Quentin Perraud

Paola Cantero

Mathilde Munier

Françoise Hoegy

Nicolas Zill

Véronique Gasser

Gaëtan L A Mislin

Laurence Ehret-Sabatier

Isabelle J Schalk
email: schalk@unistra.fr.

Phenotypic adaptation of Pseudomonas aeruginosa in the presence of siderophoreantibiotic conjugates during epithelial cell infection

Iron is an essential nutrient for bacterial growth. Thus, iron acquisition pathways have often been considered to be gateways for the uptake of antibiotics into bacteria. Bacteria excrete chelators, called siderophores, to access iron. Antibiotic molecules can be covalently attached to siderophores for their transport into pathogens during the iron-uptake process. P. aeruginosa carries genes for at least 20 different iron-uptake strategies, using various siderophores, within its genome. Although it produces the two siderophores pyoverdine and pyochelin, it is also able to use many siderophores produced by other bacteria. We investigated the phenotypic plasticity of iron-uptake pathway expression in an epithelial cell infection assay in the presence of two different siderophore-antibiotic conjugates, one with a hydroxamate siderophore and the second with a tris-catechol. Proteomic and RT-qPCR approaches showed that P. aeruginosa was able to sense the presence of both compounds in its environment and adapt the expression of its iron uptake pathways to access iron via them. In parallel, expression of the proteins of the pyochelin pathway was significantly repressed, as well as that of proteins of the pyoverdine pathway, but to a lesser extent. Interestingly, the transcription and expression of several virulence factors was also repressed in the presence of both siderophore-antibiotic conjugates. The catechol-type siderophore-antibiotic conjugates were clearly more efficient in inducing the expression of their corresponding transporter than the hydroxamate compound when both were simultaneously present in the environment of the pathogen, indicating that catechol-type siderophores are more promising vectors for antibiotic vectorization using a Trojan-horse strategy.

Introduction

Antibiotic resistance is a complex and growing problem for human health. In 2017, the World Health Organization published a list of highly resistant bacteria for which new antibiotics are urgently needed. The most critical pathogens on this list are Gram-negative bacteria, such as Acinetobacter, Pseudomonas, and various enterobacteria (including Klebsiella, E. coli, Serratia, and Proteus). The bottleneck in the development of new antibiotics for Gram-negative bacteria is the need for these drugs to cross the outer membrane. The use of nutrient-importation pathways as gateways for the uptake of antibiotics has often been proposed to bypass the low permeability of the Gram-negative cell wall (1).

Iron is a key nutrient, as this metal is involved in many crucial biological processes, and is therefore essential for bacterial growth and virulence. To access iron, bacteria produce siderophores, small molecules with a very high affinity for ferric iron (2). These iron chelators are synthesized by bacteria and released into their environment to scavenge iron. The ferric complexes that are formed are recovered by specific transporters (3). Antibiotics can be covalently linked to siderophores with the idea they will be transported into the pathogens during the ferri-siderophore uptake process -referred to as a Trojan-horse strategy (1,4,5).

One such siderophore-antibiotic conjugate (Cefiderocol), developed by Shionogi, was approved by the US Food and Drug Administration in November 2019 for the treatment of complicated urinary tract infections, showing that Trojan-horse strategies can be successful (6). Moreover, evolution has also developed natural siderophore-antibiotic conjugates (also called sideromycins), demonstrating the relevance of such a strategy. The archetypes among sideromycins are albomycins (7,8), ferrimycins (9), danomycins [START_REF] Selzer | Antibiotics: Isolation, separation, and purification[END_REF], and salmycins [START_REF] Vertesy | Salmycins A-D, antibiotics from Streptomyces violaceus DSM 8286 having a siderophore -aminoglycoside structure[END_REF], isolated mainly from streptomycetes or actinomycetes and produced to kill other microorganisms and dominate a given microbiota.

One major bottleneck limiting the development of siderophore-antibiotic conjugates is that all bacteria are equipped with several iron-uptake pathways. Consequently, bacteria can switch off the expression of the targeted iron uptake pathway and express an alternative one, possibility leading to the rapid development of resistance strategies. For example, P. aeruginosa carries genes within its genome for at least 20 different iron-uptake strategies [START_REF] Cornelis | Pseudomonas aeruginosa adapts its iron uptake strategies in function of the type of infections[END_REF]: (i) one ferrous (Fe 2+ ) iron uptake pathway, (ii) three heme acquisition pathways, (iii) ferric iron (Fe 3+ ) uptake pathways by the two siderophores pyoverdine (PVD) and pyochelin (PCH), produced by the pathogen itself, and (iv) at least 10 different "siderophore piracy" strategies to uptake Fe 3+ using exosiderophores (siderophores produced by other bacteria). Indeed, most bacteria can use siderophores produced by other bacteria in multispecies communities and during infections by expressing compatible transporters that are able to capture and import ferric complexes they cannot synthesize themselves [START_REF] Poole | Enterobactin-mediated iron transport in Pseudomonas aeruginosa[END_REF][START_REF] Hannauer | The ferrichrome uptake pathway in Pseudomonas aeruginosa involves an iron release mechanism with acylation of the siderophore and a recycling of the modified desferrichrome[END_REF][START_REF] Barber | Buried treasure: evolutionary perspectives on microbial iron piracy[END_REF][START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF]. Except for ferrous-iron uptake, bacterial iron-uptake pathways always include an outer membrane transporter (OMT) involved in ferri-siderophore or heme uptake, enzymes involved in iron release from the siderophore or heme, and inner membrane transporters (ABC transporters of protonmotive force-dependent permeases). These proteins, especially the OMTs, are always specific for one siderophore or a family of structurally-related siderophores (3,[START_REF] Schalk | Fate of ferrisiderophores after import across bacterial outer membranes: different iron release strategies are observed in the cytoplasm or periplasm depending on the siderophore pathways[END_REF]. Consequently, bacteria able to access iron via several siderophores carry genes within their genome that encode an OMT specific for each siderophore (3).

In contrast to the large amount of genomic data generated by bacterial genome sequencing, almost no data are available concerning the phenotypic plasticity associated with the expression of these various iron-uptake pathways. Uptake pathways are considered to be generally expressed at a basal level and pathogens only induce the expression of the most efficient pathway(s) for iron acquisition depending on the bacterial environment [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF]. It has also been shown that bacteria are often able to sense the presence of exosiderophores and express the corresponding transporters, allowing them to access iron via these exosiderophores [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF][START_REF] Dean | PfeR, an enterobactin-responsive activator of ferric enterobactin receptor gene expression in Pseudomonas aeruginosa[END_REF][START_REF] Visca | Iron transport and regulation, cell signalling and genomics: lessons from Escherichia coli and Pseudomonas[END_REF][START_REF] Michel | PchR-box recognition by the AraC-type regulator PchR of Pseudomonas aeruginosa requires the siderophore pyochelin as an effector[END_REF][START_REF] Llamas | The heterologous siderophores ferrioxamine B and ferrichrome activate signaling pathways in Pseudomonas aeruginosa[END_REF][START_REF] Llamas | Cell-surface signaling in Pseudomonas: stress responses, iron transport, and pathogenicity[END_REF]. Bacteria are also able to switch from the expression of one ironuptake pathway to another in response to synthetic iron chelators, as shown for P. aeruginosa cells with an analogue of the siderophore enterobactin (ENT) [START_REF] Gasser | Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore-antibiotic conjugates development[END_REF]. Recently, we showed by proteomics and RT-qPCR that tris-catechol type siderophores, such as ENT or vibriobactin (VIB), induce the transcription and expression of the genes involved in their corresponding uptake pathways in P. aeruginosa more efficiently than tris-hydroxamate siderophores, such as ferrichrome [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF]. The data also showed that the presence of different iron uptake pathways in the genome of a pathogen involves high phenotypic plasticity in response to diverse environmental stimuli [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF]. Such phenotypic plasticity plays a key role in the ability of siderophore-antibiotic conjugates to be transported efficiently into the target bacteria. If the right uptake pathway is not expressed, the drugs have no chance of being efficiently transported into the bacteria by their associated siderophore.

