Thibaut Rosay

Alexis Bazire

Suraya Diaz

Thomas Clamens

Anne- Sophie Blier

Lily Mijouin

Brice Hoffmann

Jacques-Aurélien Sergent

Emeline Bouffartigues

Wilfrid Boireau

Julien Vieillard

Christian Hulen

Alain Dufour

Nicholas J Harmer

Marc G J Feuilloley

Olivier Lesouhaitier
email: olivierlesouhaitier@univ-rouen.fr

Pseudomonas aeruginosa expresses a functional human natriuretic peptide receptor ortholog: involvement in biofilm formation

Keywords: Cystic fibrosis, Lung, Natriuretic peptides, NPR-C, AmiC, Biofilm, Bacterial adaptation, Microscale thermophoresis

Considerable evidence exists that bacteria detect eukaryotic communication molecules and modify their virulence accordingly. In previous studies, it has been demonstrated that the increasingly antibiotic resistant pathogen Pseudomonas aeruginosa can detect the human hormones Brain Natriuretic peptide (BNP) and C-type Natriuretic Peptide (CNP) at micromolar concentrations. In response, the bacterium modifies its behavior to adapt to the host physiology, increasing its overall virulence. Identifying the bacterial sensor for these hormones and interfering with this sensing mechanism offers an exciting opportunity to directly affect the infection process.

Here, we show that BNP and CNP strongly decrease P. aeruginosa biofilm formation. Isatin, an antagonist of human natriuretic peptide receptors (NPR), prevents this effect. Furthermore, the human NPR-C receptor agonist cANF 4-23 mimics the effects of natriuretic peptides on P. aeruginosa while sANP, the NPR-A receptor agonist, appears weakly active. We show in silico that NPR-C, a preferential CNP receptor, and the P. aeruginosa protein AmiC have similar 3D structures, and that both CNP and isatin bind to AmiC. We demonstrate that CNP acts as an AmiC agonist, enhancing the expression of the ami operon in P. aeruginosa.

Binding of CNP and NPR-C agonists to AmiC was confirmed by microscale thermophoresis.

Finally, using an amiC mutant strain, we demonstrate that AmiC is essential for CNP effects on biofilm formation.

In conclusion, the AmiC bacterial sensor possesses similar structural and pharmacological profiles to the human NPR-C receptor and appears to be a bacterial receptor for human hormones that enables P. aeruginosa to modulate biofilm expression.

Importance

Pseudomonas aeruginosa is a bacterium that is a highly dangerous opportunist pathogen for immunocompromised hosts, especially cystic fibrosis patients. The sites of P. aeruginosa infection are varied, with predominance in the human lung, in which bacteria are in contact with host molecular messengers such as hormones. The C-type natriuretic peptide (CNP), a hormone produced by lung cells, has been described as a bacterial virulence enhancer. In this report, we showed that the CNP hormone counteracts P. aeruginosa biofilm formation and we identified the bacterial protein AmiC as the sensor involved in the CNP effects. We showed that AmiC could bind specifically CNP. These results show for the first time that a human hormone could be sensed by bacteria through a specific protein which is an ortholog of the human hNPR-C. The bacterium would be able to modify its lifestyle by favoring virulence factors production whilst reducing biofilm formation.

INTRODUCTION

Pseudomonas aeruginosa is a well known opportunistic pathogen and a major cause of mortality among cystic fibrosis patients [START_REF] Costerton | Bacterial biofilms: a common cause of persistent infections[END_REF]. The morbidity of this bacterium and its resistance to antibiotherapy are largely attributed to its transition in host tissues (particularly in lung, soft tissues, skin and urinary bladder) from a planktonic to a biofilm lifestyle [START_REF] Bjarnsholt | Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients[END_REF][START_REF] Hoiby | Antibiotic resistance of bacterial biofilms[END_REF]. The present emergence of multi-resistant strains is now critical and is prompting the need to find new therapeutic approaches [START_REF] Shaw | Increasing resistance of the Liverpool Epidemic Strain (LES) of Pseudomonas aeruginosa (Psa) to antibiotics in cystic fibrosis (CF)--a cause for concern? Journal of cystic fibrosis[END_REF].

The high adaptability of P. aeruginosa and its rapid response to the host environment implies that it can detect a large range of eukaryotic chemical signals [START_REF] Camilli | Bacterial small-molecule signaling pathways[END_REF]. However, few bacterial sensors for eukaryotic molecules have been characterized to date [START_REF] Lesouhaitier | Gram-negative bacterial sensors for eukaryotic signal molecules[END_REF]. Studies on the E. coli quorum-sensing regulator A (QseA) led to the identification of QseC/QseB, a twocomponent regulatory system [START_REF] Sperandio | Quorum-sensing Escherichia coli regulator A: a regulator of the LysR family involved in the regulation of the locus of enterocyte effacement pathogenicity island in enterohemorrhagic E. coli[END_REF][START_REF] Sperandio | Quorum sensing Escherichia coli regulators B and C (QseBC): a novel two-component regulatory system involved in the regulation of flagella and motility by quorum sensing in E. coli[END_REF]. This system is not only activated by the bacterial quorum sensing signal autoinducer AI3, but also by eukaryotic neurohormones (epinephrine/norepinephrine) [START_REF] Hughes | Inter-kingdom signalling: communication between bacteria and their hosts[END_REF]. It was suggested that QseC could be a bacterial ortholog of eukaryotic adrenergic receptors [START_REF] Sperandio | Bacteria-host communication: the language of hormones[END_REF]. Bioinformatic screening of E. coli QseC analogs has demonstrated the presence of proteins related to this sensor in a large number of bacterial pathogens, including P. aeruginosa [START_REF] Rasko | Targeting QseC signaling and virulence for antibiotic development[END_REF], in which norepinephrine acts as a virulence inducer [START_REF] Hegde | The neuroendocrine hormone norepinephrine increases Pseudomonas aeruginosa PA14 virulence through the las quorum-sensing pathway[END_REF]. It appears that P. aeruginosa is able to sense different eukaryotic communication molecules, including interferon-γ [START_REF] Wu | Recognition of host immune activation by Pseudomonas aeruginosa[END_REF], dynorphin [START_REF] Zaborina | Dynorphin activates quorum sensing quinolone signaling in Pseudomonas aeruginosa[END_REF], gamma aminobutyric acid (GABA) [START_REF] Dagorn | Effect of GABA, a Bacterial Metabolite, on Pseudomonas fluorescens Surface Properties and Cytotoxicity[END_REF] and natriuretic peptides [START_REF] Blier | C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa[END_REF][START_REF] Veron | Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis[END_REF].

Natriuretic peptides are a family of eukaryotic hormones and neurohormones composed of three members, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP). These peptides are mainly expressed in cardiomyocytes and endothelial cells [START_REF] Drewett | The family of guanylyl cyclase receptors and their ligands[END_REF] explaining their major role in cardiovascular homeostasis. However, ANP and CNP are also produced in significant amounts by the lung bronchial and alveolar epithelia and particularly by Clara (Club) cells [START_REF] Nakanishi | Expression of C-type natriuretic peptide during development of rat lung[END_REF]. The circulating blood levels of natriuretic peptides are increased during community-acquired pneumonia [START_REF] Nowak | Direct comparison of three natriuretic peptides for prediction of short-and long-term mortality in patients with community-acquired pneumonia[END_REF], and the lipopolysaccharides (LPS) of Gram-negative bacteria induce the plasmatic release of both BNP [START_REF] Vila | Plasma NT-proBNP increases in response to LPS administration in healthy men[END_REF] and CNP [START_REF] Suga | Cytokine-induced C-type natriuretic peptide (CNP) secretion from vascular endothelial cells--evidence for CNP as a novel autocrine/paracrine regulator from endothelial cells[END_REF]. In previous studies, we demonstrated that P. aeruginosa can detect BNP and CNP at micromolar concentrations and reacts by an overall increase of virulence [START_REF] Blier | C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa[END_REF][START_REF] Veron | Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis[END_REF]. In P. aeruginosa, the effect of CNP appears to be mediated by cAMP synthesis, which subsequently activates the regulatory proteins Vfr [START_REF] Veron | Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis[END_REF] and PtxR [START_REF] Blier | C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa[END_REF], leading to a rise of HCN and exotoxin A production and a reorganization of LPS structure. For these reasons, it was suggested that CNP promotes the acute infection phenotype of P. aeruginosa [START_REF] Coggan | Global regulatory pathways and cross-talk control pseudomonas aeruginosa environmental lifestyle and virulence phenotype[END_REF], favoring the switch of P. aeruginosa towards a planktonic lifestyle, and a decrease in biofilm formation. The effects of BNP and CNP on P. aeruginosa appear to be mediated by distinct mechanisms since BNP, unlike CNP, did not modify intra-bacterial cAMP levels [START_REF] Veron | Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis[END_REF] and was without effect on P. aeruginosa virulence towards C. elegans [START_REF] Blier | C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa[END_REF]. In humans, each type of natriuretic peptide acts through a particular receptor subtype, suggesting the expression of specific bacterial orthologs of the human natriuretic peptide receptors in P. aeruginosa.

In the present study, we extended our knowledge of the effect of natriuretic peptides on P. aeruginosa by investigating for the first time the effect of these peptides on the biofilm formation activity of the bacterium. Using in silico approaches, we identified the P.

aeruginosa protein AmiC as a potential CNP binding sensor site and investigated in vitro the binding of natriuretic peptide receptor agonists to AmiC. The pharmacological profile of AmiC as a bacterial CNP receptor was completed by studying the effect of isatin, an antagonist of human CNP receptors that is also a known bacterial metabolite and signaling compound. We further determined the affinity of AmiC for several natriuretic peptide receptor agonists. Using an amiC deficient mutant and its complemented derivative, we validated the crucial role of P. aeruginosa AmiC in the effect of CNP, but not BNP, on biofilm formation. This study provides an integrated explanation of the CNP sensing system in P. aeruginosa and opens up the way to a new strategy to control P. aeruginosa biofilm formation.

