fig. 1D). Considering the distinct expression profiles of the QS molecules 3-o-C12-L-HSL, C4-L-HSL, HHQ, and PQS, we determined their ability to modulate canonical AhR signaling. Stimulation of THP-1 and A549 AhR reporter cells with the different P. aeruginosa QS molecules resulted in differential modulation of AhR signaling (Fig. 1E). 3-o-C12-L-HSL and HHQ potently inhibited AhR activation by the known Pseudomonas AhR ligand 1-hydroxyphenazine (1-HP) (10) in a dose-dependent manner (Fig. 1, F and G). Several QS molecules have been reported to induce apoptosis in host cells, depending on the concentration, cell type, and exposure time (13, 14). No major differences in cell viability were detected for the majority of the conditions tested here, as measured by lactate dehydrogenase (LDH) release (fig. S2A). An exception occurred after 24 hours of stimulation of THP-1 cells with high concentrations of 3-o-C12-L-HSL (fig. S2A). These results are in agreement with previous studies showing that epithelial cells, such as A549, are more resistant to 3-o-C12-L-HSL–induced apoptosis than macrophages (13, 14). All experiments with THP-1 cells in the presence of 3-o-C12-L-HSL were performed at earlier time points, when no differences in cell viability were detected. Yet we decided to further exclude a possible relationship between apoptosis-related effects and AhR modulation in this cell type. As shown in fig. S2, B to E, no relationship was observed, and we decided to focus on A549 cells in following experiments.

Previous studies showed that concentrations of QS molecules in P. aeruginosa, such as 3-o-C12-L-HSL, can vary profoundly according to growth status, type of culture [planktonic cultures (1 to 5 μM) or biofilms (up to 600 μM)], and sample type (sputum or murine infection samples) (1518). Notably, high concentrations of these molecules have been detected in biofilms of CF patients’ lungs, and thus in close contact with the epithelium (18). Consequently, we decided to use 50 μM of the different QS molecules in subsequent studies.

A hallmark of AhR activity is the transcriptional induction of genes that encode detoxifying enzymes, such as CYP1A1 and CYP1B1, and the AhR repressor (AhRR) (19). As previously reported (10), stimulation of A549 cells with 1-HP induces mRNA expression of these genes (Fig. 1H and fig. S3A). Intriguingly, 3-o-C12-L-HSL and HHQ inhibited 1-HP–induced gene expression (Fig. 1F and fig. S3A). Because CYP1A1 is involved in tryptophan metabolism, alterations in its expression and activity can influence AhR activation (20, 21). We took advantage of an established model using mouse liver cells (Hepa-1c1c7), which express copious levels of CYP1A1 and are therefore best suited to detect its expression and enzymatic activity (22). Similar to other cell types, AhR activation in hepatocytes was induced by 1-HP, as measured by increased luciferase activity in an AhR reporter cell line, and led to an increase in CYP1A1 enzymatic activity, as measured by the ethoxyresorufin-O-deethylase (EROD) assay (Fig. 1I and fig. S3, B and C). Intriguingly, 3-o-C12-L-HSL, HHQ, and PQS inhibited 1-HP–induced AhR activation and CYP1A1 enzymatic activity in these cells, whereas C4-L-HSL did not (Fig. 1J and fig. S3, B and C). In sum, QS molecules, including HSLs, quinolones, and phenazines, modulated AhR activity in both a stimulatory and an inhibitory direction.

