# Pseudopaline-mediated zinc uptake by Pseudomonas aeruginosa determines several clinically relevant phenotypes and infection outcomes

L Bosc, Thomas Sécher, G Ball, Déborah Le Pennec, M Tribout, M Ba, Y Bai, L Ouerdane, P Arnoux, Y Denis, et al.

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L Bosc, Thomas Sécher, G Ball, Déborah Le Pennec, M Tribout, et al.. Pseudopaline-mediated zinc uptake by Pseudomonas aeruginosa determines several clinically relevant phenotypes and infection outcomes. Infection and Immunity, 2026, 10.1101/2025.01.08.631948. https://hal-05458361

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1 **Pseudopaline-mediated zinc uptake by *Pseudomonas aeruginosa* determines several clinically**
2 **relevant phenotypes and infection outcomes.**

3 Lola Bosc¹, Thomas Sécher², Geneviève Ball¹, Deborah Le Pennec², Mathilde Tribout¹, Moly Ba³,

4 Yingjie Bai⁴, Laurent Ouerdane⁵, Pascal Arnoux⁶, Yann Denis⁷, Xiaoguang Lei⁴, Christophe Bordi³,

5 Nathalie Heuzé-Vourc'h², Susanne Häussler⁸,⁹,¹⁰, Nicolas Oswaldo Gomez¹,⁸* and Romé Voulhoux¹*.

6

7 ¹CNRS / Aix-Marseille Université LCB UMR7283, Marseille, France.

8 ²INSERM, Respiratory Disease Research Centre, U1100 / University of Tours, Tours, France.

9 ³CNRS / Aix-Marseille Université LISM UMR7255, Marseille, France.

10 ⁴Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking
11 University, Beijing, P. R. China.

12 ⁵Université de Pau et des Pays de l'Adour, CNRS, IPREM-UMR5254, Hélioparc, Pau, France.

13 ⁶CEA/CNRS/Aix-Marseille Université, BIAM UMR7265, CEA Cadarache, Saint-Paul-lez Durance,
14 France.

15 ⁷CNRS / Aix-Marseille Université IMM UMR7283, Marseille, France.

16 ⁸Helmholtz Centre for Infection Research GmbH, Braunschweig, Germany.

17 ⁹Institute for Molecular Bacteriology, Center of Clinical and Experimental Infection Research
18 (TWINCORE), a joint venture of the Hannover Medical School and the Helmholtz Center for
19 Infection Research, Hannover 30625, Germany

20 ¹⁰Department of Clinical Microbiology, Copenhagen University Hospital—Rigshospitalet,
21 Copenhagen 2100, Denmark

22

23 *Corresponding authors

24 Emails: voulhoux@imm.cnrs.fr and nicolasoswaldo.trinler@helmholtz-hzi.de

25

26 ¶These authors contribute equally to the work

27

28 **Short title:** Pseudopaline-mediated pathogenicity of *Pseudomonas aeruginosa*

29
30

31 **ABSTRACT**

32 Biological metals are vital trace elements required by metalloproteins, which are involved in virtually 33 every cellular, structural and catalytic function of the bacterial cell. Bacterial pathogenesis involves a 34 tug-of-war between the host's nutritional immunity sequestering essential metals and the invading 35 pathogens that deploy adapted high-metal affinity uptake strategies in order to efficiently overcome 36 these defence mechanisms. Metallophores are high-affinity, low-molecular mass, metal chelators 37 produced and secreted by bacteria to access chelated metals from the scarcest environments. 38 Pseudopaline is a metallophore produced and secreted by *Pseudomonas aeruginosa* to acquire zinc 39 when the bioavailability of this metal is severely restricted, as in the presence of a strong metal 40 chelator such as EDTA, or during infections when the nutritional immunity of the host is active, in 41 mammals through the production of the zinc-binding protein calprotectin. **We show that when facing 42 strong metal scarcity, the alternative Znu zinc uptake pathway becomes ineffective and only the 43 pseudopaline pathway is capable of supplying the bacteria with the necessary zinc to maintain their 44 growth,** establishing that the pseudopaline pathway is the last-resort and unique pathway for the 45 bacteria to acquire zinc under such restricted growth conditions. Based on this statement, the present 46 study explores the pleiotropic role of pseudopaline-mediated zinc acquisition on clinically relevant 47 phenotypes such as biofilm formation and antibiotic tolerance, and its capacity to determine infection 48 outcomes using murine and cell-culture models. The expression of pseudopaline-dependent 49 phenotypes in such a diversity of biological contexts demonstrates the essentiality of this specific 50 metal uptake system for *P. aeruginosa* pathogenicity during infection. We therefore identify this 51 machinery as a promising therapeutic target for *P. aeruginosa*'s infections. 52

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54 **AUTHOR SUMMARY**

55 The host-pathogen interface is a biological niche in which two entities competes for essential 56 resources. The host's nutritional immunity restrict access to metals, while a successful pathogen 57 overcomes these restrictions using dedicated uptake pathways. Pseudopaline is a high-affinity
metallophore allowing *Pseudomonas aeruginosa* to acquire zinc in chelated environments. We
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demonstrate that this pathway is the last-resort solution to acquire zinc for this dreadful pathogen. The
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capacity to provide this metal to zinc-metalloproteins drives clinically relevant phenotypes, such as the
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capacity to form a mature and antibiotic-tolerant biofilm, or to determine the outcome of an infection.
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These results place pseudopaline as a potential drug target for blocking *P. aeruginosa* pathogenic
capacity and resensitizing established biofilm to classic antibiotic treatment.
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62

63 **INTRODUCTION.**

Biological metal ions are essential across all kingdoms of life [1]. Their unique chemical
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features provide structural and catalytic properties to a wide-range of metalloproteins, broadly
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distributed in living organisms [2]. Zinc is, after iron, the second most abundant metal in living cells,
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involved in 6 to 9% of the prokaryotic and eukaryotic proteomes, respectively [3, 4]. In bacteria, zinc
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metalloproteins are involved in several functions essential to growth, such as central metabolism or
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DNA replication and repair, but also to pathogenicity and virulence alike virulence factors, antibiotic
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resistance, motility, extracellular protease activities, and modulation of the immune response [5, 6].
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71 Biological metals are inorganic resources that need to be acquired by bacteria from the environment
72 where they are often rare, either because of low concentration, restricted bioavailability, or
73 competition between the inhabitants of the same ecological niche. In the specific biotope that the host-
74 pathogen interface represents, both protagonists have developed competing strategies for either
75 sequestering or acquiring the much-coveted metal. Hence, the host synthetizes high-affinity chelators
76 which withhold essential metals through the process referred to as “nutritional immunity” [7].
77 Conversely, pathogens have evolved powerful metal uptake systems involving high affinity secreted
78 molecules called metallophores [8] that often determine their ability to survive during infections.
79