RESULTS

Effects of CNP and BNP on P. aeruginosa biofilm formation.

The influence of CNP and BNP on the biofilm formation activity of P. aeruginosa was investigated in dynamic and static conditions. Unless otherwise stated, peptides were used at a micromolar concentration since i) this corresponds to that usually used for studying the effects of peptides on eukaryotic models [START_REF] Klinger | Atrial natriuretic peptide attenuates agonist-induced pulmonary edema in mice with targeted disruption of the gene for natriuretic peptide receptor-A[END_REF], ii) they were previously shown to be active at this concentration on quorum sensing molecules and toxin productions in P. aeruginosa [START_REF] Blier | C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa[END_REF], and iii) the duration of the biofilm experiment, which required a high amount of medium (continuous flow) containing the peptides, was not compatible with the realization of a whole dose response study. When P. aeruginosa PA14 was exposed to CNP or BNP in dynamic conditions, we observed a highly significant decrease of the biofilm volume (-81.0 ± 6.6% and -80.3 ± 4.4%, p < 0.001, respectively) and of the biofilm average thickness (-72.3 ± 9.7% and -76.0 ± 4.8%, p < 0.001, respectively) (Fig. 1A). These effects were associated with a reduction of the number and mean size of mushroom-like structures in the biofilm (Fig. 1B).

Exposure of P. aeruginosa MPAO1 to CNP for 24 h in static conditions led to similar results with a marked decrease of biofilm formation (Fig. 2A) associated to a reduction of volume and thickness (-83.3 ± 1.9% and -91.7 ± 2.2% , p < 0.001, respectively) (Fig. 2B).

Effect of specific antagonist and agonists of natriuretic peptide receptors on P. aeruginosa biofilm formation and virulence activity.

The biofilm formation activity of P. aeruginosa MPAO1 was also studied in the presence of isatin, an antagonist of both A and C subtypes natriuretic peptide receptors (NPR-A and NPR-C). Administered alone for 24 h, isatin affected the organization of the biofilm which appeared more irregular with the presence of large water channels (Fig. 2A). We also noted a limited and non-significant increase of the biomass and thickness of the biofilm (Fig. 2B).

However, when P. aeruginosa MPAO1 was exposed to isatin (10 -5 M) for 20 min prior to the administration of CNP, the inhibitory effect of CNP on the biofilm formation activity of the bacterium was abolished (Fig. 2A and2B).

As we have previously shown that natriuretic peptides enhance P. aeruginosa cytotoxicity activity and global virulence [START_REF] Blier | C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa[END_REF], we tested the effect of sANP, a specific NPR-A agonist, and the artificial peptide cANF 4-23 , a specific NPR-C agonist, on bacterial cytotoxicity towards lung cells. The peptide cANF 4-23 (NPR-C agonist) caused a strong increase of P. aeruginosa cytotoxicity activity against A549 human lung epithelial cells (+ 98.3 ± 16.9%; p < 0.001) (Fig. 2C). In parallel, we observed that the sANP peptide (NPR-A agonist), also enhanced the P. aeruginosa cytotoxicity in the same cell model, but to a lower extent (+ 25.9 ± 12.1%; p < 0.05) (Fig. 2C). In silico study of a functional C-type natriuretic peptide receptor ortholog in P.

aeruginosa.

In mammalian cells, CNP preferentially binds the NPR-C receptor and has a weak affinity for the NPR-A receptor [START_REF] Potter | Natriuretic peptides, their receptors, and cyclic guanosine monophosphate-dependent signaling functions[END_REF], whereas BNP strongly binds NPR-A and has a weak affinity for NPR-C. The observations that the effects of CNP were blocked by the NPR-C antagonist isatin, and mimicked by the NPR-C agonist cANF 4-23 , suggested the presence of a bacterial ortholog of NPR-C in P. aeruginosa. This hypothesis was investigated using in silico approaches. Searching in bibliographic data, we noted a DALI structure similarity search [START_REF] Holm | Dali: a network tool for protein structure comparison[END_REF] that identified a P. aeruginosa protein, AmiC, which shows structural homologies with the binding domain of natriuretic peptides receptors [START_REF] Van Den Akker | Structural insights into the ligand binding domains of membrane bound guanylyl cyclases and natriuretic peptide receptors[END_REF][START_REF] Van Den Akker | Structure of the dimerized hormone-binding domain of a guanylyl-cyclase-coupled receptor[END_REF]. Using tools from the Protein Data Bank in Europe [http://www.ebi.ac.uk/pdbe/], we investigated in detail the structure of P. aeruginosa AmiC bound to its known ligand acetamide. We observed that it shares high structural similarity with the natriuretic peptide receptor subtype NPR-C bound to one of its agonists, the atrial natriuretic peptide (ANP; data not shown). This supports the hypothesis that AmiC could act as a bacterial natriuretic peptide sensor.

The 3D structure of P. aeruginosa AmiC (PDB ID: 1PEA) (29) was compared to that of the human NPR-C receptor (hNPR-C) (PDB ID: 1JDP) [START_REF] He | Allosteric activation of a springloaded natriuretic peptide receptor dimer by hormone[END_REF]. Using the STAMP software [START_REF] Russell | Multiple protein sequence alignment from tertiary structure comparison: assignment of global and residue confidence levels[END_REF], we superimposed an AmiC monomer onto each protomer of the structure of an hNPR-C dimer in complex with CNP. Although the sequences of these proteins share only 19% identity and 26% similarity, these two proteins present an overall similar 3D structure in the homodimeric form (Fig. 3A), particularly at the interface between each monomer which is considered to be the binding cleft of hNPR-C. The highest similarity was found in the helix of AmiC corresponding to the region involved in the binding of CNP to hNPR-C. Next, we split the AmiC monomer into two parts, corresponding to the two lobes of the protein. The first lobe consists of amino acids 8 to 123 and 261 to 338, and the second lobe of amino acids 124 to 260 and 339 to 375 of AmiC. Then, we superimposed each lobe on the 3D structure of hNPR-C (Fig. 3B). Each lobe of hNPR-C and AmiC showed a high level of secondary structure conservation with similar 3D positions, reinforcing the concept of structural homology between these two proteins. Using GASH Structural Superposition software [START_REF] Standley | GASH: an improved algorithm for maximizing the number of equivalent residues between two protein structures[END_REF] on each lobe of hNPR-C and AmiC, we observed that each of these two proteins has 254 residues estimated to be involved in equivalent secondary structures, covering 61.7% of the AmiC sequence. The hinge between the two lobes was essentially formed with flexible loops. Once superimposed, we looked at the amino acids involved in the binding of CNP, supporting the hypothesis that AmiC and hNPR-C bind the peptide in a similar fashion. S1 lists AmiC amino acids that are potentially involved in CNP binding, and the corresponding amino acids involved in the binding of hNPR-C to CNP as described by He et al. [START_REF] He | Structural determinants of natriuretic peptide receptor specificity and degeneracy[END_REF]. Amino acids located within 4 Å of CNP in the binding sites of both hNPR-C and AmiC are shown schematically in the supplemental material (Fig. S1). A total of 24 amino acids from AmiC and 21 residues from hNPR-C with equivalent physicochemical properties are located in the same regions and may stabilize the binding of CNP to AmiC and NPR-C equivalently.

Supplemental material Table

We also determined the virtual capacity of P. aeruginosa AmiC to bind natriuretic peptides (BNP and CNP) by molecular docking using AutoDock [START_REF] Morris | Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function[END_REF]. Docking revealed that CNP can bind to AmiC at the dimer interface of the bacterial protein (See Fig. S2A, B in the supplemental material). The most relevant calculated binding site of CNP on AmiC requires 13 amino-acids, Arg64, Glu68, Ala90, Met92, Pro93, Glu96, Arg97, Asp99, Glu112, Tyr113, Pro115, Asn116 and Tyr366. Since these predictions were obtained by an in silico study, the involvement of each of these residues need to be validated by constructing and studying the 13 correspondent mutant proteins, which will be addressed in a future work. In contrast, we observed in silico that BNP has no affinity to the AmiC protein. The NPR-C antagonist isatin also appeared capable of binding to the AmiC dimer interface (Fig. S2C,D). In this case, the AmiC amino-acids involved in the recognition of isatin are predicted to be Ile71, Arg72, Val94 and Arg97. These amino acids are located in the same region involved as the putative CNP binding site, and one residue (Arg97) could play a key role since it is potentially interacting with both CNP and isatin.

Effect of CNP on the expression of the amidase operon in P. aeruginosa.

In P. aeruginosa, AmiC is considered as a sensor protein. In the absence of stimulation (basal condition), AmiC binds to the AmiR protein, an antitermination factor, which down-regulates the transcription of the amiEBCRS operon [START_REF] Wilson | Cloning and DNA sequence of amiC, a new gene regulating expression of the Pseudomonas aeruginosa aliphatic amidase, and purification of the amiC product[END_REF]. Upon binding of acetamide AmiC releases AmiR, triggering the transcription of the whole ami operon [START_REF] Wilson | Antitermination of amidase expression in Pseudomonas aeruginosa is controlled by a novel cytoplasmic amide-binding protein[END_REF]. In order to verify that CNP could act in the same manner as acetamide on AmiC, we measured by qRT-PCR amiB, amiC, amiE and amiR mRNA levels 1 hour after exposure of the bacterium to CNP. The amiC, amiE, amiR mRNA levels were significantly increased (2.97 ± 0.2, 3.66 ± 0.39, and 2.73 ± 0.67 fold, p < 0.001, respectively) after 1 hour of exposure to CNP (10 -6 M) (Fig. 4), whereas the amount of lecB mRNA, encoded by a gene adjacent to the ami operon, was not modified.