Here, we investigated, for the first time, the phenotypic adaptation of P. aeruginosa to the presence of two different siderophore-antibiotic conjugates in an epithelial cell infection assay using proteomics and RT-qPCR. We used the natural sideromycin albomycin (ALBO) (Fig 1A), for which the siderophore component is similar to ferrichrome (FERRI), a siderophore that chelates iron via three hydroxamate groups. For the second conjugate, we used the synthetic compound TCVL6, corresponding to linezolid vectorized by a tris-catechol siderophore (TCV) analogue of ENT (Fig 1A). Both compounds have no antibiotic activity on the pathogen at the concentrations used here [START_REF] Braun | Sideromycins: tools and antibiotics[END_REF][START_REF] Wencewicz | Is drug release necessary for antimicrobial activity of siderophore-drug conjugates? Syntheses and biological studies of the naturally occurring salmycin "Trojan Horse" antibiotics and synthetic desferridanoxamine-antibiotic conjugates[END_REF] and could therefore be used as tools to investigate how the bacteria adapts the expression of its various iron-uptake pathways in the presence of such Trojan-horse compounds. We show that both compounds are able to promote P. aeruginosa growth in iron-restricted conditions, ALBO by the FERRI-dependent uptake pathway (Fig 1B) and TCVL6 by the ENT-dependent uptake pathway (Fig 1C). Both compounds were able to induce the transcription and expression of the genes encoding proteins involved in their corresponding uptake pathways, indicating that P. aeruginosa PAO1 is able to sense the presence of these two compounds and use them to access iron. This occurred in parallel with a decrease in the transcription and expression of the proteins of the endogenous siderophore-dependent iron uptake pathways (PVD and PCH), as well as that of virulence factors. Finally, when bacteria were simultaneously in the presence of both ALBO and TCVL6, the proteins of the ferri-TCVL6 uptake pathway were more highly expressed than those of the ferri-ALBO uptake pathway, indicating that between the two compounds, P. aeruginosa preferentially uses TCVL6 to access iron.

Results

Ability of ALBO and TCVL6 to compete for iron with PVD.

The efficient uptake of siderophoreantibiotic conjugates into bacteria requires that various conditions be met. Among them, the siderophore moiety must have a high affinity for iron and be able to compete for this metal with other siderophores that may be present in the bacterial environment, such as the siderophores produced by the pathogens themselves. P. aeruginosa produces two siderophores, PVD and PCH, with PVD having a higher affinity for iron than PCH (Ka of 10 30.8 M -1 for PVD vs 10 18 M -2 for PCH [START_REF] Blanc | Bacterial iron transport: coordination properties of pyoverdin PaA, a peptidic siderophore of Pseudomonas aeruginosa[END_REF][START_REF] Brandel | Pyochelin, a siderophore of Pseudomonas aeruginosa: Physicochemical characterization of the iron(III), copper(II) and zinc(II) complexes[END_REF]). Apo PVD also has specific spectral properties, with a characteristic absorbance peak at 400 nm (pH 7.0) and fluorescence emission at 447 nM, whereas its fluorescence is quenched when complexed with iron [START_REF] Blanc | Bacterial iron transport: coordination properties of pyoverdin PaA, a peptidic siderophore of Pseudomonas aeruginosa[END_REF][START_REF] Folschweiller | The interaction between pyoverdin and its outer membrane receptor in Pseudomonas aeruginosa leads to different conformers: a time-resolved fluorescence study[END_REF]. As we have described previously, these spectral properties can be used to compare the ability of various chelators to scavenge iron [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF]. Here we used them to monitor the competition between PVD and the compounds FERRI, ALBO, TCV, and TCVL6 for iron ( and confirmed that catechol-type compounds clearly compete more efficiently for iron with PVD than hydroxamate molecules. These data are consistent with the affinities of these families of siderophores for iron: Ka of 10 49 M -1 for ENT [START_REF] Loomis | Solution equilibria of enterobactin complexes[END_REF], an analogue of TCV, 10 29 M -1 for FERRI [START_REF] Anderegg | Hydroxamatkomplexe III. Eisen(III)-Austausch zwischen Sideraminen und Komplexonen. Diskussion der Bildungskonstanten der Hydroxamatkomplexe[END_REF], and 10 30.8 M -1 for PVD [START_REF] Blanc | Bacterial iron transport: coordination properties of pyoverdin PaA, a peptidic siderophore of Pseudomonas aeruginosa[END_REF]. Furthermore, they show that the presence of the antibiotic moiety does not significantly affect the chelation properties of the vector moiety.

ALBO and TCVL6 both transport iron into P. aeruginosa cells. Previous studies have shown that ENT and FERRI are both able to transport Fe into bacteria via the OMT PfeA for ENT and FiuA for FERRI [START_REF] Hannauer | The ferrichrome uptake pathway in Pseudomonas aeruginosa involves an iron release mechanism with acylation of the siderophore and a recycling of the modified desferrichrome[END_REF][START_REF] Gasser | Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore-antibiotic conjugates development[END_REF][START_REF] Normant | Nocardamine-dependent iron uptake in Pseudomonas aeruginosa: exclusive involvement of the FoxA outer membrane transporter[END_REF]. Moreover, previous proteomic and RT-qPCR analyses of P. aeruginosa grown in iron-restricted medium have shown that TCV is also able to enter P. aeruginosa cells via the OMT PfeA, as the presence of this compound induces the expression of PfeA and PfeE, two proteins of the ENT-Fe uptake pathway [START_REF] Gasser | Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore-antibiotic conjugates development[END_REF]. Such induction involves an interaction with the inner membrane sensor PfeS of the two-component system PfeS/PfeR [START_REF] Dean | PfeR, an enterobactin-responsive activator of ferric enterobactin receptor gene expression in Pseudomonas aeruginosa[END_REF][START_REF] Gasser | Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore-antibiotic conjugates development[END_REF]. We used the same approach here. P. aeruginosa PAO1 cells were grown in the presence of TVCL6 and the transcription of pfeA and pfeE analyzed by . We observed induction of the transcription of the pfeA and pfeE genes in the presence of TCVL6 with an efficiency equivalent to that for the vector TCV, without the antibiotic component, and higher efficiency than that for ENT. Such induction of pfeA and pfeE transcription is a proof that TCVL6 is able to cross the outer membrane of P. aeruginosa cells and interact with the two-component system PfeS/PfeR in the periplasm.

