Duchesse Lacour

Qiang Li

Philippe Ranocha

Yves Martinez

Christophe Dunand
email: dunand@lrsv.ups-tlse.fr

Mbadinga Mbadinga

Global analysis of non-animal peroxidases provides insights into the evolutionary basis of this gene family in green lineage

Keywords: Class I peroxidase, Class III peroxidase, non-animal peroxidase, ancestral genes, Spirogyra sp

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The increase of oxygen level, 2.3 billion years ago at the beginning of the Proterozoic, forced the ancestral organisms to adapt to oxidative stress [START_REF] Becker | Oxidative stress in malaria parasite-infected erythrocytes: host-parasite interactions[END_REF]. The reactive oxygen species (ROS) produced are necessary to cells, but toxic, at high levels, and are consequently managed by an ROS gene network [START_REF] Mittler | Reactive oxygen gene network of plants[END_REF]. Peroxidases are components of this network that regulate ROS homeostasis through oxidation-reduction reactions using hydrogen peroxide (H 2 O 2 ) as an electron acceptor along with different oxidized substrates as electron donors. The term "peroxidase" encompasses two major protein families that differ based on in the presence or absence of a haem, namely "haem peroxidase" and "non-haem peroxidase". The haem peroxidases are the most diverse group in terms of taxonomic distribution, and the non-animal peroxidase family belongs to this group (Passardi et al., 2007a). This family was first described by Welinder in 1992 based on structural homology [START_REF] Welinder | Plant peroxidases: structure-function relationships[END_REF] and comprises three peroxidase classes: Class I (CI Prx), Class II (CII Prx) and Class III (CIII Prx) (Fig. 1A). This large superfamily is regrouped under a unique PFAM (PF00141), which described the conserved peroxidase domain.

However, this grouping does not discriminate between the members of these three classes and can produce mis-annotation which needs to be corrected by manual annotation.

The CI Prxs are widely distributed in prokaryotes and eukaryotes and were originally described as intracellular peroxidases (lacking signal peptide). They are composed of an unglycosylated haem, without calcium ions neither disulphide bridges. This class is the most widespread of the peroxidases and is subdivided into four subclasses: Catalase Peroxidases (CPs), Cytochrome c Peroxidases (CcPs), Ascorbate Peroxidases (APxs) and Hybrid Ascorbate Peroxidase-Cytochrome c Peroxidases (APx-CcPs). CPs are predominantly found in bacteria and only occasionally in other organisms due to horizontal gene transfer (Passardi et al., 2007b). All CPs are characterized by the presence of two independent peroxidase domains. CcPs are present in organisms with mitochondria but are absent from plants. CcPs catalyse the oxidation of ferricytochrome c in the presence of H 2 O 2 [START_REF] Finzel | Crystal structure of yeast cytochrome c peroxidase refined at 1.7-A resolution[END_REF] in order to remove toxic oxygen generated during the mitochondrial respiratory process [START_REF] Kwon | Oxidative stresses elevate the expression of cytochrome c peroxidase in Saccharomyces cerevisiae[END_REF]. APx are specific to chloroplast-containing organisms with the notable exception of cyanobacteria [START_REF] Miyake | Scavenging of hydrogen peroxide in prokaryotic and eukaryotic algae: acquisition of ascorbate peroxidase during the evolution of cyanobacteria[END_REF]. They catalyse the oxidation of ascorbate in the presence of H 2 O 2 for photoprotection and regulation of photooxidative stress [START_REF] Maruta | Arabidopsis Chloroplastic Ascorbate Peroxidase Isoenzymes Play a Dual Role in Photoprotection and Gene Regulation under Photooxidative Stress[END_REF]. The last CI Prx subclass corresponds to proteins with common characteristics to both APx and CcP, and has been named hybrid Ascorbate Peroxidase-Cytochrome c Peroxidase (APx-CcP). A predominant example of this class is found in Leishmana major for which both APx and CcP activities have been detected [START_REF] Adak | Leishmania major encodes an unusual peroxidase that is a close homologue of plant ascorbate peroxidase: a novel role of the transmembrane domain[END_REF].

The CII Prxs are described as secreted fungal lignin peroxidases. They belong to large multigenic families and can oxidize molecules with high redox potential such as lignin. CII Prxs include lignin peroxidases (LiP), manganese peroxidases (MnP), and versatile peroxidases (VPs), so far only described in fungi involved in the degradation of wood [START_REF] Welinder | Plant peroxidases: structure-function relationships[END_REF]. More recently, six other classes have been identified in Ascomycetes (Asco class II type A, B and C) and in Basidiomycetes (Basidio class II type A, B and C) [START_REF] Mathé | In silico definition of new ligninolytic peroxidase subclasses in fungi and putative relation to fungal life style[END_REF]. These new classes have been primarily found in plant pathogenic and plant degrading fungi.

The CIII Prxs also known as plant secreted peroxidases have been described as potential multifunctional proteins, which can participate in auxin metabolism, cell wall elongation, stiffening and protection against pathogens [START_REF] Hiraga | A large family of class III plant peroxidases[END_REF][START_REF] Passardi | The class III peroxidase multigenic family in rice and its evolution in land plants[END_REF]. However, the precise in vivo role of any given single peroxidase is still marginal, primarily because of the wide range of peroxidase substrates (lignin subunits, lipid membranes, and some amino acid side chains [START_REF] Lazzarotto | Ascorbate peroxidase-related (APx-R) is a new heme-containing protein functionally associated with ascorbate peroxidase but evolutionarily divergent[END_REF]) and the probable functional redundancy of this large multigenic family that is highly conserved in terms of amino acid sequence. CIII Prxs have been detected in both green algae that predate land colonization and in land plants, but they are absent from Chlorophyte algae [START_REF] Mathé | Evolution and expression of class III peroxidases[END_REF]Passardi et al., 2007a). This family was subjected to numerous genes, and segmental or genome-wide duplications leading to a large increase in copy numbers from the probable first non-aquatic organisms (Bryophyte). For example, late diverging plants such as Arabidopsis thaliana [START_REF] Tognolli | Analysis and expression of the class III peroxidase large gene family in Arabidopsis thaliana[END_REF], and Oryza sativa [START_REF] Passardi | The class III peroxidase multigenic family in rice and its evolution in land plants[END_REF] contain high copy numbers generated by gene duplications (73 and 138, respectively). These explosive duplications make the genes ideal markers for the mapping of evolutionary lineages in plants.

Unlike CI Prxs, CII and CIII Prxs have more complex structures marked for several glycosylations, four to five disulfide bridges, two calcium binding sites, and one signal peptide [START_REF] Martinez | Molecular biology and structure function of lignin-degrading heme peroxidase[END_REF]. The genes encoding these two classes containing several introns at conserved positions and were subjected to numerous recent duplication events. Gene duplications, together with intronic sequences, play important roles in organism evolution.

Intronic sequences are required for various processes such as alternative splicing, translation efficiency and contain regulatory sequences, encoding other genes or non-coding RNAs [START_REF] Fawcett | Higher intron loss rate in Arabidopsis thaliana than A. lyrata is consistent with stronger selection for a smaller genome[END_REF]. The sequences of new paralogs may diverge in regulatory regions, changing its expression patterns or in coding regions may result in acquisition of new functions leading to either subfunctionalization or neofunctionallisation, [START_REF] Xu | Divergence of duplicate genes in exon-intron structure[END_REF]. If the duplicated sequences have no additional function, the accumulation of mutations will rapidly lead to pseudogenisation.

To reveal the emergence and analyse the evolution of the CIII Prxs in green plant lineage, we annotated the CI Prxs (APx, CcP and CP) and CIII Prxs families from publically available genomic and expression data. Particular attention was paid to cover most of the green lineage from the early to late divergent plants. In parallel, we confirmed the presence and absence of the three classes of proteins based on APx, CcP and CIII Prxs enzymatic activities. Finally, we focused on the evolution of the CIII Prx copy number in the green lineage, with a particular interest to the CIII Prxs from Spirogyra sp.

Results and Discussion

The 3D structure is strikingly conserved between CI, CII and CIII Prxs

The three subclasses CPs, APxs, and CcPs which belong to the CI Prxs have very similar tertiary structures, including a conserved number of -helix domains and overall organization (Fig. 1B). The CPs which contain two independent peroxidase domains, consequently as many -helices. In addition, residues proximal to the haem such as arginine, as well as distal residues such as histidine, are conserved at the same relative positions among the 3 CI Prxs subclasses (Fig. 1C). These structural similarities in term of common heam, iron, key residues and structure conservations, strongly suggest that the CPs, APxs, and CcPs originated from a common ancestral sequence. In addition, the comparison between the tertiary structures of the three classes part of the non-animal peroxidases (CI, CII and CIII Prxs) revealed similarly shared common structural elements including haem, iron, and key residues and structural conservations, strongly suggesting that the three Prx classes could have originated from the same ancestral sequence (Fig. 1) [START_REF] Lazzarotto | Revisiting the Non-Animal Peroxidase Superfamily[END_REF]Passardi et al., 2007a).

Although CII Prxs are not the primary focus of this study, it was necessary to gain insight into the evolution of this class in order to obtain a more global appreciation of the evolution of all non-animal peroxidases. The high duplication rate of CII Prxs, as well as and the absence of CII Prxs in organisms like Chytrids at the base of fungi and even in some species of basidiomycetes, hinder the clear determination of the evolutionary basis behind this class of peroxidases [START_REF] Mathé | In silico definition of new ligninolytic peroxidase subclasses in fungi and putative relation to fungal life style[END_REF]. However, the functional similarity and tertiary structure conservation of CII Prxs with CI Prxs suggest that the CII Prx sequences could have derived from CI Prxs. Therefore, it is likely that the ancestor of the CII Prxs had duplicated and emerged independently during the separation of Basidiomycetes and Ascomycetes to give rise to these different subclasses of CII Prxs [START_REF] Mathé | In silico definition of new ligninolytic peroxidase subclasses in fungi and putative relation to fungal life style[END_REF].