In contrast, BNP (10 -6 M) has no significant effect on the whole ami operon transcriptional activity (data not shown).

AmiC binding with natriuretic peptide receptor agonists.

In order to confirm that the action of CNP and isatin on P. aeruginosa is mediated directly through AmiC, and is not an indirect effect, we investigated their binding to AmiC in vitro.

Recombinant AmiC was expressed in E. coli. We first confirmed the oligomeric state of AmiC using native gel electrophoresis and analytical gel filtration (See Fig. S3 in the supplemental material). These methods showed an apparent molecular weight of 120 ± 30 kDa and 136 ± 3 kDa, respectively. These results are most consistent with AmiC (protomer MW: 45.5 kDa) forming a dimer with an irregular shape, as predicted by the model (Fig. S3).

We then tested the interaction of AmiC with natriuretic peptide receptor agonists and isatin using microscale thermophoresis (Fig. 5A). AmiC bound to CNP tightly, showing a dissociation constant (K D ) of 2.0 ± 0.3 μM (Fig. 5). In contrast, we observed that BNP possessed no affinity for AmiC (Fig. 5A). Isatin could also bind to AmiC, although in this case the interaction was not so strong, showing a K D of 600 ± 200 μM (Fig. 5A and5B).

Additionally, we measured the interactions of other natriuretic peptide receptor agonists. sANP, a specific NPR-A agonist, showed a weak interaction with AmiC, which had clearly not proceeded to completion at 900 μM (Fig. 5B), the highest concentration point that we were able to assess. These data clearly predict that the K D is greater than 600 μM. Conversely, our initial experiments on osteocrin (a specific NPR-C agonist) showed a very strong interaction with AmiC, apparently tighter than we were able to determine using the available instrument. This indicates a K D value below 100 nM. In the same way, cANF 4-23 , another NPR-C specific agonist presented a K D value of 40 ± 10 µM (Fig. 5).

Involvement of AmiC in CNP-regulated P. aeruginosa biofilm formation

In dynamic conditions, the biofilm formed by the PA14 amiC-strain appeared reduced in comparison to wild-type strain PA14 WT (Fig. 6A). Conversely, the morphology of the biofilm formed by the PA14 AmiC+ strain (PA14 containing the wildtype amiC gene plus extra amiC plasmid-borne copies) or by the PA14 EV strain carrying the empty vector was similar to that of the wild-type (data not shown). Complementation of the amiC-strain with the amiC gene carried by a vector restored entirely the ability of the bacteria to form a biofilm (Fig. 6A and6B). The measure of the biofilm biovolumes revealed 64.5 ± 12.5 % reduction for the PA14 amiC-strain in comparison to the PA14 wild-type (p < 0.001) whereas the biovolume of the biofilm generated by the complemented strain is similar to that of PA14 wild-type (+13.7 ± 14.6 %) (Fig. 6B). The biofilm formed by the strain PA14 AmiC+ was slightly, but non significantly, enhanced (Fig. 6B).

We observed that the ability of CNP to reduce biofilm formation is abolished in the amiCstrain (Fig. 6A and6C), and is restored using the complemented strain (-71.9 ± 12.7 %; (p < 0.01) (Fig. 6C). In contrast, BNP, which strongly reduced biofilm formation as well (-75.1 ± 9.9 %; (p < 0.001) (Fig. 6C), continued to have the same impact on biofilm formation on amiC-mutant strain (-70.8 ± 12.1 %; (p < 0.05) (Fig. 6C).

In parallel, since it has been previously shown that CNP also modifies P. aeruginosa virulence, we tested the effect of AmiC on cytotoxicity towards lung cells. We observed that whereas the PA14 wild-type strain showed an increase in cytotoxic activity after CNP exposure (+76.6 ± 17 %; p < 0.001), the amiC-strain did not significantly modify its cytotoxicity in the presence of CNP when compared with controls (amiC-strain without CNP exposure) (+14.7 ± 6.0 %) (Fig. S4C). In accordance with these data, we observed that the increased production of HCN triggered by CNP on PA14 wild-type strain disappeared in the amiC-mutant strain (Figure S4D). It is interesting to note that the strain over-expressing AmiC (AmiC+) strongly lost its ability to kill lung cells (Figure S4A), confirming a role for AmiC in the regulation of P. aeruginosa virulence.

DISCUSSION

We have shown that P. aeruginosa is sensitive to micromolar concentrations of natriuretic peptides (i.e. BNP and CNP) and responds to these eukaryotic factors by a major increase in virulence [START_REF] Blier | C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa[END_REF][START_REF] Veron | Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis[END_REF]. Previously, the intra-bacterial cascade involving Vfr, PtxR, RhlR and LasR proteins, leading to a remodeling of the lipopolysaccharide (LPS) structure and an increase of HCN and exotoxin A production, was identified ( 16), but the nature of the P. aeruginosa natriuretic peptide sensors remained a central question.

P. aeruginosa is an opportunistic pathogen and in cystic fibrosis patients it is known to form biofilms in lung alveoli [START_REF] Bjarnsholt | Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients[END_REF]. As an exchange surface between air and blood, the alveolar epithelium formed by pneumocytes is particularly thin (0.2 µm). If bacteria are developing on this epithelium, they are certainly exposed to diffusible blood containing endocrine factors such as natriuretic peptides. These are also produced locally by endothelial cells [START_REF] Suga | Cytokine-induced C-type natriuretic peptide (CNP) secretion from vascular endothelial cells--evidence for CNP as a novel autocrine/paracrine regulator from endothelial cells[END_REF], by lung airway epithelium cells and by nonciliated Clara cells [START_REF] Nakanishi | Expression of C-type natriuretic peptide during development of rat lung[END_REF]. The growth of bacteria in biofilms is generally associated with a decrease of diffusible virulence factor synthesis [START_REF] Balasubramanian | A dynamic and intricate regulatory network determines Pseudomonas aeruginosa virulence[END_REF]. This assists in establishing a chronic infectious status, which progressively weakens the host leading to a potential lethal outcome [START_REF] Hoiby | Antibiotic resistance of bacterial biofilms[END_REF]. Conversely, an anticipated release of bacterial virulence factors, particularly when the bacterium is not protected by the biofilm, should trigger the host defence responses and the clearance of the pathogen and increase antibiotic sensitivity. Due to the effect on biofilm formation in P. aeruginosa, CNP and related molecules could potentially be interesting for therapy. CNP strongly decreased the biofilm formation activity of both P. aeruginosa PAO1 and PA14 strains confirming the hypothesis that virulence factor production and biofilm formation are inversely related. This is consistent with our previous results showing that CNP enhances cAMP production in P. aeruginosa [START_REF] Veron | Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis[END_REF]. Indeed, cAMP and c-di-GMP synthesis appear to be antagonistically regulated in bacteria. Whilst c-di-GMP promotes the growth of biofilms [START_REF] Coggan | Global regulatory pathways and cross-talk control pseudomonas aeruginosa environmental lifestyle and virulence phenotype[END_REF], cAMP prevents P. aeruginosa biofilm formation [START_REF] Ono | cAMP signaling affects irreversible attachment during biofilm formation by Pseudomonas aeruginosa PAO1[END_REF]. The present results are also in agreement with the observation that CNP induces a rearrangement of the Pseudomonas LPS structure [START_REF] Veron | Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis[END_REF] and enhances algC mRNA synthesis [START_REF] Blier | C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa[END_REF], since LPS contributes to the bacterial surface properties and AlgC is involved in the synthesis of the LPS core [START_REF] Goldberg | Avirulence of a Pseudomonas aeruginosa algC mutant in a burned-mouse model of infection[END_REF].

In order to identify the potential natriuretic peptide sensor in P. aeruginosa, we started from the hypothesis that this protein should show structural homology with the binding domain of eukaryotic natriuretic peptide receptors. Van den Akker et al. [START_REF] Van Den Akker | Structure of the dimerized hormone-binding domain of a guanylyl-cyclase-coupled receptor[END_REF] have shown that the binding domain of the eukaryotic natriuretic peptide receptor subtype A (NPR-A), a receptor which binds ANP and BNP, exhibits a type-I periplasmic binding protein fold and that the closest structural neighbour of this ANP binding domain is the AmiC protein from P. aeruginosa. Analysis of the crystal structure of the dimerized hormone binding domain of NPR-A and of the structure of the AmiC-AmiR complex showed that in NPR-A and AmiC, the same residues in helixes α3 and α7 interact respectively with ANP and AmiR. Furthermore, the pocket forming the ANP binding site on NPR-A overlaps with the recognition site of AmiR on AmiC [START_REF] Van Den Akker | Structural insights into the ligand binding domains of membrane bound guanylyl cyclases and natriuretic peptide receptors[END_REF][START_REF] Van Den Akker | Structure of the dimerized hormone-binding domain of a guanylyl-cyclase-coupled receptor[END_REF]. In eukaryotes, NPR-A is known as a receptorenzyme coupled to cGMP synthesis. However, CNP does not lead to an increase of cGMP in P. aeruginosa whereas it stimulates cAMP synthesis [START_REF] Veron | Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis[END_REF]. Then, we looked for possible similarities between AmiC and human NPR-C (hNPR-C), the sole ANP and CNP eukaryotic receptor coupled to an adenylate cyclase activity [START_REF] Anand-Srivastava | Cytoplasmic domain of natriuretic peptide receptor-C inhibits adenylyl cyclase. Involvement of a pertussis toxin-sensitive G protein[END_REF]. We observed in silico that hNPR-C and P. aeruginosa AmiC proteins present 19% identity and 26% similarity, confirming the structural homology of eukaryotic natriuretic peptides receptors and AmiC [START_REF] Van Den Akker | Structural insights into the ligand binding domains of membrane bound guanylyl cyclases and natriuretic peptide receptors[END_REF]. The AmiC binding domain helix α3 is delineated by amino-acids 85 to 99 [START_REF] O'hara | Crystal structure and induction mechanism of AmiC-AmiR: a ligand-regulated transcription antitermination complex[END_REF]. In the present study, we noted that the binding of CNP to AmiC would occur in the same region; 6 of the 13 CNP binding amino acids (Ala90, Met92, Pro93, Glu96, Arg97 and Asp99) are in helix α3, suggesting that CNP could enter into competition with AmiR for binding on AmiC. We compared all the amino acids putatively involved in the AmiC-CNP interaction to those observed in the interaction of CNP with NPR-C in eukaryotes [START_REF] He | Structural determinants of natriuretic peptide receptor specificity and degeneracy[END_REF]. The physico-chemical properties of amino-acids involved in these two interactions (i.e. CNP/AmiC in bacteria and CNP/NPR-C in eukaryotes) showed a remarkably high level of conservation (this study). We determined that AmiC (like NPR-C) [START_REF] Anand-Srivastava | Cytoplasmic domain of natriuretic peptide receptor-C inhibits adenylyl cyclase. Involvement of a pertussis toxin-sensitive G protein[END_REF] is functionally expressed as a dimer reinforcing the hypothesis that AmiC could have a CNP binding activity.