Thus, invading bacterial pathogen face a particularly dire restriction of metals, and specifically zinc
80 due to its sequestration by the host zinc-binding protein calprotectin [7, 9-12]. During pathogen driven
81 inflammation in general and in cystic fibrosis (CF) patients in particular, the levels of calprotectin in
82 bronchoalveolar lavage fluid (BAL) and serum are elevated and used as a biomarker of diseases
83 correlating with exacerbation [13-15]. To responsively overcome the growth-limiting zinc deficiency
84 in tissues, imposed by nutritional immunity, the human pathogen, *Pseudomonas aeruginosa* has
developed effective zinc import mechanisms judiciously de-repressed under zinc deficiency by the Zur regulator (formerly known as np20). Zur is a cytoplasmic repressor protein, that prevents gene transcription by binding to the zur box of its promoter region and magnitude of repression is proportional to the intracellular zinc concentration [16]. Thus, the lower the intracellular zinc concentration, the less active Zur is, and the greater the transcription of the initially repressed gene [17]. P. aeruginosa is therefore able to sense zinc scarcity in its environment in order to react by gradually de-repressing the Zur regulon, comprising, among others, the Znu and Cnt (also called Zrm) zinc import mechanisms [10, 18]. The Znu pathway, widely distributed in bacteria, recognizes and imports free zinc in the cytoplasm via the periplasmic substrate-binding protein ZnuA and the inner membrane importer ZnuBC [18, 19]. In order to acquire zinc when it is not free but sequestered by a powerful chelator such as Ethylenediaminetetraacetic acid (EDTA) or calprotectin, P. aeruginosa relies on the dedicated Cnt pathway involving the high zinc affinity zincophore pseudopaline [20-23]. Utilizing this pathway, pseudopaline is first synthesized in the cytoplasm in a two-step process by CntL and CntM. In the first step, CntL produces the intermediate yNA using S-adenosylmethionine (SAM) and L-histidine as substrates which exhibits chemical homology with xNA in staphylopine biosynthesis and nicotianamine (NA). This is followed by a reductive condensation of the yNA intermediate with a molecule of α-ketoglutarate, catalyzed by CntM, to produce pseudopaline [21, 24]. The metallophore is then secreted in the milieu through the bacterial envelope by the CntI inner membrane exporter coupled to the outer membrane MexAB-OprM efflux pump. The extracellular zinc-bound pseudopaline is finally recovered by the bacteria through its specific outer membrane receptor, CntO. Insofar, the fate of pseudopaline after import in the periplasm remains unknown, and could involve a dedicated ABC importer as suggested by the genetic organization observed for other "opine-type" metallophores found in γ proteobacteria [25]. Numerous transcriptomic studies have reported a systematic up-regulation of the cnt operon under the infection conditions of CF patients or in medium reproducing these conditions [10, 26-32]. Moreover, the cnt genes are particularly conserved in P. aeruginosa clinical isolates [33], and the promoter of these genes is the target of mutations during adaptation to CF patients which often increase the expression of the cnt genes in evolved strains [34]. Together with the requirement of the Cnt pathway
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for *P. aeruginosa* growth in airway mucus secretion [35], these data indicate that the Cnt pathway is
not only induced but also critical for *P. aeruginosa* to successfully establish an infection.

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We have developed a minimal growth medium called Minimal Chelated Media (MCM) consisting of
minimal succinate (MS) medium supplemented with 100µM of the divalent metal chelator EDTA
[21]. **We show that under such severe zinc sequestering conditions, the alternative Znu zinc uptake
pathway becomes ineffective, rendering the pseudopaline mandatory for efficient zinc acquisition
from the medium since a *P. aeruginosa* ΔcntL strain - with a functional Znu pathway - is unable to
maintain a normal intracellular zinc homeostasis.** In line with this statement, we demonstrate in this
study the pleiotropic role played by pseudopaline in *P. aeruginosa* infections to overcome metal
scarcity. We first establish its central role to secure the intracellular zinc concentration required for
optimal planktonic growth of the pathogen in MCM medium, **independently of the Znu machinery**.

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Moreover, we show that in such zinc limiting conditions, the Cnt pathway is necessary to establish a
mature biofilm which provides protection from antibiotic exposure. We further explored the role of
pseudopaline in environments encountered during *P. aeruginosa* infection of the host. Specifically,
during bacterial interaction with macrophages, we demonstrate its role in adherence, phagocytosis,
intracellular survival and induction of immune responses. Additional *in vivo* experiments in a mouse
model of *P. aeruginosa* lung infection confirmed the requirement for this zincophore to colonize the
lung and develop a full virulent phenotype. Altogether, our data revealed that the pseudopaline
metallophore produced and secreted by *P. aeruginosa* gives it the exceptional ability to maintain a
proper intracellular zinc homeostasis under conditions of extreme metal deficiency, conditions
encountered on several occasions during the nutritional immunity imposed by the host during
infection.

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## RESULTS

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### 1. The Cnt pathway is the last resort pathway safekeeping zinc homeostasis in severe metal-sequestered growth condition.

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We previously showed that under conditions of low zinc bioavailability due to extracellular
sequestration by EDTA, as the one created in MCM [21], the normal intracellular zinc level and

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optimal planktonic bacterial growth are ensured by pseudopaline since in its absence - i.e. in the ΔcntL strain - the intracellular zinc content decreases and bacterial growth is impaired compared to the parental wild type (WT) strain (Fig. 1A, 1B and as previously shown [20]). Considering the described role of the Znu machinery as a Zn importer [18, 19], we further question whether this pathway supports or at least contributes to the residual growth of the ΔcntL strain in MCM. The similar MCM intracellular zinc concentration and generation times, reported figure 1A and 1B between the WT and the single ΔznuA strain on the one hand and the single ΔcntL and the double ΔcntL/ΔznuA strains on the other hand, indicate that this is not the case. These data demonstrate that under conditions of extremely low zinc availability, such as those created in MCM, the free zinc Znu import pathway is no longer operational, and the necessary zinc for the optimal growth of P. aeruginosa is exclusively imported by the zincophore pseudopaline. We next confirmed that the growth defect of the ΔcntL strain depends exclusively on the presence of pseudopaline as supplementation by exogenous addition of 1μM of synthetic pseudopaline restores the growth defect of the ΔcntL strain (Fig. 1C, grey curve). This synthetic supplementation of the ΔcntL strain with externally acquired pseudopaline not only excludes any polar effect of the deletion but also confirms that the mode of action of pseudopaline involves an extracellular acquisition stage. This agrees with the three-step cycle of synthesis, secretion and recovery that we previously proposed for the pseudopaline-dependent zinc uptake pathway [20]. Additional experiments performed with different concentrations of externally added synthetic pseudopaline indicate that optimal WT growth is already achieved at 1 μM (Fig. 1C), revealing that in the metal limiting conditions encountered in MCM, the natural extracellular concentration of pseudopaline do not exceed 1 μM. Moreover, the observation that the addition of higher concentrations of pseudopaline leads to better growth than that observed in the WT strain also indicates that zinc recovered by pseudopaline under these conditions is the limiting factor for growth in MCM.

Despite the ability of pseudopaline to bind other divalent metals [23], we also showed that the growth defect of the ΔcntL strain in MCM is effectively due to zinc deficiency since it is specifically restored by extracellular zinc supplementation (Fig. 1D) and not by other divalent metals (Fig. S1).
Altogether, our data based on planktonic growth indicate that in MCM, zinc is scarce and only the secreted pseudopaline can import enough of this metal to guaranty its intracellular levels, needed to feed the zinc proteome and ensure optimal planktonic bacterial growth.

## 2. Pseudopaline is required for proper biofilm formation in MCM.

In order to evaluate the implication of pseudopaline in clinically relevant conditions, we explored zinc acquisition in biofilm formation, the most common lifestyle reported during infections [36]. This multicellular organisation protects the bacterial community from external onslaughts such as phagocytosis or killing by antibiotics and allows for establishing chronic recalcitrant infections. We compared biofilm formation of WT and ΔcntL strains grown in MCM using several quantitative and qualitative approaches. We first examined biofilm formation in 24-wells polystyrene plates after 24h of static growth in MCM at 30°C. The ΔcntL strain, unable to produce pseudopaline, shows a significant 30% reduction of crystal-violet stained biofilm compared to the WT (Fig. 2A). Adding synthetic exogenous pseudopaline to the ΔcntL strain showed biofilm restoration, confirming that alike in planktonic growth, this biofilm phenotype is also exclusively due to the absence of pseudopaline.

To correlate the biofilm default with intracellular zinc deficiency and since intracellular zinc concentration cannot be precisely measured in 24-wells polystyrene plates, we indirectly evaluated intracellular zinc level by monitoring CntO production as reporter of intracellular zinc concentration. Planktonic transcriptional data, together with metal and protein level measurements, all presented figure S2, indeed revealed a direct relationship between intracellular zinc concentration and the amounts of CntO protein produced (Fig. S2A) or the corresponding transcripts (Fig. S2B). The use of this read-out in biofilm samples first confirmed the zinc default in the ΔcntL strain as well as the import of zinc by the exogenously added pseudopaline respectively illustrated by higher and absence of CntO protein detection compared to the wild type level (Fig. 2A, bottom). The restoration of biofilm in the mutant by 100 µM pseudopaline to a level significantly higher than that observed in the WT also supports that, similar to the planktonic growth (Fig. 1C), pseudopaline-dependent zinc import is also the limiting factor for biofilm formation. This result shows that in *P. aeruginosa* biofilms
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grown under zinc-limited conditions, the zinc homeostasis required for its optimal formation is
exclusively ensured by pseudopaline.

