Zinc (Zn), commonly found as the divalent cation Zn²⁺, is a trace element primarily involved as a cofactor of many enzymes and therefore essential for life. It is considered as the second most important trace metal after iron (Fe) (Blencowe and Morby, 2003). In prokaryotes, Zn is principally found bound to proteins (approximately 6% of the proteome) and the free cellular fraction is kept at a concentration as low as femtomolar (Outten and O’Halloran, 2001). In excess, this metal becomes toxic, especially by competing with other trace elements, giving rise to protein mismetallization (Foster et al., 2014). The equilibrium of cellular Zn concentration is therefore tightly regulated and numerous systems involved in Zn homeostasis (and that of other divalent cations) are well-described in several bacteria (Chandrangsu et al., 2017).

The toxic properties of Zn are widely exploited in the context of infection control. Upon bacterial invasion, one of the host defense mechanisms is the sequestration of essential metal ions, in particular Fe, Zn, and manganese (Mn), by proteins and molecules, leading to so called “nutritional immunity” (Kehl-Fie and Skaar, 2010; Capdevila et al., 2016; Lonergan and Skaar, 2019). Conversely, during phagocytosis, toxic concentrations of Zn and copper (Cu) are delivered into the phagolysosome, participating in the destruction of the pathogen (Stafford et al., 2013; Gao et al., 2018). Thus, to successfully infect, microorganisms not only must adapt to environments of Zn deprivation or excess, but also rapidly alternate between these two extreme conditions. The opportunistic Gram-negative pathogen Pseudomonas aeruginosa is an ubiquitous and versatile bacterium that possesses several systems for Zn homeostasis (Figure 1A). P. aeruginosa is responsible for a wide array of severe infections, particularly in cystic fibrosis and immunocompromised patients (Kerr and Snelling, 2009), and Zn homeostasis systems are highly relevant to its pathogenicity (Gonzalez et al., 2019). Notably, they modulate virulence by acting on quorum sensing (Dieppois et al., 2012) as well as antibiotic resistance by repressing the expression of the route of entry for carbapenem antibiotics (Perron et al., 2004).

Numerous systems are involved in the import of Zn into P. aeruginosa (Pederick et al., 2015). At first, the TonB-dependent receptors ZnuD and PA2911 interact with its energizing TonB-ExbBD protein complex to translocate extracellular Zn into the periplasmic space (Gonzalez et al., 2019). Subsequently, the metal crosses the cytoplasmic membrane via either the P-type ATPase HmtA or via an ATP-binding cassette (ABC) transporter such as ZnuABC, the most common Zn uptake pathway in bacteria (Pederick et al., 2015). Revealed for the first time in Escherichia coli, this transporter consists of the solute-binding protein (SBP) ZnuA, which scavenges Zn in the periplasm with a high affinity and brings it to the inner membrane permease ZnuB. The energy required for this transport is provided by the cytoplasmic ATPase ZnuC (Patzer and Hantke, 1998).

Two additional ABC transporters have been described in P. aeruginosa: PA4063-PA4066 and PA2912-PA2914 (Pederick et al., 2015). Although these transporters have been shown to be overexpressed under conditions of Zn limitation, they remain poorly characterized and their exact contribution to Zn uptake remains to be elucidated.

The ZrmABCD system has recently been designated as a new Zn acquisition system, involving a nicotianamine-related zincophore called pseudopaline by similarity to the Staphylococcus aureus staphylopine (Lhospice et al., 2017; Mastropasqua et al., 2017). The molecule is synthetized by the ZrmB and ZrmC cytoplasmic enzymes and then released outside the cell by the EamA-like transporter ZrmD. Internalization of the Zn-pseudopaline complex is mediated by the TonB-dependent receptor ZrmA, in a siderophore-like manner (McFarlane and Lamb, 2017). Though many gaps remain to be filled at the mechanical level, the importance of this system under conditions of Zn limitation and its involvement in bacterial pathogenicity appear evident, especially in a cystic fibrosis context (Hermansen et al., 2018).

A considerable reservoir of Zn is represented by ribosomal proteins. Indeed, during nutrient deprivation, P. aeruginosa copes with metal deficiency by expressing additional ribosomal paralogous proteins, annotated as C−, which have lost their Zn finger domain (Haas et al., 2009; Gonzalez et al., 2019). A release of Zn probably results from this exchange phenomenon, that can be used by other proteins such as DNA polymerase, primase, etc. The P. aeruginosa PA3600-PA3601 operon encodes two C− paralogs, RpmE2 and RpmJ2, able to substitute in place and in function the ribosomal proteins RpmE and RpmJ, respectively. Recent data from E. coli have shown that RpmE2 and RpmJ2 are of comparable efficiency to their Zn-ribbon paralogs (Ueta et al., 2020). Such a strategy is also observed with the global transcription regulator DksA, involved in the stringent response. Under Zn-scarce conditions, the alternative protein DksA2, devoid of a Zn finger domain, is induced and takes over the functional relay from its paralog DksA (Blaby-Haas et al., 2011; Furman et al., 2013). The dksA2 gene is found in a Zn-regulated cluster composed of approximately 10 genes including two encoding C− paralog enzymes, pyrC2 and folE2 (Pederick et al., 2015).