QS molecules are not only expressed by P. aeruginosa; several other Gram-negative bacteria also produce HSLs, with subtle modifications, mostly in the carbon side chain (3, 4) (table S1). Because the crystal structure of AhR has not yet been solved, it is challenging to predict ligands that bind to AhR. Taking advantage of the AhR-modulatory properties of a vast number of HSLs and their tested analogs (fig. S4, A to C), we optimized an existing in silico model (10) to interrogate whether and how these QS molecules from P. aeruginosa can be accommodated in the AhR-binding pocket (Fig. 2A). The ligands were divided by impact on agonistic or competitive behavior and sorted according to increasing molecular mechanics generalized Born surface area (MM-GBSA) binding energies (ΔGBind). This revealed 3-o-C12-L-HSL as the strongest binder and C4-L-HSL as the weakest binder in this study (fig. S4D). In this model, all residues previously found to interact with the bona fide AhR ligand 2,3,7,8-tetrachlorodibenzodioxin (TCDD) by mutagenesis experiments (23, 24) are predicted to be involved in forming the binding pocket. The key residues, Thr289, His291, Phe295, Ser365, and Gln383, form hydrogen bonds with most of the ligands investigated here (fig. S4, D and E). Furthermore, and in agreement with data retrieved from ligand-selective modulation of AhR ligand binding, AhR complexes with bound competitors showed additional hydrophobic interactions with Phe287, Leu308, and Leu315 (fig. S4, D and E) (25). 1-HP is predicted to contact Phe324 via interactions of the aromatic rings (fig. S4E). This residue is known to mediate agonist/antagonist switching upon mutation (Phe324 → Ala/Leu) and converts agonists such as 3-methylcholanthrene (3-MC) or β-naphthoflavone (BNF) into antagonists (25). Predictions were validated by ligand-binding studies (10) that confirmed the binding of 3-o-C12-L-HSL and HHQ, with dissociation constant (Kd) values of 4.67 μM and 3.77 μM, respectively (Fig. 2B and fig. S4F). In addition, we developed a complementary method to detect AhR binding of different ligands, including TCDD (26), using purified AhR and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins in a microscale thermophoresis (MST) assay (fig. S4G). This approach also demonstrated AhR binding to QS molecules including 3-o-C12-L-HSL, PQS, and 1-HP, but not to C4-L-HSL (Fig. 2C). (HHQ binding could not be analyzed by MST because of its intrinsic fluorescence properties, which interfere with the assay.) Together, these findings show that various QS molecules other than phenazines bind to AhR and modulate its activity; hence, this pathway is appropriate as a potential target for sensing bacterial infection dynamics in the host.

Fig. 2 Binding of P. aeruginosa QS molecules to AhR.

(A) In silico docking of P. aeruginosa QS molecules into the AhR ligand-binding pocket. (B) Binding of QS molecules to AhR, as measured by displacement of radioactive [3H]2,3,7,8-tetrachlorodibenzodioxin ([3H]TCDD) from AhR in wild-type mouse liver cytosol. Kd values: 3-o-C12-L-HSL, 4.67 μM; HHQ, 3.77 μM; 1-HP, 4.48 μM. Data are pooled from n = 3 (3-o-C12-L-HSL), n = 2 (C-4-L-HSL), n = 4 (HHQ), n = 2 (PQS), or n = 3 (1-HP) independent experiments. (C) Binding of QS molecules to AhR, as measured by microscale thermophoresis assay. Kd values: 3-o-C12-L-HSL, 2.69 μM; PQS, 130 μM; 1-HP, 1.18 μM. Data are pooled from n = 4 (3-o-C12-L-HSL), n = 3 (C4-L-HSL), n = 4 (PQS), or n = 4 (1-HP) independent experiments.

AhR QS ligand interactions were further defined using an A549 AhR CRISPR knockout (KO) cell line (Fig. 3A and fig. S5A). Induction of AhR-dependent genes was detected upon 1-HP stimulation of CRISPR scramble control, and was absent in the AhR-KO cells (fig. S5B). In contrast, and as previously shown in wild-type A549 cells, 3-o-C12-L-HSL and HHQ caused AhR inhibition (fig. S5, B and C). Major functions of AhR include xenobiotic metabolism, toxin degradation, and excretion (26). Previously, we demonstrated that AhR mediates the degradation of bacterial molecules such as P. aeruginosa phenazines and Mycobacterium tuberculosis naphthoquinone phthiocol (10). Using an established P. aeruginosa 3-o-C12-L-HSL luminescence reporter strain (PA14-R3) (27) to detect 3-o-C12-L-HSL levels (fig. S5D), we evaluated its degradation profile upon exposure to AhR-proficient and AhR-deficient cells (fig. S5E). Bioluminescence emitted by the bacterial reporter cells decreased in a time-dependent manner, indicating reduced abundance of 3-o-C12-L-HSL (Fig. 3B). In contrast, no differences were detected between scramble control and AhR-KO cells (Fig. 3B). These results were confirmed by HPLC (Fig. 3C). A similar approach was used to determine the metabolism of HHQ (fig. S5E), using the PAO1 pqsA CTX-lux::pqsA reporter strain (fig. S5F) (28) and HPLC. Surprisingly, no degradation of HHQ was observed with any of the methods when exposing cells to 50 μM HHQ (fig. S5, G and H). However, when cells were exposed to a lower concentration of HHQ (0.5 μM), diminished levels of HHQ were detected at late time points, although no differences between AhR-proficient and AhR-deficient cells were observed (Fig. 3D).