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In order to ensure that this biofilm defect in absence of pseudopaline is not only due to the growth
defect (Fig. 1A) and to assess the qualitative impact of the absence of pseudopaline on biofilm
formation in MCM, we used confocal microscopy to follow its formation and organization in flow cell
experiments using genetically engineered strains expressing constitutively the green fluorescent
protein (GFP), in *cis* at a neutral site on the chromosome. We imaged biofilms formed by WT and
ΔcntL strains after 4, 7 and 10 days of incubation in a flow cell chamber. Biomass ratios and thickness
measurements presented figure 2B show significant reductions in cell number and thickness of the
biofilm in the absence of pseudopaline after 7 and 10 days of incubation. From a qualitative point of
view, the ΔcntL strain formed biofilms with an impaired morphology compared to the parental WT
strain, unable to form the classic pilar- and mushroom-shaped architecture (Fig. 2B). We wondered if
these morphological alterations were indicative of variation in antibiotic tolerance, a property
described for *P. aeruginosa* biofilms [37]. To do so, we measured the capacity of these biofilms to
withstand the offence of tobramycin treatment, an aminoglycoside antibiotic commonly used by
inhalation with CF patients suffering from chronic infection [38]. D11 biofilms were treated for 24h
with 20 µg/mL of the aminoglycoside. The live (GFP-green) and dead (propidium iodine-red) dies
applied at D13 highlight a significantly higher sensitivity to tobramycin for the ΔcntL strain compared
to the WT (Fig. 2B right panels). **This observation was not reported in planktonic growth in MCM**
**where WT and ΔcntL strains were both sensitive from 4 µg/mL (Fig. S3).** Together, we infer that the
absence of pseudopaline not only delays biofilm formation but also significantly impairs its structural
properties and its ability to confer tolerance to an antibiotic. The negative effect of the absence of
pseudopaline on the volume and quality of the biofilm in metal-scarce conditions is most likely
attributable to the impact of the reduced intracellular zinc level - reported by the higher amount of
CntO detected (Fig. 2A lower panel) - on the proper function of bacterial zinc-dependent
metalloproteins involved in this process [39].

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The pseudopaline-dependent growth and biofilm phenotypes observed in this study (Figs. 1 & 2)
highlight the broad and exclusive importance of this metallophore in environments where zinc is
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scarce, such as the one created in MCM or reported during infections where nutritional immunity leads to serious metal deficiencies in general, and zinc in particular [7, 9-11]

### 3. The impact of pseudopaline on the sequence of events during macrophage infection suggests multiple roles for this molecule

To further explore the role of the pseudopaline at different steps of a host-cell infection process, we used RAW 264.7 macrophage-like murine cells [40]. We started by quantifying phagocytic uptake through a gentamycin-protection assay and observed that the ΔcntL strain pre-cultured in MCM is significantly less phagocyted than the parental WT strain, a phenotype mostly recovered in the complemented strain (Fig. 3A). We hypothesized that this reduced phagocytic uptake could be due to a reduced adherence of the ΔcntL strain to the cell membrane of macrophages. To address this hypothesis, we blocked phagocytosis by adding 10 µM cytochalasin D to the culture medium before infection, and determined adherent bacterial cells. We observed that the ΔcntL strain indeed showed a decreased adherence to the macrophages (Fig. 3B), thus explaining, at least in part, the reduced phagocytic uptake.

Following phagocytosis, macrophages employ several strategies to clear bacterial cells, including both nutrient starvation and metal ion toxicity [41]. We evaluated the role of pseudopaline on bacterial survival in the phagosome by measuring the number of colony-forming-units (CFU) recovered 6 hours post-infection (hpi). To account for the decreased phagocytosis of the ΔcntL strain compared to the WT strain, we normalized the survival to the number of CFU phagocyted 2 hpi. Under these phagocytosis-independent conditions, the ΔcntL strain shows a significant reduced survival than the WT (Fig. 3C). Surprisingly, while all our phenotypes insofar where complemented in the ΔcntL::cntL strain, this was not the case for bacterial survival in macrophages, although the complemented strain produces enough pseudopaline to satisfy the operational requirements of this uptake pathway and supply enough zinc to maintain metal-homeostasis. We also previously observed a significant reduced production of pseudopaline in this strain [21], which may result from the transcriptional reduction of its corresponding cntL biosynthesis gene, observed in the present study (Fig S4). To assess whether pseudopaline could have an alternative role as a buffering agent against metal intoxication in the phagolysosome, we quantified the survival of a ΔcntO strain. Indeed, in the absence of its outer
membrane receptor, pseudopaline is produced but cannot be recovered by the bacteria hereby phenocopying the zinc deficiency of our ΔcntL strain but maintaining the role of the secreted pseudopaline as a buffering agent. We observe that a ΔcntO mutant has a better survival than the ΔcntL strain and no statistical difference with the parental WT strain (Figure 3C). All together, these informations suggest that, unlike all the other phenotypes described in this manuscript, the impact of pseudopaline on intracellular survival could arise from a dual use of this molecule, and in this specific case results from a buffering strategy to counter metal ion toxicity. Of note and considering the ability of pseudopaline to bind other divalent metals [23], we suppose that this role could extend beyond its zinc binding properties to other transition metals and therefore extend the spectrum of its buffering property.

Additionally, we also observe that, during macrophage infection, a ΔcntL strain is less cytotoxic (Fig 3D) and less immunogenic than the parental one. This is shown by the decreased production of TNF-α (Fig. 3E) and IL6 (Fig. 3F) produced by macrophages when infected by the ΔcntL strain. Owing to our knowledge of the reduced Zn-metallome of this strain, the missing cofactor of several proteins involved in these processes are impacting these properly-complemented phenotypes.

These results demonstrate the multiple roles of pseudopaline on the sequence of events during macrophage infection. They also confirm that the nutritional zinc deficiency imposed on the pathogen by the macrophage triggers the induction of the pseudopaline pathway, which therefore proves to be mandatory for the expression of its full pathogenic power throughout the macrophagic infection. Pseudopaline acts as a zinc-cargo able to displace this atom from otherwise bio-unavailable source to feed the operating-requirement of various zinc-binding proteins which are particularly relevant during the interaction with host-cells. Pseudopaline can also be used as a buffering agent against burst of ions, as it is the case in the phagosome. Either roles are key during the interaction with the host and underline the critical role of pseudopaline in establishing pathogenicity.

### 4. Pseudopaline plays a critical role during *P. aeruginosa* pulmonary mouse infection.

To further extend our cell culture-based findings to an *in vivo* animal model with a fully functional immune system, we used a well-established mouse pulmonary infection model [42]. Mice were
infected by pulmonary instillation of MCM-grown WT and a mutant strain devoid of the whole
*cntOLMI* operon, referred as *Δcnt*. Several markers including body weight, clinical score and survival
were recorded during the infection. While all mice infected with the WT strain suffered from severe
conditions characterized by continuous body-weight loss (Fig. 4A), increased clinical score (Fig. 4B)
and > 50% of death 4 days post-infection (Fig. 4C), the mice infected with the *Δcnt* strain exhibited
significantly milder evidence of susceptibility, including regain of body-weight loss 2 days post-
infection (Fig. 4A), less severe clinical symptoms (Fig. 4B) and almost full survival after 7 days post-
infection (Fig. 4C). To gain insight into the mechanisms that contributed to the lower virulence
associated with the *Δcnt* mutant, we analysed the host's antibacterial responses 48 hpi. Mice infected
with the WT strain showed significantly higher bacterial load in broncho-alveolar fluid (BAL) and
lungs as compared to *cnt* mutant infected animals (Figure 4D). Given that pneumonia is intimately
associated with lung inflammation, we evaluated the production of IL6, a prototype pro-inflammatory
cytokine, in the BAL and in the lungs (Fig. 4E). As observed with macrophage infections, the present
analysis revealed that IL6 was significantly reduced in the animals infected by the *cnt* mutant as
compared to the WT. Altogether, these data confirm the attenuation of virulence of the pseudopaline-
deficient *P. aeruginosa* strain in a mouse model of lung infection.

