Salmonella enterica serovar Typhimurium growth is ...

Biochimica et Biophysica Acta 1860 (2016) 534–541

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Salmonella enterica serovar Typhimurium growth is inhibited by the concomitant binding of Zn(II) and a pyrrolyl-hydroxamate to ZnuA, the soluble component of the ZnuABC transporter Andrea Ilari a,⁎, Luca Pescatori d, Roberto Di Santo d, Andrea Battistoni e, Serena Ammendola e, Mattia Falconi e, Francesca Berlutti c, Piera Valenti c, Emilia Chiancone b,⁎⁎ a CNR-Institute of Molecular Biology and Pathology and Pasteur Institute Fondazione Cenci Bolognetti c/o Department of Biochemical Sciences, ‘Sapienza’ University of Rome, Piazzale A. Moro, 5, 00185 Rome, Italy b Department of Biochemical Sciences ‘Sapienza’ University of Rome, Piazzale A. Moro, 5, 00185 Rome, Italy c Department of Public Health and Infectious Diseases, ‘Sapienza’ University of Rome, Piazzale A. Moro, 5, 00185 Rome, Italy d Department of Chemistry and Technology of Drugs, Pasteur Institute Fondazione Cenci Bolognetti, ‘Sapienza’ University of Rome, Piazzale A. Moro, 5, 00185 Rome, Italy e Department of Biology, University of Rome ‘Tor Vergata’, Via della Ricerca Scientifica 1, 00133 Rome, Italy

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Article history: Received 10 August 2015 Received in revised form 4 November 2015 Accepted 11 December 2015 Available online 12 December 2015 Keywords: ZnuA ZnuABC zinc transporter Salmonella enterica X-ray structure Lead compounds

a b s t r a c t Background: Under conditions of Zn(II) deficiency, the most relevant high affinity Zn(II) transport system synthesized by many Gram-negative bacteria is the ZnuABC transporter. ZnuABC is absent in eukaryotes and plays an important role in bacterial virulence. Consequently, ZnuA, the periplasmic component of the transporter, appeared as a good target candidate to find new compounds able to contrast bacterial growth by interfering with Zn(II) uptake. Methods: Antibacterial activity assays on selected compounds from and in-house library against Salmonella enterica serovar Typhimurium ATCC14028 were performed. The X-ray structure of the complex formed by SeZnuA with an active compound was solved at 2.15 Å resolution. Results: Two di-aryl pyrrole hydroxamic acids differing in the position of a chloride ion, RDS50 ([1-[(4chlorophenyl)methyl]-4-phenyl-1 H-pyrrol-3-hydroxamic acid]) and RDS51 (1-[(2-chlorophenyl)methyl]-4-phenyl-1 H-pyrrol-3-hydroxamic acid) were able to inhibit Salmonella growth and its invasion ability of Caco-2 cells. The X-ray structure of SeZnuA containing RDS51 revealed its presence at the metal binding site concomitantly with Zn(II) which is coordinated by protein residues and the hydroxamate moiety of the compound. Conclusions: Two molecules interfering with ZnuA-mediated Zn(II) transport in Salmonella have been identified for the first time. The resolution of the SeZnuA-RDS51 X-ray structure revealed that RDS51 is tightly bound both to the protein and to Zn(II) thereby inhibiting its release. These features pave the way to the rational design of new Zn(II)binding drugs against Salmonella. General Significance: The data reported show that targeting the bacterial ZnuABC transporter can represent a good strategy to find new antibiotics against Gram-negative bacteria. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Zn(II) is essential for all living organisms since it is used as cofactor in a very wide number of enzymes and proteins involved in central metabolic pathways, in the protection against oxidative damage, in the control of gene expression, and in immune response [1–4]. Abbreviations: SeZnuA, Salmonella enterica ZnuA; RDS library, Roberto Di Santo library; PDB, Protein Data Bank. ⁎ Correspondence to: A. Ilari, CNR-Institute of Molecular Biology and Pathology and Istituto Pasteur Fondazione Cenci Bolognetti c/o Department of Biochemical Sciences “A. Rossi Fanelli”, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy. ⁎⁎ Corresponding author. E-mail addresses: [email protected] (A. Ilari), [email protected] (E. Chiancone).

http://dx.doi.org/10.1016/j.bbagen.2015.12.006 0304-4165/© 2015 Elsevier B.V. All rights reserved.