CI, CII and CIII Prxs have specific taxonomic distribution

Several taxonomic distributions of CI, CII and CIII Prxs have already been described [START_REF] Lazzarotto | Revisiting the Non-Animal Peroxidase Superfamily[END_REF]Passardi et al., 2007a), but more recent advances in available comprehensive genomic resources [START_REF] Mukherjee | 1,003 reference genomes of bacterial and archaeal isolates expand coverage of the tree of life[END_REF], allowed us to perform exhaustive data mining and expert sequence annotation in order to define the evolutionary basis giving rise to the actual taxonomic distribution of the non-animal Prxs in the different kingdoms.

To do this, we performed data mining for peroxidase sequences and organelle gain or loss events from different eukaryotic taxonomic groups that yielded a fine distribution of the different classes (Fig. 2, Table 1 andSupplementary Table 1). We employed two complementary strategies to perform exhaustive and expert data mining. First, NCBI, JGI and 1KP databases were queried using the PFAM annotation (PF00141). As this PFAM encompasses the entire set of haem containing peroxidases (animal and non-animal peroxidases), it allowed us to collect most of the non-animal peroxidase sequences, but did not allow to discriminate between the different classes and subclasses. To complete this approach, we used a homology-based strategy to determine the class/subclass memberships and to detect new sequences not already annotated. All the sequences detected were available from the RedoxiBase [START_REF] Savelli | RedoxiBase: A database for ROS homeostasis regulated proteins[END_REF].

Thanks to the exhaustive collection and analysis of sequences, we have obtained a fine distribution of non-animal peroxidases (Fig. 2 and Table 1). Our results confirm and strongly support the concept that APx sequences are only detected in the eukaryotic groups that having acquired and maintained chloroplasts. In the other way around, all the chloroplastic containing organisms analysed (based on available genome sequences) possessed at least one to ten APx sequence (Table 1). Several duplications occurred in Streptophytes but without significant variations of the copy number between streptophyte algae and land plants. A more divergent sequence, APx-related (APx-R) proteins have only been detected in the green lineage (Chlorophyceae and Streptophyte) and not in the other chloroplastic organisms. Only two diatoms (Phaeodactylum tricornutum and Thalassiosira pseudonana) and one early divergent fungus (Gonapodya prolifera) possessed one APx-R encoding sequences, which may have resulted from lateral gene transfers from a green lineage organism (Table 1). Furthermore, the APx-R may have emerged more recently in the green lineage from an existing APX (neofunctionalisation) and still presented a high identity level. The sequence has been probably lost in Charales (missing sequence or short DNA fragment in Nitella hyaline and Chara braunii respectively). In addition, CcPs are present in all eukaryotic organisms but absent from Streptophytes and organisms that have undergone a mitochondrial loss event such as Microsporida (obligate intracellular parasite fungi) or which have developed parasitic style of life (Apicomplexa animal parasites, Pyromyces commensal organisms of the herbivore gut) (Fig. 2 and Table 1). Interestingly, we can notice that all the organisms having acquired a chloroplast, have preserved their mitochondria. On the other hand, a loss of mitochondria is never associated with a gain of chloroplast. The majority of the detected CP sequences belong to the prokaryotes (bacteria) while the rest was distributed punctually in several eukaryotic genomes due to probable lateral gene transfer (Passardi et al., 2007a) (Table 1). We found that all CI Prxs are distributed throughout the eukaryotic kingdom confirming the hypothesis of a common ancestral sequence for the different members of the CI Prx subclasses. The APx-CcP hybrid sequences were detected in several kingdoms without taxonomic relations (Table 1). The APx-CcP have conserved key residues from the Class I but are clustered together within the kingdoms meaning that they diverged independently from an ancestral APx or CcP sequence.

Next, with the use of a larger dataset, we confirmed and supported, the hypothesis that APxs are specific to chloroplast containing organisms, and that CcPs are specific to mitochondrial containing organisms with the exception of all embryophytes. In the Excavata, two phyla perfectly illustrated these gain or lost events. On the one hand, Giardia intestinalis, Spironucleus barkhanus and Spironucleus salmonicida belonging to the Diplomonadida phylum have lost their mitochondria and lack the CcP sequence. On the other hand, Euglena mutabilis, E. gracilis and Eutreptiella gymnastica part of Euglenida phylum, have gained chloroplasts and contained both APx and CcP sequences.

A marginal presence of APx sequences was found in non chloroplast-containing organisms, likely due to lateral gene transfer (LGT) (Table 1). This phenomenon is most often observed in living water organisms (proximity with algae containing APx) and for symbiotic organisms. Hydra viridis, belonging to animals contained a non-animal Prx with both characteristic of APx and traits of CIII Prx. Based on the homology with APx sequences found in green algae, a LGT from Chlorella can be postulated, and that some subsequent evolutionary convergence or divergence occurred, leading to the homology with CIII Prx. We also note the marginal presence of CI Prxs in organisms belonging to animal kingdoms, even though these proteins are known to be generally absent from the animal kingdom. For example, the presence of APx and CcP encoding sequences in the order of Choanoflagellates demonstrates this.

In addition, we observed a lack of CcP for several parasitic organisms containing mitochondria. Among them, is Apicomplexa, a unicellular animal parasite containing a mitochondria and an organelle referred to as the apicoplast (a relic of chloroplasts obtained by secondary endosymbiosis of red algae, [START_REF] Lim | The evolution, metabolism and functions of the apicoplast[END_REF]. More than 10 Apicomplexa genomes are available from NCBI and JGI but no Class I has been detected in these organisms. A chloroplast-containing Apicomplexa, Chromera (small EST library) was analysed and an APx like protein was found but no CcP.

Regarding the CII and CIII Prxs, the taxonomic distribution is more precise and has recently been described [START_REF] Mathé | In silico definition of new ligninolytic peroxidase subclasses in fungi and putative relation to fungal life style[END_REF]. However, we found that it was necessary to include CII and CIII Prxs in our analysis of non-animal peroxidase evolution, because it allowed us to draw a more integrative evolutionary scenario.

Briefly, the set of CII Prxs sequences was only detected in Ascomycetes and Basidiomycetes that interact with living or dead plants. As mitochondrial containing organisms, Ascomycetes and Basidiomycetes contained CcP in two copies in most cases. This is likely not due to recent gene duplication, as the two copies are phylogenetically distant. We observed that most of the fungi containing CII Prxs lack the second CcP copy, which suggest that CII Prx could have originated from one CcP copy. Microsporidia, obligate intracellular parasite fungi, and Pyromyces (Neocallimastigomycota order), a commensal organism of the herbivore gut, have both lost mitochondria during evolution and did not contain CcP sequence (absence confirmed from more than 10 available genomes).

Similarly to the CII Prxs evolutive scenario, CIII Prxs sequences were only found in Streptophytes and are absent from Chlorophyceae. Such as CII Prxs, CIII Prxs belong to multigenic families whose size has largely increased along the green lineage (from 0 in Mesostigma viride or 1 in Chlorokybus atmophyticus to 191 E. grandis, Fig. 2 and Table 1).

The CIII Prxs copy number found in Streptophyte algae is low in comparison to land plants.

The late diverging plants contain more CIII Prxs copies than early diverging plants due to explosive and recent duplications (tandem (TDs), segmental (SDs) and whole genome duplications (WGDs), [START_REF] Li | Explosive tandem and segmental duplications of multigenic families in Eucalyptus grandis[END_REF], which make the size of CIII family quite variable (Fig. 2) including a significant number of pseudogene (as also found for the CII Prxs). Since Streptophytes are the only mitochondria containing organisms lacking CcP, and because CI and CIII Prxs present high level of similarity and several conserved key residues, we raise a hypothesis that the ancestral CIII Prx was derived from the ancestral CcP sequence.

Other non-animal peroxidases, not belonging to any of the well described CI, CII and CIII Prxs, are non-specifically distributed. Therefore, they can be considered as the result of an evolutive divergence or convergence from an ancestral CI Prxs.

In land plants, the CIII Prx activities are greater than those of other plants

APxs, CcP and CIII Prx specific activities have been tested in different species including early to late divergent plants, several fungi, and chloroplast-containing protists (Fig. 3).

Significant variations of APx activities were observed between species likely due to the organ variability or different substrate affinity. However, the different activities are in the same order of magnitude between the different organisms from the green lineage tested (Fig. 3A), which is in agreement with the conserved number of APx sequences detected with the in silico analysis (Fig. 2). As expected, no APx activity was detected in non chloroplastcontaining organisms. CcP activity was detected in all mitochondria-containing organisms excepted in the eight organisms belonging to the Streptophytes (Fig. 2B). This was in agreement with the presence or absence of CcP encoding sequences (Fig. 2). CIII Prxs activity was only detected in the Streptophytes (Fig. 3C) which is consistent with our in silico analysis. The differences between early divergent plants (K. flaccidum, S. pratensis and C. orbicularis) and late divergent plants were significant (P = 0.01), and the range of values was very high (a ratio of 1000:1 between C. orbicularis and E. grandis).

Between the terrestrial streptophytes tested, we observed that the activities were also significantly different possibly due to certain variability in plant ages or stages development. This is in agreement with the finding that the activities of different-aged A. thaliana were significantly different (data not shown [START_REF] Cosio | Transcriptome analysis of various flower and silique development stages indicates a set of class III peroxidase genes potentially involved in pod shattering in Arabidopsis thaliana[END_REF]). Surprisingly, low activity was detected from K. flaccidum, S. pratensis and C. orbicularis protein extracts despite the presence of CIII Prx sequences in these organisms (Fig. 2, 3C and Supplementary Fig. S1).

This low activity could be due to the poor cell wall protein extractability and/or to the lower affinity for in vitro substrate, to the reduced sequence similarity when compared with A. thaliana sequences (36 to 42% of identity for the sequences of K. flaccidum, S. pratensis and C. orbicularis when compared with the closest A. thaliana orthologous sequence) or to the low expression level of the CIII Prx in Streptophyte algae In Zygnematales, the 12 species analysed contained similar number of APx sequences, but 5 did not present CIII Prx suggesting a lower expression of CIII Prxs than APx.