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DISCUSSION

Zinc fulfils multiple functions in living cells and its intracellular concentration must therefore
constantly be finely regulated, whatever the environment. In *P. aeruginosa*, at least three different
levels of zinc import processes are operating, depending on its scarcity. When zinc is abundant in the
milieu, its entry is mediated by passive diffusion across the outer membrane followed by low affinity
import mechanisms to reach the cytoplasm, as already reported for *E. coli* [43]. When zinc starts to run
out, the Zur regulon is consequently induced leading to the activation of the dedicated Znu and Cnt
high affinity zinc uptake systems. Under moderate zinc starvation conditions and as soon as free zinc
is still available in enough quantity, zinc uptake is mainly mediated by the dedicated Znu free zinc
uptake pathway since intracellular level of zinc is significantly affected in the corresponding *znuA*
mutant [18, 19, 22]. This observation also means that the Cnt pseudopaline-mediated zinc import
pathway cannot fully complement the Znu pathway in such moderate zinc scarcity. This could be
explained by a lower induction of this pathway at this level of zinc deficiency, in agreement with the
levels of cntO induction proportional to zinc deficiency reported in figure S2. Nevertheless, while the
widely distributed Znu system ensures high-affinity free zinc uptake under conditions of moderate
deficiency, we are demonstrating in this study that the pseudopaline-dependent Cnt zinc uptake
pathway of P. aeruginosa confers this bacterium the ability to deal with highly pronounced zinc
deficiency conditions such as those imposed by high level of metal chelator in MCM or by the host's
nutritional immunity during infection, where the Znu pathway, specialized in the import of free zinc, is
no longer adapted. In line with this hypothesis and as already proposed by Mastropasqua and
colleagues [22], the significant contribution of the Znu pathway in the bacterial zinc homeostasis
reported in previous studies [18, 19, 22] is probably due to the lower degree of metal chelation
achieved with either citrate or only 10µM EDTA or TPEN whereas the MCM medium used in the
present study contains 10 times more EDTA. This explanation is supported by planktonic growth
examination of P. aeruginosa strains in various metal chelation conditions. Using optimal growth in
MCM under various EDTA concentrations as a read-out for the necessity of both pathways, we are
showing figure S5 that in the severe zinc scarcity of the MCM medium (100µM EDTA), the Znu
pathway is unable to rescue the CntL negative growth phenotype while it is able to do it when a less
severe zinc scarcity (10µM EDTA) is applied. The ZnuA-dependent growth phenotype observed in the
ΔcntL strain at 10 µM EDTA but not at 100µM agrees with an involvement of this pathway only at
moderate zinc starvation and also confirm the effective inactivation of znuA gene in the corresponding
mutant strain.

Given that zinc import is essential for the bacterium and that the cnt/znu double mutant still grows,
albeit poorly, in MCM medium (Fig. 1), it is highly likely that this bacterium possesses an additional
and yet to be identified mechanism for zinc import. Candidates includes the pyochelin siderophore,
which has recently been proposed to play a role in zinc import [44]. Several other potential
machineries, produced in zinc deficiency via the Zur regulon, have also been proposed by Pederick
and colleagues [18]. These include the two TonB-dependent receptors PA2911 and PA1922,
characteristic of this metal import systems and which could, with their respective genetically encoded
partners subjected to the same Zur regulation, form additional zinc import machineries [45]. Unlike other bacterial species, *P. aeruginosa* therefore seems to be particularly well equipped to acquire zinc in all circumstances, which could give it a singular advantage in the course of the infection [46].

We used synthetic pseudopaline to perform supplementation experiments and confirmed that the pseudopaline zinc uptake pathway is a cyclic process where the metallophore is first synthetized and secreted by the bacterium to then be regained by the bacterium in its holo form loaded with zinc. This observation could already have been deduced from two independent studies demonstrating firstly that pseudopaline is produced by CntL and then secreted into the supernatant [21] and secondly that the addition of supernatants harvested from the wild type rescued the growth of a strain lacking cntL but did not rescue the growth of a ΔcntO strain [22].

Despite indirect evidences suggesting a release of the metal from the pseudopaline in the periplasm [20], the fate of the pseudopaline-metal complex once imported by CntO remains elusive. As the znuA mutant does not show any growth defect in MCM (Fig. 1), we are ruling out the existence of an interplay between the two pathways and the import in the cytoplasm of the pseudopaline-imported zinc by the Znu machinery. Thus, the molecular journey of pseudopaline after crossing the outer membrane involves either a metal release in the periplasm or a translocation of holo-pseudopaline through the inner-membrane. In both cases, either the metal alone or the holo-pseudopaline needs a dedicated inner membrane transporter that remains to be discovered.

In addition to its general role in bacterial planktonic growth, zinc also promotes biofilm formation in *P. aeruginosa* [39]. It is therefore not surprising to find that zinc uptake systems become important for biofilm formation in zinc limited environment such as the one encountered during lung infection. In line with this, Mastropasqua and colleagues report significant alteration of biofilm formation and its associated traits such as swimming and swarming motility in the double ΔznuA/ΔcntO strain compared to the wild type grown in moderate VB-MM zinc-deplete medium [27]. We also previously showed that under more severe zinc depletion - growth in MCM - where zinc uptake in only mediated by the Cnt pathway and its metallophore pseudopaline, the ΔcntL strain presents a reduced quantitative amount of biofilm [20]. In the present study we are expending the pseudopaline-mediated CntL biofilm phenotype in MCM to fine qualitative alteration and the consequential antibiotic sensitivity.
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These results demonstrate the importance of pseudopaline in biofilm formation under the conditions of high zinc deficiency encountered during infection, where this metallophore becomes necessary for proper zinc import.

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The protective role of pseudopaline at different infection stages of the macrophage observed in this study may counteract several metal-related processes set up by the macrophage to fight the pathogen.

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Much of the research on nutritional immunity has indeed shown a general sequestration by the host of metal ions to limit microbial growth, a process also observed in activated macrophages to limit zinc availability to intracellular pathogens [47] which, the pseudopaline-mediated zinc import pathway logically mitigates. It is now also clear that additional and complementary processes, centered on

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TLR-regulated metal ion trafficking, induce metal intoxication of the pathogen inside the macrophagic vacuole by modulating macrophage-mediated metal ion transport systems [6]. Our data unravel a

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protective role of pseudopaline inside the vacuole, which is explained by a dual extracellular role of pseudopaline allowing to buffer large burst of metal, as already reported for copper by the mycobacterial metallothionein MymT [48]. Such extracellular buffering function of pseudopaline in the macrophage vacuole is supported by the lack of complementation of the intracellular survival phenotype, shown in Figure 3C. The genetic engineering of this strain, relying on the insertion of a *cnt*

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promoter fusion with the *cntL* gene at a neutral chromosomal site leads to a reduced production of pseudopaline in the supernatant [20]. We identified herein that this is a direct consequence of a six-

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fold transcriptional down-regulation compared to its parental strain (Figure S4). It is remarkable that the reduced production of pseudopaline in our complemented strain seems to concern only the intracellular survival phenotype (Fig 3C). We rationalized that this Cnt phenotype is the only one for

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which an optimal level of extracellular pseudopaline is necessary to counteract, through a buffering activity, intoxication by sudden divalent-metals burst considering its high affinity for most divalent

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metal [23]. Thus, a reduced production of pseudopaline decreases bacterial survival in the phagolysosome. This use of pseudopaline as a metal-buffering agent is also supported by the improved survival of a Δ*cntO* strain, not restricted in pseudopaline production and secretion but unable to import

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it (Figure 3C).