Nonetheless, as for other metals, both overloads and deficiencies of zinc may be harmful such that all organisms have developed homeostatic mechanisms to finely control the intracellular content of this metal ion. A theme emerged clearly from recent microbial pathogenesis studies, namely, that bacteria compete with their hosts for the binding of Zn(II) [5]. In fact, in spite of the apparent abundance of Zn(II) in host tissues, most of the metal is tightly bound to proteins and is not easily available to infectious bacteria. In addition, mammalian hosts adopt a series of responses, now collectively described as “nutritional immunity,” which further reduce metal availability and prevent bacterial multiplication [6]. In particular, at mucosal sites, the availability of Zn(II) is decreased by the release of the neutrophilic protein calprotectin, whereas its systemic distribution is regulated by changes

A. Ilari et al. / Biochimica et Biophysica Acta 1860 (2016) 534–541

in the expression of zinc transporters that lead to a decrease in the plasma levels of the metal and its storage in intracellular deposits [7–10]. In bacteria, specific transport systems allow Zn(II) acquisition when its availability in the environment is scarce or, in the case of pathogenic bacteria, when the host Zn(II)-sequestration mechanisms have to be neutralized. Under conditions of Zn(II) deficiency, the most relevant and conserved Zn(II) transport systems synthesized by many Gramnegative bacteria are the high affinity Zn(II) ZnuABC transporter and ZupT [5,11,12]. The former has no homolog in eukaryotes, whereas ZupT is related to the members of the ZIP family of eukaryotic divalent metal transporters. Notably, the ZnuABC transport system plays a major role in bacterial virulence as evidenced by the observation that its inactivation results in reduced virulence and colonization of the host in several bacteria including Salmonella enterica, Campylobacter jejuni, Haemophilus ducreyi, Moraxella catharralis, uropathogenic Escherichia coli [10,13–17]. In addition, Zn(II) chelation inhibits biofilm development and represents a potential therapeutic approach for combating biofilm growth in a wide range of biofilm-related infections [18]. Conversely, free Zn(II) increases the virulence of Streptococcus pyogenes [19] and induces intercellular adhesion of Staphylococcus epidermidis and Staphylococcus aureus, the main sources of hospitalacquired infections [18]. Several ABC transporters have a central role in bacterial pathogenesis and show poor homology with eukaryotic proteins, suggesting that they could be targets for antimicrobial therapies [20,21]. Interestingly, a proof of concept that it is possible to modulate microbial virulence by interfering with metal homeostasis has been provided through the identification of new antifungal drugs targeting Zn(II) homeostasis in Candida albicans [22]. Even though a few Gram-negative bacteria, including Neisseria meningitides [23], Yersinia pestis [24], and Pseudomonas aeruginosa [25] have been reported to possess multiple zinc uptake systems, in many other pathogens ZnuABC is the unique importer ensuring efficient Zn(II) uptake in the infected host, thereby suggesting that, in these pathogens, ZnuABC represents a most promising candidate target to interfere with Zn(II) uptake [5]. ZnuABC is composed of three proteins: ZnuA is a soluble periplasmic Zn(II) binding protein which transfers the metal to the channel ZnuB, whose opening is regulated by the ZnuC ATPase. Our previous studies on Salmonella enterica sv Typhimurium have clearly indicated that there is no detectable difference in virulence between strains lacking the whole znuABC operon or the single znuA gene [15]. In turn, this observation suggests that interfering with the role of ZnuA could suffice to inhibit Zn(II) uptake. Moreover, the structure of S. enterica ZnuA (SeZnuA) with the Zn(II) binding site either partially or fully occupied by the metal has been solved at high resolution [26]. In principle, the SeZnuA crystals could lend themselves to the preparation of crystals with an inhibitor molecule and provide information on the structural basis of the inhibitory action. The present paper reports a first attempt to identify molecules interfering with ZnuA-mediated Zn(II) transport in Salmonella. The availability of an in-house library comprising a number of compounds endowed with Zn(II) binding capacity has led to the identification of two di-aryl pyrrole hydroxamic acids capable to inhibit both Salmonella growth and its invasion ability in Caco-2 cells. The resolution of the SeZnuA Xray structure containing one of the two inhibitors revealed its presence at the Zn(II) binding site concomitantly with the metal, thus providing the likely structural basis of its biological action. These findings open the way to the rational design of new Zn(II)-binding drugs against S. enterica-caused infections. 2. Materials and methods 2.1. Compounds All tested compounds were selected from the RDS library, an inhouse chemical library synthesized during the decennial activity of