In the green lineage, it appeared that the activities of CIII Prxs increased in the land plant vs the green algae while no significant difference of APx between plants or CcP activity has been detected (Fig. 3). The explosion of CIII Prx activities can be correlated with the explosion of the gene copy number. Although no direct evidence has been demonstrated yet, this explosion could be related to the variation of environmental oxygen concentration, to the organs and cell wall complexification, to the climatic changes, to the colonization of new biotopes, to the constant appearance of new pathogens and, most recently, to human impact on cultivated plants [START_REF] Passardi | The class III peroxidase multigenic family in rice and its evolution in land plants[END_REF].

The features of CIII Prx in early divergent plants

In order to confirm the increase in gene number from early to late divergent plants, Spirogyra sp. genome and S. pratensis RNA-seq data were analysed [START_REF] Delaux | Algal ancestor of land plants was preadapted for symbiosis[END_REF][START_REF] Van De Poel | Transcriptome Profiling of the Green Alga Spirogyra pratensis (Charophyta) Suggests an Ancestral Role for Ethylene in Cell Wall Metabolism, Photosynthesis, and Abiotic Stress Responses(1[OPEN])[END_REF]. Spirogyra belongs to the Zygnemophyceae, a close relative of land plants. At least seven independent CIII Prx and five independent APx encoding sequences were detected from Spirogyra sp and S. pratensis. In parallel, a search of K. flaccidum and Closterium peracerosum available sequences allowed for the identification of 3 CIII Prx from the two species and seven and six APx, respectively. The CIII Prxs from the two Spirogyra sp encode for proteins with regular predicted 3D structure containing 12 alpha-helixes (Supplementary Fig. S2). Although the sequences were not shorter, no predicted signal peptide or C-terminal extension can be detected with various prediction programs (SignalP 4.1 [START_REF] Petersen | SignalP 4.0: discriminating signal peptides from transmembrane regions[END_REF], PrediSi [START_REF] Menne | A comparison of signal sequence prediction methods using a test set of signal peptides[END_REF], Signal-3L [START_REF] Shen | Signal-3L: A 3-layer approach for predicting signal peptides[END_REF] and SOSUIsignal [START_REF] Hirokawa | SOSUI: classification and secondary structure prediction system for membrane proteins[END_REF]) for two CIII Prxs of the seven (considering that two sequences are partial). The canonical CIII Prx sequences have eight conserved cysteine (Cys) residues (C1-C8) that are necessary for disulfide bond formation (Fig. 1B). CIII Prx genes lacking some Cys residues can be found all along the green lineage with a frequency lower than 5 % (41 CIII Prx with missing cysteine for 959 CIII Prx analysed). No genes lacking more than two disulfide bonds have been identified in this study. Despite the absence of data concerning the influence of the lack of disulfide bonds on the protein stability or activity, we could assume that the thermo stability could be affected due to missing disulfide bonds. In the seven CIII Prxs from Spirogyra sp, the cysteines C2 and C3 surrounding the distal histidine are missing while they are present in the CIII Prx from K. flaccidum and C. peracerosum. The transient expression in tobacco leaves of the native SsPrx03 and SsPrx03 with the two missing cysteines have been performed in N. benthamiana. No fluorescence was detected in any cell compartments of N. benthamiana leaves transformed with SsPrx03 fused to the TagRFP. However, the cell wall localisation was detected when leaves were transformed with sequence containing the two cysteines C2 and C3 (SsPrx03m1, Fig. 4A).

However, similar signal was detected in the leaves transformed with CIII Prx from A. thaliana (native AtPrx36 and AtPrx36 mutated for the cysteines C2 and C3 (AtPrx36m3)).

In parallel, total CIII Prx activity was measured in the infected areas of the leaves.

First, the lack of the cysteines C2 and C3 did not significantly affect the CIII Prx activity detected in the leaves of the AtPrx36m3 vs AtPrx36. The addition of the cysteins C2 and C3 in the Spirogyra sp sequence did not increase the initial activity (SsPrx03 vs SsPrx03m1) and was not significantly different from the activity detected with the empty vector in both cases (Fig. 4B). The low activity detected with SsPrx03, lacking the cysteines C2 and C3, could support the very low activity detected from Spirogyra sp fresh material (Fig. 3).

Conclusions

Based on previous studies [START_REF] Lazzarotto | Revisiting the Non-Animal Peroxidase Superfamily[END_REF]Passardi et al., 2007a), we have performed exhaustive data mining and expert sequence annotation. We have greatly increased the number of sequences belonging to underrepresented kingdoms. We conclude that the three CI Prx subclasses (APx, CcP and CP) have originated from the same ancestral CI Prx sequence initially present in the Neomura (Fig. 5). This sequence is no longer present in all of the current prokaryotes and only the bifunctionnal CP containing two fused peroxidase domains has been marginally found. The CP are also detected in some eukaryotes with a chaotic distribution due to LGT. The punctual distribution of CP in most of the kingdoms and the lack of other non animal Prx in prokaryotes strongly supports the existence of a mono functional ancestral CI Prx. The presence of APx in most of photosynthetic organisms (uni, multi and pluricellular organisms) suggests that sequence gains followed independent primary endosymbiosis. The evolutive scenario leading to the modern CII and CIII Prxs appeared to be similar. They probably both emerged independently from an ancient CcP in the ascomycetes and bascidiomycetes for the CII Prx and in the streptophyte algae for the CIII Prxs. The emergence of the CII and CIII Prxs was accompanied by the acquisition of a secretion signal and several cysteine residues allowing disulfide bridge formation, which was coherent with the extracellular activity of these proteins. These common innovations could strongly support a similar evolutionary of CII and CIII Prxs in relation with the plant cell wall loosening and/or stiffening. In the perspective of the better understanding of CIII Prxs evolution and functions, the use of new steptophyte algae containing few CIII Prxs copies will be of major interest.

Materials and Methods

Cultivated and collected organisms

The protist (Euglena mutabilis (Euglenida), Chlorarachnion reptans (Cercozoa),

Synura petersenii (Other

Total protein extraction and enzyme assays of APx, CcP and CIII Prxs

For multicellular organisms, approximately 100 mg fresh material was ground into fine powder with a mortar and pestle under liquid nitrogen. For cell culture, to extract proteins 200 µl extraction buffer containing potassium phosphate buffer (pH 7.0, 50 mM/L), EDTA (5 mM/L), PVPP (16 g/L) was used for extraction. The homogenate was centrifuged at 10, 000 g for 10 min at 4 °C and the supernatant was used for enzyme assays and total protein assay.

The APx activity was determined based on the oxidation of ascorbic acid (ASA).

Oxidation was determined based on measurements of the decrease in absorbance at 290 nm between 30 sec to 5 min. one ml reaction mixture contained potassium phosphate buffer (pH 7.0, 50 mM/L), ASA (0.5 mM/L), H 2 O 2 (0.1mM/L) and 20 µl of protein extract already containing ASA, 2 mM/L. The reaction was initiated with the addition of the of protein extract. A correction was made for the low, non-enzymatic oxidation of ascorbate by H 2 O 2 [START_REF] Nakano | Induction of leucocyte migration inhibitory factor (LIF) by stimulation with free hapten and water-insoluble epoxy resin[END_REF]. The APx unit is defined as the decrease in 1 min of 1 µg total proteins under the above assay conditions.

The CcP activity was measured by monitoring at 550 nm the oxidation of Cytochrome c (from the bovine heart, Sigma-Aldrich) previously reduced. The reduction was carried out by mixing a solution of 0.22 mM Cytochrome c with a 0.5 mM final concentration DTT solution for 15 min at room temperature. After this time, the color of the solution should change from red to pale pink-red for a good reduction. Nevertheless, the reduction must be estimated by spectrometry by measuring the ratio of OD 555 nm / OD 565 nm of an aliquot of the solution diluted 20-fold in the assay buffer. For a good reduction, the ratio should be between 10 and 20. The assay conditions for the CcP activity are a few microliters of protein, 50 mM sodium phosphate buffer pH = 7, 200 μM H 2 O 2 and 20 μM Cytochrome c in a plastic tub. After adding the proteins or H 2 O 2 , the OD is measured between 10 and 30 seconds.

The CIII Prx activity was assayed in a 1 ml reaction mixture consisting of potassium phosphate buffer (50 mM/L, pH 6.0), 0.125% guaiacol (v/v) and 20 µL of protein extract. The reaction was initiated by adding 125 µL H 2 O 2 (11 mM/L), and the oxidation of guaiacol was determined based on the increase in A470. One CIII Prx unit is defined as the increase N. benthamiana leaves overexpressing SsPrx03-TagRFP, SsPrx03m-TagRFP, and AtPrx36-TagRFP were dissected, weighed (40-60 mg), and frozen in liquid nitrogen. Leaves not transformed or transformed with the pEAQ empty vector were used as negative controls.

Tissues were grinded twice for 3 min at 30 Hz using a Retsch MM400 ball mill, with a quick spin between the two cycles. Soluble proteins were extracted in the following extraction buffer (50 ml for 100 mg leaves): 20 mM HEPES, pH 7.0, 1 mM EGTA, 10 mM vitamin C, and PVP PolyclarAT (100 mg•g -1 fresh weight). The extract was centrifuged twice 10 min at 10,000 g (4°C) to remove insoluble material. The protein content was determined using the Bradford reagent (Serva) with BSA (Sigma-Aldrich) as a standard (Bradford, 1976). CIII Prx activity kinetic was measured at 25°C by following the oxidation of 8 mM guaiacol (Fluka) at 470 nm in the presence of 2 mM H 2 O 2 (Carlo Erba) in a phosphate buffer (200 mM, pH 6.0).