Fig. 3 AhR-dependent responses.

(A) Western blot detection of AhR protein expression in A549 CRISPR scramble control and CRISPR AhR-KO cells. (B and C) Degradation of 3-o-C12-L-HSL measured in the supernatants of stimulated A549 CRISPR cells compared to control without cells. Expression of 3-o-C12-L-HSL was detected by bacterial PA14-R3 bioluminescence reporter assay (data pooled from n = 3 independent experiments) (B) or HPLC (data pooled from n = 3 independent experiments) (C). (D) Degradation of HHQ measured and detected as in (C) (data pooled from n = 4 independent experiments). (E and F) Gene expression analysis of different cytokines and chemokines in A549 CRISPR cells upon 24 hours of stimulation with P. aeruginosa QS molecules. Data are pooled from 3-o-C12-L-HSL (n = 6), HHQ (n = 5), or 1-HP (n = 7) independent experiments. Data are means ± SEM [(B), (C), (D), (F)]. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 [two-way ANOVA in (B), two-tailed Student t test in (F)]; n.s., not significant.

Together, under the conditions tested, our results argue against an involvement of AhR in the degradation of P. aeruginosa 3-o-C12-L-HSL or HHQ. In addition to its role in xenobiotic metabolism, AhR participates in the regulation of different immune mediators (10, 19, 26, 29). Accordingly, we evaluated whether AhR regulates cytokine and chemokine expression upon exposure to different QS molecules. Different bacterial ligands induced different gene expression patterns (Fig. 3, E and F). It was previously reported that infection with P. aeruginosa, or exposure to 3-o-C12-L-HSL, leads to IL-6 and IL-8 expression (30, 31). Consistently, among the genes induced by 3-o-C12-L-HSL, IL-6 and IL-8 were highly induced in the AhR-KO cells as compared to scramble control. Elevated induction of IL-8 was also observed upon exposure of AhR-KO cells to HHQ, whereas 1-HP stimulation led to reduced induction. A similar profile was observed for CXCL1, CXCL2, and CXCL3 (Fig. 3, E and F). These results emphasize differential AhR modulation of host responses, where sensing the different levels of QS molecules expressed along the infection process can differentially regulate the composition of multiple cytokines and chemokines. Thus, sensing of QS molecules by AhR shapes immunity to infection.

AhR senses bacterial QS molecules in vivo

As mentioned above, AhR is conserved among different species (including human, mouse, and zebrafish) and few amino acid positions differ in the ligand-binding site of AhR proteins (fig. S6A). However, subtle amino acid differences have been reported to affect binding to specific ligands (26). For example, the human Val381, corresponding to Ala in mouse and zebrafish, is implicated in species-related differences regarding binding affinity to TCDD; specifically, mouse AhR has higher binding affinity to TCDD than does human AhR (26, 32). Consistently, using our in silico modeling, higher TCDD binding affinities of mouse and zebrafish AhR were detected relative to human AhR (fig. S6B). A similar approach was chosen for the P. aeruginosa QS molecules, and MM-GBSA ΔGBind values were calculated starting from the same ligand docking pose as obtained for the human AhR (fig. S6C). Strikingly, no species-specific differences were predicted to occur, further pointing to a conserved mechanism of sensing of P. aeruginosa infection.

The zebrafish (Danio rerio) has become a powerful model in developmental biology and genetics, and more recently in toxicology and immunology (3336). The AhR pathway is conserved in zebrafish and has also been shown to be involved in xenobiotic metabolism (36). As a result of genome-wide duplication events, teleosts express various co-orthologs of mammalian genes, although not all are functional. Zebrafish express three AhR isoforms (ahr1a, ahr1b, and ahr2), and AhR2 is the primary isoform for recognition of toxic ligands such as TCDD (36). Upon ligand activation, AhR2 drives the expression of hallmark genes such as cyp1a, ahrra, and ahrrb (36).