In P. aeruginosa, the amiC gene appears highly conserved (99.57% sequence homology and total preservation of the amino acid sequence between PAO1 and PA14 strains) suggesting that a crucial role is played by this protein in this species. The amiC gene is encoded in an operon including the amiL, amiE, amiB, amiC, amiR and amiS genes [START_REF] Wilson | Antitermination of amidase expression in Pseudomonas aeruginosa is controlled by a novel cytoplasmic amide-binding protein[END_REF] [www.pseudomonas.com]. AmiC activation by acetamide, its classical ligand in bacteria, is known to provoke the release of AmiR leading to the transcriptional activation of the whole ami operon [START_REF] Wilson | Cloning and DNA sequence of amiC, a new gene regulating expression of the Pseudomonas aeruginosa aliphatic amidase, and purification of the amiC product[END_REF][START_REF] O'hara | Crystal structure and induction mechanism of AmiC-AmiR: a ligand-regulated transcription antitermination complex[END_REF]. We therefore hypothesized that, if CNP acts on AmiC in a similar fashion to acetamide, it should also induce the ami operon expression. This was confirmed by our observations. CNP actually induced amiERC transcription, and the specificity of this action was reinforced by the lack of effect of BNP on the ami operon transcription (data not shown). As mentioned elsewhere, P. aeruginosa AmiC appears functionally related to hNPR-C and it is interesting to note that, in eukaryotic cells, NPR-C preferentially binds to CNP with a higher affinity than to BNP [START_REF] He | Structural determinants of natriuretic peptide receptor specificity and degeneracy[END_REF]. Such highly conserved specific recognition modes between eukaryotic and bacterial sensors have been previously observed in Helicobacter pylori for the human hormone somatostatin whose bacterial sensor can only recognize one agonist sub-type [START_REF] Yamashita | Inhibitory effect of somatostatin on Helicobacter pylori proliferation in vitro[END_REF].

The issue of how CNP contacts AmiC in P. aeruginosa needs to be addressed, as AmiC is predicted to be located in the bacterial cytoplasm [www.pseudomonas.com]. This means that CNP must enter into the bacterium before binding to AmiC. Such an assumption is conceivable since another eukaryotic peptide, dynorphin, was shown able to cross freely the membrane of P. aeruginosa, inducing an increased virulence [START_REF] Zaborina | Dynorphin activates quorum sensing quinolone signaling in Pseudomonas aeruginosa[END_REF]. This behaviour was explained by the high percentage of basic and hydrophobic amino acid residues present in dynorphin (76%) [START_REF] Marinova | Translocation of dynorphin neuropeptides across the plasma membrane. A putative mechanism of signal transmission[END_REF]. Similarly, CNP contains 73% basic or hydrophobic amino acids.

Moreover, natriuretic peptides have been shown to form pores in phospholipid bilayers [START_REF] Kourie | Synthetic mammalian C-type natriuretic peptide forms large cation channels[END_REF] and could therefore manage their own entrance into the bacterium. However, we cannot exclude that AmiC protein could, in some conditions, reach the P. aeruginosa inner membrane. Indeed, AmiC belongs to the type I periplasmic binding fold protein family and the Uniprot data base claims that AmiC is located in the periplasmic space [www.uniprot.org; reference P27017]. One protein being located in multiple bacterial compartments to undertake unrelated functions is not unprecedented. Indeed, the EF-Tu protein, which acts in the bacterial cytoplasm as a ribosomal elongation factor, was shown to migrate upon stress to the plasma membrane and acquires new functions as a sensor molecule [START_REF] Dallo | Elongation factor Tu and E1 beta subunit of pyruvate dehydrogenase complex act as fibronectin binding proteins in Mycoplasma pneumoniae[END_REF]. In P. aeruginosa, EF-Tu, after migration into bacteria membrane also behaves as a plasminogen binding protein [START_REF] Kunert | Immune evasion of the human pathogen Pseudomonas aeruginosa: elongation factor Tuf is a factor H and plasminogen binding protein[END_REF]. To support this hypothesis, we observed, using AmiC antibodies, that whilst AmiC protein is mainly located into the bacterial cytosol, a proportion of AmiC is associated with the inner membrane compartment (See supplemental material Fig. S6). In this case, we assumed that CNP is delivered to AmiC proteins that have reached the inner membrane through protein transporters, for example. To reinforce this hypothesis we observed that a protein encoded by the gene PA14_64270 (ortholog of PA4858 in PAO1) possesses a high structural similarity with AmiC, could virtually bind CNP, and is necessary for the CNP effect on bacterial biofilm (See supplemental material Fig. S5).

Although a couple of bacterial sensors for eukaryotic messengers have been identified [START_REF] Lesouhaitier | Gram-negative bacterial sensors for eukaryotic signal molecules[END_REF], to date none of the bacterial sensors structurally identified possess a similarity with the eukaryotic counterpart receptor. For example, E. coli QseC, the sensor protein for epinephrine, is an histidine sensor kinase that twice crosses the bacterial membrane, and which appears structurally very different from the G-coupled protein receptor [START_REF] Hughes | Inter-kingdom signalling: communication between bacteria and their hosts[END_REF][START_REF] Xie | Structure of the cytoplasmic segment of histidine kinase receptor QseC, a key player in bacterial virulence[END_REF]. In the same vein, interferon-γ, which binds the eight transmembrane P. aeruginosa OprF protein [START_REF] Sugawara | Pseudomonas aeruginosa porin OprF exists in two different conformations[END_REF], in mammals activates a single transmembrane receptor. Since our data suggest that AmiC and hNPR-C possess a homologous 3D-structure, and that CNP acts as an agonist on P. aeruginosa, we undertook to study the pharmacological profile of AmiC, as happens in eukaryotic receptor studies.

Results from our pharmacological studies carried out using cANF 4-23 , a NPR-C agonist, and isatin, an NPR-A/NPR-C antagonist (49) also support the hypothesis that AmiC acts as an ortholog of eukaryotic NPR-C. In silico molecular modeling revealed that isatin binding to AmiC would require only 4 amino acids but, as expected for an antagonist, these amino acids are located in the potential CNP binding pocket of AmiC. The antagonist effect of isatin was confirmed experimentally. Isatin had no intrinsic activity on the biofilm formation activity of P. aeruginosa but inhibited the effect of CNP. Conversely, the artificial peptide cANF 4-23 , a specific NPR-C agonist in eukaryotes, was able to reproduce the effects of CNP on P. aeruginosa global virulence on lung cells, as observed by Blier et al. [START_REF] Blier | C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa[END_REF]. These data reinforced the hypothesis that AmiC acts as a sensor for natriuretic peptides, but do not exclude the possibility that these peptides bind to another protein that in turn affects AmiC. In order to validate our hypothesis, we purified P. aeruginosa AmiC recombinantly produced in E. coli and checked the interactions of CNP, BNP, isatin, osteocrin (an NPR-C agonist) (50), cANF 4-23 (an NPR-C agonist) and sANP (an NPR-A agonist) [START_REF] Olson | Novel natriuretic peptide receptor/guanylyl cyclase A-selective agonist inhibits angiotensin II-and forskolin-evoked aldosterone synthesis in a human zona glomerulosa cell line[END_REF] with AmiC. As expected, AmiC bound strongly to CNP, showing a K D of 2.0 ± 0.3 μM, consistent with the effective concentrations of CNP on P. aeruginosa. In contrast, BNP does not interact with AmiC suggesting that BNP acts on P. aeruginosa through another bacterial target, a hypothesis strongly reinforced by the fact that BNP retains an effect on biofilm formation in the amiCmutant strain (this study). Isatin also showed a specific interaction, although at a significantly higher concentration (600 ± 200 μM). As normal blood isatin concentrations are usually of the order of 0.3-1.3 μM (52), this implies that in vivo, isatin is unlikely to be at a sufficiently high concentration to antagonise the effects of CNP on AmiC. Finally, we attempted to test specific agonists of NPR-A and NPR-C, to confirm that AmiC reflects NPR-C specificity. sANP showed some interaction with AmiC, but this interaction was weak, and indeed was not complete at the highest ligand concentration available, indicating a K D (likely considerably) in excess of 600 μM, explaining probably the weak effect of this peptide on bacteria cytotoxicity (this study). In contrast, initial experiments on osteocrin showed that it bound sufficiently strongly that it was beyond the limit of detection of our experiment (100 nM), and cANF 4-23 possesses a K D in the same range as CNP. These observations suggest that AmiC does indeed show the same specificity as NPR-C, and is selective against NPR-A agonists. Finally, using an amiC deficient mutant strain and its complemented derivative, we physiologically validated the crucial role of AmiC sensor in CNP effects on P. aeruginosa virulence expression and biofilm formation.