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drug discovery in the Roberto Di Santo laboratories at “Sapienza” University of Rome. In particular, for the screening against Salmonella, a total of 36 compounds characterized by zinc binding groups were chosen (Table S1). The Di Santo laboratory produced, as detailed in [27], about 20 mg of all the compounds given in Table S1, specifically of 1-[(2chlorophenyl)methyl]-4-phenyl-1 H-pyrrol-3-hydroxamic acid, named RDS51, and of [1-[(4-chlorophenyl)methyl]-4-phenyl-1 H-pyrrol-3hydroxamic acid], named RDS50. The amount produced allowed all biological assays to be performed.

2.2. Bacterial strains and culture media S. enterica serovar Typhimurium ATCC14028 was used throughout. Vogel–Bonner minimal medium E (MM), that contains Zn(II) as contaminant, was employed (0.04 g/l anhydrous MgSO4, 2 g/l citric acid, 10 g/l anhydrous K2HPO4, 3.5 g/l NaNH4HPO4 4H2O, 2 g/l glucose). To maintain Zn(II) contaminations at submicromolar levels, MM was prepared avoiding the use of glassware [28]. Such low concentrations of zinc do not support the growth of Salmonella strains lacking znuA and ensure expression of znuABC in the wild strain [15]. To verify the absence of higher concentrations of contaminating Zn(II), each batch of MM was tested before use by monitoring the growth of the znuAdeleted mutant strain SA123 [15]. MM with zinc was prepared by adding this ion as ZnSO4 at different concentrations ranging from 100 to 500 μM. The wild-type strain was grown in static conditions at 37 °C for 18 h in MM with or without added zinc, reaching an absorbance at 600 nm of 0.65 and 0.60 corresponding to 1.0 × 109 and 8.0 × 108 colony-forming units (CFU)/ml, respectively. For the experiments, bacterial cultures were diluted in MM with or without added zinc to obtain an inoculum corresponding to about 1.0 × 106 CFU/ml.

2.3. Cell cultures Caco-2 cells, derived from a human colon carcinoma cell line, were grown as semi-confluent monolayers in Minimal Essential Medium (MEM), purchased from EuroClone, supplemented with 2 mM glutamine, 100 μg/ml penicillin, 0.1 mg/ml streptomycin, and 10% fetal bovine serum (FBS) at 37 °C for 48 h in a humidified 5% CO2 incubator. Two hours before the invasion assay experiments, cell monolayers were washed with phosphate-buffered saline (PBS) without Ca2 + and Mg2 + prior to the addition of fresh medium with 2% FBS and antibiotics.

2.4. Antibacterial activity assay of selected RDS compounds against S. enterica serovar Typhimurium The antibacterial activity of selected compounds against S. enterica Typhimurium ATCC14028 (about 1.0 × 106 bacteria/ml) has been assessed in MM at 37 °C for 18 h. The compounds tested were able to bind one Zn(II)/molecule and were employed at concentrations ranging from 100 to 500 μM either in Zn(II)-free form or in the Zn(II)-saturated form obtained by addition of equimolar Zn(II). MM without zinc was used to assess the antibacterial activity of Zn(II)-free compounds; in the case of Zn(II)-saturated ones, MM containing the Zn(II) concentration that saturates each compound was used as control of a presumed antibacterial activity of Zn(II). The antibacterial activity of each RDS compound was evaluated after 18 h of incubation by measuring the optical density at 600 nm and by enumerating viable bacteria through CFU counts.