It was previously reported that static immersion of zebrafish larvae in a bacterial suspension, including P. aeruginosa, increases cyp1a expression (37, 38). Similar results were obtained from microarray analysis of larvae at 2 days post-fertilization (2 dpf) infected with PA14 WT for 5 or 24 hours (fig. S7A and tables S2 and S3). Moreover, in addition to cyp1a, increased expression of additional AhR-related genes such as ahrra and cyp1c1 was observed (36). Therefore, we evaluated whether we could recapitulate our in vitro findings using this in vivo model organism. Here, 5 hours of exposure of 2-dpf larvae to PA14 WT collected from different phases of bacterial growth with distinct expression patterns of QS molecules (e.g., 3-o-C12-L-HSL and Pyo; Fig. 4A) resulted in distinct AhR activation, as measured by cyp1a mRNA expression (Fig. 4B). To mimic the course of infection, we collected bacteria from different growth phases, and washed and further resuspended them in E3 medium to a final optical density (OD) similar to the point of collection (i.e., early log, OD600 = 0.2; mid-log, OD600 = 0.7; late log, OD600 = 1). Exposure of larvae to these bacterial suspensions led to increasing cyp1a expression along the growth phase (Fig. 4B). Still, this could be the result of higher expression of QS molecules and/or increasing bacterial density. To exclude the latter option, we exposed zebrafish larvae to bacterial supernatants after filtration and dilution in E3 medium (1:25 ratio) or to similar bacterial numbers collected from the different growth stages. Exposure of 2-dpf larvae to filtered supernatants or to infection by immersion resulted in elevated cyp1a expression toward late stages of bacterial growth (Fig. 4, C and D). These results are in agreement with our in vitro findings (Fig. 1, B and C, and fig. S1, A and E), thereby confirming that P. aeruginosa molecules expressed during diverse growth phases modulate AhR differentially.

Fig. 4 AhR activation by P. aeruginosa QS molecules in zebrafish larvae.

(A) Expression of 3-o-C12-L-HSL and Pyo in supernatants of PA14 WT, collected at different growth phases in LB medium; 3-o-C12-L-HSL determined by PA14-R3 bioluminescence reporter assay and Pyo concentrations evaluated by spectrophotometry (data pooled from n = 9 independent experiments). (B to D) cyp1a expression in 2-dpf zebrafish larvae infected by immersion with PA14 WT [(B) and (D)] or exposed to bacterial supernatants for 5 hours (C) (data pooled from n = 7 independent experiments). In (B), zebrafish were infected with different bacterial loads collected from various phases of PA14 WT growth, according to the defined final OD600 in E3 medium (adjusted to early log-OD600 = 0.2, mid log-OD600 = 0.7, late log-OD600 = 1; data pooled from n = 3 independent experiments). In (D), zebrafish were infected with 1 × 109 CFU/ml, with PA14 WT collected from various phases of bacterial growth (data pooled from n = 7 independent experiments). (E) Gene expression analysis of cyp1a, ahrra, and ahrrb transcripts from zebrafish larvae (2 dpf) treated (red) or untreated (blue) for 2 hours with 5 μM CH223191 (AhR inhibitor) followed by a further 4 hours of exposure to 5 μM 1-HP or DMSO vehicle control. One representative experiment of at least three independent experiments is shown. Triplicates of 12 larvae are depicted at each data point. (F) Cyp1a protein expression detected by Western blot analysis in 2-dpf zebrafish larvae treated for 24 hours with DMSO, 5 μM 1-HP, 5 μM CH223191, or both 1-HP and CH223191. (G) Cyp1a enzymatic activity expressed as total intensity of resorufin (EROD assay) detected per 2-dpf larva treated (red) or not (blue) for 2 hours with 5 μM CH223191 followed by a further 4 hours of exposure to 5 μM 1-HP or DMSO vehicle control (each dot represents one larva; data are median values). One representative experiment of at least three independent experiments is shown. (H and I) Microarray analysis of 2-dpf larvae preexposed to DMSO or 5 μM CH223191 for 2 hours, followed by 4 hours of exposure to 5 μM 1-HP or DMSO, in the presence or absence of 5 μM CH223191. Data are pooled from n = 5 independent experiments. (H) Venn diagram depicting the differentially expressed genes; (I) AhR gene enrichment curve. Data are means ± SEM [(A) to (D)] or means ± SD (E). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way ANOVA).