The last puzzling question is the reason why a bacterium such as P. aeruginosa should express a sensor system for peptides which are naturally labile molecules only present in the inner medium of vertebrates. The fact that the CNP sensor should also be a binding site for isatin provides some clues on a possible explanation. Isatin, or indole-2,3-dione, is an interspecies signal molecule produced in large amounts by bacteria such as Escherichia coli [START_REF] Lee | Indole is an inter-species biofilm signal mediated by SdiA[END_REF]. Until now, Pseudomonads were not thought to synthesize isatin or isatin related compounds but as shown by Lee et al. [START_REF] Lee | Enterohemorrhagic Escherichia coli biofilms are inhibited by 7-hydroxyindole and stimulated by isatin[END_REF] and in the present study, P. aeruginosa can detect this molecule. Isatin is also produced in large amounts by vegetals where it is a precursor in auxin biosynthesis [START_REF] Zou | Determination of indican, isatin, indirubin and indigotin in Isatis indigotica by liquid chromatography/electrospray ionization tandem mass spectrometry[END_REF] and by mammalian tissues including lungs [START_REF] Watkins | Isatin, regional distribution in rat brain and tissues[END_REF][START_REF] Medvedev | Biological targets for isatin and its analogues: Implications for therapy[END_REF]. Therefore, in the host, P. aeruginosa is regularly exposed to significant amounts of isatin. As is the case for GABA [START_REF] Dagorn | Effect of GABA, a Bacterial Metabolite, on Pseudomonas fluorescens Surface Properties and Cytotoxicity[END_REF], it will gain an advantage by adapting its behavior in response to the host physiology. Nevertheless, even if this explanation is correct, the fact that AmiC showed common properties with the eukaryotic NPR-C, including CNP recognition, remains to be explained.

In conclusion, the present work provides the first demonstration that bacteria are not only sensitive to eukaryotic messengers but could express eukaryotic receptor-like structures to detect human hormones. The AmiC sensor protein acts as an ortholog of the eukaryotic receptor NPR-C, acting as a CNP/Isatin sensor in P. aeruginosa that modulates bacterial biofilm formation. Since the CNP hormone is produced in the lung, the effects of CNP on the biofilm formation activity of P. aeruginosa could represent a natural defense mechanism against lung colonization. Furthermore, a natriuretic peptide receptor agonist has been used as bronchodilator drug [START_REF] Edelson | In vitro and in vivo pharmacological profile of PL-3994, a novel cyclic peptide (Heptcyclo(Cys-His-Phe-d-Ala-Gly-Arg-d-Nle-Asp-Arg-Ile-Ser-Cys)-Tyr-[Arg mimetic]-NH(2)) natriuretic peptide receptor-A agonist that is resistant to neutral endopeptidase and acts as a bronchodilator[END_REF]. This could on the one hand pose a risk of enhancing P. aeruginosa virulence; and on the other it may prevent biofilm formation. This opens up the tantalising prospect that compounds in development for other indications could be re-targeted for use against biofilms.

MATERIALS AND METHODS

Reagents and test substances.

The CNP peptide was obtained from Polypeptide (Strasbourg, France). Isatin and hBNP were purchased from Sigma-Aldrich (St Quentin Fallavier, France). Osteocrin, sANP and cANF 4-23 were synthesized by Dr J. Leprince (PRIMACEN Platform, University of Rouen, France).

DMSO was purchased from Acros Organics (Geel, Belgium).

Bacterial strains and bacterial cultures.

Pseudomonas aeruginosa PAO1 was obtained from an international collection [START_REF] Veron | Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis[END_REF]. P. aeruginosa MPAO1 was originally from Iglewski lab. A transposon library was constructed from the MPAO1 strain at the University of Washington ( 59). The P. aeruginosa PA14 was from Harvard Medical School (Boston, MA) [START_REF] Liberati | An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants[END_REF] and kindly provided by the Biomerit Research Center (Univ. Cork, Ireland). All strains used in this work are listed in Supplemental material Table S2. All strains were grown at 37°C in Luria Bertani medium (LB). For treatment with CNP, an overnight culture was diluted to OD 600 = 0.08 using a ThermoSpectronics (Cambridge, UK) spectrophotometer. CNP was added two hours after the onset of the culture, a time corresponding to the middle of the exponential growth phase. For all studies performed using isatin, because of its hydrophobicity this molecule was initially dissolved in a solution of dimethyl sulfoxide (DMSO) in water (5/95). The final concentration of DMSO in bacterial culture medium was kept under 0.2%. In this case, control experiments were conducted in the presence of the same final concentration of DMSO. The final bacterial density and the absence of contamination were controlled by plating.

Biofilm formation in dynamic conditions.

After 3 h of pre-culture in LB at 37°C, P. aeruginosa PA14 was inoculated at OD 600 = 0.08 in LB medium and sub-cultured for 2 h. CNP (1 µM final concentration) was added and bacteria were grown for additional 3 h. Bacteria were then washed and adjusted to OD 600 = 0.1 in 0.9% NaCl supplemented with CNP (1 µM). The bacterial suspensions were then used to study biofilm formation under dynamic conditions at 37°C in a three-channel flow cell as described by Bazire et al. [START_REF] Bazire | The sigma factor AlgU plays a key role in formation of robust biofilms by nonmucoid Pseudomonas aeruginosa[END_REF]. Briefly, each bacterial suspension was injected into a flow cell channel and bacteria were allowed to attach onto the glass coverslip for 2 h. A flow (2.5 ml/h) of LB medium containing CNP (1 µM) was then applied for 24 h. At the end of the experiments, the biofilms were stained with 5 µM Syto 61 Red dye or 5 µM SytoX (Molecular probes).

Observations were made using a confocal laser scanning microscope (LSM 710 confocal laser-sanning microscope, Zeiss). The biofilm thicknesses and corresponding biovolumes were estimated by measuring field samples from at least 3 independent experiments using the COMSTAT software [START_REF] Heydorn | Quantification of biofilm structures by the novel computer program COMSTAT[END_REF].

Biofilm formation in static conditions.

The static bacterial biofilm formation activity was determined after 24 h of development. Precultures of P. aeruginosa, containing the pSMC2.1 plasmid harbouring the GFP gene, were grown overnight at 37°C. An aliquot of pre-culture (100 µl) was then added to 5 ml of LB medium supplemented with 400 µg/mL kanamycin. Tested molecules or the corresponding excipient were added after 2 hours of sub-culture. Three hours later, bacteria were collected by centrifugation (7,000 x g, 10 min). The supernatant was then removed and the pellets were resuspended in 25 ml of physiological solution (0.9% NaCl) and adjusted to OD 600 = 0.1.

Sterile glass slides, previously cleaned with 4% TFD4 in water (Dutscher, Brumath, France), were layered in Petri dishes and covered by the bacterial suspension. The slides were then incubated for 2 h at 37°C in static condition. At the end of the attachment period the physiological solution was replaced by LB medium and incubated for 24 h at 37°C in static condition. The slides were then rinsed twice in 25 ml sterile phosphate buffered saline (PBS 0.1 M, pH=7.4) and bacteria were heat fixed. Biofilms were observed using a confocal laser scanning microscope (LSM 710, Zeiss). Images were treated using a 3 median filter and segmented using Zen® 2009 software (Zeiss). The volumes and thicknesses of the biofilms were estimated as described elsewhere [START_REF] Heydorn | Quantification of biofilm structures by the novel computer program COMSTAT[END_REF]. All 2D images are representative from 15 observations.

In silico studies.

The STAMP software [START_REF] Russell | Multiple protein sequence alignment from tertiary structure comparison: assignment of global and residue confidence levels[END_REF] was used within VMD (63) on a single computer. The GASH Structural Superposition software [START_REF] Standley | GASH: an improved algorithm for maximizing the number of equivalent residues between two protein structures[END_REF] was used on the web server (http://sysimm.ifrec.osakau.ac.jp/gash/). Potential ligand/protein interactions were investigated in silico by the molecular docking technique using the amidase sensor protein of P. aeruginosa AmiC (Protein Data Bank 1QO0). Essential hydrogen atoms, Kollman united atom type charges and solvation parameters were added with the aid of AutoDock tools [START_REF] Morris | Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function[END_REF]. Affinity (grid) maps of 20X20X20 Å grid points and 0.375 Å spacing were generated using the Autogrid program [START_REF] Morris | Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function[END_REF]. AutoDock parameter set-and distance-dependent dielectric functions were used in the calculation of the van der Waals and electrostatic terms, respectively. Docking simulations were made using the Lamarckian genetic algorithm (LGA). Initial position, orientation and torsions of the ligand molecules were set randomly. Each docking experiment was derived from three different runs that were set to terminate after a maximum of 1,000,000 energy evaluations. The population size was set to 100.

Quantitative reverse transcription-PCR (qRT-PCR) mRNA assays.

RNA extraction and quantification were realized as previously described (61) using bacteria grown for 3 h in LB. The primers employed are presented in supplemental material Table S3.

The transcription level of 16S rRNA was used as the endogenous control. PCR reactions were performed in triplicate and the standard deviations were lower than 0.15 CT. Relative quantification of mRNA was obtained according to Bazire et al. [START_REF] Bazire | Effects of osmotic stress on rhamnolipid synthesis and time-course production of cell-to-cell signal molecules by Pseudomonas aeruginosa[END_REF] using the comparative CT (2 -ΔΔCT ) method [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[END_REF].

Lung epithelial cells cytotoxicity tests.