Results were expressed in nanokatals / mg proteins ± SD (n ≥ 4). P-value of one-way anova followed by Tukey's significance test, (***) P < 0.0001, (**) P < 0.001, (ns) non significanly different to leaves transformed with empty vector.

Figure legends

  Stramenopiles), Moneuplotes crassus (Alveolates), Tetrahymena thermophila (Ciliophora, ciliates), Emiliania huxleyi (Isochrysidales)) and the algae (Chlamydomonas reinhardtii (Chlorophyceae), Klebsormidium flaccidum, Coleochaete. Orbicularis, Spirogyra pratensis) were provided by the Culture Collection of Algae and Protozoa (CCAP, https://www.ccap.ac.uk/) and cultivated in vitro with culture media compositions detailed in supplementary Table 2. Illustrations of cultivated cultures are visible in supplementary Fig. S3 Saccharomyces cerevisiae (Saccharomycetes) is baker yeast and protein extraction was made directly without in vitro culture. The two diatoms (Moneuplotes crassus and Phaeodactylum tricornutum (Bacillariophyta) were provided by the LISBP laboratory and their growth conditions are also describe in supplementary Table2.The Oomycete (Aphanomyces euteiches) and the Glomeromycete (Glomus irregularis were available from our laboratory and have been cultivated as indicated in supplementary

Fig. 1 .

 1 Fig. 1. Non-animal peroxidases: 3D structures and catalytic domain are conserved. (A) Class composition and taxonomic distribution of the 3 non-animal peroxidase classes. Among the CI Prx, CP are mainly found in bacteria but also marginally in other organisms due to LGT, APX are only detected in chloroplastic organisms, CcP in all organism with mitochondria with the exception of plants and finally the hybrid APx-CcP found in most of the eukaryotes (plants excepted). The CII Prxs and the CIII Prxs are only detected in fungi and viridiplantae respectively (B) The available tertiary structure of 3 CI Prxs (CP: 5L05 from Burkholderia pseudomallei, APX: 1APx from Pisum sativum and CcP: 2CYP from Saccharomyces cerevisiae), 1 CIII Prx (1HCH from Armoracia rusticana) and 1 CII Prx (3Q3U from Trametes cervina) were visualized and compared using Swiss-PdbViewer. The 3D models show strong conservation in the number of -helixes (blue) and their overall spatial organisation. (C) Moreover, the key residues necessary for haem binding and electron transfer are also highly conserved amongst the 3 main Prx classes.

Fig. 2 .

 2 Fig. 2. Evolution of CI and CIII Prxs isoform numbers in green lineage. The number of genes encoding CI Prxs (CP, APx and CcP) and CIII Prx was analysed for 21 species from viridiplantae and Chlorophyceae. All the sequences detected and included in the table are

Fig. 4 .

 4 Fig. 3. APx, CcP and CIII Prx activities were correlated with the presence of the corresponding encoding proteins. Total protein was extracted from 19 species belonging to green plants, fungi and protists. (A) APx activity was determined by the oxidation of ascorbic acid based on the decrease of the absorbance at 290 nm. (B) CcP activity was measured by monitoring the oxidation of Cytochrome c previously reduced at 550 nm. (C) CIII Prx activity was assayed at 470 nm to the follow the oxidation of guaiacol. (D) Table summarizing, the presence of mitochondria (Mito), CcP encoding sequences (CcP seq), chloroplast (Chloro), APx encoding sequences (APx seq) and CIII Prx encoding sequences (CIII Prx seq) ) for the 19 studied species. : organite or sequences have been detected, no: organite or sequence has not been detected, a : Presence of CI, CII and CIII Prxs in C.reptans is based on the analysis of related species Bigelowiella natans, b : presence of CI, CII and CIII Prxs in Moneuplotes (Euplotes) crassus is based on the analysis of two closely related species E.octocarinatus (http://ciliates.ihb.ac.cn/database/home/#eo) and E.vannus (http://evan.ciliate.org). The enzymatic activities are expressed in nanokatal per mg of total proteins. Activities are the mean of 3 independent assays ± SD. Fig. 4. The lack of cysteine C2 and C3 could affect the cell wall localisation of Spirogyra CIII Prx. (A) Alignment of SsPrx03, SsPrx03m1 and AtPrx36 partial region surrounding the conserved distal histidine (Hd) and containing the cysteine C2 and C3 (missing in Spirogyra sequence). (B) Pieces of Nicotiana benthamiana leaves transformed with constructs

Fig. 5 .

 5 Fig. 5. An evolutionary model to explain the current distribution of the different nonanimal peroxidase classes. *: Ancestral CI Prx sequence probably existing in Neomura and lost in the Archebacteria, Proteobacteria and Cyanobacteria; **: chaotic and marginal distribution of CP resulting from LGT; ***: several independent events of CcP lost.

Table 2 .

 2 The land plants Marchantia polymorpha, Physcomitrella patens, Selaginella moellendorffii, Gingko biloba, Brachypodium distachyon, Arabidopsis thaliana, Eucalyptus grandis were available directly from the growth room facility.

Acknowledgments

The authors are thankful to the Paul Sabatier Toulouse 3 University and to the Centre National de la Recherche Scientifique (CNRS) for granting their work. QL was supported by PhD grants from the China Scholarship Council (CSC). The authors especially thank the Delwiche Lab for the unpublished sequence data available for our analysis, Endymion D.

Cooper for the spirogyra DNA and Aurélie Le Ru (UMR5546 CNRS/UPS, Castanet-Tolosan, France) for their valuable help in performing and imaging the transient expression experiments.

between 1 min and 2 min of 1 µg total proteins under the above assay conditions [START_REF] Jia | Class III peroxidases are activated in proanthocyanidin-deficient Arabidopsis thaliana seeds[END_REF].

The concentration of total proteins, used to determine the enzyme activity was detected using Coomassie Protein Assay (Bradford).

Identification and annotation of CI Prxs and CIII Prx genes in green lineage

The presence or numbers of CI Prxs and CIII Prxs were analyzed from various organisms belonging to the green lineage including early and late divergent plants. First an exhaustive data mining was performed from available annotated repositories (Phytozome v12 (http://www.phytozome.net/), NCBI [START_REF] Benson | The National Center for Biotechnology Information[END_REF])) using PFAM annotation (PF00141) and keywords searches. All the collected peroxidase sequences were included in the RedoxiBase [START_REF] Fawal | PeroxiBase: a database for large-scale evolutionary analysis of peroxidases[END_REF] and classified based on homology, phylogeny and homemade Prosite profile. In parallel, non-annotated repositories (1 kP and EST libraries from NCBI) were also analyzed thank to homology based strategy.

Subcellular localization of CIII Prx protein SsPrx03

The full-length ORF frame of SsPrx01 (without stop codon) was amplified by PCR from synthetic sequence using 5'-CCGGAATTCATGTCTCTTCTTCCCC-3' and 5'-CCGCCCGGGAAGATACAAGCAATAC-3'. The PCR product was digested with EcoRI and SmaI, and cloned into TagRFP-AS-N vector (Evrogen). The SsPrx03 and SsPrx03m1-TagRFP C-terminal fusion was then subcloned (LR reaction, Gateway Technology, Invitrogen) into pEAQ-HT-DEST1 vector [START_REF] Sainsbury | pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants[END_REF]. This construct was confirmed by restriction analysis and sequencing, and transferred into Agrobacterium tumefaciens (strain GV3101). The YFP fusion protein pm-yb CD3-1006 [START_REF] Nelson | A multicolored set of in vivo organelle markers for colocalization studies in Arabidopsis and other plants[END_REF] was used as a plasma membrane control. Both constructs were co-infiltrated into N. benthamiana 30-day old leaves at an optical density of 0.5 at 600 nm. 36 hours after infiltration leaves were detached and used for image acquisition on a Leica TCS SP2

Confocal Laser Scanning Microscope with the following excitation wavelengths: 561 nm for TagRFP and 514 nm for YFP.

SsPrx03-TagRFP, SsPrx03m-TagRFP activity measurement


Gang W Wang

Jean-Michel Brunel

Jean-Michel Bolla

Françoise Van Bambeke
email: francoise.vanbambeke@uclouvain.be

Revision for Antimicrobial Agents and Chemotherapy The polyaminoisoprenyl R2.1potentiator NV716 revives old disused antibiotics against intracellular forms of infection by Pseudomonas aeruginosa

Active efflux confers intrinsic resistance to multiple antibiotics in Pseudomonas aeruginosa, including old disused molecules. Beside resistance, intracellular survival is another reason for failure to eradicate bacteria with antibiotics. We evaluated the capacity of polyaminoisoprenyl R2.1.potentiators (designed as efflux pump inhibitors [EPIs]) NV716 and NV731 compared to PAβN to restore the activity of disused antibiotics (doxycycline, chloramphenicol [substrates for efflux], rifampin [not substrate]) in comparison with ciprofloxacin against intracellular P. aeruginosa (strains with variable efflux levels) in THP-1 monocytes exposed during 24h to antibiotics alone (0.003-100x MIC) or combined with EPIs.

Pharmacodynamic parameters (apparent static concentrations [C s ]; maximal relative efficacy [E max ])

were calculated using the Hill equation of concentration-response curves. PAβN and NV731 moderately reduced (0-4 doubling dilutions) antibiotic MICs but did not affect their intracellular activity.

NV716 markedly reduced (1-16 doubling dilutions) the MIC of all antibiotics (substrates or not for efflux; strains expressing efflux or not); it improved their relative potency and maximal efficacy (lower C s ; more negative E max ) intracellularly. In parallel, NV716 reduced the persister fraction in stationary cultures when combined with ciprofloxacin. In contrast to PAβN and NV731 that act as EPIs against extracellular bacteria only, NV716 can resensitize P. aeruginosa to antibiotics whether substrates or not for efflux, both extracellularly and intracellularly. This suggests a complex mode of action that goes beyond a simple inhibition of efflux and reduces bacterial persistence. NV716 may appear as a useful adjuvant, including to disused antibiotics with low antipseudomonal activity, to improve their activity, including against intracellular P. aeruginosa.