Zinc uptake and storage systems are regulated by the one-component regulator Zn uptake regulator (Zur) protein, that belongs to the ferric uptake regulatory (FUR) protein family. Zur senses the cytoplasmic concentration of the metal and its regulatory function is directly related to its ability to reversibly bind Zn (Ellison et al., 2013; Gonzalez et al., 2019). Under conditions of Zn sufficiency or excess, the regulator is found in a dimeric form containing four Zn atoms (Zur2-Zn4 conformation), which promotes DNA binding and ensures the repression of target genes by preventing RNA polymerase from initiating transcription. In P. aeruginosa, nine Zur binding sites were predicted, characterized by a 17-nucleotide palindromic motif overlapping the −10 of the promoter region (Pederick et al., 2015).

Pseudomonas aeruginosa has the ability to counter high toxic concentrations of Zn. Three families of export systems have been described in this bacterium, including the CzcCBA efflux pump, a homolog of the system found in the metal-resistant bacterium Cupriavidus metallidurans (Nies et al., 1989). CzcCBA belongs to the Resistance-Nodulation-Division (RND) group of the Heavy Metal Efflux (HME) family, capable of expelling excess Zn, cadmium (Cd) and cobalt (Co) from the cytoplasmic or periplasmic compartments directly outside the cell (Goldberg et al., 1999). This pump is regulated by the CzcRS two-component system (TCS), where CzcS is the transmembrane sensor and CzcR is the response regulator. CzcS detects and becomes active under conditions of excess periplasmic Zn or Cd. It then activates CzcR by phosphorylation, which in turn acts as a transcriptional activator of the efflux system, ensuring detoxification of the cell. Additionally, CzcRS TCS has been shown to activate its own transcription, promoting a positive regulation loop, but also to directly repress the OprD porin, the route of carbapenem entry, thus rendering the bacterium resistant to these antibiotics (Perron et al., 2004; Dieppois et al., 2012).

Moreover, the bacterium possesses two cation diffusion facilitators (CDF), CzcD, and YiiP. These systems act as a homodimer or heterodimer to expel Zn from the cytoplasm to the periplasm by way of a proton gradient. They also appear to be involved in maintaining membrane integrity, which would explain why mutants deleted for these CDFs are more sensitive to several antibiotics (Salusso and Raimunda, 2017).

A final layer consists of the P-type ATPase CadA, which is also responsible for detoxifying the cytoplasm by expelling Zn to the periplasm. In P. aeruginosa, CadA was first described as a system involved in Cd resistance (Lee et al., 2001). Similar to the E. coli ZntA, this protein has recently been shown to be essential for Zn resistance (Ducret et al., 2020). CadA is regulated by CadR that belongs to the MerR family of response regulators. CadR is constitutively expressed and has a high affinity for the cadA promoter in the apo and holo forms. The activity of this regulator is directly linked to its ability to bind Zn since it plays the dual role of repressor and activator of cadA transcription, depending on whether it is in conditions of Zn limitation or excess, respectively (Ducret et al., 2020).

Numerous strategies allowing Zn homeostasis have been described and characterized in P. aeruginosa. All these systems serve to ensure the survival of the bacterium in environments that are limited or contaminated with Zn and, interestingly, make the link between metallostasis, virulence and antibiotic resistance. Knowledge of how these systems interact with each other, however, is scarce.

Our lab previously highlighted a dynamic in the expression of Zn export systems in P. aeruginosa. Indeed, we had shown that the P-type ATPase CadA was not only the first line of protection against a boost in Zn, but also facilitated the induction of the major export system, the CzcCBA efflux pump (Ducret et al., 2020). These results support the hypothesis that the different systems involved in Zn resistance are not redundant, but follow a precise strategic plan, guaranteeing a rapid adaptation of the cell to variations in Zn concentrations.

The purpose of this study was to follow up on the “strategic plan” hypothesis and assess the dynamics of all the systems involved in Zn homeostasis (import/export/storage) when the bacterium transitions from starvation to surfeit, as it happens during an infection. To achieve this aim, we made use of the NanoString technology, which allows the direct counting of several mRNAs simultaneously by using digital bar-coded probe pairs (Geiss et al., 2008). We could therefore quantify transcripts of all Zn homeostasis systems, rather than following a single element of the system, and follow their dynamics over time. While we clearly observed a global import systems repression, a hierarchical induction of the export systems appeared. An additional novel partner of the Zn resistance, PA2807, a CzcE-like protein, was also discovered and characterized. Finally, the rapid induction of carbapenem resistance via OprD porin repression in the presence of Zn was precisely analyzed at the transcriptomic and proteomic levels.