Nothing has been described in the literature about the ability of ALBO to transport iron into P. aeruginosa cells. Consequently, we carried out an 55 Fe uptake assay on iron-starved P. aeruginosa cells that were unable to produce PVD or PCH (∆pvdF∆pchA strain [START_REF] Gasser | Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore-antibiotic conjugates development[END_REF]) with ALBO as a siderophore (Fig 3B). The ∆pvdF∆pchA mutant was used to avoid any iron uptake via the siderophores produced by the bacteria itself. We observed an uptake rate of 150 pmol 55 Fe transported per OD600 nm per min, showing that ALBO is able to transport iron into P. aeruginosa cells. Uptake was completely abolished in the presence of CCCP, a protonmotive force inhibitor [START_REF] Clément | The binding mechanism of pyoverdin with the outer membrane receptor FpvA in Pseudomonas aeruginosa is dependent on its iron-loaded status[END_REF], indicating that such uptake is not due to diffusion via porins across the outer membrane but is TonB-dependent. TonB is an inner-membrane protein that spans into the periplasm and provides the energy to the ferri-siderophore OMTs for uptake across the outer membrane [START_REF] Postle | TonB-dependent energy transduction between outer and cytoplasmic membranes[END_REF][START_REF] Celia | Structure and Stoichiometry of the Ton Molecular Motor[END_REF]. The deletion of fiuA (FERRI OMT) resulted in 40% inhibition of 55 Fe uptake at 1 h and 15% at 3 h. FoxA has also been described as a OMT for hydroxamate siderophores [START_REF] Hannauer | The ferrichrome uptake pathway in Pseudomonas aeruginosa involves an iron release mechanism with acylation of the siderophore and a recycling of the modified desferrichrome[END_REF][START_REF] Normant | Nocardamine-dependent iron uptake in Pseudomonas aeruginosa: exclusive involvement of the FoxA outer membrane transporter[END_REF] but its deletion had no effect on 55 Fe accumulation via ALBO. These data show that ferric iron-loaded ALBO uptake occurs via FiuA and at least one other OMT.

We evaluated the ability of ALBO and TCVL6 to provide bacteria with iron by also carrying out growth assays on P. aeruginosa cells unable to produce PVD or PCH (∆pvdF∆pchA strain) in iron-restricted CAA medium, in the presence or absence of 10 µM ENT, TCV, TCVL6, FERRI, or ALBO. The same assays were also carried out with various OMT mutants to identify the transporter involved in such uptake. As these growth assays were carried out using strains unable to produce siderophores, all iron present in the media was scavenged by ENT, TCV, TCVL6, FERRI, or ALBO. Consequently, bacteria only grew if they expressed the OMT necessary to import the ferric forms of these compounds. The ∆pvdF∆pchA strain grew in the presence of all tested compounds (Fig 4 ), showing that P. aeruginosa is able to access iron by using them as siderophores and that neither ALBO nor TCVL6 had antibiotic activity at the concentrations tested. Growth was still observed in the presence of ENT, TCV, and TCVL6 with the ∆pvdF∆pchA∆pfeA and ∆pvdF∆pchA∆pirA mutants, but not ∆pvdF∆pchA∆pfeA∆pirA cells, indicating that both PfeA and PirA are involved in iron uptake by these three compounds. Both transporters had to be deleted to stop the bacteria from growing in the presence of the catechol compounds. Concerning FERRI and ALBO, we observed growth for the four strains tested ∆pvdF∆pchA, ∆pvdF∆pchA∆fiuA, ∆pvdF∆pchA∆foxA and ∆pvdF∆pchA∆foxA∆fiuA (FoxA being involved in the uptake of the ferri-nocardamine complex, a tris-hydroxamate siderophore (32)), showing that although FiuA is able to transport iron into bacteria (Fig 3B ), at least one other unidentified OMT can perform the same function and it is not FoxA.

In conclusion, ENT, TCV, and TCVL6 are able to transport iron into P. aeruginosa cells via the OMTs PfeA and PirA and FERRI and ALBO by FiuA and another unidentified OMT.

The presence of siderophore-antibiotic conjugates induces the transcription and expression of their corresponding OMTs in P. aeruginosa cells infecting epithelial cells. It is well

documented that bacteria are able to sense the presence of exosiderophores in their environment, consequently leading to induction of the transcription and expression of their corresponding OMTs [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF][START_REF] Dean | PfeR, an enterobactin-responsive activator of ferric enterobactin receptor gene expression in Pseudomonas aeruginosa[END_REF][START_REF] Visca | Iron transport and regulation, cell signalling and genomics: lessons from Escherichia coli and Pseudomonas[END_REF][START_REF] Michel | PchR-box recognition by the AraC-type regulator PchR of Pseudomonas aeruginosa requires the siderophore pyochelin as an effector[END_REF][START_REF] Llamas | The heterologous siderophores ferrioxamine B and ferrichrome activate signaling pathways in Pseudomonas aeruginosa[END_REF][START_REF] Llamas | Cell-surface signaling in Pseudomonas: stress responses, iron transport, and pathogenicity[END_REF]. We have previously shown that the presence of either TCV or ENT in planktonic cultures of P. aeruginosa induces the transcription and expression of the OMT pfeA and the presence of FERRI, the OMT fiuA [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF][START_REF] Gasser | Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore-antibiotic conjugates development[END_REF]. Proteomic and RT-qPCR approaches have shown that P. aeruginosa is also able to sense the presence of exosiderophores and subsequently adapt its phenotype in even more complex systems, such as an epithelial infection assay [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF]. We used the same approach here to investigate whether P. aeruginosa PAO1 senses the presence of ALBO and TCVL6 in an epithelial cell infection assay.

We first used a proteomic approach to identify the bacterial proteins, especially the OMTs, showing differential expression in the presence of ALBO or TCVL6 (Fig 5) and then RT-qPCR to confirm the proteomic results and compare the levels of transcription of the different genes (Fig 6). Epithelial A549 cells were infected with P. aeruginosa PAO1 cells grown overnight in LB (conditions under which the iron-uptake pathways are poorly expressed ( 16)).

The bacteria/epithelial cell ratio used resulted in 40 to 50% viability of the cells 10 h after infection, as we previously described in Perraud et al., 2020 (16). Only bacteria associated with the epithelial cells were harvested for proteomic analysis, after 3 h of infection, when we started to observe apoptosis of the epithelial cells. The sample preparation and proteomic workflow used allowed the identification of 1,300 proteins from P. aeruginosa in the presence of a large number of proteins from the epithelial cells.

Proteomic and RT-qPCR data (Figs 5 and6) showed the presence of either TCVL6, TCV, or ENT to strongly induce the expression of PfeA (log2 fold change of 4.61 for proteomics and 4.71 for RT-qPCR for TCVL6). The expression of PfeE (gene number PA2689, esterase hydrolyzing ENT -Fig 1C) was also induced, with a log2 fold change of 2.60, 2.58 and 0.91 in the proteomic data in the presence of TCVL6, TCV, and ENT. There was also an effect on PirA transcription or expression, by ENT, TCV, and TCVL6 (Figs 3 and4) with a log2 fold change of 1.83 for TCVL6, 1.70 for TCV and 0.70 for ENT. All three molecules (ENT, TCV, and TCVL6) had a similar effect on the P. aeruginosa phenotype, proving that TCVL6 is able to enter the bacterial periplasm, like ENT and TCV, and bind to PfeS, the sensor of the two-component system involved in the regulation of the expression of the ENT-dependent iron-uptake pathway in P. aeruginosa cells [START_REF] Dean | PfeR, an enterobactin-responsive activator of ferric enterobactin receptor gene expression in Pseudomonas aeruginosa[END_REF][START_REF] Gasser | Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore-antibiotic conjugates development[END_REF], and showing that the linezolide component linked to TCV does not affect this detection mechanism.

The presence of ALBO or FERRI induced the expression of FiuA (log2 fold change of 5.68 for proteomics and 3.03 for RT-qPCR for ALBO) and no other OMT, suggesting that the other unidentified OMT that is involved in iron acquisition by ALBO (Figs 3 and4) must be either expressed constitutively at sufficiently high levels to allow uptake, regardless of the growth conditions, or its expression is induced only if FiuA cannot be expressed. In addition, in the case of ALBO, the antibiotic component linked to FERRI did not interfere with the molecular mechanisms involving the sigma/anti-sigma system FiuI/FiuR [START_REF] Llamas | The heterologous siderophores ferrioxamine B and ferrichrome activate signaling pathways in Pseudomonas aeruginosa[END_REF] and required for the detection of these ferri-hydroxamate compounds by P. aeruginosa.