INTRODUCTION

Pseudomonas aeruginosa is one of the so-called ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens (1) that harbor multiple mechanisms of drug resistance. It is considered by the World Health Organization (WHO) as a critical priority species for the search of innovative therapies [START_REF] Shrivastava | World health organization releases global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics[END_REF].

P. aeruginosa has acquired a series of resistance mechanisms to all classes of commonly used antibiotics (-lactams, fluoroquinolones, aminoglycosides, and, more recently, polymyxins). Moreover, it is also innately resistant to many antibiotics, mainly due to poor outer membrane permeability and/or active efflux [START_REF] Mesaros | Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium[END_REF]. Four main efflux systems, named MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM, have been associated with increased MICs for a broad range of antimicrobial agents [START_REF] Poole | Multidrug efflux pumps and antimicrobial resistance in Pseudomonas aeruginosa and related organisms[END_REF], bringing the active concentrations above the levels that can be achieved in the serum of patients. This explains why drugs like tetracyclines or chloramphenicol cannot be used against P. aeruginosa or other Gram-negative bacteria, although they are capable of binding to their respective target in these bacteria when tested in acellular systems [START_REF] Olson | Functional, biophysical, and structural bases for antibacterial activity of tigecycline[END_REF][START_REF] Vazquez | Uptake and Binding of Chloramphenicol by Sensitive and Resistant Organisms[END_REF].

Another, often neglected, reason for antibiotic failure against P. aeruginosa is the capacity of this bacterium to adopt specific lifestyles, such as intracellular survival. Although considered as an extracellular pathogen, P. aeruginosa has been shown by many researchers as capable of invading and surviving inside epithelial [START_REF] Fleiszig | Pseudomonas aeruginosa invades corneal epithelial cells during experimental infection[END_REF][START_REF] Emam | Glycolipid-Dependent, Protease Sensitive Internalization of Pseudomonas aeruginosa Into Cultured Human Respiratory Epithelial Cells[END_REF] and phagocytic cells [START_REF] Porto | Dysfunctional CFTR alters the bactericidal activity of human macrophages against Pseudomonas aeruginosa[END_REF][START_REF] Garai | Killing from the inside: Intracellular role of T3SS in the fate of Pseudomonas aeruginosa within macrophages revealed by mgtC and oprF mutants[END_REF][START_REF] Buyck | Pharmacodynamic evaluation of the intracellular activity of antibiotics towards Pseudomonas aeruginosa PAO1 in a model of THP-1 human monocytes[END_REF] in vitro, as well as in lung epithelial cells and macrophages in vivo [START_REF] Schmiedl | Bacterial distribution in lung parenchyma early after pulmonary infection with Pseudomonas aeruginosa[END_REF][START_REF] Zaas | Counteracting signaling activities in lipid rafts associated with the invasion of lung epithelial cells by Pseudomonas aeruginosa[END_REF]. In these intracellular niches, bacteria are protected from the host humoral immune defenses but also from antibiotics, which need to have access, accumulate, and express their activity in the infected subcellular compartment [START_REF] Carryn | Intracellular pharmacodynamics of antibiotics[END_REF][START_REF] Van Bambeke | Cellular pharmacodynamics and pharmacokinetics of antibiotics: current views and perspectives[END_REF]. Moreover, they can adopt dormant phenotypes that do not respond anymore to antibiotics, like persisters. These are subpopulations of otherwise antibiotic-susceptible bacteria that switch to a transient non-dividing phenotype surviving to high concentrations of bactericidal antibiotics [START_REF] Balaban | Definitions and guidelines for research on antibiotic persistence[END_REF]. We have recently provided evidence for another species, Staphylococcus aureus, that bacteria surviving to antibiotics intracellularly are persisters, explaining why antibiotics fail to eradicate them [START_REF] Peyrusson | Intracellular Staphylococcus aureus persisters upon antibiotic exposure[END_REF]. Preliminary data suggest that the same may hold true for P. aeruginosa [START_REF] Khandekar | The Putative De-Nacetylase DnpA Contributes to Intracellular and Biofilm-Associated Persistence of Pseudomonas aeruginosa Exposed to Fluoroquinolones[END_REF].

As P. aeruginosa resistance is worryingly increasing, few new antibiotics have been approved over the last years, and are essentially new derivatives in existing classes with improved activity or reduced susceptibility to resistance mechanisms [START_REF] Burrows | The Therapeutic Pipeline for Pseudomonas aeruginosa Infections[END_REF]. In this context, reviving old and disused antibiotics may appear as an attractive strategy [START_REF] Theuretzbacher | Reviving old antibiotics[END_REF], which should be accompanied by an in-depth reassessment of their efficacy according to the modern standards imposed by pharmacokinetic/pharmacodynamic (PK/PD) concepts, including in models of persistent infections like intracellular survival.

One of the approaches proposed to increase the potency of drugs that are substrates for efflux is to combine them with efflux pump inhibitors (EPIs) [START_REF] Burrows | The Therapeutic Pipeline for Pseudomonas aeruginosa Infections[END_REF]. PAβN (MC 207,110; Phenylalanine-arginine β-naphthylamide ( 21)) and CCCP (Carbonyl cyanide m-chlorophenyl hydrazone [START_REF] Pages | Inhibitors of efflux pumps in Gram-negative bacteria[END_REF]) have been widely used in vitro to increase the accumulation of antibiotics within Gram-negative bacteria, but their toxicity R1.2.and inadequate pharmacokinetic properties preclude clinical applications [START_REF] Lomovskaya | Practical applications and feasibility of efflux pump inhibitors in the clinic--a vision for applied use[END_REF][START_REF] Mahamoud | Antibiotic efflux pumps in Gramnegative bacteria: the inhibitor response strategy[END_REF]. Efforts have been made to synthesize new efflux pumps inhibitors with improved safety profile [START_REF] Douafer | Antibiotic Adjuvants: Make Antibiotics Great Again![END_REF]. Among them, polyaminoisoprenyl compounds NV716 and NV731 (see chemical structures in Figure S1) demonstrated their capacity to restore chloramphenicol activity against Enterobacteria spp (26) and one of them (NV716), that of chloramphenicol and doxycycline against Pseudomonas aeruginosa [START_REF] Borselli | Polyamino-Isoprenic Derivatives Block Intrinsic Resistance of P. aeruginosa to Doxycycline and Chloramphenicol In Vitro[END_REF], or of florfenicol against Bordetella bronchiseptica [START_REF] Borselli | Polyamino-Isoprenyl Derivatives as Antibiotic Adjuvants and Motility Inhibitors for Bordetella bronchiseptica Porcine Pulmonary Infection Treatment[END_REF].

The aim of the present study was to evaluate the effects of NV716 in comparison with that of NV731 (less potent against P. aeruginosa (27)) and PAβN (reference EPI) in combination with old and disused antibiotics against the intracellular forms of P. aeruginosa, using strains expressing or not efflux pumps to infect THP-1 monocytes. We exploited a previously established in vitro pharmacodynamic model [START_REF] Buyck | Pharmacodynamic evaluation of the intracellular activity of antibiotics towards Pseudomonas aeruginosa PAO1 in a model of THP-1 human monocytes[END_REF] that allows comparing key pharmacological descriptors of antibiotic intracellular activity, namely their relative potency and maximal efficacy [START_REF] Buyck | In Vitro Models for the Study of the Intracellular Activity of Antibiotics[END_REF]. As antibiotics, we selected doxycycline, chloramphenicol (both substrates for efflux (see Table S1) and for which preliminary data do exist regarding the beneficial effect of NV716 [START_REF] Borselli | Polyamino-Isoprenic Derivatives Block Intrinsic Resistance of P. aeruginosa to Doxycycline and Chloramphenicol In Vitro[END_REF]), rifampin (not substrate for efflux), and ciprofloxacin, selected as the most active drug against intracellular P. aeruginosa in this pre-established in vitro model [START_REF] Buyck | Pharmacodynamic evaluation of the intracellular activity of antibiotics towards Pseudomonas aeruginosa PAO1 in a model of THP-1 human monocytes[END_REF]. The results were examined in the light of the effect of these EPIs on antibiotic MIC and on the selection of persisters in broth culture.

We showed that NV716, contrarily to NV731 and PAβN, was capable of increasing the relative potency and maximal efficacy of these antibiotics against intracellular P. aeruginosa, whether substrates of not for efflux and in strains expressing or not efflux pumps. These effects were related to drastic reductions in MIC and in persister fractions. Altogether, these data suggest that NV716 does not simply act by inhibiting efflux but could be a useful adjuvant, including to disused antibiotics with low antipseudomonal activity, to improve their activity also against intracellular P. aeruginosa.

RESULTS

Antimicrobial susceptibility

Table 1 shows the MICs of antibiotics against PAO1, PAO509 (deleted in the genes encoding 5 efflux pumps), 205-2 and BV1 (two clinical isolates expressing respectively a non-functional and a functional MexAB-OprM efflux pump), in the absence of or in the presence of 20 mg/L (38 μM) PAβN, or 4 mg/L (10 μM) NV731 or NV716. These concentrations were selected because commonly used in previous works [START_REF] Lomovskaya | Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy[END_REF][START_REF] Borselli | Polyamino-Isoprenic Derivatives Block Intrinsic Resistance of P. aeruginosa to Doxycycline and Chloramphenicol In Vitro[END_REF]. In control conditions (no inhibitor), ciprofloxacin, doxycycline, and chloramphenicol showed much lower MICs against PAO509 than against PAO1, in accordance with the fact they are substrates for efflux (see Table S1). Conversely, the MIC of rifampin, which is not a substrate, remained unchanged. BV1 was less susceptible than PAO1 to ciprofloxacin and doxycycline and more susceptible to chloramphenicol, while 205-2 was less susceptible to ciprofloxacin only. PAβN and NV731 reduced antibiotic MICs in few cases, while NV716 systematically decreased them, except for ciprofloxacin in PAO509, the MIC of which was already very low. Of note, the effect of NV716 was as (or even more) important when testing strains that do not express efflux pumps or those that do, as well as when using rifampin (not substrate for efflux) or other antibiotics (all substrates for efflux).