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2.5. Invasion assays of selected RDS compounds against Salmonella enterica serovar Typhimurium S. enterica. S. enterica serovar Typhimurium ATCC14028 was grown in MM without zinc for 18 h at 37 °C. A total of 1.0 × 106 CFUs/ml, collected by this overnight culture, was added to MM without zinc or with 500 μM added Zn(II), in the presence or absence of selected RDS compounds at 500 μM concentration in Zn-(free) or Zn(II)-saturated form. After incubation for 18 h at 37 °C, bacteria were harvested by centrifugation at 10,000 rpm, washed three times in sterile saline, and re-suspended in MEM without antibiotics for the invasion assay. At the outset, cytotoxicity of the compounds was assayed on Caco-2 cell monolayers using the Cell Proliferation Kit I (MTT, Sigma–Aldrich, Milan, Italy) following the manufacturer's instructions. Briefly, cells were seeded in 96-well plates and incubated for 48 h. Different concentrations of the compounds ranging from 100 to 500 μM were added to the cells. After 24 h of incubation, the MTT assay was performed. The invasion assay was performed by infecting Caco-2 cell monolayers (for 2 h at 37 °C in a humidified 5% CO2 incubator) with S. enterica serovar Typhimurium ATCC14028, pre-incubated in the absence or presence of 500 μM Zn-(free) or Zn(II)-saturated RDS compounds, at a multiplicity of infection (MOI) of 100 CFUs/cell. After a 2 h incubation, infected cells were washed extensively with PBS and fresh medium containing 100 μg/ml gentamicin was added to each well to kill extracellular bacteria. Thereafter, the monolayers were washed with PBS, treated with 0.05% trypsin-0.02% EDTA in PBS and lysed by the addition of 0.1% (w/v) sodium deoxycholate. The mixture of lysed Caco-2 cells and bacteria was properly diluted and counted on Luria Bertani (LB) agar by CFU counts to determine the number of viable intracellular bacteria. Invasion efficiency was calculated as percentage of the ratio between intracellular bacteria and inoculum. Controls for antibiotic killing of extracellular bacteria were included in all experiments.

2.6. Statistics The results are expressed as mean values ± standard deviations (SD) of at least three independent experiments. Statistical analysis was performed using the Student's t test for unpaired data. P values of b0.05 were considered significant.

2.7. Protein crystallization, data collection, and data reduction Purified SeZnuA was prepared according to published procedures [29] in 20 mM Hepes, 10 mM NaCl at pH 7.0 and concentrated to 25 mg/ml. The protein was crystallized at 298 K by the hanging-drop vapor diffusion method. Aliquots (0.8 μl) of the protein sample were mixed with an equal amount of reservoir solution containing 1.5% (w/v) polyethylene glycol 400, 2.6 M ammonium sulfate, and 0.1 M Hepes at pH 7.5 and were allowed to equilibrate with a 500 μl volume of reservoir solution. Crystals grew in 1 week and reached dimensions of 0.05 × 0.05 × 0.3 mm. SeZnuA crystals were soaked with RDS51, at a final concentration in the drop of ~2 mM, for 3 h to yield crystals of the complex. These were mounted in nylon loops and flash frozen by quick submersion into liquid nitrogen for transport to the synchrotron-radiation source. A single-wavelength data set (λ = 0.918 Å) was collected from a single RDS51-SeZnuA crystal at the BL-14.1 beamline of the Synchrotron Radiation Source BESSY (Berlin, Germany), using a CCD detector at a temperature of 100 K. The data processed with Denzo [30] and scaled with Scalepack [30] indicate that the crystal is hexagonal and belongs to the P63 space group. Crystal parameters and data collection statistics for the measured crystal are listed in Table S2.