Next, we verified in the zebrafish model our in vitro findings that P. aeruginosa expresses QS molecules that either activate or inhibit the canonical AhR pathway. We previously demonstrated that P. aeruginosa phenazines (e.g., 1-HP) activate the AhR pathway in human and mouse (10). Here, exposure of 2-dpf zebrafish larvae to TCDD induced the expression of AhR-dependent genes (36) (fig. S7B). AhR dependency was confirmed by reduced gene expression in the presence of the AhR inhibitor CH223191 (39) (fig. S7, B and C). Similarly, we observed AhR modulation upon exposure to the P. aeruginosa phenazine 1-HP at the transcriptional level (Fig. 4E) and Cyp1a protein expression in response to 1-HP (Fig. 4F). To determine whether increased Cyp1a expression translates into enhanced enzymatic activity, we measured its activity in vivo in a semi–high-throughput assay (fig. S7, D and E). An increment in fluorescence, as readout of increased Cyp1a enzymatic activity, was detected upon exposure to 1-HP or TCDD and was inhibited by CH223191 (Fig. 4G and fig. S7, E and F). AhR was the major sensor of P. aeruginosa phenazines in vivo, because microarray analysis of larvae exposed to 1-HP in the presence or absence of the AhR inhibitor revealed that AhR-dependent genes (36) were among the top 10 1-HP–induced genes and that their induction was reverted by the CH223191 inhibitor (Fig. 4, H and I, fig. S7G, and table S4). Not all of the differentially 1-HP–induced genes had been previously shown to be transcriptionally regulated by AhR in zebrafish. Therefore, we performed an in silico analysis to identify xenobiotic responsive elements (XREs) in their promotor regions (40). We identified putative XREs in the promoter regions of all evaluated genes (fig. S7H).

Given that our in vitro studies demonstrated that P. aeruginosa also expresses QS molecules that inhibit the AhR pathway, we exposed larvae in vivo to 3-o-C12-L-HSL or HHQ in the presence or absence of 1-HP. Simultaneous exposure to 3-o-C12-L-HSL or HHQ together with 1-HP reduced induction of AhR-related genes by 1-HP (Fig. 5A and fig. S8, A to D). Moreover, Cyp1a enzymatic activity was diminished when zebrafish larvae were co-exposed to 3-o-C12-L-HSL, HHQ, and PQS together with 1-HP, whereas C4-L-HSL did not affect 1-HP–induced activation (Fig. 5B and fig. S8B). Remarkably, 3-o-C12-L-HSL and HHQ even inhibited AhR activation by TCDD (Fig. 5, A and B, and fig. S8, A and B). Microarray analysis further confirmed that 3-o-C12-L-HSL inhibited AhR activation by 1-HP (table S5). None of the ligands induced toxicity in zebrafish larvae under the conditions tested (fig. S8E). Overall, our results demonstrate that zebrafish AhR recognizes diverse P. aeruginosa QS molecules.

Fig. 5 AhR modulation by P. aeruginosa QS molecules in zebrafish larvae.

(A and B) cyp1a gene expression (A) and Cyp1a enzymatic activity (B) upon 4 hours of exposure of 2-dpf larvae to diverse P. aeruginosa QS molecules or TCDD. One representative experiment of at least three independent experiments is shown. In (A), triplicates of 12 larvae are depicted at each data point. In (B), each dot represents one larva; data are median values. (C) Expression of 3-o-C12-L-HSL (determined by PA14-R3 bioluminescence reporter assay) and Pyo (evaluated by spectrophotometry) in the supernatants of different PA14 strains collected at mid-log growth phase; pooled data from n = 6 independent experiments. (D and E) Infection of 2-dpf zebrafish larvae by immersion for 5 hours with 1 × 109 CFU/ml of different P. aeruginosa strains collected at mid-log growth phase. (D) cyp1a gene expression; triplicates of 12 larvae are depicted at each data point. (E) Cyp1a enzymatic activity. Each dot represents one larva; data are median values. Data are from one representative experiment of at least three independent experiments. Data are means ± SD [(A) and (D)] or means ± SEM (C). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way ANOVA).