The human A549 lung epithelial cell type II line (ATCC-CCL185TM) (American Type Culture Collection, Manassas, VA) was grown at 37°C in 5% CO 2 atmosphere in Dulbecco's modified Eagle's medium (DMEM, Lonza) supplemented with 10% fetal calf serum (Lonza) and 1% of penicillin and streptomycin (Penistrep, Lonza). For cytotoxicity assays, cells were seeded in 24 well plates at a final density of 3x10 5 cells per well, and grown for 48 h before use. A minimum of 24 h before infection assays, cells were deprived of antibiotics and fetal calf serum by addition of a fresh serum-and antibiotics-free medium. For the assays, A549 cells were incubated for 2 h with control or pre-treated P. aeruginosa PA14 at a multiplicity of infection of 10. Bacterial cytotoxicity was determined by measurement of lactate dehydrogenase (LDH) release. LDH is a stable cytosolic enzyme that diffuses into the culture medium upon cell lysis. This marker of cytotoxicity was assayed using the Cytotox 96 enzymatic assay (Promega, Charbonnières, France) as previously described [START_REF] Veron | Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis[END_REF].

P. aeruginosa AmiC purification.

To express P. aeruginosa AmiC in E. coli, a synthetic codon optimized gene was prepared (MWG Operon) with a leader sequence of MHHHHHHSSGVDLGTENLYFQS to provide a cleavable 6*His tag. This was cloned into the NcoI and HindIII restriction sites of the pET-Duet1 vector (Novagen). This construct was transformed into Rosetta2 (DE3) cells (Novagen), and grown in 500 mL of ZYM-5052 media (66) at 37°C until mid-log phase, and then at 20°C overnight. Cells were harvested by centrifugation at 4,750 ×g for 30 min, and resuspended in 20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole pH 8.0 (buffer A). Cells were lysed using a sonicator, and clarified by centrifugation at 20,000 × g. The protein was purified using a 1 mL HisTrap crude FF column and a Superdex 200 16/600 hr column (GE Healthcare), using an ÄKTAxpress system (GE Healthcare) at 4°C. Lysate was loaded over the IMAC column at 0.25 ml.min -1 , and washed with buffer A. AmiC was eluted in buffer A supplemented with 250 mM imidazole pH 8.0 and loaded onto the gel filtration column. The protein was then eluted isocratically in 10 mM Hepes-NaOH, 0.5 M NaCl pH 7.0. DMSO was added to the purified protein to 1% (v/v), and the protein was concentrated using a Vivaspin 20 centrifugal concentrator (Sartorius; 10 kDa molecular weight cut-off) at 4°C.

Microscale thermophoresis.

AmiC was labeled using the RED-NHS Labeling kit (NanoTemper Technologies). The labeling reaction was performed according to the manufacturer's instructions in the supplied labeling buffer applying a concentration of 20 μM protein (molar dye:protein ratio ≈ 2:1) at RT for 30 min. Unreacted dye was removed with the supplied dye removal columns equilibrated with MST buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM MgCl 2 ). The label:protein ratio was determined using photometry at 650 and 280 nm. Typically, a ratio of 0.8 was achieved. AmiC, either labelled (CNP, isatin, BNP and cANF 4-23 ) or unlabeled (sANP, osteocrin), was adjusted to 100 nM with MST buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM MgCl 2 ) supplemented with 0.05 % Tween-20. CNP, isatin, sANP or osteocrin were dissolved in MST buffer supplemented with 0.05 % Tween-20. A series of 1:1 dilutions were prepared in the identical buffer, producing ligand concentrations ranging from 13 nM to 434 μM (CNP), 3.12 μM to 3 mM (isatin), 892 nM to 1.8 mM (sANP), 28 nM to 450 μM (BNP), 68 nM to 1.1 mM (cANF 4-23 ) and 120 pM to 4 μM (osteocrin). For thermophoresis, each ligand dilution was mixed with one volume of AmiC, which leads to a final concentration of AmiC Where f(c) is the observed thermophoresis signal, unbound is the signal in the absence of ligand (normalized to 0), bound is the signal at infinite ligand concentration, c is the concentration of ligand in the same units as AmiC concentration, and K D is the dissociation constant. The data were fitted using GraphPad Prism version 5, and figures for thermophoresis were generated using this software. For cANF 4-23 , incubation with AmiC resulted in a dose-dependent increase in AmiC fluorescence, which confounds the MST signal. This increase was therefore measured using the MST device, and the data fitted to the above equation. To confirm that the fluorescence increase was related to binding, samples were heated to 95°C for 10 min with 2% (w/v) SDS and 20 mM DTT, and retested: addition of cANF 4-23 no longer increased fluorescence. AmiC was fluorescently labeled and incubated with varying concentrations of natriuretic peptides receptor agonists and isatin(A). These mixtures were then analysed using by microscale thermophoresis. The results were normalized to a fitted zero for unbound AmiC, and the thermophoresis signal shown as parts per thousand. Addition of cANF 4-23 caused AmiC fluorescence to increase in a dose dependent fashion: this increase was used as the signal rather than thermophoresis, as the effect confounds the thermophoresis signal. The results were fit to the dissociation constant formula from the law of mass action. The fitted values for K D are shown for isatin, BNP, sANP, cANF 4-23 and osteocrin (B). We showed that these compounds bound either too weakly (sANP), more tightly (osteocrin) or presented no interaction (BNP). For each compound tested, data are the mean of three independent experiments.

Figure legends

  of 50 nM and final ligand concentrations at half of the ranges above. After 5 min incubation, followed by centrifugation at 10,000 × g for 10 min, approximately 4 μL of each solution was filled into Monolith NT Standard Treated Capillaries (NanoTemper Technologies GmbH). Thermophoresis was measured using a Monolith NT.115 or Monolith NT.LabelFree instrument (NanoTemper Technologies GmbH) at an ambient temperature of 25°C with 5 s/30 s/5 s laser off/on/off times, respectively. Instrument parameters were adjusted with 50 % LED power and 20-40% MST power. Data of three independently pipetted measurements were analyzed (NT.Analysis software version 1.5.41, NanoTemper Technologies) using the signal from Thermophoresis + T-Jump. Following data analysis, the thermophoresis signals were fitted to formula for K D from the law of mass action:

Figure 1 .

 1 Figure 1. Biofilm formation by Pseudomonas aeruginosa PA14 exposed to C-type Natriuretic Peptide (CNP) or Brain Natriuretic Peptide (BNP) in dynamic condition. (A) COMSTAT analyses of biofilms of P. aeruginosa PA14 control (PA14) or exposed to CNP (10 -6 M) or BNP (10 -6 M). ( = p < 0.001). Data are the means of twelve samples from six independent experiments for control bacteria, ten samples from six independent experiments for CNP-exposed bacteria and six samples from two independent experiments for BNP-exposed bacteria. (B) 3D-shadow representations of the biofilm structures developed under dynamic condition by P. aeruginosa PA14 control, exposed to CNP or exposed to BNP at 37 °C for 24 h in LB broth. Biofilms were stained with the Syto 61 Red dye or SytoX dye and observed by confocal laser scanning microscopy.

Figure 2 .

 2 Figure 2. Effect of antagonists and agonists of natriuretic peptide receptors. (A) 2D black and white images resulting from CLSM observations of MPAO1 24-h biofilms obtained in static condition after growth in control condition, exposed to CNP (10 -6 M), exposed to isatin (10 -5 M) or exposed to both isatin and CNP. (B) COMSTAT analyses of biofilms of P. aeruginosa MPAO1 exposed to CNP (+ CNP), exposed to isatin (+ Isa) or exposed both to isatin and CNP (+Isa + CNP). Data are the mean of three independent experiments for CNP exposition, and two independent experiments for isatin and isatin + CNP exposition. (= p < 0.001; NS: Not significantly different). (C) Cytotoxic activity of PA14 alone (control), PA14 exposed to NPR-C agonist cANF 4-23 (10 -6 M) or PA14 exposed to NPR-A agonist sANP (10 -6 M). The cytotoxic effect of the bacterium was determined by measuring lactate dehydrogenase (LDH) accumulation in the medium. Data are the means of nine samples from three independent experiments for cANF 4-23 and six samples from two independent experiments for sANP. : Significantly different (p< 0.001). : Significantly different (p< 0.01). : Significantly different (p< 0.05).

Figure 3 .

 3 Figure 3. Cartoon representation of AmiC (pdb:1PEA), in red, and hNPR-C (pdb:1JDP), in blue, superimposed using STAMP algorithm. (A) Overall superimposition of AmiC and NPR-C, shown in cartoon form. CNP peptide is colored green. (B) Superimposition by lobes of AmiC and hNPR-C. The lobe on the left corresponds to amino acids 8 to 123 and 261 to 338 of AmiC. The lobe on the right corresponds to amino acids 124

Figure 4 .

 4 Figure 4. Effect of C-type Natriuretic Peptide (CNP) on expression level of genes from the ami operon. Expression levels of ami genes in PAO1-treated bacteria relative to those in PAO1 non-treated (control). RNAs were extracted 1 h after PAO1 exposure to physiologic solution (control) or CNP (10 -6 M) and assayed by quantitative real-time reverse transcription-PCR (qRT-PCR). These data are the mean of two independent experiments.  = p < 0.001; NS: Not significantly different.

Figure 5 .

 5 Figure 5. CNP, BNP , NPR agonists and isatin affinities for AmiC in vitro. Recombinant

Figure 6 .