Cytotoxicity of inhibitors and antibiotics

In a first set of experiments, we evaluated the cytotoxicity of the EPIs and antibiotics in the conditions of their use for intracellular infection by trypan blue exclusion test after 24 h incubation. Infection of THP-1 cells by PAO1 caused 85% cell mortality in the absence of antibiotic. The inhibitors alone caused concentration-dependent cell mortality, which was minimal at the concentration used for intracellular infection studies (Figure 1, left panel). Doxycycline, chloramphenicol, and rifampin showed toxic effects at high concentrations, with IC 50 (i.e. concentration causing 50% cell mortality) reaching approx. 30 times their respective MIC in infected cells (Figure 1, right panel). Ciprofloxacin was much less toxic at equivalent multiples of its MIC (which corresponds to much lower mass concentrations). IC 50 values for toxicity of inhibitors, antibiotics or their combinations in non-infected and infected cells are detailed in supplementary Figure S2 and Table S2 but do not differ from what is described in Figure 1.

Activity of antibiotics alone or combined with EPIs against intracellular P. aeruginosa in an in vitro pharmacodynamic model

Figure 2 shows the concentration-response curves against intracellular PAO1 in the absence of or in the presence of NV716. The activity of antibiotics alone developed following a sigmoidal concentration-response curve, as previously described [START_REF] Buyck | Pharmacodynamic evaluation of the intracellular activity of antibiotics towards Pseudomonas aeruginosa PAO1 in a model of THP-1 human monocytes[END_REF]. At low, subMIC concentrations, a ~3 log 10 increase in CFUs was noticed over the 24 h incubation period. A static effect was observed at extracellular concentrations close to the MIC of each drug. At the highest concentration tested, the reduction in bacterial counts ranged from 2 log 10 CFU decrease for chloramphenicol, doxycycline, and rifampin, to 2.6 log 10 CFU decrease for ciprofloxacin. In the presence of NV716 at 10 µM, all curves (except that of ciprofloxacin) were shifted to the left, meaning that the corresponding antibiotic was more potent, a static effect being reached at lower extracellular concentrations. Furthermore, the maximal reduction in intracellular counts was also increased, indicating that the antibiotics were more effective.

The same type of experiment was performed with the other EPIs and the other strains. Figure 3 illustrates the pharmacodynamic parameters calculated based on the Hill equation of these concentration-response curves (see methods for a definition of these parameters), namely the C s (a measure of the relative potency of the drug or the drug combination) and the E max (a measure the relative maximal efficacy of the drug alone or in combination). C s of antibiotics alone were close or slightly higher than the MICs and systematically reduced of 1 doubling dilution or more by NV716, except for ciprofloxacin against PAO1 and PA0509, which were already highly susceptible to this drug. NV731 and PAN did not show any significant effect on C s values. Similarly, NV716 was the only EPI capable of increasing the efficacy of the 4 drugs against all strains, with E max values being at least 0.5 log 10 CFU more negative than those measured for the antibiotics alone.

Concentration-response curves of NV716 combined with fixed concentrations of antibiotics against intracellular P. aeruginosa

NV716 being the only adjuvant capable of improving both the relative potency and the relative maximal efficacy of antibiotics, we examined the concentration-dependency of its effect when combined with antibiotics at fixed concentrations corresponding to their respective C min and C max in human serum (Table S1 for these values). The data are illustrated in Figure 4. Considering first the activity of NV716 alone, a ~3 log 10 increase in CFU was observed after 24 h incubation with low NV716 concentrations ( 10 μM) for all strains except PAO509, for which a slight reduction in CFU was already observed at 10 µM. A static effect was obtained for NV716 concentrations close to its MIC in RPMI-1640 (25 µM) for all strains. At 50 μM, NV716 caused a reduction in bacterial counts that ranged from ~1 log 10 CFU decrease for PAO1, 205-2 and BV-1, to ~2 log 10 CFU decrease for PAO509. These data are coherent with those obtained in broth (Figure S3), showing that NV716 did not prevent PAO1 growth for concentrations  10 µM but caused a rapid decrease in bacterial counts at 50 µM. In the presence of antibiotics at their C min , we noticed that the intracellular concentration-response curves were globally parallel to those obtained with NV716 alone, reflecting thus essentially the activity of the EPI.

Three exceptions need however to be highlighted. First, the activity of chloramphenicol was increased for NV716 concentrations > 3 µM against all strains. Second, an improvement in antibiotic activity was noticed against PAO509, as soon as NV716 concentration was > 3 µM for all drugs that are substrates for efflux (ciprofloxacin, chloramphenicol and doxycycline). Third, at the highest concentration of NV716 tested (50 µM), the reduction in CFU counts was 0.5-1.8 log 10 more important than that observed with NV716 alone for all drugs, denoting some sort of synergy. Combining NV716 with antibiotics at their C max caused a drastic improvement of activity, with CFU counts being reduced from 0.5-2.2 log 10 at NV716 concentrations ≤ 10 µM to 1.4-5.6 log 10 at higher NV716 concentrations in comparison with CFU counts measured for the antibiotic alone.

Persister assay

The fact that NV716 increases the intracellular efficacy of antibiotics (improved killing capacity) suggests it may decrease the proportion of antibiotic persisters in the population, recently identified as a cause of failure to eradicate intracellular S. aureus with antibiotics [START_REF] Nguyen | The persister character of clinical isolates of Staphylococcus aureus contributes to faster evolution to resistance and higher survival in THP-1 monocytes: a study with moxifloxacin[END_REF]. We therefore examined the persister fraction surviving to ciprofloxacin (selected as highly bactericidal antibiotic) alone or combined with NV716 in stationary phases cultures. As shown in the left panel of Figure 5A, ciprofloxacin at 50x MIC caused a rapid drop in CFU counts of PAO1 over the 2 first hours of incubation, after which no further change was observed, leaving a persisting fraction of 0.005% of the initial inoculum. The same type of experiment was then performed with PAO1 and PAO509 exposed to NV716 at increasing concentrations and used alone or combined with 50x MIC of ciprofloxacin. The right panels of Figure 5A show the proportion of persisters after 5 h of incubation, expressed in percentage of the CFU counts measured in the absence of drugs. NV716 alone had no effect on persisters for concentrations  100 µM but caused a marked reduction in bacterial counts at higher concentrations (3-5 log 10 CFU for concentrations > 300 µM for PAO1 and > 100 µM for PAO509).

Strikingly, NV716 at lower concentrations (10-100 µM) was already capable to reduce up to 11-(PAO1) to 28-(PAO509) fold the percentage of persisters selected by ciprofloxacin.

R2.3To investigate whether NV716 needs to be administered simultaneously with ciprofloxacin to obtain a maximal activity, we repeated killing experiments with stationary phase cultures exposed to 50 x MIC of ciprofloxacin alone or combined with NV716 added at different time points (Figure 5B) [START_REF] Liebens | Identification of 1-((2,4-Dichlorophenethyl)Amino)-3-Phenoxypropan-2-ol, a Novel Antibacterial Compound Active against Persisters of Pseudomonas aeruginosa[END_REF].

CFUs were significantly reduced after the addition of NV716 at any time point, for concentrations ≥ 10 µM and in both PAO1 and PAO509, indicating that NV716 does not need to be added at the same time as the drug to be able to increase its activity. In a next step, we aimed at examining whether NV716 action was related to a direct capacity to kill persisters or rather to resensitize them to the killing effect of ciprofloxacin. To this end, we isolated the persisters surviving after 5 h of incubation with ciprofloxacin at 50 x MIC and reexposed them to ciprofloxacin at 50 x MIC, NV716 at different concentrations, or a combination thereof (Figure 5C) [START_REF] Liebens | Identification of 1-((2,4-Dichlorophenethyl)Amino)-3-Phenoxypropan-2-ol, a Novel Antibacterial Compound Active against Persisters of Pseudomonas aeruginosa[END_REF]. As expected, incubation of the isolated persisters with ciprofloxacin alone caused only a marginal decrease in CFUs for both strains, confirming the effective isolation of persister cells. When used alone, NV716 caused a significant decrease in CFUs only at the highest concentration tested (250 μM). In contrast, a significant reduction in CFUs was already observed when it was combined at 10 µM with ciprofloxacin for both strains.

DISCUSSION

In this study we show that, in contrast with PAβN and NV731 that act as efflux inhibitors against planktonic bacteria only, NV716 is capable to re-sensitize P. aeruginosa to antibiotics whether substrates (doxycycline, chloramphenicol, ciprofloxacin) or not (rifampin) for efflux, not only in broth but also intracellularly.

Considering first susceptibility data in broth, we observed a remarkable effect of NV716 on the antibiotic intrinsic activity. While PAβN and NV731 moderately reduced the MICs of antibiotics (essentially those that are substrates for efflux), NV716 caused a marked decrease in the MICs of all drugs, comprising rifampin (not substrate for efflux; see Table S1) against all strains, including PAO509 that does not express efflux pumps or BV1 that expresses a non-functional MexAB-OprM efflux pump. This suggests that its mode of action is not related to the inhibition of efflux only, but probably also relies on its capacity to permeabilize the outer membrane of the P. aeruginosa [START_REF] Borselli | Polyamino-Isoprenic Derivatives Block Intrinsic Resistance of P. aeruginosa to Doxycycline and Chloramphenicol In Vitro[END_REF]. Of note, the synergy with antibiotics is obtained at sub-inhibitory concentrations of NV716, R1.3.indicating that, if a direct effect on bacteria takes place, it is not sufficient to cause bacterial death. PAβN has also been shown to permeabilize the outer membrane of Gram-negative bacteria by displacing Mg 2+ and Ca 2+ (32), but at concentrations higher than the one we used here, established as adequate to inhibit efflux in princeps studies with this molecule [START_REF] Lomovskaya | Identification and characterization of inhibitors of multidrug resistance efflux pumps in Pseudomonas aeruginosa: novel agents for combination therapy[END_REF]. The activity of NV716 and of the two other EPIs is observed at concentrations that do not cause cytotoxicity for eukaryotic cells, suggesting no or minimal interaction with eukaryotic membranes. This contrasts with the cytotoxicity noticed for the three old and disused antibiotics, which caused significant loss of viability of THP-1 monocytes at the highest concentrations tested here, which are largely above those measured in the plasma of treated patients, owing to the high MIC of these drugs against P. aeruginosa. We had thus to limit the range of antibiotic concentrations tested in our concentration-response curves against intracellular bacteria in order to avoid a too massive loss of cells.