Taking advantage of P. aeruginosa mutants producing dissimilar levels of distinct QS molecules, we tested whether AhR is differentially modulated in vivo in response to bacteria expressing different QS molecules. We used the mutant PA14 ΔrsaL and PA14 09480 P. aeruginosa strains, which overproduce 3-o-C12-L-HSL (27, 41) or phenazines (10), respectively. No differences in bacterial growth or in the sequential expression of QS molecules were observed among these strains (fig. S9, A and B), whereas the levels of 3-o-C12-L-HSL and phenazines differed as previously documented (fig. S9C). Consistent with earlier studies (41), Pyo levels were also elevated in the PA14 ΔrsaL relative to PA14 WT (fig. S9C). Therefore, we focused on bacteria collected from one distinct growth phase (mid-log phase) with consistent differences in the levels of 3-o-C12-L-HSL and Pyo (Fig. 5C). Static immersion of larvae to similar bacterial numbers [1 × 109 colony-forming units (CFU)/ml; fig. S10A] led to distinct Cyp1a expression and activity (Fig. 5, D and E), apparently related to the proportions of the AhR activators and inhibitors (Fig. 5C and fig. S10B). Relative to PA14 WT, higher expression of phenazines (PA14 09480) increased Cyp1a activity, whereas higher expression of 3-o-C12-L-HSL (PA14 ΔrsaL) decreased Cyp1a activity (Fig. 5E). We conclude that AhR recognition of these molecules, whose expressions are tightly regulated in P. aeruginosa, allows for quantitative sensing of the course of infection.

Recognition of phenazines by AhR is important for clearance of P. aeruginosa (10). Infection of wild-type and AhR–/– mice with a Pyo-overexpressing strain (PA14 09480) (10) (figs. S9C and S11A) confirmed the importance of AhR in bacterial clearance in responses to these molecules (Fig. 6A). Intriguingly, infection with bacteria from earlier stages of growth, not expressing phenazines (fig. S11A), had detrimental consequences mediated by AhR (Fig. 6A). These results further illustrate that distinct P. aeruginosa molecules expressed at different growth stages modulate AhR signaling differentially. To evaluate the impact of AhR sensing of QS molecules expressed at early stages, focusing on the AhR inhibitor 3-o-C12-L-HSL identified here, we infected mice with the P. aeruginosa strain (PA14 ΔrsaL). We focused on bacteria from mid-log growth phase to exclude differences in lung CFUs between the two mouse strains (WT and AhR–/–) after 8 hours of infection (fig. S11, B and C). Differential expression of various cytokines and chemokines depended not only on the mouse strain, but also on the P. aeruginosa strain (Fig. 6, B and C, and fig. S11D). These in vivo results are consistent with our in vitro experiments (Fig. 3, E and F), where AhR differentially regulated the expression of distinct cytokines and chemokines, depending on the presence of distinct QS molecules. Previously we reported a critical role of AhR in the recruitment of neutrophils to the lungs of P. aeruginosa–infected mice (10). Likewise, lower numbers of neutrophils were detected in the lungs of AhR–/– mice upon infection with PA14 WT (Fig. 6D). Strikingly, these differences were lost when infecting mice with PA14 ΔrsaL, where we observed comparable numbers of neutrophils in the lungs of wild-type and AhR–/– mice (Fig. 6D).

Fig. 6 AhR-mediated responses upon P. aeruginosa infection in mice.

(A) Bacterial clearance in the lungs of WT and AhR-knockout (AhR–/–) mice after 8 hours of infection with P. aeruginosa PA14 09480 (2 × 106 CFU administered per mouse). Bacterial growth phases: early log, OD600 < 0.3; mid-log, 0.5 < OD600 < 0.8; late log, OD600 > 1. Each dot represents one mouse (data pooled from n = 2 independent experiments; median values are shown). (B to D) Infection of WT and AhR–/– mice for 8 hours with PA14 WT or PA14 ΔrsaL strains (data pooled from n = 2 independent experiments). (B) Gene expression analysis of different cytokines and chemokines in the lungs of infected mice, compared to the respective noninfected mouse strain (WT, n = 8 mice; AhR–/–, n = 6 mice). Data are means ± SEM. (C) Cytokine and chemokine median protein levels in lung homogenates after infection. Each dot represents one mouse (data pooled from n = 2 independent experiments). (D) Neutrophil numbers (medians) in the lungs of infected and non-infected mice. Each dot represents one mouse (data pooled from n = 2 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 [Mann-Whitney U test in (A) and (D); two-tailed Student t test in (B); two-way ANOVA in (C)].