 6 Figure 6. Involvement of AmiC protein on P. aeruginosa biofilm formation. (A) 3Dshadow representations of the biofilm structures developed by P. aeruginosa PA14 control (PA14), amiC-strain and amiC-complemented strain (amiC-Comp), not exposed to peptide or exposed to CNP (10 -7 M) in dynamic condition, at 37 °C for 24 h in LB broth. Biofilms were stained with the SytoX and observed by confocal laser scanning microscopy. (B) COMSTAT analyses of biofilms of P. aeruginosa PA14 control, amiC-, amiC-complemented and AmiC+ strain. (C) COMSTAT analyses of biofilms of P. aeruginosa PA14 control, amiC-and amiC-complemented exposed to CNP (10 -7 M) or to BNP (10 -7 M) or neither of them. Data are the means of twenty-seven samples from eleven independent experiments for PA14 control, eleven samples from four independent experiments for PA14 exposed to CNP, nine samples from three independent experiments for PA14 exposed to BNP, nineteen

  Fig.1

  Fig.2

  Fig.3

ACKNOWLEDGEMENTS

We wish to thank Magalie Barreau and Olivier Maillot for technical assistance. We wish to thank Christine Farmer for linguistic insight for this manuscript. T. Rosay is a recipient of a doctoral fellowship from the French Ministry of Research (MRE). This work was supported by grants from the Communauté d'Agglomération d'Evreux, the Conseil Général de l'Eure, European Union (FEDER n°31970), the French Association "Vaincre la Mucoviscidose" and the InterReg IVA PeReNE project.

Statistical analysis.

The non parametric Mann-Whitney test was used to compare the means within the same set of experiments. S1. List of amino acids potentially involved in the binding of CNP to NPR-C and AmiC. S2. List of Strains and plasmids used in this study. S3. Primers used in this study.

Supplemental material figures and tables

Supplemental Table

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Moustafa Abdel Hafiz

Gregoire Coget

Emeric De Clercq

Rodolphe Boudot

Doppler-free spectroscopy on Cs D 1 line with a dual-frequency laser

Keywords:

We report on Doppler-free laser spectroscopy in a Cs vapor cell using a dual-frequency laser system tuned on the Cs D1 line. Using counter-propagating beams with crossed linear polarizations, an original sign-reversal of the usual saturated absorption dip and large increase in Doppler-free atomic absorption is observed. This phenomenon is explained by coherent population trapping (CPT) effects. The impact of laser intensity and light polarization on absorption profiles is reported in both single-frequency and dual-frequency regimes. In the latter, frequency stabilization of two diode lasers was performed, yielding a beat-note fractional frequency stability at the level of 3 × 10 -12 at 1 s averaging time. These performances are about an order of magnitude better than those obtained using a conventional single-frequency saturated absorption scheme.

In gas cell experiments, saturated absorption spectroscopy or Doppler-free spectroscopy [1,[START_REF] Letokhov | Saturation Spectroscopy[END_REF] is an elegant technique to circumvent Doppler broadening and to allow the detection of natural-linewidth resonance dips in the bottom of absorption profiles. This method, frequently used to stabilize the frequency of a laser to a particular atomic line, is applied in atomic frequency standards [START_REF] Vanier | The quantum physics of atomic frequency standards[END_REF], magnetometers [START_REF] Budker | [END_REF], laser-cooling experiments [5] or atom interferometry. Laser fractional frequency stability in the 10 -13 -10 -11 range at 1 s integration time can be obtained by combining saturated absorption techniques and narrow-linewidth diode lasers [6][7][8][9][10]. In a common saturated absorption scheme, two counter-propagating laser beams of same frequency, a pump-beam and a probe beam, derived from a single laser beam, overlap one another in an atomic vapor cell. Since both beams have the same frequency, the Doppler effect brings different velocity groups into resonance with each beam. On resonance, only atoms presenting zero velocity along the light propagation axis actually experience the two beams with the same laser frequency. If the pump-beam intensity is high enough, the ground state is depleted and therefore the absorption of the probe beam is reduced compared to the case without pump beam. In this case, a so-called Lamb dip, with lorentzian profile, appears at the resonance frequency of the atomic transition in the bottom of a gaussian Doppler-broadened absorption profile. The full-width at half maximum (FWHM) of the Dopplerfree Lamb dip can be at low laser intensity as small as the natural linewidth of the atomic transition (4.6 MHz for the Cs D 1 line). This process is relatively easy to understand in the case of a simple two-level atom with a single-frequency laser system. In real experience, alkali atoms exhibit a complex multi-level energy structure with hyperfine structure splitting and numerous Zeeman sub-levels, especially in the Cs case (see Fig. 1). Such multi-level structures can lead, under appropriate conditions, to a sign reversal of saturation resonances [11][12][13].

In the present article, we report on a Doppler-free laser frequency stabilization setup on Cs D 1 line using a coherent bi-chromatic optical field. This setup was initiated in the frame of the development of a high-performance Cs vapor cell atomic clock based on coherent population trapping (CPT) [14]. In many Cs CPT clocks, including chip-scale atomic clocks [START_REF] Knappe | Emerging topics: MEMS Atomic Clocks[END_REF], the laser is modulated at 4.6 GHz (half of the Cs atom clock frequency). In this configuration, both first-order optical sidebands induce the CPT process and the laser carrier frequency needs to be frequency-stabilized 4.6 GHz away from Cs resonance transition. In CSACs, the laser frequency is currently stabilized onto the bottom of a homogeneously broadened optical line detected in the buffer-gas filled CPT cell, leading to modest laser frequency stabilization. In a high-performance CPT clock [14], enhanced stabilization of the laser frequency is required. In that sense, we investigated the possibility to perform Doppler-free spectroscopy in a Cs vapor cell with a dual-frequency laser field. Here, in this two-frequency configuration, two different transitions are involved in the saturated absorption scheme, |F = 3⟩ → |F ′ = 4⟩ and |F = 4⟩ → |F ′ = 4⟩, where F (F ′ ) are the hyperfine quantum numbers of the ground (excited) states, respectively. Using counter-propagating beams with crossed linear polarizations, the detection of high-contrast natural-linewidth reversed dips is demonstrated. The impact of polarization and laser intensity on absorption profiles is shown, supported by a qualitative theoretical approach. We demonstrate that the dual-frequency configuration allows to enhance greatly the dip-based frequency discriminator slope and the laser frequency stabilization compared to the usual single-frequency Doppler-free spectroscopy. Figure 1 shows the experimental setup. A single laser beam is and reflected back through a Cs vapor cell to produce both pump and probe beams.

The laser source is a 1-MHz Distributed Feedback (DFB) diode laser tuned on the Cs D 1 line at 894.6 nm. An optical isolator is used to prevent optical feedback. A pigtailed Mach-Zehnder intensity electro-optic modulator (MZ EOM), driven at 4.596315 GHz by a low noise microwave frequency synthesizer, allows the generation of two first-order optical sidebands frequency-split by 9.192631 GHz. The optical carrier suppression can be actively stabilized thanks to the technique presented in [16]. The contribution of second-order and third-order optical sidebands, rejected at a level 25 to 35 dB lower, is neglected in the article. A fiber collimator is used to extract a free-space collimated laser beam with a diameter of 1.5 mm from the EOM output. A fraction of the laser power is used to perform the Doppler-free spectroscopy setup. The laser beam is sent and reflected back in a 2-cm-diameter and 2-cm long Cs vapor cell. A quarter wave plate can be adjusted such that propagating and counter-propagating beams are linearly and parallel or mutually orthogonally polarized. The power of the reflected beam is measured by a photodiode. All tests were performed at ambient temperature (about 22.5 • C). The cell is surrounded by a mu-metal magnetic shield. No static magnetic field is applied. Figure 2 shows typical transmission signals detected by the photodiode (PD in Fig. 1) in the single-frequency configuration and the dual-frequency regime. The total laser power incident in the cell is 400 µW in all cases. In the single-frequency case, the transition doublet |F = 4⟩ → |F ′ = 4⟩ and |F = 4⟩ → |F ′ = 3⟩ is shown. With parallel polarizations, the usual narrow Lamb dip, with lorentzian profile, appears in the bottom of both Doppler-broadened absorption gaussian-profile lines. With crossed polarizations, the dip is totally vanished in the bottom of the |F = 4⟩ → |F ′ = 3⟩ transition. In the dual-frequency regime, with crossed polarizations, a significant sign-reversal of the dip and large increase in Doppler-free atomic absorption is observed, especially at high laser power.

The dip reversal can be explained by CPT effects which are cancelled for counter-propagating waves. We give here a simple qualitative explanation. A detailed theoretical investigation should take into account all the 25 involved Zeeman sub-levels and can be only numerically computed. When a multilevel atom is irradiated by a wave connecting in a coherent way two sub-levels of the ground state to a same excited level, forming a Λ scheme, the atom can be placed in a light-uncoupled linear superposition of both sub-levels. The atom no longer absorbs the light in this so-called dark state [17]. A linearly polarized monochromatic wave propagating along the quantification z-axis is σ polarized. In null magnetic field and at optical resonance, a Zeeman sub-level of the excited state |e, m⟩ will then be linked to two sub-levels of the ground state |g, m -1⟩ and |g, m + 1⟩. These conditions allow the existence of a dark state that will disappear for increasing magnetic field, as soon as the Zeeman splitting is larger than the CPT resonance linewidth. This effect is known as the non-linear Hanle effect [18]. An example of full density matrix numerical calculation is reported in [START_REF] De Clercq | Proc. European Freq. Time Forum EFTF[END_REF]. According to the linear polarization along the x-axis or y-axis, the dark state is given by:

|ds x ⟩ =d e,m;g,m+1 |g, m -1⟩ + d e,m;g,m-1 |g, m + 1⟩ , |ds y ⟩ =d e,m;g,m+1 |g, m -1⟩ -d e,m;g,m-1 |g, m + 1⟩ , ( 1
)
where d e,m;g,m±1 are the dipole moments of the involved transitions. Equal field amplitudes are assumed. Note that both dark states are orthogonal. This means that the atoms driven in a dark state by a polarized wave will absorb the perpendicularly polarized wave (bright state).