The data also show that all five compounds strongly repress the expression of the various proteins of the PCH pathway, such as the OMT FptA (mean log2 fold change of -3.16 for the proteomic data) as well as the enzymes involved in PCH biosynthesis (mean log2 fold change of -2.92 for all enzymes) (Fig 5B). The expression of the proteins of the PVD pathway was more strongly repressed by ALBO than the other compounds tested, with FERRI and TCVL6 having almost no effect on the transcription and expression of these genes (Fig 5B). As most of the proteins in the Fur regulon appeared to also be under-expressed in the presence of ALBO relative to the other compounds tested (S1 Fig), this phenotype is certainly due to low-level iron contamination of ALBO that probably had an impact on the Fur regulon as a whole.

In conclusion, P. aeruginosa is able to detect the two siderophore-conjugates used in this study (ALBO and TCVL6) in its growth environment, even in a complex system such as an epithelial infection assay, and clear phenotypic adaption is observed. Both compounds were able to induce the transcription and expression of their corresponding OMT in P. aeruginosa cells: FiuA for ALBO and PfeA for TCVL6. The phenotypic adaptation of P. aeruginosa in this infection assay also involved repression of the transcription and expression of the proteins of the PCH and PVD pathways.

Siderophore-antibiotic conjugates also affect the expression of virulence factors.

Transcription of the genes of the PVD pathway is regulated by two sigma factors, FpvI and PvdS, the second also regulating the transcription of virulence factors such as exotoxin A (ToxA) [START_REF] Pollack | The role of exotoxin A in Pseudomonas disease and immunity[END_REF][START_REF] Chaudry | Pseudomonas aeruginosa exotoxin A[END_REF], a major virulence factor of P. aeruginosa and Piv (also called PrpL) endoprotease [START_REF] Thibodeaux | Pseudomonas aeruginosa protease IV: a corneal virulence factor of low immunogenicity[END_REF][START_REF] Oh | Post-secretional activation of Protease IV by quorum sensing in Pseudomonas aeruginosa[END_REF]. The alkaline protease AprA is regulated by OxyR and is thus dependent on redox stress, which can be induced by high iron concentrations [START_REF] Laarman | Pseudomonas aeruginosa alkaline protease blocks complement activation via the classical and lectin pathways[END_REF]. Thus, we also performed proteomic analysis of the expression of all virulence factors expressed by P. aeruginosa cells during the infection of A549 cells (Fig 7A). However, all secreted virulence factors could not be detected with the protocol used to harvest the P. aeruginosa cells after infection. Consequently, we used RT-qPCR to evaluate the infected samples for the transcription of the genes encoding virulence factors for which their transcription is regulated or not by PvdS or OxyR (Fig 7B ), as well as several other virulence factors, such as AmrZ, a transcriptional regulator that controls the expression of many genes involved in environmental adaptation [START_REF] Muriel | AmrZ is a major determinant of c-di-GMP levels in Pseudomonas fluorescens F113[END_REF], PhzA2, a flavin-containing monooxygenase involved in phenazine biosynthesis [START_REF] Mavrodi | Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1[END_REF][START_REF] Recinos | Redundant phenazine operons in Pseudomonas aeruginosa exhibit environment-dependent expression and differential roles in pathogenicity[END_REF], and, finally, secreted virulence factors such as ExoY, a protease injected into eukaryotic cells by the type III secretion system [START_REF] Duong | The AprX protein of Pseudomonas aeruginosa: a new substrate for the Apr type I secretion system[END_REF][START_REF] Wilderman | Characterization of an endoprotease (PrpL) encoded by a PvdS-regulated gene in Pseudomonas aeruginosa[END_REF][START_REF] Casilag | The LasB elastase of Pseudomonas aeruginosa acts in concert with alkaline protease AprA to prevent flagellinmediated immune recognition[END_REF][START_REF] Yahr | ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system[END_REF] and the LasB elastase [START_REF] Wolz | Pseudomonas aeruginosa LasB mutant constructed by insertional mutagenesis reveals elastolytic activity due to alkaline proteinase and the LasA fragment[END_REF].

Proteomic analyses showed repression of the expression of many virulence factors, mostly in the presence of ALBO, but also FERRI, ENT, and TCV, and, to a lower extent, TCVL6 (Fig 7). The lower effect of TCVL6 can be explained by the slightly lower solubility of this compound. Indeed, in some cases, a small amount of precipitate of TCVL6 could be observed.

In addition, RT-qPCR showed different transcriptional profiles of the tested virulence factors in the presence of ENT, TCVL6, and ALBO. The presence of ENT mostly repressed the transcription of aprA and toxA, as the expression of these virulence factors is regulated by PvdS. TCVL6 also repressed the transcription of aprA (more strongly than ENT) and toxA, as well as the transcription of piv and phzA2. Finally, ALBO repressed the transcription of aprA, exoY, piv, and toxA.

A tris-catechol conjugate more efficiently induces the transcription of its OMT than a hydroxamate conjugate in P. aeruginosa cells infecting epithelial cells. We have previously

shown that P. aeruginosa cells infecting epithelial A549 cells increase the transcription and expression of catechol siderophore-dependent iron-uptake pathways, and not those of hydroxamate siderophores, in the presence of a mixture of four different siderophores [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF].

Here, the infection assay was repeated with a mixture of either TCV and FERRI or TCVL6 and In conclusion, the data show that conjugates with a catechol-type siderophore moiety have more chances to induce their corresponding OMT and consequently their corresponding uptake pathway than hydroxamate vectors.

Long-term effect of TCVL6 compounds on P. aeruginosa cells. A recurrent question in the field

of siderophore-antibiotic conjugates is how long bacteria can be treated with such compounds before resistance appears. This issue is complex because resistance can appear at different levels. Here, we investigated how long P. aeruginosa retains a phenotype that allows the uptake of iron via TCV without switching to the expression of another phenotype. This assay could not be carried out for TCVL6 and ALBO because of problems of availability of these compounds in larger amounts. TCV is a vector that can chelate iron (Fig 2 ) and transport it into P. aeruginosa cells [START_REF] Gasser | Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore-antibiotic conjugates development[END_REF].

We followed the expression of the ENT-dependent uptake pathway in P. aeruginosa cells induced by the presence of 10 µM ENT or TCV for 11 days of culture using a strain that expresses a fluorescently tagged PfeE (mcherrypfeE [START_REF] Perraud | A key role for the periplasmic PfeE esterase in iron acquisition via the siderophore enterobactin in Pseudomonas aeruginosa[END_REF]). The mcherrypfeE strain expresses mCherry tagged PfeE, with the fluorescent tag inserted in the chromosome at the C-terminus of the protein to maintain expression levels close to those of the wildtype strain [START_REF] Perraud | A key role for the periplasmic PfeE esterase in iron acquisition via the siderophore enterobactin in Pseudomonas aeruginosa[END_REF]. Each day, mcherrypfeE cells were harvested and resuspended in new CAA medium (iron restricted medium) in the presence of either ENT or TCV. Bacterial growth was followed in parallel with the emission of mCherry fluorescence (emission at 610 nm with excitation at 570 nm).

Fluorescence due to mCherry increased in the presence of both ENT and TCV, with exactly the same intensity for both compounds during the 11 days of culture (Fig 9), indicating that PfeE, and consequently PfeA, proteins of the ENT-dependent iron uptake pathway, are expressed at same level in the presence of ENT and TCV. During the 11 days, the phenotype of the P. aeruginosa cells, in terms of the expression of the ENT-dependent iron uptake pathway, remained the same.