2.8. Structure solution and refinement The structure of SeZnuA in complex with RDS51 was determined by molecular replacement using SeZnuA with the Zn(II) binding site partially occupied by the metal (PDB entry 2XY4) as search model. The rotational and translational searches performed with the program MOLREP [31], CCP4 suite, in the resolution range 10.0–3.0 Å produced a clear solution, corresponding to a monomer in the asymmetric unit. Refinement was performed using the maximum-likelihood method with the program REFMAC [32] while the Coot program [33] was employed for model building (see Table 1). The quality of the model was assessed using the program PROCHECK [34]; all the residues of the structure were within the allowed or generously allowed regions of the Ramachandran plot (see Table 1). The RDS51-SeZnuA structure was refined to 2.15 Å resolution. The final Rcrys value was 19.8%, and the Rfree one was 23.1%. The final model contains 266 residues (residues 27–117 and 139–314), 136 water molecules, 1 sulfate ion with an occupancy of 1, two sulfate ions with an occupancy of 0.7, one molecule of RDS51 with an occupancy of 0.8, and one zinc ion with an occupancy of 1. Residues 118–138 are not visible in the structure. To identify the bound metal ion, a single-wavelength anomalous diffraction data set was collected at 1.282 Å, the zinc anomalous peak wavelength. A peak search in an anomalous difference Fourier map identified a peak with a height of 17 σ located at the Zn(II) binding site.

3. Results 3.1. Effect of zinc on the growth of Salmonella enterica serovar Typhimurium and its znuA-deleted SA123 mutant strain Preliminary experiments were carried out in MM on S. enterica serovar Typhimurium ATCC14028 and its znuA-deleted SA123 mutant strain both in the absence or presence of added zinc (at concentrations between 100 and 500 μM). At the different zinc concentrations tested, the metal did not show antibacterial activity against both strains. The addition of 500 μM zinc to MM only decreases the length of the latent phase of ATCC 4028 from about 480 min to about 130 min. Importantly, the mutant strain did not grow in the absence of added Zn(II) to MM thereby confirming the relevance of ZnuA as major Zn(II) transporter and the absence of contaminating Zn(II) in the medium. The growth curves of both strains are reported in Fig. 1.

Table 1 Effect of Zn(II) and of Zn(II)-free or Zn(II)-saturated RDS51 on the growth of Salmonella enterica serovar Typhimurium ATCC14028 and its znuA-deleted mutant strain SA123. Conditions

S. enterica Serovar Typhimurium ATCC14028 CFUs/ml

znuA-deleted Mutant strain SA123 CFUs/ml

MM MM + Zn(II) 100 μM MM + Zn(II) 250 μM MM + Zn(II) 500 μM MM + RDS51 100 μM MM + RDS51 250 μM MM + RDS51 500 μM MM + Zn(II)-RDS51 100 μM MM + Zn(II)-RDS51 250 μM MM + Zn(II)-RDS51 500 μM

1.5 ± 0.1 × 109 1.9 ± 0.2 × 109 2.0 ± 0.2 × 109 2.2 ± 0.4 × 109 1.2 ± 0.2 × 109 3.6 ± 0.4 × 108⁎⁎ 4.7 ± 0.4 × 107⁎⁎ 8.9 ± 0.4 × 108

1.5 ± 0.2 × 106 1.6 ± 0.2 × 109⁎⁎⁎ 2.2 ± 0.4 × 109⁎⁎⁎ 2.3 ± 0.4 × 109⁎⁎⁎ 4.8 ± 0.2 × 106 1.7 ± 0.2 × 106 1.0 ± 0.2 × 106 1.7 ± 0.2 × 109⁎⁎⁎

6.4 ± 0.2 × 107⁎⁎

1.2 ± 0.4 × 109⁎⁎⁎

1.0 ± 0.2 × 106⁎⁎⁎

8.6 ± 0.2 × 109⁎⁎⁎

The inoculum corresponded to 1.4 ± 0.1 × 106 CFUs/ml. Surviving bacteria were counted by CFU counts after 18 h of incubation. Statistical significance was evaluated with respect to MM. ⁎⁎ P b 0.01. ⁎⁎⁎ P b 0.005.


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Fig. 1. Growth curves of Salmonella enterica serovar Typhimurium ATCC14028 (gray) and of the znuA-deleted SA123 mutant (black) in minimal medium (MM) with or without 500 μM zinc.