In order to monitor the dynamics of Zn transport systems when P. aeruginosa switches from a situation of Zn starvation to Zn excess, we designed an experimental procedure described in Figure 1B. Briefly, P. aeruginosa PAO1 strain was cultivated at 37°C in a Zn-depleted medium (M-LB) containing 30 μM TPEN [N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine] to chelate all residual Zn for 2 h 30 min (Ellison et al., 2013). Then, 2 mM ZnCl₂ (final concentration) was added to the culture. Previous to Zn addition (t0) and at different times afterward, cells were sampled for proteomic, transcriptomic, and Zn content analysis.

At protein levels we observed, as expected, a significant increase of all export systems during the first hour of high Zn exposure, while surprisingly the level of only two proteins involved in Zn import, ZrmB and PA1925, decreased more than 50% (Figure 1C and Supplementary Table 1). Indeed, the large majority of uptake systems significantly dropped only between 1 and 5 h after Zn addition. During this time frame, the amount of the CadA P-type ATPase decreased slightly in agreement with our previous observations on its role of early player in case of a sudden Zn excess (Ducret et al., 2020). The 5 h-period was also marked by the increasing of the CzcD protein. This very late induction took place after the CzcCBA efflux rise and may explain why this CDF was not essential for Zn resistance (Ducret et al., 2020).

When looking at the intracellular Zn concentration over time, the values changed from 0.014 to 0.14 μM in 15 min, then returned to equilibrium at a concentration of approximately 0.04 μM after 60 min (Figure 1D). It thus appears that the rapid induction of efflux systems prevails over the slow repression of import systems and it is sufficient to counteract the Zn overflow.

Finally, to decipher early events of regulation of the Zn homeostasis network, we directly monitored the transcript levels of genes involved in Zn transport using the NanoString technology (Geiss et al., 2008). Twenty-five oligonucleotide probes targeting mRNAs of the known Zn import/export/storage systems of P. aeruginosa were designed (Table 1). In addition, four probes targeting housekeeping genes were also designed for data normalization (Supplementary Table 2). In case of a system encoded by operon, we selected at least one gene probe to represent the whole transcription unit (e.g., czcA and czcC were chosen to monitor the expression of the czcCBA operon). For this analysis, RNA was extracted at t0 and 30 s, 1, 2, 5, 15, 30, and 60 min (t0.5, t1, 2, 5, 13, and 60, respectively) after Zn addition (Figure 1B). The mRNA copy number for each of the target genes was then calculated in each sample, as shown in Supplementary Table 3.

To infect and take advantage of its host, a pathogen must be able to adapt quickly to extreme conditions, including rapid changes in Zn concentration. P. aeruginosa is typically a bacterium armed to counter this kind of situations, as shown by the numerous systems involved in Zn homeostasis. These systems, together with the ability to express them in a timely fashion, contribute to P. aeruginosa ability to infect hosts (Gonzalez et al., 2019). In this study, we carried out a systematic investigation of the expression dynamics of the entire P. aeruginosa Zn homeostasis network when switching from a Zn depleted environment to a Zn excess situation, a transition mimicking what happens during an infectious process (Djoko et al., 2015). Our analysis allowed us to draw a global model of Zn homeostasis gene expression organization in P aeruginosa, as illustrated in Figure 8.

As a first reaction, we observed a quick induction of cadA expression (after 1 min). Presumably, this serves to rapidly evacuate the Zn excess out of the cytoplasm into the periplasm (Ducret et al., 2020). Downregulation of transcripts for uptake systems is also occurring fast, probably to prevent new Zn ions from entering the cell. Surprisingly, at the protein level this repression is visible only after 5 h, suggesting a slow turnover of Zn uptake proteins. Given the importance of Zn for survival, this might be the result of a “prudential regulatory strategy,” to assure a prompt Zn uptake in case of the metal concentration decreases suddenly.

In a second step, the CzcCBA major efflux system is induced to detoxify all the cellular compartments, thus allowing the bacterium to thrive in an environment highly contaminated with Zn. In agreement with this, a decrease in total cellular Zn is observed as the expression of czcCBA increases (Figure 1D). The CzcD CDF is not an essential component in metal resistance (Ducret et al., 2020), but we show here to be part of the czcRS regulon (Figure 2) and therefore responding to the periplasmic Zn concentration. Considering its late induction, CzcD may contribute to the maintenance of a steady state level of Zn resistance by continuing to pump Zn into the periplasm and allowing the CzcS to remain active while cadA expression decreases. One may consider that the P-type ATPase system CadA, despite being very efficient, uses ATP as an energy source while CzcD is driven by proton-motive forces and, therefore, might be more profitable for the cell in terms of energy optimization.