Discussion

Introducing antibiotics into Gram negative bacteria, such as P. aeruginosa, via a Trojanhorse strategy using siderophores as vectors is a promising approach. However there are still several bottlenecks in its development. The high antibiotic activity of natural sideromycins clearly shows that the strategy works and the fact that cefiderocol has been approved by the FDA for use in the treatment of urinary infections is a sign that antibiotic vectorization by siderophores should be explored further by pharmaceutical companies. However, developing such compounds requires extensive knowledge of the molecular mechanisms and regulatory network involved in bacterial siderophore-dependent iron acquisition. Most bacteria carry genes encoding several iron uptake pathways in their genome, allowing very high phenotypic plasticity, depending on the presence or not of exosiderophores or sideromycins. Such phenotypic plasticity of P. aeruginosa has been investigated in various growth media and in the presence of several exosiderophores [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF]. The data show that if siderophores produced by other bacteria (exosiderophores) are present, bacteria induce the expression of the uptake pathway corresponding to the exosiderophore(s) (corresponding OMT and enzymes necessary to release the iron from the siderophore) and, in parallel, repress those corresponding to the endogenous siderophores [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF].

Here, we show that this also occurs in the presence of the two siderophore-antibiotic conjugates ALBO and TCVL6 in a complex system (an epithelial cell infection assay) with rapid kinetics (3-h infection with the bacteria having been previously grown in iron-rich media). Both compounds have no antibiotic activities on P. aeruginosa on the concentrations used. The bacteria sense the presence of both conjugates in their environment and adapt their phenotypes to access iron via the sideromycins that are present. This occurs via auto-loop regulating systems: the two-component system PfeS/PfeR for TCVL6 and the sigma and anti-sigma factors FiuI/FiuR for ALBO. Comparison of the transcription induction levels between FERRI and ALBO and between TCV and TCVL6 for their corresponding OMTs shows that the presence of the antibiotic group on each sideromycin had no inhibitory effect on the regulatory mechanism: the presence of the conjugates was sensed by P. aeruginosa with the same efficiency as the vector or corresponding siderophore alone.

Such induction of FiuA and PfeA expression occurred in parallel with a decrease in the transcription and expression of (i) the proteins involved in PVD and PCH biosynthesis and ferri-PVD and ferri-PCH uptake and (ii) several virulence factors, especially those regulated by PvdS, the sigma factor that regulates the transcription of the genes involved in PVD production.

When the bacteria are transferred from LB medium to the infection assay (A459 cells in RIPM medium) in the presence of ALBO and TCVL6, the bacteria prefer to induce the expression of FERRI and ENT uptake pathways and much less those of their own siderophores PVD and PCH.

Moreover, the phenotypic effect induced by the presence of the siderophore-conjugates was not only limited to the expression or not of the iron uptake pathways but also that of virulence factors, as their transcription and expression are tightly interconnected. The ability of siderophore-conjugates to repress the transcription and expression of virulence factors is clearly an asset that has to also be considered in such a Trojan-horse strategy using siderophores as vectors.

When the infection assay was carried out in the simultaneous presence of both sideromycins, ALBO and TCVL6 (present in equivalent amounts), the catechol-type vector was clearly more efficient in inducing the expression of its corresponding transporters, PfeA, than that for ferrichrome, because of its strong affinity for iron. Catechol-type compounds are clearly more efficient in competing for iron with PVD than hydroxamate molecules (Fig 2 ), consistent with the affinities of such families of siderophores for iron: Ka of 10 49 M -1 for ENT [START_REF] Loomis | Solution equilibria of enterobactin complexes[END_REF], 10 29 M -1 for FERRI [START_REF] Anderegg | Hydroxamatkomplexe III. Eisen(III)-Austausch zwischen Sideraminen und Komplexonen. Diskussion der Bildungskonstanten der Hydroxamatkomplexe[END_REF], and 10 30.8 M -1 for PVD [START_REF] Blanc | Bacterial iron transport: coordination properties of pyoverdin PaA, a peptidic siderophore of Pseudomonas aeruginosa[END_REF]. Because of their higher affinity for iron, tris-catecholate vectors scavenge iron more efficiently in the bacterial environment than hydroxamate compounds (Fig. 2) and consequently have a higher chance to induce the transcription of their corresponding uptake pathways in the bacteria via the auto-loop regulating systems.

In conclusion, the ability of siderophore-conjugates to be detected by the pathogen in its growth environment, even in a complex system, such as an epithelial infection assay, is a great advantage in Trojan-horse strategies, because it ensures the expression of the needed uptake pathway and suggests that such induction should occur as well in the host during an infection. Moreover, the data show that conjugates with a catechol-type siderophore moiety have a greater chance to induce their corresponding OMT, and consequently their corresponding uptake pathway, than hydroxamate vectors and are potentially more efficient Trojan-horse vectors.

Materials and Methods

Chemicals. Enterobactin (ENT) and ferrichrome (FERRI) were purchase from Sigma-Aldrich. Albomycin δ2 (ALBO) was purchased from EMC Microcollection. Pyoverdine (PVD) was purified from P. aeruginosa PAO1 culture supernatants as previously described [START_REF] Demange | Bacterial siderophores: structure and NMR assigment of pyoverdins PaA, siderophores of Pseudomonas aeruginosa ATCC 15692[END_REF]. TCV was synthesized according to previously published protocol [START_REF] Baco | Diphenyl-benzo[1,3]dioxole-4-carboxylic acid pentafluorophenyl ester: a convenient catechol precursor in the synthesis of siderophore vectors suitable for antibiotic Trojan horse strategies[END_REF] and TCVL6 was synthesized as described in Supporting information. The protonophore CCCP (carbonyl cyanide mchlorophenylhydrazone) was purchased from Sigma-Aldrich. 55 FeCl3 was obtained from Perkin Elmer Life and Analytical Sciences (Billerica, MA, USA), at a concentration of 71.1 mM, with a specific activity of 10.18 Ci/g. RPMI was purchased from Thermo-Fisher.

Bacterial strains, plasmids and growth conditions. The P. aeruginosa strains used in this study are all listed in 1SM Table in Supporting information. P. aeruginosa strains were grown at 30°C in LB broth. When P. aeruginosa strains were grown in iron-restricted conditions, CAA medium was used (casamino acid medium, composition: 5 g L -1 low-iron CAA (Difco), 1.46 g L -1 K2HPO4 3H2O, 0.25 g L -1 MgSO4 7H2O).

Iron scavenging from PVD-Fe. Compounds were prepared in solution at 10 mM in DMSO (TCV and TCVL6) or water (FERRI, ALBO and PVD). The competition assay with PVD-Fe was carried out as described previously [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF]. PVD-Fe is prepared in solution at 10 µM in 100 µL of 100 mM HEPES buffer pH 7.4, and incubated at 25 °C in the presence of increasing concentrations of the tested molecules for 48 h. The fluorescence at 447 nm corresponding to formed apo PVD was monitored (excitation at 400 nm) in a TECAN Infinite M200 plate reader. For the kinetic of iron scavenging from PVD-Fe, PVD-Fe at 10 µM in 100 µL of HEPES buffer was incubated at 25 °C in the presence of 100 µM FERRI, ALBO, ENT or TCVL6. The fluorescence was monitored in function of time during 1 h in a TECAN Infinite M200 plate reader.

Growth assays in iron-restricted conditions. P. aeruginosa strains were first grown overnight at 30°C in LB broth and were then washed, resuspended and cultured overnight at 30°C in CAA medium. Afterwards, bacteria were washed, resuspended in CAA medium at 0.02 OD at 600 nm and distributed in the wells of a 96-well plate (Greiner, U-bottomed microplate) in the absence or presence of 10 µM of FERRI, ALBO, ENT, TCV or TCVL6. The plate was incubated at 30 °C, with shaking, in a Tecan microplate reader (Infinite M200, Tecan) and bacterial growth was monitored at OD600 nm. We calculated the mean of three replicates for each measurement.