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391 Induction of the *cnt* pathway in the macrophage vacuole is demonstrated by the phenotype reported in 392 its absence (Fig. 3C) as well as by the DualSeq detection of specific *cntO* and *cntL* transcripts in the 393 macrophage [40]. Such induction of the *cnt* operon implies zinc deficiency in this compartment, where 394 zinc bursts have nevertheless been reported upon bacterial infection [49]. This apparent contradiction 395 could be explained by the various subversion mechanisms enabling pathogen survival within the 396 macrophage [50]. Indeed, the authors report in this study that only a small proportion (~20%) of the 397 intracellular pathogen experience zinc stress during human macrophage infection. This phenomenon 398 could therefore extend the buffering effect of extracellular pseudopaline to zinc. 399

400 The pseudopaline-deficient phenotype in the mouse infection model reported figure 4 indicates an 401 important role for the pseudopaline Cnt pathway in *P. aeruginosa* pulmonary pathogenesis. While 402 using an intraperitoneal sepsis mouse model, Mastropasqua and colleagues also report an improve 403 survival upon intra-abdominal infection of the ΔcntO strain compared to the wild type strain that also 404 support our findings [22]. More generally, the numerous mice or human transcriptome data showing a 405 systematic up-regulation of the *cnt* genes under similar pulmonary infection conditions therefore 406 extend the role played by the pseudopaline to pulmonary infections in general [26, 27, 30]. The 407 pseudopaline pathway is therefore important for *P. aeruginosa* survival in a mammalian host when it 408 must cope with severe metal deficiencies such as those encountered in pulmonary infections and 409 possibly due to sequestration by calprotectin [51]. 410

411 Results presented in this study revealed that under metal scarce and chelating conditions such as the 412 one encountered during pulmonary infections, among the two zinc import pathways described in *P. 413 aeruginosa*, the Cnt pseudopaline zinc uptake pathway suggests to be the last resort and unique 414 pathway allowing sufficient zinc uptake into the bacterium to preserve the proper intracellular zinc 415 concentration necessary to maintain the pool of zinc metalloproteins fully functional. We are reporting 416 in this study numerous phenotypes affected by zinc privation due to the absence of pseudopaline, that 417 can be explained by the presence of a large number of zinc metalloproteins, previously shown to be 418 important for many cellular functions including transcription, translation, motility or protease activity.
419 The identification of the essential and multifactorial role of pseudopaline in *Pseudomonas* infections 420 paves the way for the development of new antibacterials against *P. aeruginosa* infections. Such
molecules hold promises to act as synergetic pathoblockers potentially able to resensitize established biofilms to current and available antibiotic therapies. In addition, the observation that the Cnt pathway is at play during infection and the fact that pseudopaline is taken up in the periplasm by its specific CntO receptor offers the perspective of using pseudopaline as a scaffold for Trojan horse inspired chemistry, as previously tried with other metallophores [52].

Nicotianamine-like metallophores structurally similar to pseudopaline have also been reported in other bacteria [25]. Interestingly, our finding that pseudopaline is the last resort mechanism to overcome the severe zinc scarcity encountered during infection is consistent with the similar function described for the opine-type metallophore staphylopine which enables *Staphylococcus aureus* to compete with the host's calprotectin to overcome nutritional immunity during infection [53]. Together with our findings, these data strongly suggest that this family of metallophores may plays an important role in the pathogenesis of several microbes. Finally, and more broadly, the fact that *P. aeruginosa* are found in non-pathogenic environments and that numerous non-pathogenic environmental bacteria appear to encode for nicotianamine-like metallophores [25, 54] extend their range of action and competence well beyond the host-pathogen relationship.

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## 435 MATERIALS & METHODS

### 436 Bacterial strains and preculture or planktonic growth conditions.

Bacterial strains used in this study are listed in Table 1. All *P. aeruginosa* strains used in this study are derivatives of the parental PA14 strain. In macrophage infection experiments, the absence of *cntL* in the PA14ΔcntL strain was complemented by *cis* chromosomal expression of the *cntL* gene under the *cnt* operon promotor, inserted at the *attB* site of the *P. aeruginosa* chromosome [21]. Unless otherwise specified, precultures and planktonic growths of all *P. aeruginosa* strains were performed in minimal succinate (MS) medium (per liter: 6g K₂HPO₄, 3g KH₂PO₄, 1g (NH₄)₂SO₄, 0.2g MgSO₄, 4g Succinic acid and 3.1g NaOH, pH 7.0) and minimal chelated medium MCM (MS medium supplemented with 100µM EDTA), respectively, both at 37 °C with horizontal shaking in sterile disposable polycarbonate Erlenmeyer flasks or 96 well plates in order to avoid any metal contamination and monitored by OD₆₀₀ measurements.
Construction of znuA and znuA-cntL deletion mutant strains of P. aeruginosa.

Two DNA fragments corresponding to upstream and downstream regions of znuA gene were amplified
from PA14 chromosomal DNA with PCR primers Mut-znuA-1/Mut-znuA-2 and Mut-znuA-3/Mut-
znuA-4 (Table 2). Upstream and downstream regions were ligated by overlapping PCR and cloned into
linearized pKNG101 [55] by the SLIC method [56]. The resulting constructs were transformed into E.
coli CC118λpir and introduced into P. aeruginosa PA14 by conjugation. The strains in which the
chromosomal integration event occurred were selected on Pseudomonas isolation agar plates
supplemented with 2 mg/mL streptomycin. Excision of the plasmid, resulting in the deletion of the
chromosomal target gene was performed after selection on Luria-Bertani (LB) plates containing 6%
sucrose. Sucrose resistant and streptomycin sensitive clones were confirmed to be deleted for znuA
gene by PCR analysis using Mut-znuA-5/Mut-znuA-6. The ΔznuA ΔcntL strain was constructed by
knocking-out znuA in the ΔcntL strain.

Intracellular Zn quantification by ICP-MS

Pre-cultures in MS medium were inoculated from fresh MS agar plates and grown overnight at 37°C
under agitation. MCM cultures were then inoculated at OD = 0.05 and incubated for 9h in the same
conditions. OD600 were measured before cells harvested by centrifugation (4000g, 20min, 4°C). The
pellets were washed twice with 1mL of fresh MS + 1mM EDTA followed by a wash with 1mL of
fresh MS. The pellets were dried overday at 95°C. After acidic digestion, the metal quantification was
determined by inductively coupled plasma mass spectrometry (ICP-MS) as described elsewhere [57].

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Pseudopaline and metal supplementation experiments

MCM cultures of the pseudopaline deficient strain (PA14ΔcntL) used in planktonic and biofilm
growths were supplemented with various externally added concentrations of chemical S-pseudopaline
synthetized as described in [23] and resuspended in ultrapure water at 100 mM stock solution. For
metal supplementation, MCM cultures of PA14ΔcntL were supplemented with ZnSO4, CuSO4, CoCl2,
MnCl2 or NiCl2 (10 mM stock solution, solubilized in ultrapure water) at 5 µM final.
474 RNA Preparation and Reverse Transcription.

475 RNAs were prepared from 25 mL culture of PA14 grown in LB or MCM after 10h shaking at 37°C.

476 The cells were harvested by centrifugation and frozen at -80°C. Total RNAs were isolated from the
477 pellet using RNeasy mini Kit (Qiagen) according to the manufacturer's instructions; and using
478 TURBO DNAse (Invitrogen) to eliminate the contaminating DNA. The RNA quality was assessed by
479 the TapeStation 4200 system (Agilent). RNA was quantified spectrophotometrically at 260 nm
480 (NanoDrop 1000; Thermo Fisher Scientific). For cDNA synthesis, 1 µg total RNA and 0.5 µg random
481 primers (Promega) were used with the GoScript™ Reverse transcriptase (Promega) according to the
482 manufacturer's instruction.