In sum, these results reveal differential modulation of AhR during the course of infection, depending on the relative abundances of distinct QS molecules. Taken together, our data show that AhR not only detects P. aeruginosa QS molecules in a qualitative way, but also quantifies their relative levels. This quantitative assessment endows the host with the capacity to sense bacterial community densities, and consequently infection dynamics. Thus, our findings emphasize a crucial role of AhR as master regulator of host defense responses, capable of tuning immunity according to the stage of infection and disease and hence to their threat to the host.

Discussion

Recently we showed that AhR, by binding bacterial pigmented virulence factors such as P. aeruginosa phenazines, regulates host resistance to infection (10). Our present findings show that in addition to phenazines, AhR recognizes QS molecules comprising different chemical entities including homoserine lactones and quinolones. In contrast to phenazines, the QS cognates 3-o-C12-L-HSL and HHQ inhibit the canonical AhR signaling by competing and antagonizing the effects of known AhR activators, such as P. aeruginosa 1-HP (10) or the bona fide AhR ligand TCDD (19, 42). Strikingly, AhR sensing of QS molecules is not restricted to a particular cell type or a specific in vitro model: (i) Mammalian macrophages, hepatocytes, and epithelial cells responded in a similar fashion, and in all cases subtle alterations in the ratios of bacterial ligands influenced the outcome of AhR activation and its downstream responses, such as cytokine and chemokine expression. (ii) These results are reciprocated in vivo using zebrafish, where exposure of larvae to different concentrations of P. aeruginosa QS molecules modulated AhR activation and elicited downstream responses. (iii) Exposure of zebrafish larvae to different P. aeruginosa mutants producing distinct QS molecules at different abundances at a given point of infection resulted in a specific AhR activation profile. (iv) Complementing these findings, an experimental mouse infection model with P. aeruginosa strains expressing variable levels of QS molecules revealed that AhR regulates bacterial elimination upon sensing bacterial quorum. In sum, AhR resembles a “processing hub” that integrates the information linked to the abundance of different QS molecules, both activators and inhibitors, thereby mobilizing the most appropriate host defense mechanisms at a given stage of infection.

QS is used by certain bacteria to coordinate their gene expression in response to changes in their population density or their stage of infection (1, 3, 4). Accordingly, direct correlation between different QS molecules and severity of infection has been observed (7). In P. aeruginosa, QS regulates different virulence and adaptation mechanisms and is therefore crucial for coordinated colonization of a new environment (1, 3, 4, 12). Differences in P. aeruginosa virulence and transition from acute to chronic infection have been linked to altered expression of QS molecules and their regulated genes (1, 6, 8). For instance, the expression of phenazines plays a critical role in biofilm formation and development (7, 43), and P. aeruginosa QS mutants producing thinner and less developed biofilms, are more sensitive to antibiotics and eradication (1, 5, 44). Furthermore, high concentrations of P. aeruginosa phenazines are detected in the sputum of CF patients, who are severely affected by this pathogen (2, 7). Therefore, depending on its metabolic state, mirrored by a distinct composition of QS molecules, the bacteria may pose different threats to the host, and the host needs to adapt its response accordingly. Interestingly, inter– and intra–P. aeruginosa species differences in virulence and expression of secreted molecules have been reported to occur, not only among clinical isolates but also among laboratory strains (e.g., between PAO1 sublines or between PA14 and PAO1). For example, expression levels of Pyo, rhamnolipids, PQS, exopolysaccharides, and elastase have been reported to differ between PA14 and PAO1 or among diverse PAO1 sublines (4548). It is tempting to speculate that as a result of its capacity to detect different levels of P. aeruginosa QS molecules, including Pyo or PQS, AhR is also well suited to detect strain-related differences during the course of infection, and consequently regulate host responses accordingly. However, further studies are needed to evaluate this hypothesis.