Iron uptake. ALBO- 55 Fe complexes were prepared at 55 Fe concentrations of 50 µM, with a ALBO:iron (mol:mol) ratio of 20:1. P. aeruginosa strains were first grown overnight at 30°C in LB broth and were then washed, resuspended and cultured overnight at 30°C in CAA medium. Afterwards, bacteria were washed and again grown in CAA medium in the presence of 10 µM of ALBO in order to induce the expression of the corresponding iron uptake pathways. The bacteria were then used for 55 Fe uptake kinetics as described previously [START_REF] Gasser | Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore-antibiotic conjugates development[END_REF].

The experiments were repeated with cells pretreated with 200 µM CCCP. This compound inhibits the protonmotive force across the bacterial cell membrane, thereby inhibiting TonBdependent iron uptake [START_REF] Clément | The binding mechanism of pyoverdin with the outer membrane receptor FpvA in Pseudomonas aeruginosa is dependent on its iron-loaded status[END_REF].

Infection assay. A549 (ATCC ® CCL-185 ™ ) human pulmonary epithelial cells were routinely cultivated at 37 °C, 5 % CO2 in RPMI 1640 medium (Gibco) supplemented with 10 % vol/vol FBS (Gibco) and passaged every 3 to 4 days as described previously [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF]. A549 cell viability was estimated using the Real Time-Glo™ MT cell viability assay kit (Promega™) as we described previously for equivalent infection conditions using P. aeruginosa [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF]. A549 cells infections were also carried out as we described previously [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF]. The epithelial cells were seeded at a density of 1 x 10 6 cell per plate in 10 cm tissue culture dishes (Corning), one day before infection with P. aeruginosa PAO1 cells. P. aeruginosa was grown overnight in LB medium at 30°C under shaking, before being diluted to an OD600 nm of 0.1 and incubated for 2 h 30, until the OD600 nm of the suspension reached 0.4 to 0.6 indicating that bacteria are in exponential phase of growth. A549 cells were infected with a volume of this P. aeruginosa suspension equivalent to 50 x 10 6 CFU (multiplicity of infection of 50). FERRI, ALBO, ENT, TCV or TCVL6 were added to the A549 cells at the same moment as P. aeruginosa cells.

The infection was allowed to carry on for 3 h at 37°C, 5 % CO2. Afterwards, the plates were washed with cold PBS 1X buffer and the A549 cells and bacteria still adhering to the bottom of the plate harvested with a cell scraper. A549 cell and bacteria were then harvested by centrifugation and the dry pellet was re-suspended in one volume of PBS 1X buffer and stored at -80°C for proteomic analyses. For RT-qPCR analyses two volumes of RNAprotect Bacteria Reagent (Qiagen) were added before centrifugation and storage at -80°C overnight.

Quantitative real-time PCR on bacteria infecting epithelial cells. A549 cells were infected as described above. These analyses were carried out as previously described by our team [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF]. Samples were prepared as described above. On the day following sample preparation, lysis of the sample was carried out using Tris-EDTA pH 8 buffer containing 15 mg.mL -1 lyzozyme, the lysates were homogenized using the QIAshredder kit (Qiagen) and RNA was extracted using RNeasy Mini kit (Qiagen). Genomic DNA digestion was performed with DNase (RNase-Free DNase Set, Qiagen) and purified with a RNeasy Mini Elute cleanup kit (Qiagen). In order to assess for homogeneity of the amount of A549 cell RNA present in each sample, a quantification of GAPDH was used as a control. 1 µg of total RNA was then reversetranscribed with a High-Capacity RNA-to-cDNA Kit, in accordance with the manufacturer's instructions (Applied Biosystems). The amounts of specific cDNAs were assessed in a StepOne Plus instrument (Applied Biosystems) with Power Sybr Green PCR Master Mix (Applied Biosystems) and the appropriate primers (Table 2SM), with the uvrD mRNA used as an internal control. The transcript levels for a given gene in a given strain were normalized with respect to those for uvrD and are expressed as a base two logarithm of the ratio (fold-change) relative to the reference conditions.

Label-free proteomic analysis on bacteria infecting epithelial cells. A549 cells were infected as described above. The pellets of A549 cells and bacteria (five independent biological replicates) in PBS were diluted in Laemmli buffer before undergoing lysis as previously described [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF]. Proteins in lysate were cast into a 7.5 % acrylamide tube-gel as described [START_REF] Muller | Benchmarking sample preparation/digestion protocols reveals tube-gel being a fast and repeatable method for quantitative proteomics[END_REF] and digested with a 1:25 enzyme /protein ratio for 14h at 37°C. The resulting peptides were submitted to nanoLC-MS/MS analyses (750 ng injected) on a nanoACQUITY Ultra-Performance-LC systems hyphenated to a Q-Exactive Plus mass spectrometer as previously described [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF].

The raw data obtained were converted into ".mfg" files with MSConvert software (ProteomeWizard, version 3.0.6090). Peaks were assigned with Mascot (Matrix Science, version 2.6.2) against an in-house database containing human entries from SwissProt database (17 March 2017, 20194 entries) and P. aeruginosa PAO1 entries from UniProtKB database (17 March 2017, 5677 entries). Common contaminant proteins such as human keratins and trypsin were added to the database and concatenated with the reverse copies of all sequences. Trypsin was selected as enzyme, one missed cleavage was allowed. Database searches parameters in Mascot search engine and generated PSM validation were performed as previously described [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF]. For quantification purpose, the ".raw" files were converted into ".mzDB" files with MS Angel software (version 1.6.2). XIC quantification was performed in Proline software (53) using 5 ppm as m/z tolerance for the peptides abundance extraction.

Loess smoothing was performed for the LC-MS runs alignments. Cross assignments of peptide ions were performed using 5 ppm as m/z tolerance and 60 s as retention time tolerance. Only proteins identified with at least one unique peptide were considered and only specific peptides were kept for the sum of protein abundances. The contaminants were excluded from the protein list prior to statistical analysis.

Experimental design and Statistical Rationale: Statistical analysis was performed using the ProStaR software (version 1.16.6) [START_REF] Wieczorek | DAPAR & ProStaR: software to perform statistical analyses in quantitative discovery proteomics[END_REF]. Proteins identified in at least 4 replicates per condition were considered, normalization was performed using median quantile centering mode for all conditions. Imputation was performed at the protein level SLSA mode for partial observed values per condition and DetQuantile (2.5 %, factor 1) mode for values missing in entire condition. Welch's T-test was used to identify statistically differentially expressed proteins between two conditions. For each comparison, the p-value calibration was performed using the Benjamini-Hochberg procedure. Proteins with p-value below 0.001 were considered as differentially expressed, corresponding to a FDR between 1-5 %. Following values were obtained: 2.35% FDR for ENT, 2.26 % FDR for TCV, 7.33 % FDR for TCVL6, 0.72 % FDR for ALBO and 4.24% for FERRI.

The MS data were deposited to the ProteomeXchange Consortium via the PRIDE partner repository [START_REF] Vizcaíno | ProteomeXchange provides globally coordinated proteomics data submission and dissemination[END_REF] with the PXD020378. Reviewer account details: Usernam: reviewer98509@ebi.ac.uk Password: . 3lVkMvDs.