483 Quantitative real-time retro transcriptional PCR (qRT-PCR)

484 qRT-PCR analyses were performed on a CFX96 Real-Time System (Bio-Rad, France). The reaction
485 volume was 15 µL and the final concentration of each primer was 0.5 µM. The cycling parameters of
486 the qRT-PCR were 98°C for 2 min, followed by 45 cycles of 98°C for 5 s, 60°C for 10 s. A final
487 melting curve from 65°C to 95°C was added to check the specificity of the amplification. To
488 determine the amplification kinetics of each product, the fluorescence derived from the incorporation
489 of EvaGreen into the double-stranded PCR products was measured at the end of each cycle using the
490 SsoFast EvaGreen Supermix 2X Kit (Bio-Rad). The results were analyzed using Bio-Rad CFX
491 Manager software, version 3.1 (Bio-Rad). The 16S RNA and *uvrD* genes were used as a reference for
492 normalizations. For each point a technical triplicate was performed. The amplification efficiencies for
493 each primer pairs were comprised between 80 and 100%. All of the primer pairs used for qRT-PCR
494 are shown Table 2.

495 Biofilm formation

496 Biofilm formation for quantitative evaluation was measured in clear, flat bottom, 24-well plates
497 (Corning™ Costar 3526). Each overnight culture was inoculated at OD₆₀₀ of 0.2 in 1 mL fresh MCM
498 and incubated at 30°C for 24 hours. Pseudopaline complementation was performed with addition of
499 100µM of synthetic S-pseudopaline to the medium before bacteria inoculation. Following incubation,
500 planktonic bacteria and media were rinsed away with non-sterile deionized water. The wells were
501 filled with 0,25% crystal violet solution and after 15 min at room temperature, they were washed three
502 times with deionized water. Any crystal violet staining on the bottom of the well were cleaned, and the
503 plate was let to air-dry overnight at room temperature. Crystal violet rings were then solubilized in
504 30% glacial acetic acid. Adherent biofilm was quantified by measuring optical density of all samples
505 at 550 nm and normalized to mean absorbance from wells containing WT strain. Images used for
506 biofilm illustration are representative of three independent experiments. For analysis of CntO
507 production by western-blot, biofilms were resuspended within the medium and cells centrifuged in
508 order to resuspend the pellet in a volume of 1X Laemmli buffer allowing a final cell concentration of
509 0.02 OD units/µL.

510 Time-lapse qualitative biofilm formation was performed in flow chambers as previously described
511 [58]. Each experiment was repeated two times. The *P. aeruginosa* PA14 WT::gfp and
512 PA14ΔcntL::gfp were tagged with green fluorescent protein (GFP), following the procedure described
513 in [59] except that conjugation instead of electroporation was used to transfer plasmids into *P.
514 aeruginosa*. Bacteria from MS overnight precultures were inoculated at OD₆₀₀ of 0.1 in MCM in flow
515 chambers with individual channels (dimensions of 1 × 4 × 40 mm³) and incubated for 3h at 30°C
516 before flow start. At day 11, tobramycin at 20µg/mL was added and incubated for 24 h with stopped
517 flow. At day 12, the flow was turned on for 30 min, before addition of propidium iodide and
518 incubation for 30 min with stopped flow. The flow was started again for 24 h before observation at
519 day 13. Observation was performed after 4, 7, 10 and 13 days with an Olympus FV-1000 microscope
520 equipped with detectors and a filter set for monitoring GFP and propidium iodide. **Z-stack images
521** were used to reconstruct the 3D architecture of the biofilms, which were then analyzed using Fiji [60].
522 Quantitative parameters such as biovolume (µm³/µm²), average thickness, and surface coverage were
523 extracted. To account for potential differences in cell density, the number of cells per imaging field
524 was estimated from GFP fluorescence intensity, allowing normalization of biofilm biovolume to cell
525 number.

526 **Western blot analysis**

527 For analysis of CntO production, protein samples from bacteria corresponding to 1 OD unit were
528 resuspended in 50µl 1X SDS-PAGE loading Laemmli buffer containing β-mercaptoethanol, heated at
529 95 °C for 10 min and 5 µl was loaded and separated on 12% acrylamide SDS-PAGE. After separation,
the proteins were transferred onto nitrocellulose membranes for western blot analysis. Transfers were performed with semi-dry technic using Power Blotter Station (Invitrogen). The membranes were washed briefly in Tris-buffered saline, 0.10 % Tween 20 (TBS-T) and then blocked in TBS-T + 10 % milk for 1 hour at RT. The membranes were incubated for 1 h at RT in TBS-T + 10 % milk containing the primary (mouse) antibody directed against CntO (laboratory collection) at 1/1,000 dilution. This was followed by 4 washes in TBS-T before an incubation of 45 min at RT in TBS-T + 10 % milk containing the secondary antibody anti-mouse-HRP conjugated (1/5,000). Finally, after five more washes with TBS-T, the peroxidase reaction was revealed by chemiluminescence (SuperSignal™ West Dura Extended Duration Substrate, Thermo Scientific) and scanned with ImageQuant LAS 4,000.

## 540 Macrophage infections

### 541 Phagocytic uptake and survival

RAW264.7 (ATCC number: TIB-71TM) cells were routinely grown in DMEM (4.5 g/l Glucose) and supplemented with 10% FCS, 20 mM HEPES and 1% BSA at 37°C, 5% CO₂. All cell cultures used in this study were tested and confirmed as mycoplasma-free. Invasion assays were performed as described previously [40]. Briefly, 5x10⁵ RAW 264.7 cells were seeded in DMEM in a 24 well plate for phagocytic uptake or intracellular replication measurements. Before the start of the experiments, the supernatant of each well was discarded and replaced with 400 µL of fresh DMEM without phenol red. Eukaryotic cells were infected at a MOI (multiplicity of infection) of 1 with selected P. aeruginosa strains grown 10 h in MCM, washed with PBS, and adjusted to a final volume of 100 µL in fresh DMEM. Number of CFU in the inoculum dose is verified by serial plating on LB plates. Immediately after start of the infection, a centrifugation step at 1200 x g for 5 min was used to ensure cell-to-cell contact. To kill extracellular bacteria, one-hour post-infection (hpi), 100 µL of DMEM containing gentamicin was added to the final concentration of 50 µg/mL. At 2hpi, cells were washed with PBS and lysed with PBS containing 0.5% (v/v) TritonX-100. CFUs were determined by plating of serial dilutions and compared to the parental strains. These CFU represent the number of cells phagocyted, and normalized to the inoculum. A replicate plate was used for survival assay, using the same inoculum and cells batch. After 6h, cells were washed with PBS and lysed with PBS containing
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0.5% (v/v) TritonX-100. CFUs were determined by plating of serial dilutions and compared to the parental strains. Intracellular survival is evaluated by the ratio of CFU recovered after 6h, and normalized to the number of cells phagocyted after 2 h.

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### Adherence assay

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Adherence assay was performed as described before [40]. Briefly, RAW234.7 cells were seeded in DMEM as detailed in the phagocytic uptake and survival assays, and infected by *P. aeruginosa* strain grown in MCM for 10 h. 10 µM of Cytochalasin D was added to cell cultures 2 h prior to infection to block phagocytosis. Directly after infection, the bacteria were centrifuged at 1200 x g for 5 min to ensure cell-to-cell contact. 2 hpi, the supernatant was removed. The cells were then gently washed twice with PBS and lysed using PBS containing 0.5% (v/v) TritonX-100. Serial dilutions were used to determine the adherent bacteria by plating and counting CFUs.

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### Cytotoxicity

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50 µL of supernatant of infected RAW264.7 cells were collected 6 hpi and LDH levels were measured using the CytoTox 96 Non-radioactive Cytotoxicity Assay (Promega) according to the manufacturer's protocol. PBS serves as negative control and lysis solution of the manufacturer was used as killing control. Values are expressed as % of the value obtained with the PA14 WT strain infection.

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### Measure of cytokine production

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Macrophage supernatants were sampled 6 hpi. The TNF-α and IL-6 ELISA Max Standard Kit (Biolegend) were used to determine the TNF-α and IL-6 levels according to the manufacturer's manual. Three different biological replicates were analysed in technical triplicates, and a PBS-treated group served as negative control.