Consider now atoms of a gas irradiated by this wave and the reflected counterpropagating wave. At non-zero values of the optical detuning ∆, because of the Doppler shift, one travelling wave is resonant for atoms of the axial velocity class v = ∆/k (k is the wave number) while the reflected wave is resonant for atoms of the velocity class -v. In that way, a fraction of atoms interacting with only one of the two waves is trapped in a dark state whatever the polarization of the other wave. At optical resonance the atoms of velocity class v = 0 are resonant for both counter-propagating waves. When the polarization of the two fields is the same, dark states are common, less atoms can absorb the probe wave and the expected saturated absorption dip is observed. According to (1), when the polarization vectors are orthogonal, atoms in the dark states of the pump beam become bright and absorbing for the probe, and vice-versa. In this case, the absorption of both beams is increased and the Lamb dip is reversed. A similar effect has already been investigated for linear and elliptical polarizations in the Hanle configuration [START_REF] Brazhnikov | [END_REF]21]. In this study, the monochromatic laser was tuned at resonance and the probe beam power was recorded versus the magnetic field. In Hanle configuration, the linewidth is limited by the CPT resonance width, inversely proportional to the Zeeman coherence lifetime. In the present experiment, the magnetic field is kept null. Then, the sub-Doppler resonance cancels when no more atoms interact with both waves simultaneously, i. e. when ∆ > Γ, with Γ the linewidth of the optical transition. It can be shown that this CPT effect occurs when F ′ = F -1 [22]. It explains why we observe this phenomenon only on the (|F = 4⟩ → |F ′ = 3⟩) transition (see Fig. 2). The same effect occurs with a bichromatic beam carrying two optical frequencies frequency-split by the hyperfinetransition frequency. Since the Doppler-shift difference related to both frequencies is negligible in this case, hyperfine coherences between Zeeman sub-levels of both hyperfine states are also built. The previous argument is still valid. If the pump and probe beams have the same linear polarization, dark states are dark for both beams and the saturated absorption dip is observed. If pump and probe beams are orthogonally polarized, dark states are no more identical. For example, at ∆ = 0, assuming an optically thin medium and equal field amplitudes, the low-frequency σ + component of pump beam (x polarized) and high frequency σ -component of probe beam (ŷ polarized) induce the dark states:

|DS x ⟩ = d e,m; 4,m-1 |3, m + 1⟩ + d e,m; 3,m+1 |4, m -1⟩ , |DS y ⟩ = d e,m; 4,m-1 |3, m + 1⟩ -d e,m; 3,m+1 |4, m -1⟩ , ( 2
)
where 3(4) holds for F = 3 (F = 4), respectively, and e for F ′ = 3 or F ′ = 4. Dark states of Eq. ( 2) are orthogonal. A higher absorption is expected at optical resonance. This occurs for Zeeman dark states and (|∆F | = 1, |∆m| = 2) dark states, but not for (|∆F | = 1, |∆m| = 0) dark states. In monochromatic and bichromatic cases, several state linear superpositions with |∆m| = 2n, n ∈ N * , can coexist. However, since the atomic transit time across the beam is short, the steady-state is not reached and these dark states should be poorly populated.

In the case of crossed polarizations, we measured the evolution of the lorentzian dip versus the total laser power incident in the cell for both single-frequency and dualfrequency regimes. For each laser power, the absorption profiles of the Cs D 1 line spectrum are each fitted by the sum of a gaussian (Doppler broadened profile) and a lorentzian function (Doppler-free dip). Figures 3(a), 3(b) and 3(c) report the full-width at half maximum (FWHM), height and height/FWHM ratio of the Lorentzian dip signal detected in the bottom of the Doppler-broadened profile versus the total laser power incident in the cell. We observe on Fig. 3(a) that the power broadening of the one-frequency dip is larger than in the two-frequency case. Simultaneously, the amplitude of the dip is dramatically increased in the dual-frequency case (see Fig. 3(b)). These observations are attributed to optical pumping effects. In the single-frequency case, the atomic system is an open system, causing the atoms to be pumped into the other hyperfine ground state and leading to a higher effective saturation parameter [23]. At the opposite, in the dual-frequency regime, the atomic system is closed, avoiding the loss of atoms. In the latter regime, a significant result is the important increase of the dip height with laser power, particularly for transitions connecting the (F ′ = 4) excited level. On Fig. 3(c), we observe that the ratio signal/FWHM of the dip (slope of the frequency discriminator for laser frequency stabilization) is significantly increased the dual-frequency regime. For a laser power of 500 µW, in the case of the transition to F ′ = 4, the dip-based frequency discriminator slope is about an order of magnitude higher than in the single-frequency case. Laser frequency stabilization experiments were performed to compare single and dual-frequency regimes. For this purpose, two laser systems, named LS1 and LS2, were implemented. Both systems are mounted on distinct optical tables separated by about 2 meters and linked by an optical fiber. LS1 is similar to the system described in Fig. In the free-running regime, the laser beat-note fractional frequency stability is 1.5 × 10 -9 at 1 s averaging time. In test 2 where one of the laser is stabilized through a standard single-frequency saturated absorption scheme, results are similar to those reported in [9] and improved at the level of 2 × 10 -11 at 1 s and 3.5 × 10 -11 at 100 s. In test 3, where both lasers are stabilized through a dual-frequency Doppler-free spectroscopy setup, the laser beat-note frequency stability is improved by almost 10 yielding 3 × 10 -12 at 1 s and 5 × 10 -12 at 1000 s. For a laser power of 500 µW, this improvement is in correct agreement with the increased frequency discriminator slope reported in Fig. 3(c). The bump at 500 s on Allan deviation plots is suspected to come from the room air conditioning cycle period. Degradation of the frequency stability at higher time scales could be explained by temperature variations, fiber perturbations or laser amplitude variations along time.

In conclusion, we reported the detection of naturallinewidth and high-signal inverted resonance dips through Doppler-free spectroscopy in a Cs vapor cell using a dual-frequency laser system. This technique has demonstrated excellent laser frequency stabilization at the level of 10 -12 at 1 s and 100 s integration time for a single laser, a gain of an order of magnitude compared to performances obtained with a conventional singlefrequency laser system in similar conditions. This system can be used in various applications including CPT atomic clocks, laser spectroscopy, non-linear optics and laser-cooling experiments.

FIG. 1 :

 1 FIG. 1: Experimental setup for Doppler-free Cs D1 line spectroscopy. EOM: electro-optic modulator, ISL: optical isolator, HWP: half-wave plate, PBS: polarizing beam splitter, BS: beam splitter, QWP: quarte-wave plate, M: mirror, PD: photodiode, LO: local oscillator. The Cs D1 line energy structure is reminded on the top left.

FIG. 2 :

 2 FIG. 2: Spectroscopy signals detected in the Cs vapor cell in single-frequency (parallel or orthogonally polarized counterpropagating beams) and dual-frequency (crossed polarizations) regimes. For clarification of the figure, a background of 0.45 V was subtracted to each curve. 3, 4, 3' and 4' stand for |F = 3⟩, |F = 4⟩, |F ′ = 3⟩ and |F ′ = 4⟩ respectively.

FIG. 3 :

 3 FIG. 3: Full-width at half maximum (a), height (b) and height/FWHM ratio (c) of the Lorentzian dip signal detected in the bottom of the Doppler-broadened profile versus the total laser power incident in the cell. Squares: single-frequency regime (transition 3-4'). Dual-frequency regime (Stars: → F ′ = 4, Circles: → F ′ = 3).

FIG. 4 :

 4 FIG.4: Allan deviation of the laser beat-note frequency. Test1: free-running lasers, Test2: LS1 in the DFSR versus LS2 in the SFSR, Test 3: LS1 and LS2 are in the DFSR. For the latter, the total laser power incident in the cell is 500 µW.

  1. LS2 uses a Distributed Bragg Resonator (DBR) laser and a phase EOM (PEOM) driven at 9.192 GHz. In this case, hyperfine CPT is induced by the carrier and one of the first-order sideband. The second first-order sideband is 9.192 GHz out of resonance and neglected here. We note that the gain on the frequency discriminator slope in the dual-frequency regime compared to the standard single frequency regime was found to be about the same for MZ EOM and PEOM-based setups. Different tests were performed. In test 1, each laser is in the free-running regime. In test 2, LS1 operates in the dual-

	frequency stabilization regime (DFSR) whereas LS2 is
	in the single-frequency stabilization regime (SFSR). For
	LS1, the MZ EOM is driven at 4.6 GHz with carrier
	suppression. The laser direct output (carrier) is shifted
	4.6 GHz away from Cs resonance and the dual-frequency
	MZ EOM output beam is sent into the cell for laser sta-
	bilization. For LS2, the PEOM microwave modulation
	is turned off and the PEOM output beam is sent into
	the cell for laser stabilization onto a Cs transition. In
	test 3, both laser systems operate in the DFSR. LS1 is
	configured as in test 2 and the PEOM of LS2 is driven
	at 9.192 GHz. In all tests, the single-frequency direct
	output of each laser (before EOMs) is sent into a beam
	splitter cube. A laser beat-note at 4.596 GHz is detected
	by a fast photodiode, amplified and band-pass filtered.
	This signal is compared to a 4.621 GHz signal from a
	high-performance microwave synthesizer driven by a hy-
	drogen maser. A final beat-note at 25 MHz is low-pass fil-
	tered and counted by a frequency counter (HP 53132A).
	Laser frequency is stabilized by modulating the laser in-
	jection current and demodulating it synchronously with
	a lock-in amplifier at the output of the photodiode. The
	bandwidth of laser frequency servos is about 1 kHz. Fig-
	ure 4 reports the laser beat-note stability for each test.

Acknowledgments

This work has been funded by LabeX FIRST-TF, Région de Franche-Comté and the EMRP program (IND55 Mclocks). The EMRP is jointly funded by the EMRP participating countries within EURAMET and the European Union.