Interactions of P. aeruginosa QS molecules with different host receptors and signaling pathways have been reported (9). For example, 3-o-C12-L-HSL has been found to be sensed by the Ras GTPase-activating–like protein IQGAP1 or the peroxisome proliferator–activated receptors (PPAR β/δ/γ) (9, 49, 50). Additionally, P. aeruginosa HSLs (e.g., 3-o-C12-L-HSL) and quinolones (HHQ and PQS) modulate different host signaling pathways involving NF-κB or PPAR (9, 5052). Curiously, interactions between AhR and the indicated signaling pathways (e.g., NF-κB and PPAR) have been described (19), but their interplay and elicited responses upon P. aeruginosa infection remain unknown and should be the focus of future studies. Nonetheless, the capacity of AhR to bind and recognize three distinct types of QS molecules (HSLs, quinolones, and phenazines), as well as its capacity to monitor and integrate their relative expression levels, supports the identification of this receptor as a major host sensor of bacterial quorum and infection dynamics. It is tempting to speculate that host AhR and bacterial QS systems can actively spy on each other by recognizing similar molecules, even beyond the QS molecules described here. Recently, Ismail et al. (53) described how host epithelia can produce QS-like molecules, including an autoinducer-2 mimic, enabling it to interfere with bacterial QS circuits. However, the host AhR can sense P. aeruginosa QS molecules and has vast ligand-binding properties, so we cannot exclude the possibility that it senses and modulates the expression of different host molecules (such as host QS-like molecules) that may be involved in this host-bacteria interkingdom cross-talk during infection (Fig. 1A, gray arrows).

Given that AhR acts as a host sensor that monitors different QS molecules and their expression profiles along the course of infection and disease, the host can tune immune defense according to the stage and density of the bacterial community and the threat of infection. This mechanism would be particularly apt for nosocomial pathogens, which can be tolerated by the immunocompetent host at low density but become harmful once a threshold of tolerability has been exceeded. In this way, cost of energy for defense would be focused on the harmful trait only, with the harmless trait being ignored. Because P. aeruginosa is an opportunistic pathogen, defense mobilization is avoided at low bacterial densities, which can be tolerated, and it kicks in only with increasing population densities, which can harm the host. We propose that by spying on interbacterial communication, AhR is capable of sensing the status quo of the P. aeruginosa community during infection, allowing the host to mobilize the most appropriate defense mechanism according to the severity of threat.

Supplementary Materials

science.sciencemag.org/content/366/6472/eaaw1629/suppl/DC1

Materials and Methods

Figs. S1 to S11

Tables S1 to S11

References (5485)

References and Notes

Acknowledgments: We thank B. Stockinger (Francis Crick Institute) for the AhR–/– mice; C. Grabher (Karlsruhe Institute of Technology) and D. Panakova (Max Delbruck Center) for the zebrafish AB WT strain; L. Leoni (University Roma Tre) for P. aeruginosa strains PA14 ΔrsaL and PA14-R3; B. Tuemmler (Medizinische Hochschule Hannover) for PA14 WT and PA14 09480; F. Ausubel (Harvard Medical School/Massachusetts General Hospital) for PA14-GFP; P. Williams (University of Nottingham) for PAO1 WT, PAO1 pqsA, and CTX-lux::pqsA; U. Klemm for mouse breedings; N. Fielko, J. Otto, A. Fadeev (Max Planck Institute for Infection Biology), and M. Simões (Max Delbruck Center) for zebrafish breedings; and A. Diehl (Leibniz-Institut für Molekulare Pharmakologie) for technical help to prepare mouse liver lysates. Special thanks to A. Meijer and V. Torraca (University of Leiden) for support in setting up a zebrafish facility, zebrafish handling, and experimental design. Funding: Supported by the European Research Council under the Horizon 2020 program of the European Commission, grant 311371 (A.S. and M.K.B.), the Helmholtz BioInterfaces in Technology and Medicine (BIFTM) program of Karlsruhe Institute of Technology (F.K. and G.B.W.), and the Max Planck Society. Author contributions: P.M.-A. and S.H.E.K. conceived and designed the study and wrote the manuscript; P.M.-A. designed and performed experiments and data analysis; A.P., G.P., U.G., and M.K. provided technical help for in vitro and in vivo experiments; A.D., P.S., and C.P. performed mouse infection experiments; L.L. performed and analyzed Fluidigm experiments; M.K.B., A.S., and J.F. performed binding studies; R.H., F.K., and G.B.W. performed and analyzed HPLC experiments; A.K., J.P., G.K., and H.O. performed virtual docking studies; and H.J.M. and J.W. performed and analyzed microarray experiments. All authors commented on the paper. Competing interests: Authors declare no competing interests. Data and materials availability: All data are available in the main text or the supplementary materials. Data are deposited in GEO under accession number GSE121101.

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