The PA2807 gene was previously shown to be induced by the CopRS system in the presence of Cu (Quintana et al., 2017). Here, we show that it encodes a CzcE-like protein involved in Zn resistance and also induced by the CzcRS systems in the presence of Zn. The role of this periplasmic protein in Zn and Cu resistance requires further investigation, but we could confirm the link between Zn and Cu resistance that has already been observed in other bacteria. For instance, in Pseudomonas stutzeri, a common overlapping response to Cu and Zn by common regulator DNA binding motifs has been elegantly demonstrated (Garber et al., 2018). In P. aeruginosa, the induction of CopRS induces the expression of the CzcRS TCS (Caille et al., 2007). In general, both Zn and Cu resistance may allow bacteria to resist to the toxic boost of metals that are discharged into the phagosome of macrophages to kill the pathogens (Botella et al., 2012; Djoko et al., 2015). Shift assays have shown a greater affinity of Zur for promoters controlling the expression of PA1922, PA2911, zrmA, and dksA2 genes. Interestingly, all these genes are also involved in cobalt (Co) homeostasis, since PA1922 is located within an operon that contains a cobN-like gene (PA1923), PA2911 is co-transcribed with PA2914, a homolog of the cobalamin ABC permease (Pederick et al., 2015) and pseudopaline, involving the zrm system, has been shown to transport metals such as Zn, Co, Fe, and nickel (Ni) (Zhang et al., 2019). Similarly, dksA2 is also part of an operon including PA5535, a cobW-like gene, involved in cobalamin biosynthesis. These results might reveal a putative Co-dependent regulatory activity of Zur, which could be linked with vitamin B12 (cobalamin) synthesis (Osman et al., 2021). Our in vitro conditions, by creating a Zn deficiency using TPEN, could displace the filling of Zur with Co (Osman et al., 2017). Some promoters would thus also appear to react first to a Co boost. The analysis of the sequences of the different Zur boxes did not allow us to highlight any particular signature. The DNA-Zur-Co interaction would deserve further investigation.

In E. coli, each of the 50,000 ribosomes present in exponentially growing cell contains about 3 Zn²⁺ ions, resulting in the recruitment of 75% of all the intracellular Zn available (Blaby-Haas et al., 2011). Under conditions of Zn deficiency, the ribosomal proteins RpmE and RpmJ, along with the transcription factor DksA and the two enzymes FolE and PyrC all of which contain Zn-ribbon motifs (and are thus called C+) are replaced by their C− paralog proteins (RpmE2, RpmJ2 DksA2, FolE2, and PyrC2), which lack the key Cys residues and do not require Zn for their function. This replacement frees Zn, which becomes available for other essential biological tasks (Gabriel and Helmann, 2009). A similar mechanism is found also in B. subtilis, in which the RpmE protein is degraded under Zn depleted conditions (Akanuma et al., 2006). As expected we found a rapid drop of C− proteins upon Zn excess in P. aeruginosa. However, unlike B. subtilis, P. aeruginosa C+ proteins are constitutively present even in conditions of Zn deficiency (t0). It is possible that the presence of these proteins reflects a regulatory strategy that guarantees a pool of proteins rapidly functional as soon as Zn is available.

In summary, this work shows for the first time the dynamics of the components of the Zn homeostasis network in P. aeruginosa. The use of the NanoString technology allowed us to precisely quantify several transcripts simultaneously and to follow their dynamics over time. The complementary proteomic analyses allowed us to observe the final outcome of this regulation and to visualize the Zn homeostasis systems at steady state (i.e., after 5 h of growth in presence of Zn excess). Our findings are important to identify the molecular mechanisms favoring host colonization and infection, as well as to understand the environmental signals leading to the insurgence of antibiotic resistance. Specifically, the CzcRS-dependent repression of the oprD porin renders P. aeruginosa resistant to carbapenem in presence of Zn (Perron et al., 2004). According to our data, the OprD protein cannot be detected by western blot after 3 h of Zn treatment (Figure 7C). We have recently reported OrpD repression during phagocytosis (Ducret et al., 2020). Macrophages, and in general the immune system response, could therefore represent an important underestimated cause of carbapenem phenotypic resistance. Understanding the molecular mechanism favoring the appearance of carbapenem resistance will be thus fundamental to optimize the use of these last resort antibiotics.

The data presented in the study are deposited in the Gene Expression Omnibus (GEO) repository, accession number GSE183060.

VD, MV, and KP contributed to conception and design of the study, and wrote the manuscript. All authors contributed to formal analysis, investigation, manuscript revision, read, and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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