3.2. Antibacterial activity of Zn(II)-free and Zn(II)-saturated RDS compounds against Salmonella enterica serovar Typhimurium and its SA123 znuA-deleted mutant The compounds of the RDS library selected for their ability to bind zinc (see Table S1) were dissolved in dimethyl sulfoxide (DMSO) to yield concentrations ranging from 100 to 500 μM and were added to Zn(II)-free MM. The inoculum of S. Typhimurium ATCC14028 was adjusted through appropriate dilutions followed by CFU counts. The antibacterial activity was evaluated during 18 h of incubation of S. Typhimurium ATCC14028 with a given compound by measuring the optical density at 600 nm as well as by enumerating the surviving bacteria after incubation by means of CFU counts. DMSO at the concentration used had no antibacterial activity (data not shown). Among the RDS compounds tested, only two molecules were shown to be active in inhibiting S. Typhimurium ATCC14028 growth, at concentrations lower than about 500 μM, namely RDS51 [1-[(2chlorophenyl)methyl]-4-phenyl-1 H-pyrrol-3-hydroxamic acid] and RDS50 [1-[(4-chlorophenyl)methyl]-4-phenyl-1 H-pyrrol-3-hydroxamic acid], which belong to the class of aryl-arylmethyl pyrrole hydroxamic acids and differ only in the position of the chloride atom. RDS51 showed a significant growth inhibiting activity at 500 μM even though an effect was evident also at half this concentration (Table 1). The effect is a bacteriostatic one as bacteria re-grow in subcultures in the absence of the two compounds. The other RDS compounds tested were unable to inhibit bacterial growth significantly (data not shown). The ability of RDS compounds to bind one Zn(II)/molecule prompted us to study the effect of adding Zn(II) at equimolar concentrations relative to the compound itself so as to saturate its metal binding capacity. The antibacterial activity against S. enterica serovar Typhimurium ATCC14028 compounds was tested over the concentration range 100– 500 μM in MM. When Zn(II)-saturated RDS51 becomes significantly more effective at 250 and 500 μM in inhibiting bacterial growth as shown in Table 1 where the CFU values and the number of surviving bacteria after incubation at 37 °C for 18 h are reported. Therefore, the effect of Zn(II) in the presence of RDS51 is exactly the opposite of that observed when only the metal is added to the growth medium, an

indication that the most biologically active form of RDS51 is the Zn(II)-saturated one. Indeed, the IC50 for Zn(II)-saturated RDS51 and (Zn)-free RDS51 in MM was 221 μM and 128 μM, respectively, demonstrating that Zn(II) binding improves the efficacy. In further tests, the growth-inhibiting activity of RDS51 on S. enterica serovar Typhimurium ATCC14028 and its znuA-deleted mutant SA123 was compared. The data, included in Table 1, clearly point to a specific involvement of SeZnuA in the biological action of the RDS compound. As anticipated above, RDS50 both in the Zn(II)-free and in the Zn(II)-saturated form behaves similarly to RDS51 in inhibiting Salmonella growth (Table 2). The IC50 measured for RDS50 and Zn(II)-saturated RDS50 in MM are 233 μM and 186 μM, respectively. It follows that, just as observed for RDS51, RDS50 improves its efficacy when bound to Zn(II) and that it is less effective than RDS51 in inhibiting Salmonella growth. 3.3. Invasion efficacy on Caco-2 cells by S. enterica serovar Typhimurium ATCC14028 grown in the absence or presence of Zn(II)-free or Zn(II)-saturated RDS50 and RDS51 The possible activity of RDS50 and RDS51 in inhibiting the invasion efficiency of S. Typhimurium ATCC14028 was tested on Caco-2 cells as

Table 2 Antibacterial activity of Zn(II)-free or Zn(II)-saturated RDS50 and RDS51 at 500 μM against Salmonella enterica serovar Typhimurium ATCC14028. Minimal medium plus Zn(II)-free RDS compound

Minimal medium plus Zn(II)-saturated RDS compound

Compound

CFUs/ml

Ng

CFUs/ml

Ng

None RDS50 RDS51

1.2 ± 0.1 × 109 7.9 ± 0.2 × 107⁎ 4.7 ± 0.4 × 107⁎⁎

10 6 5

1.4 ± 0.2 × 109 7.5 ± 0.3 × 106⁎⁎⁎ 3.8 ± 0.6 × 106⁎⁎⁎⁎

10 2 1

The inoculum corresponded to 1.4 ± 0.1 × 106 CFUs/ml. The CFU values and the number of generations (Ng) refer to 18 h of incubation. Statistical significance was evaluated with respect to control. ⁎ P b 0.05. ⁎⁎ P b 0.01. ⁎⁎⁎ P b 0.005.