Growth and quantification of mCHERRY fluorescence intensity. P. aeruginosa PAO1 strain were first grown overnight at 30°C in LB broth and were then washed, resuspended and cultured overnight at 30°C in iron-deficient CAA medium. Afterwards, bacteria were washed and again grown in CAA medium in 96-well plate (Greiner, U-bottomed microplate) in the presence or absence of 10 µM of ENT or TCV. The plate was incubated at 30 °C, with shaking, in a Tecan microplate reader (Infinite M200, Tecan) for measurements of OD600 nm and mCherry (excitation/emission wavelengths: 570 nm/610 nm) fluorescence, at 30 min intervals, for 11 days. Every 24 h, the bacteria were centrifuged and resuspended in new CAA media with or without 10 µM ENT or TCV. We calculated the mean of three replicates for each measurement. Ten micromoles of PVD-Fe in 100 mM HEPES pH 7.4 was incubated with increasing concentrations of FERRI, ALBO, TCV, or TCVL6 for 48 h (until equilibrium was reached), as described previously [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF]. Metal-free PVD is fluorescent, with absorbance typically at 447 nm, and PVD-Fe is not [START_REF] Blanc | Bacterial iron transport: coordination properties of pyoverdin PaA, a peptidic siderophore of Pseudomonas aeruginosa[END_REF][START_REF] Folschweiller | The interaction between pyoverdin and its outer membrane receptor in Pseudomonas aeruginosa leads to different conformers: a time-resolved fluorescence study[END_REF]. Apo PVD formation was thus followed by monitoring its fluorescent at 447 nm (excitation at 400 nm). The data were normalized using the formula (FMEASURED -FPVD-Fe)/(FPVD -FPVD-Fe), FMEASURED being the fluorescence measured for each experimental condition, FPVD-Fe the fluorescence of 10 µM PVD-Fe, and FPVD the fluorescence of 10 µM PVD.

B. Kinetics of PVD-Fe dissociation in the presence of FERRI, ALBO, TCV, or TCVL6.

Ten micromoles of PVD-Fe in 100 mM HEPES buffer pH 7.4 was incubated with 100 µM FERRI, ALBO, TCV, or TCVL6, as described in Materials and Methods. The kinetics of apo PVD formation were followed by monitoring the fluorescence emission at 447 nm (excitation at 400 nm), as previously described [START_REF] Perraud | Phenotypic adaption of Pseudomonas aeruginosa by hacking siderophores produced by other microorganisms[END_REF]. The data were normalized relative to the reference gene uvrD and are representative of three independent experiments performed in triplicate (n = 3). Results are given as the ratio between the values obtained in the presence of siderophores or conjugates over those obtained in their absence. amrZ encodes a transcriptional regulator controlling many genes involved in environmental adaptation [START_REF] Muriel | AmrZ is a major determinant of c-di-GMP levels in Pseudomonas fluorescens F113[END_REF], aprA, an alkaline protease [START_REF] Laarman | Pseudomonas aeruginosa alkaline protease blocks complement activation via the classical and lectin pathways[END_REF], exoY [START_REF] Yahr | ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system[END_REF], lasB, an elastase [START_REF] Wolz | Pseudomonas aeruginosa LasB mutant constructed by insertional mutagenesis reveals elastolytic activity due to alkaline proteinase and the LasA fragment[END_REF], phzA2, a flavin-containing monooxygenase involved in phenazine biosynthesis [START_REF] Mavrodi | Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1[END_REF][START_REF] Recinos | Redundant phenazine operons in Pseudomonas aeruginosa exhibit environment-dependent expression and differential roles in pathogenicity[END_REF], piv, a lysyl-endopeptidase (also called PrpL) [START_REF] Thibodeaux | Pseudomonas aeruginosa protease IV: a corneal virulence factor of low immunogenicity[END_REF][START_REF] Oh | Post-secretional activation of Protease IV by quorum sensing in Pseudomonas aeruginosa[END_REF] and toxA, an exotoxin [START_REF] Pollack | The role of exotoxin A in Pseudomonas disease and immunity[END_REF][START_REF] Chaudry | Pseudomonas aeruginosa exotoxin A[END_REF]. encodes the OMT of ferri-citrate, feoA, the ferrous transporter, and fiuA, the OMT of FERRI, foxA, that of ferrioxamine B, fptA, that of PCH, fpvA, that of PVD, hasR and phuR, that of heme, pfeA and pirA, that of ENT, and piuA, that of an unknown siderophore. (kinetics in orange) (10 µM) was added and a control experiment with no chelator was carried out in parallel (kinetics in black). OD600 nm measurements were used to assess growth over time. The fluorescence of mCherry was measured at 610 nm (excitation at 570 nm). The measurements were performed at 30-min intervals in a TECAN microplate reader at 30°C, with shaking, for 11 days. Every 24 h, the plate was centrifuged, the supernatant removed, and new CAA media, with or without TCV or ENT, added and the plate returned to the microplate reader at 30°C with shaking. Each curve corresponds to the mean of three replicates. quality, Air products). Solvents used were of analytical grade purity (>99.9%). When necessary, solvents and bases were purchased extra-dry. All other chemicals were obtained from commercial suppliers and were used as received, unless otherwise stated. All reactions were monitored by thin-layer chromatography (TLC) using Merck precoated silica gel 60F 254 (0.25 mm). TLC are visualized using UV (254 nm/365 nm, Vilber Lourmat, VL-4LC) and/or using classical revelation mixtures (sulfuric vaniline, potassium permanganate, ninhydrin reagent).

Final conjugate 3 was also detected on TLC using a 2% hydromethanolic solution of FeCl3 (2%).

Protocol

Linezolid azide 2 (1 eq.) and alkyne-vector 1 (1,2 eq.) were solubilized in THF (final concentration 0.1 M). An aqueous solution of CuSO4 (200 mg/mL, 1 eq.) and sodium ascorbate (5 eq.) were successively added (9). The mixture was sonicated and further stirred at 20°C

  Fig 2). PVD-Fe (non-fluorescent) at 10 µM was incubated in the presence of increasing concentrations of apo FERRI, ALBO, TCV, and TCVL6 (Fig 2A). The complete removal of iron from PVD was only observed for the catechol-type compounds (TCV and TCVL6) at concentrations of approximately 20 µM (twofold excess compared to PVD-Fe; Fig 2A). Neither FERRI nor ALBO were able to completely remove iron from PVD at the concentrations tested, (70 -80% of the PVD was still in its ferric form, Fig 2A), indicating that these compounds compete less efficiently for the scavenging of iron than catechol-type siderophores or conjugates. We also monitored the kinetics of PVD-Fe dissociation by incubating 10 µM PVD-Fe with 100 µM FERRI, ALBO, TCV, or TCVL6 (Fig 2B)

  ALBO and analyzed by. The presence of a mixture of FERRI and TCV in the infection assay strongly induced the transcription of pfeA (ENT OMT), with a log2 fold change of 5; fiuA (FERRI OMT) transcription being almost not induced (Fig8A). In addition, we observed repression in the range of a 0.95 -1.8 log2 fold change for fptA, fpvA, and hasR expression (the OMTs of PCH, PVD, and the HasA hemophore, respectively). In the presence of a mixture of ALBO and TCVL6, only pfeA transcription was strongly induced (log2 fold change of 5.2), that of fiuA almost not at all, and that of fptA, fpvA, and hasR repressed, with a of 0.5 -0.9 log2 fold change. As the various genes involved in iron acquisition by a given siderophore are always in an operon, a change in the transcription and expression of the gene encoding an OMT implies a similar change for the transcription and expression of the other genes present in the operon.

Fig 1 .

 1 Fig 1. A. Structure of ferrichrome (FERRI), albomycin (ALBO), enterobactin (ENT), triscatechol vector (TCV), and tris-catechol-linezolid vector (TCVL6). B and C. ENT-and FERRIdependent iron-uptake pathways in P. aeruginosa, respectively. FERRI and ENT loaded with iron are recognized at the bacterial surface by the OMTs FiuA and PfeA, respectively, and transported across the outer membrane. Iron release from ENT occurs in the bacterial periplasm and involves hydrolysis of the siderophore by the esterase PfeE and an ironreduction step by a non-identified reductase. ENT-Fe does not enter the bacterial cytoplasm in P. aeruginosa cells. Concerning FERRI-Fe, once in the bacterial periplasm, the complex is transported further across the inner membrane by the protonmotive-dependent permease FiuB and iron is released from FERRI in the bacterial cytoplasm by a mechanism involving acetylation of the siderophore by FiuC and iron reduction by an unknown reductase (14).