Considering then intracellular activity data, we noticed that all antibiotics displayed a bacteriostatic activity at concentrations close or slightly higher than their MIC (C s being considered as a measure of the intracellular MIC), despite the fact they all accumulate to some extent in eukaryotic cells [START_REF] Tulkens | Intracellular distribution and activity of antibiotics[END_REF] (Table S1). This is in line with our previous observations with other antibiotics in the same model [START_REF] Buyck | Pharmacodynamic evaluation of the intracellular activity of antibiotics towards Pseudomonas aeruginosa PAO1 in a model of THP-1 human monocytes[END_REF] or in similar models of intracellular infections by other bacterial species [START_REF] Barcia-Macay | pharmacodynamic evaluation of the intracellular activities of antibiotics against staphylococcus aureus in a model of thp-1 macrophages[END_REF][START_REF] Lemaire | Activity of finafloxacin, a novel fluoroquinolone with increased activity at acid pH, towards extracellular and intracellular Staphylococcus aureus, Listeria monocytogenes and Legionella pneumophila[END_REF], and was interpreted as denoting a poor intracellular bioavailability. While PAβN and NV731 did not increase this relative potency (minimal modification in C s ), NV716 caused a shift of the concentrations-response curves to lower concentrations, with a commensurate reduction in the C s , except for ciprofloxacin against PAO1 and PAO509, the MIC of which was already rather low. Thus, NV716 can decrease the MIC of antibiotics not only extracellularly, but also intracellularly. We did not measure the cellular concentration of NV716 in the cells, but these activity data suggest that it has access to intracellular bacteria in sufficient concentrations to exert its synergistic effect. The absence of gain in relative potency observed with PAβN and NV731 can be consecutive to their lower intrinsic effects on MICs (as observed in broth) or possibly also to a lower penetration inside the cells, which has not been investigated.

Turning then our attention towards the intracellular maximal efficacy, we observed, for antibiotics used alone, reductions of 2-2.5 log 10 CFU with ciprofloxacin, in accordance with our previous results [START_REF] Buyck | Pharmacodynamic evaluation of the intracellular activity of antibiotics towards Pseudomonas aeruginosa PAO1 in a model of THP-1 human monocytes[END_REF], and of 1-2 log 10 CFU for the other antibiotics, the lowest effect being observed for chloramphenicol and doxycycline against 205-2, indicating individual differences in responsiveness among strains. In a broader context, the extent of intracellular killing may also differ depending on the bacterial species. An intracellular E max of -1.8 log 10 CFU was noticed for rifampin against a small colony variant of S. aureus [START_REF] Nguyen | Intracellular activity of antibiotics in a model of human THP-1 macrophages infected by a Staphylococcus aureus small-colony variant strain isolated from a cystic fibrosis patient: pharmacodynamic evaluation and comparison with isogenic normal-phenotype and revertant strains[END_REF], but the activity of chloramphenicol and doxycycline has not been investigated in this model. Chloramphenicol demonstrated bacteriostatic activity against intracellular Salmonella Typhimurium at 10x MIC (37) while doxycycline reduced of 1.5 log 10 the intracellular CFU of Francisella tularensis after 24 h of incubation at a concentration close to its MIC [START_REF] Maurin | Bactericidal activities of antibiotics against intracellular Francisella tularensis[END_REF]. For fluoroquinolones, a maximal reduction of 2 to 4 log 10 CFU was observed in concentration-response experiments similar to those performed here against S. aureus, Listeria monocytogenes, Legionella pneumophila, Burkholderia thailandensis, Yersinia pseudotuberculosis, or Francisella philomiragia [START_REF] Barcia-Macay | pharmacodynamic evaluation of the intracellular activities of antibiotics against staphylococcus aureus in a model of thp-1 macrophages[END_REF][START_REF] Lemaire | Activity of finafloxacin, a novel fluoroquinolone with increased activity at acid pH, towards extracellular and intracellular Staphylococcus aureus, Listeria monocytogenes and Legionella pneumophila[END_REF][START_REF] Chalhoub | Influence of pH on the activity of finafloxacin against extracellular and intracellular Burkholderia thailandensis, Yersinia pseudotuberculosis and Francisella philomiragia and on its cellular pharmacokinetics in THP-1 monocytes[END_REF]. While PAβN and NV731 did not affect the efficacy of any antibiotic against none of the strains, NV716 systematically improved it (more negative E max ), suggesting an effect on bacterial responsiveness to antibiotics. We can most likely preclude interference related to the release of bacteria out of the cells before lysis or to the R1.6.remaining amount of antibiotics accumulated in cells if considering that (a) NV716 did not increase the cytotoxicity of antibiotics, and (b) charcoal was added to culture plates to adsorb residual antibiotic. Thus, the gain in intracellular efficacy observed in the presence of NV716 is rather suggestive of improved bacterial responsiveness to the drugs. We previously showed an increase in the intracellular efficacy of fluoroquinolones against an insertion mutant in a putative deacetylase in PAO1, which showed a lower persister character in planktonic cultures [START_REF] Khandekar | The Putative De-Nacetylase DnpA Contributes to Intracellular and Biofilm-Associated Persistence of Pseudomonas aeruginosa Exposed to Fluoroquinolones[END_REF]. Here, we found that NV716 was capable of decreasing the proportion of persisters selected by ciprofloxacin in a stationary phase culture at concentrations for which it did not cause any bacterial killing by itself R2.3and also of reducing at subMIC concentrations the number of persisters when combined with ciprofloxacin but not when used alone, suggesting a mode of action unrelated to a direct bactericidal effect but rather to a resensitization of persisters to the antibiotic [START_REF] Liebens | Identification of 1-((2,4-Dichlorophenethyl)Amino)-3-Phenoxypropan-2-ol, a Novel Antibacterial Compound Active against Persisters of Pseudomonas aeruginosa[END_REF]. Of interest, other molecules capable of disrupting membrane integrity also reduced the residual fraction of persisters upon exposure to antibiotics for different bacterial species [START_REF] Defraine | 1-((2,4-Dichlorophenethyl)Amino)-3-Phenoxypropan-2-ol Kills Pseudomonas aeruginosa through Extensive Membrane Damage[END_REF][START_REF] Nicol | Anti-persister activity of squalamine against Acinetobacter baumannii[END_REF][START_REF] Naclerio | Multiple ways to kill bacteria via inhibiting novel cell wall or membrane targets[END_REF]. One of them (SPI009; 1-((2,4-Dichlorophenethyl)Amino)-3-Phenoxypropan-2-ol)) was also shown to increase the efficacy of ciprofloxacin against intracellular P. aeruginosa [START_REF] Defraine | Antibacterial Activity of 1-[(2,4-Dichlorophenethyl)amino]-3-Phenoxypropan-2-ol against Antibiotic-Resistant Strains of Diverse Bacterial Pathogens, Biofilms and in Preclinical Infection Models[END_REF]. This work suffers from some limitations. Firstly, we used a single phagocytic cell line (THP-1 monocytes), which does not necessarily represent the repertoire of cells infected by P. aeruginosa in vivo. The reason for selecting this permissive cell line was that it allows assessing the effect of antibiotics without interference of cell defense mechanisms. Secondly, we did not measure the cellular accumulation of antibiotics and EPIs in our experimental conditions, but setting-up the necessary methodologies would represent a work by itself. Thirdly, we did not study in details the occurrence of intracellular persisters, the aim of the study being rather oriented towards an in-depth pharmacodynamic evaluation of the possible interest of EPIs to restore the activity of old and disused antibiotics against intracellular P. aeruginosa.

If we thus examine our data in a more clinically-oriented perspective, we see that NV716 is capable of improving the activity of all antibiotics in the range of concentrations achieved in the serum of patients as soon as its extracellular concentration exceeds 3 µM. Ten times higher concentrations allow to reach a 2-3 log 10 CFU decrease when combined with antibiotics at their human C max , pleading for examining the possible interest of this compound in vivo.

MATERIALS AND METHODS

Bacterial strains and culture media

Table 1 shows the reference and clinical strains used in the study. R2.m2BV-1 has been isolated in 2006 at the Queen's University Belfast, Belfast, UK, from a patient with cystic fibrosis. It shows a truncation in MexB (672 amino acids instead of 1046) that drastically reduced the activity of efflux transporter and its impact of the MIC of the preferential MexAB-OprM antibiotic substrate temocillin [START_REF] Chalhoub | Mechanisms of intrinsic resistance and acquired susceptibility of Pseudomonas aeruginosa isolated from cystic fibrosis patients to temocillin, a revived antibiotic[END_REF]. 205 has been isolated in 2012 at the University Hospital Münster, Münster, Germany, from a patient with cystic fibrosis [START_REF] Mustafa | Antimicrobial Susceptibility of Pseudomonas aeruginosa Isolated from Cystic Fibrosis Patients in Northern Europe[END_REF], and shows a fully active MexAB-OprM efflux pump, based on the elevated MIC of temocillin.

Bacteria were R2.m1incubated overnight at 37°C in Tryptic Soy Agar (TSA; VWR; Radnor, PA). A single colony was then R2.m1added in 10mL cation-adjusted Mueller-Hinton Broth (CA-MHB; BD Life Sciences, Franklin Lakes, NJ) and incubated at 37°C overnight under gentle agitation (130 rpm). TSA supplemented with 2g/L charcoal (Sigma-Aldrich, St Louis, MO) was used for CFU counting (colonyforming unit). Cell culture media (RPMI-1640) and human and fetal bovine sera were from Gibco/ThermoFisher Scientific (Waltham, MA).