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### Pulmonary mouse infections

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#### Mice

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Adult male C57BL/6jrj (B6) mice (6-8 weeks old) were obtained from Janvier (Le Genest Saint-Isle, France). All mice were housed under specific-pathogen-free conditions at the “PST Animaleries” animal facility (Université de Tours, France) and had access to food and water *ad libitum*. All
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experiments complied with the French government's ethical and animal experiment regulations (APAFIS#7608-2016111515206630).

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*P. aeruginosa* infections

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MCM precultures of WT and Δcnt strains were used in this study. Mice were anesthetized with isoflurane 4% and an operating otoscope fit with intubation specula was introduced to both maintain tongue retraction and visualize the glottis. A fiber optic wire threaded through a 20G catheter and connected to a torch stylet (Harvard Apparatus, France) was inserted into the mouse trachea. Correct intubation was confirmed using lung inflation bulb test and 40µL of the bacterial inoculum (2-8x10⁶ cfu) was applied using an ultrafine pipette tip. Inoculum size for infections were confirmed after each experiment by counting colony-forming unit (cfu) on *Pseudomonas* isolation agar plates. Mortality, body weight and clinical score of animals were monitored daily. Clinical score was calculated by recording the following symptoms: ruffled fur, hunched posture, motor impairment, breathing impairment, closed eyes, purulent eyes and allocating 1 point to each. In all experiments, moribund animals - animals with a weight loss of more than 20% or clinical score of 7 - were sacrificed for ethical reasons and considered as dead due to the infection.

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Broncho-alveolar lavage, organ sampling and bacterial load assay

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At day 2 after infection, mice were sacrificed with intra-peritoneal injection of pentobarbital. Broncho-alveolar lavage fluid (BAL) was collected after *P. aeruginosa* infection by introducing a catheter into the trachea under deep pentobarbital anaesthesia and washing sequentially the lung with 2 x 1mL of 1X PBS at room temperature. The lavage fluid was centrifuged at 400 g for 10 min at 4°C and the supernatant was stored at -20°C for ELISA analysis. Lungs were perfused with 10mL of PBS1X and harvested in GentleMACS M tubes (Miltenyi Biotec, Germany) containing 2 mL of 1X PBS for microbiology assay. Bacterial load in lung homogenates or BAL (before centrifugation) was determined by plating tenfold serial dilutions on *Pseudomonas* Isolation Agar (PIA) plates were incubated at 37°C in a 5% CO₂ atmosphere, and the CFUs were counted after 24 hours.

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Measure of cytokine production

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Concentration of IL-6 in BAL supernatants and lung homogenates were measured using specific ELISA Max Standard Kit (Biolegend) according to the manufacturer's manual.
612 **Statistics**

613 Statistics were determined using one-way or two-way analysis of variance (ANOVA) followed by a
614 Tukey's or Sidak's post-test (respectively for Fig. 1, 2, 3ACDEF, 4DE and Fig. 3B, 4AB). Log-rank
615 test was used for survival analysis (Fig. 4C). Figures and statistics were performed using GraphPad
616 Prism 10.4.1.

617 **ACKNOWLEDGEMENTS**

618 We are gratefully to Pr. Steve Lory for careful reading of the Manuscript. We acknowledge the
619 support of Sebastian Felgner for introduction and training to the RAW 264.7 cell-culture model and
620 Anja Kobold, Astrid Dröge, Tanja Nicolai for technical assistance. We also thank the whole RV group
621 for constant support in the project. This work was supported by the grants: RF20210502871 from
622 "Vaincre la Mucoviscidose" and "Gregory Lemarchal" associations allocated to RV, NHV and TS;
623 2019-00131897 NOVANTINH from the "Région Centre-Val-de-Loire" allocated to NHV, TS and
624 RV, ANR-20-CE11-0018 "Pseudo-Traffic" allocated to RV and PA and NR-24-CE44-7195-04
625 allocated to PA and RV. S.H. received funding from the DFG in the DFG SPP 2389 program, under
626 Germany's Excellence Strategy - EXC 2155 "RESIST"-Project ID 390874280, from the Lower
627 Saxony Ministry for Science and Culture (BacData ZN3428), and from the Novo Nordisk Foundation
628 (NNF 18OC0033946).

629

630 AUTHOR CONTRIBUTIONS

631 Conceive the experiments: NOG, TS, LB, MB, RV. Perform, analyse and interpret the experiments:
632 NOG, TS, LB, GB, DLP, MT, MB, YD, LO, PA, MB, CB, RV. Write the paper: NOG, RV. Review
633 the paper: TS, LB, GB, MT, PA, LO, XL, YB, SH, NHV. Secure funding's: NHV, TS, SH, PA and
634 RV.

635 The authors declare no conflict of interest

636

637 FIGURE LEGENDS
638

**FIGURE 1: The pseudopaline pathway but not the Znu one is required for optimal intracellular zinc homeostasis and planktonic growth in MCM.**

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(A) Intracellular zinc concentrations and (B) growth curves and corresponding generation times of PA14 wild-type (WT), ΔcntL, ΔznuA and ΔznuA/ΔcntL strains in MCM. (C) Impact of externally added pseudopaline on cell growth of wild-type (WT) and ΔcntL strains, supplemented or not with various concentrations (from 1 to 100 µM) of exogenous pseudopaline, grown in MCM. Complete growth curve (left) as well as generation times (right) are presented. (D) Impact of externally added zinc on cell growth of PA14 wild-type (WT) and ΔcntL strains, supplemented or not with 5 µM of exogenous zinc (Zn) and grown in MCM. Error bars, mean ± standard deviation (sd) of at least three independent biological replicates. Not significant (ns), ****, ***, ** and * correspond to p < .0001; < .001; < .001 and < .005 respectively (one-way ANOVA, Tukey post-hoc test).

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**FIGURE 2: The pseudopaline pathway is required for biofilm formation and antimicrobial resistance in MCM.**

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Impact of pseudopaline on biofilm formation of (A) PA14 wild-type (WT) and ΔcntL strains or (B), Gfp-labelled PA14 wild-type (WT::gfp) and ΔcntL (ΔcntL::gfp) strains, supplemented or not with exogenous pseudopaline (Pp) at 100µM and grown in MCM. (A) Biofilm formation was measured by quantifying bacterial mass adhering to the walls of a microtiter plate well as described in the M&M section. A representative illustration of crystal violet rings obtained with the different strains during the biofilm formation assay is presented in the middle panel. CntO productions in the different protein samples visualized by immunoblot experiments are presented in the lower panel. (B) Qualitative biofilm formation was monitored at days 4 (D4), 7 (D7) and 10 (D10) post-incubation. Stacked confocal scanning laser microscopy images of biofilms and corresponding extracted z images and their respective xy and xz planes as well as their thickness (in µm) are presented. The ratios (in %) indicate the average biomass of the ΔcntL strain (PA14ΔcntL::gfp) compared to the wild type (PA14wt::gfp) strains at the three different days of incubation. Antimicrobial tolerance of PA14wt::gfp and PA14ΔcntL::gfp strains from 11 days aged biofilms, exposed to 20 µg/mL of tobramycin for 24 h and observed at day 13 (D13) after 24 h of additional growth. Cells with a compromised membrane that are dead or dying stain red with propidium iodide, whereas cells with an intact membrane stain green
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with GFP fluorescence. The live cells (in %) indicate the biomass of the live cells compared to the total live and dead cells obtained from two independent experiments. The experiment was repeated twice, independently, and the images presented correspond to the typical results observed in both experiments. Errors bar, mean ± standard deviation (sd) of at least three independent biological replicates. **** correspond to p < .0001 (one-way ANOVA, Tukey post-hoc test).