Fig 2 .

 2 Fig 2. A. Ability of FERRI, ALBO, TCV, and TCVL6 to scavenge iron from PVD-Fe complexes.

Fig 4 .

 4 Fig 4. Bacterial growth in the presence of FERRI, ALBO, ENT, TCV, or TCVL6. Various strains (∆pvdF∆pchA and its corresponding pfeA, pirA, fiuA and foxA deletion mutants) were grown in iron-restricted CAA medium, with or without 10 µM FERRI, ALBO, ENT, TCV, or TCVL6, at 37°C. The growth curves are the mean of three independent biological triplicates.

Fig 5 .

 5 Fig 5. Analysis of changes in the expression of proteins involved in iron-uptake pathways in P. aeruginosa cells during A549 epithelial cell infection, in the presence or absence of ALBO, TCVL6, FERRI, ENT or TCV. A-B. Proteomic analyses were performed on P. aeruginosa PAO1

Fig 8 .

 8 Fig 8. Analysis of the changes in the transcription of genes encoding OMTs involved in ironuptake pathways in P. aeruginosa cells during A549 epithelial cell infection, in the presence or absence of a mixture of either FERRI and TCV or ALBO and TCVL6. RT-qPCR analyses were performed on P. aeruginosa PAO1 cells after 3 h of incubation with A549 epithelial cells in RPMI medium, with or without a mixture of two compounds: either FERRI and TCV or ALBO and TCVL6 (each of the compounds being present at a concentration of 10 µM). The data were normalized relative to the reference gene uvrD and are representative of three independent experiments, each performed in triplicate (n = 3). Results are given as the ratio between the values obtained in the presence of the conjugates over those obtained in their absence. fecA

Fig 9 .

 9 Fig 9. Monitoring the ENT-Fe uptake pathway via PfeEmCherry fluorescence during bacterial growth in the presence or absence of ENT or TCV. Fresh CAA medium was inoculated with pfeEmcherry (cells expressing fluorescent tagged PfeE) grown in the same medium and the resulting culture dispensed into the wells of a 96-well plate. ENT (kinetics in pink) or TCV

  Before chromatographic purification reaction mixtures were adsorbed on silica gel (60-200 µm, VWR Chemicals). Chromatographic purifications were performed on a Reveleris (Grace Davison Discovery Sciences) purification device using PuriFlash® pre-packed silica gel columns (Interchim, Montluçon, France). NMR spectra were recorded on Bruker Avance 400 ( 1 H: 400 MHz,13 C: 100 MHz,19 F : 376 MHz) or Avance 500 ( 1 H: 500 MHz,13 C: 125 MHz,19 F : 470 MHz), using the residual non-deuterated solvent as reference. The chemical shifts (δ) and coupling constants (J) are expressed in ppm and hertz respectively. Multiplicity is indicated as follow: s for singlet, d for doublet, t for triplet, q for quadruplet, quint for quintuplet and m for a multiplet. The letter "b" before the multiplicity indicate a broaden signal. Mass spectra were recorded in the Service Commun d'Analyses (SCA) de la Faculté de Pharmacie de l'Université de Strasbourg and were measured in ES-TOF experiments on a Bruker Daltonic MicroTOF mass spectrometer. LC/HRMS were measured after calibration on an Agilent QToF.

Acknowledgments

This work was partially funded by the Centre National de la Recherche Scientifique and grants from the associations Vaincre la Mucoviscidose and Association Gregory Lemarchal. In addition, these results were generated as part of the work of the Translocation Consortium (www.translocation.com), supported by the Innovative Medicines Joint Undertaking under Grant Agreement no. 115525, through financial contributions from the European Union's Seventh Framework Program (FP7/2007-2013) and contributions in kind from EFPIA companies. The equipment at the IPHC was partly funded by the French Proteomics Infrastructure (ProFI; ANR-10-INSB-08-03).

Author contributions

QP and PC performed all proteomic experiments QP, PC and LS analyzed the proteomic data QP also performed all the qRT-PCR, growth and 55 Fe uptake assays and wrote the paper MM, FH and NZ performed the synthesis of TCV and TCVL6 VG performed the molecular biology and the experiments with PfeE mCherry GLAM designed the syntheses of TCV and TCVL6 LS and IJS, secured funding, directed project, analyzed data and wrote paper B. 55 Fe uptake in P. aeruginosa strains using ALBO as a siderophore. ∆pvdF∆pchA and ∆pvdF∆pchA∆fiuA cells grown in iron-restricted CAA medium, in the presence of 10 µM ALBO to induce any needed OMT, were incubated with 500 nM ALBO- 55 Fe and the kinetics of 55 Fe uptake measured as previously described [START_REF] Gasser | Catechol siderophores repress the pyochelin pathway and activate the enterobactin pathway in Pseudomonas aeruginosa: an opportunity for siderophore-antibiotic conjugates development[END_REF]. As a control, the experiment was repeated in the presence of 200 µM CCCP protonophore for ∆pvdF∆pchA cells. Errors bars were calculated from three independent biological replicates. cells after a 3-h infection of A549 epithelial cells in RPMI medium, in the presence or absence of 10 µM ALBO or TCVL6. Median values measured in the infection assay supplemented with either 10 µM ALBO or TCVL6 were plotted against those measured in the infection assay in the absence of supplementation with siderophore-antibiotic compounds. The median values represent the median of the relative intensity of each protein normalized against all proteins detected by shotgun analysis (n = 5).

C-E. Heat maps of various proteins involved in the PVD (E) and PCH (D) pathways and of OMTs (C): the darker the shade of green, the higher the induced expression of the protein. Only the proteins for which a change in the level of expression was observed are shown. The nd note indicates that the protein hasn't been identified, ns indicates that the p-value is higher than 0.05; the *, ** and *** symbols indicates a p-value lower than 0.05, 0.01 and 0.001 respectively. All siderophore or siderophore-antibiotic compounds were added at 10 µm. values obtained in the presence of the siderophores or conjugates over those obtained in their absence. fecA encodes the OMT of ferri-citrate, feoA, the ferrous transporter, and fiuA, the OMT of FERRI, foxA, that of ferrioxamine B, fptA, that of PCH, fpvA, that of PVD, hasR and phuR, that of heme, pfeA and pirA, that of ENT and piuA, that of an unknown siderophore.

Supporting information

S2 Table. Primers used for RT-qPCR analysis

Synthesis of TCVL6.

General informations

TCVL6 was synthesized as described in 1S Scheme. Linezolid was purchased from Sigma-Aldrich. Protected TCV 1 and linezolid-azide derivative 2 were synthesized according previously described protocols (7,8). All reactions were carried out under argon (technical under argon till the total consumption of limiting reagent (linezolid azide). The mixture was then filtered through a celite pad and the filtrate was adsorbed on silical gel. The crude mixture was then purified on silica gel using a gradient of EtOH in CH2Cl2. The resulting white solid was dissolved in the mixture CH2Cl2/TFA/Triisopropylsilane/EtOH (70/20/5/5, 5 mL for 0.1 mmol of starting linezolid azide 2). The solution was stirred at 20°C till the total consumption of the starting material. Solvents were then evaporated from the mixture under reduced pressure. The oily residue was dissolved in a minimum volume of THF then cyclohexane was added dropwise till the precipitation of the expected conjugate as a thin powder. The pure conjugate was filtered off on a Hirsch funnel. Spectroscopic analysis of TCVL6.