Antibiotics and Inhibitors

Chloramphenicol (potency, 98%), doxycycline (potency, 98%), and rifampin (potency, 98%) were obtained as microbiological standards from Sigma-Aldrich (St Louis, MO), ciprofloxacin HCl (potency, 89%) from Bayer (Leverkusen, Germany) and gentamicin sulfate (potency, 60.7%), from PnReac AppliChem (Darmstadt, Germany). Table S1 shows relevant properties of the antibiotics used in this study. The reference efflux pump inhibitor Phe-Arg β-Naphthylamide (PAβN; potency, 98%) was purchased from Sigma-Aldrich. The hydrosoluble hydrochloride polyamino-isoprenic salt derivatives NV716 and NV731 were synthesized at Aix-Marseille University [START_REF] Brunel | Polyamino geranic derivatives as new chemosensitizers to combat antibiotic resistant gram-negative bacteria[END_REF]. Stock solutions of these inhibitors were prepared in dimethyl sulfoxide (DMSO) at concentrations of 96 mM [50 mg/mL] for PAβN or in water at 10 mM [4 mg/L] for NV716 and NV731, respectively, and stored at -20°C until use.

Susceptibility Testing

MICs of antibiotics against P. aeruginosa were measured by serial 2-fold microdilution in MHB-CA according to CLSI guidelines in control conditions or in the presence of 38 µM [20 mg/L](21) PAβN or of 10 µM [4 mg/L] NV731 or NV716 [START_REF] Borselli | Polyamino-Isoprenic Derivatives Block Intrinsic Resistance of P. aeruginosa to Doxycycline and Chloramphenicol In Vitro[END_REF].

Cytotoxicity assessment (Trypan blue exclusion assay)

THP-1 cells (7.5 x 10 5 cells per mL) were incubated for 24 hours in 96-wells plates with the antibiotics or inhibitors under study over a wide range of concentrations. At the end of the incubation, viability was assessed using trypan blue exclusion test (vital colorant excluded from viable cells) using a previously described method [START_REF] Buyck | In Vitro Models for the Study of the Intracellular Activity of Antibiotics[END_REF], with specific adaptations to the purpose of our study. Briefly, 50 μL of trypan blue reagent was added to 50 μL of cell suspension. After 10 min of incubation at 37°C, non colored (viable) cells were counted using a Fuchs-Rosenthal counting chamber (Tiefe 0.2mm). Cells were counted in the same surface for all conditions (1 big square made of 16 single squares (0.0625 mm 2 for each single square). The percentage of cytotoxicity was evaluated based on the reduction in the number of living cells according to the following equation:

.

Intracellular Infection

All experiments were performed using human THP-1 monocytes cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) based on a previously established protocol [START_REF] Buyck | Pharmacodynamic evaluation of the intracellular activity of antibiotics towards Pseudomonas aeruginosa PAO1 in a model of THP-1 human monocytes[END_REF]. In brief, bacteria were opsonized by incubation during 1 h in RPMI-1640 medium supplemented with 10% human serum at 37°C with gentle agitation (130 rpm). Phagocytosis of opsonized bacteria was then allowed for 2 h using a bacterium:cell ratio of 10:1, after which non-phagocytosed bacteria were eliminated by incubation for 1 h with gentamicin at 50x MIC. After 3 washings with PBS, infected cells were resuspended in the original volume of RPMI-1640 with 10% FBS. The post-phagocytosis inoculum was consistently 5 to 7 x 10 5 CFU/mg of cell protein (defined as time zero). Infected cells were then incubated for 24 h with antibiotics over a broad range of concentrations (0.003-100x their MIC) in 12well plates. After 24 h of incubation, infected cells were pelleted by centrifugation, washed once with PBS at room temperature to eliminate extracellular bacteria and collected in 1 mL distilled water, to achieve complete cell lysis. Lysates were used for determining CFU counts by spreading on agar supplemented with 2g/L charcoal (to avoid carry-over effect, especially in samples that had been incubated with high antibiotic concentrations [R1.5.no difference in colony counts was observed between plates supplemented or not with charcoal for samples exposed to low antibiotic concentrations, ruling out any interfering effect of charcoal in the assay]) and protein content using a commercially available kit (Bio-Rad DC Protein Assay, Bio-Rad Laboratories, Hercules, CA). The activity was expressed as the change in CFU (normalized by mg of cell protein) from the initial inoculum after 24 hours of incubation. The data were used to fit a sigmoidal function and calculate pharmacodynamic parameters based on the corresponding Hill-Langmuir equation (apparent static concentrations [C s ], i.e.

extracellular concentration resulting in no apparent intracellular growth, and maximal relative efficacy [E max ], i.e. maximal decrease in bacterial counts compared to the post-phagocytosis inoculum as extrapolated for an infinitely large antibiotic concentration).

Persister assay and isolation

A single colony from overnight culture of PAO1 and PAO509 on TSA was R2.m1.added to in 200mL MHB-CA in a 1 L flask and incubated for 24 h under agitation (130 rpm) at 37°C. 10 mL of the bacterial suspension were then incubated with 50x MIC ciprofloxacin for 5 hours at 37°C under agitation (130 rpm), in the presence of or in the absence of NV716. Aliquots were serially diluted in PBS and spread on TSA supplemented with 2 g/L charcoal for CFU counting. The persister fraction was defined as the number of CFU growing at the end of this experiment as compared to the initial inoculum [START_REF] De Groote | Novel persistence genes in Pseudomonas aeruginosa identified by high-throughput screening[END_REF]. R2.3For experiments using isolated persisters, stationary-phase cultures were incubated with ciprofloxacin (50x MIC) during 5 hours, i.e. condition for which a plateau of killing was reached. The remaining bacteria were washed twice with sterile Phosphate Buffered Saline (PBS), centrifuged (5,200 g, 15 min, 4°C) and used for killing assays [START_REF] Liebens | Identification of 1-((2,4-Dichlorophenethyl)Amino)-3-Phenoxypropan-2-ol, a Novel Antibacterial Compound Active against Persisters of Pseudomonas aeruginosa[END_REF].

Curve fittings and statistical analyses.

Curve S1).

The combination with CIP at its C max could not be studied, CFU counts being already close to the limit of detection with the antibiotic alone. The horizontal lines highlight respectively a static effect (black dotted line), the effect of the antibiotic alone at its C min (red dotted line) or at its C max (red plain line) and the vertical dotted line, the MIC of NV716 in RPMI-1640 (25 µM). All data are means ± SEM (triplicates from 3 experiments).

Figure 2 .Figure 3 .Figure 4 .

 234 Figure 2. Concentration-response curves of antibiotics alone or combined with NV716 against intracellular PAO1 in a model of THP-1 monocytes. The graphs show the changes in CFU counts per mg of cell protein from the initial, post-phagocytosis inoculum after 24 h of incubation with increasing extracellular concentrations of antibiotics alone (ciprofloxacin [CIP], doxycycline [DOX], chloramphenicol [CHL] or rifampin [RIF]) or combined with a fixed concentration (10 µM; 4 mg/L) of NV716. The horizontal dotted line highlights a static effect and the vertical dotted line, the MIC of each antibiotic. All data are means ± SEM (triplicates from 3 experiments).

Table 1 . MICs of antibiotics and adjuvants (EPIs) alone or combined against reference strains and clinical isolates in broth.

 1 fittings were performed with GraphPad Prism (version 8.4.3) software for Windows (GraphPad Prism Software, San Diego, CA). Pharmacodynamic parameters were calculated based on Hill equations of concentration-response curves and statistical analyses were executed with GraphPad InStat 3 version 3.10 (GraphPad Prism Software) or Graphpad Prism version 8.4.3.

					MIC (mg/L) a and (fold-dilution decrease vs antibiotic alone)
	Strains Phenotype	Reference Antibiotics b	AB alone (mg/L)	+PAβN (38μM) [20mg/L]	PAβN alone (μM)	+NV731 (10µM) [4mg/L]	NV731 alone (μM)	+NV716 (10µM) [4mg/L]	R1.6NV716 alone (μM)
			CIP	0.25	0.06 (2)		0.06 (2)		0.03 (3)
	PAO1	Wild-type	(47)			>200		>250

Cytotoxicity of EPIs and antibiotics as determined by trypan blue exclusion test.

  Left: cytotoxicity of efflux inhibitors in non-infected THP-1 cells incubated with increasing concentrations of NV716, NV731, or PAβN during 24 h. Right: cytotoxicity of antibiotics in THP-1 cells infected by PAO1 and incubated with increasing concentrations of ciprofloxacin (CIP), doxycycline (DOX), rifampin (RIF), or chloramphenicol (CHL) during 24 h. R1.6.Antibiotic concentrations are expressed in multiples of their MIC against PAO1 (Log 10 scale)The tick horizontal dotted lines show the percentage of cell mortality measured in untreated infected cells. The thin dotted line shows a 50% cell mortality threshold, allowing to estimate IC 50 values for each compound (see FigureS2and TableS2for these values). The vertical arrows in the left panel show the inhibitor concentrations used in intracellular experiments R1.2.(10 µM for NV716 and 38 µM for PAβN). All data are means ± SEM (triplicates from 3 independent experiments).

Acknowledgements

We are thankful to V. Mohymont, K. Santos-Saial, and V. Yfantis for proficient technical assistance. W.W.G. received a PhD grant from the China Scholarship Council (CSC). F.V.B. is Research Director from the Belgian Fonds de la Recherche Scientifique (FRS-FNRS).

Funding

This work was supported by the Belgian FRS-FNRS (grant T.0189.16). We thank Campus France and Wallonie Bruxelles International (WBI) for financing a PHC Tournesol program (France-Belgium 2018-2019).