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**FIGURE 3: The pseudopaline pathway is sequentially required at different stages of macrophage infection by P. aeruginosa.**

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Strains PA14 WT (WT), ΔcntL, ΔcntO and ΔcntL cis-complemented (ΔcntL::cntL) strains, precultured in MCM were compared in different macrophage infection assays. (A) Phagocytic uptake into RAW 264.7 cells. The results of three independent experiments with three biological replicates are presented. **** correspond to p<0.0001 (One-way ANOVA, Tukey Post-hoc test). (B) Adherence measurement to RAW 264.7 cells, following treatment of cells with cytochalasin D to stop phagocytosis. The inoculum (grey) were set as 100% for each strain and the uptake without cytochalasin D (corresponding to phagocytosis) was also run as internal control (red), as well as a negative control with cytochalasin and gentamicin in which no CFU were recovered (blue). Mean ± standard deviation of three biological replicates are shown. Phagocytic uptake **** correspond to p<0.0001 (Two-way ANOVA, Dunnett Post-hoc test). (C) Bacterial survival in RAW 264.7 macrophages. Macrophages were infected by P. aeruginosa strains and treated with gentamicin 1 hpi. Ratio of CFU recovered 6 hpi were normalized to CFU recovered 2 hpi, and expressed as percentage. The results of three independent experiments with three biological replicates are depicted. * and ** correspond to p<0.0332 and p<0.0021 respectively (One-way ANOVA, Tukey Post-hoc test). (D) Cytotoxicity on RAW 264.7 macrophages. Cells were infected at MOI of 1 after culture in MCM and treated with gentamicin 1 hpi. Supernatant was sampled 6 hpi, and lactate dehydrogenase (LDH) activity measured by a colorimetric assay. Values are normalized to LDH activity upon infection with PA14 WT strain, expressed as percentage. The results of three independent experiments with three biological replicates are depicted. ** correspond to p<0.0021 (One-way ANOVA, Tukey Post-hoc test). Quantification of Tumor necrosis factor α (TNF-α) (E) and interleukin 6 (IL-6) (F) produced 6 hpi by RAW 264.7 macrophages infected by P. aeruginosa strains at MOI 1 after growth in MCM and
treated with gentamycin 1 hpi. The results of two independent experiments with three biological replicates are depicted. * and ** correspond to p<0.0332 and p<0.0021 respectively (One-way ANOVA, Tukey Post-hoc test).

FIGURE 4: The pseudopaline pathway is key to *P. aeruginosa* pulmonary infection.

Mice were infected intratracheally with PA14 wild-type (WT) and Δcnt strains precultured in MCM. Animal weight (A), clinical score (B) and survival (C) were recorded throughout the experiment as described in the M&M section. (D) In a separate experiment, mice were sacrificed at 2 days post-infection and the number of PA14 in the bronchoalveolar lavage fluid (BAL) and lungs was determined. (E) The amount of interleukin 6 (IL-6) was measured in the bronchoalveolar lavage and lungs. Each result corresponds to at least 3 independent experiments (n=17/group) for A-C and 1 experiment (n=5-7/group) of D and E. *, ** and *** correspond to p<0.05, p<0.01 and p<0.001 respectively (One-way ANOVA, Tukey Post-hoc test).

TABLES

<table><caption>**Table 1:** Bacterial strains used in this study</caption><thead><tr><td><i>P. aeruginosa</i> Strains</td><td>Descriptions</td><td>References</td></tr></thead><tbody><tr><td>PA14 WT</td><td>Wild type strain</td><td>[61]</td></tr><tr><td>PA14 WT::gfp</td><td>Wild type PA14 strain harboring the <i>gfp</i> gene under constitutive promoter at <i>att</i> site</td><td>(This study)</td></tr><tr><td>PA14ΔcntL</td><td>ΔcntL deletion strain</td><td>[21]</td></tr><tr><td>PA14ΔcntL::gfp</td><td>PA14ΔcntL strain harboring the <i>gfp</i> gene under constitutive promoter at <i>att</i> site</td><td>(This study)</td></tr><tr><td>PA14ΔcntL::cntL</td><td>PA14ΔcntL harboring a wild type copy of <i>cntL</i> gene at <i>att</i> site.</td><td>[21]</td></tr><tr><td>PA14ΔcntO</td><td>ΔcntO deletion strain</td><td>[21]</td></tr><tr><td>PA14 ΔznuA</td><td>ΔznuA deletion strain</td><td>(This study)</td></tr><tr><td>PA14 ΔcntLΔznuA</td><td>ΔznuA ΔcntL double deletion strain</td><td>(This study)</td></tr><tr><td>PA14Δcnt</td><td>PA14 strain deleted for the complete <i>cntOLMI</i> operon.</td><td>[20]</td></tr></tbody></table>

<table><caption>**Table 2:** Primers used in this study</caption><thead><tr><td>Name</td><td>Sequence (5'-3')</td></tr></thead><tbody><tr><td>Mut znuA-1</td><td>CAGGTCGACGGATCCCCGGGATCAGGCGTCCTTCTGGTC</td></tr></tbody></table>
<table><tr><td>Mut znuA-2</td><td>CACGGGTTTTCACACGGCGGCACT</td></tr><tr><td>Mut znuA-3</td><td>AGTGCCGCCGTGTGAAAACCCGTGACT</td></tr><tr><td>Mut znuA-4</td><td>TATGCATCCGCGGGCCCGGGTTTCTCGAGGATGTTGTCCA</td></tr><tr><td>Mut znuA-5</td><td>CGATCAGGGTGACGATCTG</td></tr><tr><td>Mut znuA-6</td><td>TGACGGTGAAGCACATCTTG</td></tr><tr><td>qRTcntOFor1</td><td>CGCCATTTCTCGTTGAACTC</td></tr><tr><td>qRTcntORev1</td><td>ACGAACTGATCCTGCGTAGC</td></tr><tr><td>QRTcntLFor1</td><td>ATTTGTGCTGCCTGGACATC</td></tr><tr><td>QRTcntLRev1</td><td>AGCGAAGCGATCAGGAAAT</td></tr><tr><td>uvrD1</td><td>CACGCCTCGCCCTACAGC</td></tr><tr><td>uvrD2</td><td>GGATCTGGAAGTTCTGCTCAGC</td></tr><tr><td>16S For</td><td>CAGCTCGTGTCGTGAGATGT</td></tr><tr><td>16S Rev</td><td>GATCCGGACTACGATCGGTT</td></tr></table>

710

711

SUPPLEMENTARY FIGURE LEGENDS

FIGURE S1: The growth of ΔcntL is not restored by the addition of other external metals

Impact of externally added metals on cell growth of PA14 wild-type (WT) and ΔcntL strains, supplemented or not with 5 µM of exogenous copper (Cu), cobalt (Co), manganese (Mn) or nickel (Ni) and grown in MCM.

717

FIGURE S2: Direct correlation between intracellular zinc concentration and cntO gene expression and CntO protein detection.

(A) Intracellular zinc concentration and (B) cntO gene expression of PA14 wild-type (WT) and ΔcntL strains grown in MCM or LB medium. For all panels, CntO protein levels in the different protein samples were estimated by immunoblot experiments using CntO antibodies (lower panels). Error bars, mean ± standard deviation (sd) of at least three independent biological replicates. *** Correspond to p < .001. (one-way ANOVA, Tukey post-hoc test).

725

FIGURE S3: The deletion of cntL does not impact the tobramycin sensitivity of PA14 in planktonic growth in MCM.
728 Cell growth in MCM of PA14 wild-type (■) and ΔcntL (Δ) strains grown in presence of increasing concentrations of tobramycine. Error bars, mean ± standard deviation (sd) of at least three independent replicates.
729
730

731
732 FIGURE S4: cntL expression is significantly reduced in the PA14 ΔcntL::cntL complemented strain.
733

734 cntL gene expression quantified by qRT-PCR of WT or ΔcntL::cntL strains grown in MCM or LB medium. Error bars, mean ± standard deviation (sd) of at least three independent replicates.
735
736

737 FIGURE S5: The Znu and Cnt zinc import pathways are hierarchically required under low, moderate and high zinc scarcity.
738
739

740 Planktonic cell growth of PA14 wild-type (WT), ΔcntL, and ΔcntL/ΔznuA strains in MS medium supplemented or not (0) by 10 (10) or 100 (100) µM of EDTA.
741

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