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Table 3 Number of intracellular Salmonella enterica serovar Typhimurium ATCC14028 in Caco-2 cells after incubation in minimal medium (MM) without Zn(II), with 500 μM Zn(II) or with Zn(II)-free or Zn(II)-saturated RDS compounds at 500 μM.

3.4. X-ray structure of SeZnuA in complex with RDS51

in vitro model. Preliminary experiments showed that both RDS50 and RDS51 did not demonstrate cytotoxicity; cell viability ranging from 100 to 90% was recorded (data not shown). Caco-2 cells (9 × 105 cells/ml) were infected with 9 × 107 S. Typhimurium ATCC14028/ml pre-incubated in MM without added zinc, with 500 μM Zn(II), with Zn(II)-free or Zn(II)-bound RDS50, and RDS51 at 500 μM. Table 3 brings out that bacteria grown in the presence of RDS51 or Zn(II)-saturated RDS51 are characterized by a noteworthy decrease in invasion ability, while those grown in the presence of RDS50 do not change their invasion efficacy irrespective of the presence of Zn(II). Significantly, addition of solely Zn(II) to the bacteria did not affect their invasion capacity.

To arrive at the structural basis of the biological actions just described, both the co-crystallization of SeZnuA with RDS50 and RDS51 and soaking of SeZnuA crystals with the two compounds at different concentrations, in the presence of Zn(II), were attempted. It was possible to collect X-ray diffraction data only for the SeZnuA crystals soaked with RDS51. Their structure was refined to 2.15 Å resolution and showed the concomitant binding of RDS51 and Zn(II) at the SeZnuA metal binding site (Fig. S1, panels A and B). The structure of the RDS51-Zn(II)-SeZnuA complex was compared with the known SeZnuA structures with the Zn(II) binding site occupied either partially or fully by the metal (PDB codes: 2XY4 and 2XQV; ref. 26). However, prior to the analysis of RDS51 binding, a description of the SeZnuA molecule itself is in order. The molecular architecture resembles that of other metal binding transporters and consists of two topologically similar (β/α)4 domains that are connected by a long backbone helix and form a hydrophobic pocket at their interface (Fig. 2A). The Zn(II) binding site is placed at the inter-domain interface and the metal is coordinated by Glu59, His147, His211, and His140 (Fig. 2B). The so-called histidine-rich loop (His-rich loop, residues 118–141), known to play a role in Zn(II) sequestration and transport to the binding site, is not visible with the exception of His140, which coordinates Zn(II) both in the fully and in the partially metal-saturated protein. The structure of the RDS51-Zn(II)-SeZnuA complex was superimposed on the two SeZnuA structures just mentioned and on that of SeZnuA Δ118–141, a deletion mutant lacking a large part of the His-

Fig. 2. A X-ray structure of the RDS51-Zn(II)-SeZnuA complex. The RDS51 compound and the residues establishing electrostatic interactions with it are represented as sticks. B Blow-up of the SeZnuA active site. The residues establishing electrostatic interactions with RDS51 are indicated.

Fig. 3. A Superimposition of the RDS51-Zn(II)-SeZnuA complex (in slate, PDB code: 4BBP) with the other SeZnuA structures determined so far, namely, SeZnuA with the Zn(II) binding site occupied either partially (in orange, PDB code: 2XY4) or fully (in pink, PDB code: 2XQV) and the structure of SeZnuA Δ118–141, a deletion mutant lacking a large part of the His-rich loop (in green, PDB code: 2XH8). The residues of the RDS51-Zn(II)-SeZnuA complex which participate in metal coordination are indicated. B Blow-up of the metal binding site. The 51–65 loop is circled.

Bacterial inoculum deriving from different culture conditions

Number of intracellular bacteria (CFU/ml)

MM MM + Zn(II) MM + Zn(II)-free RDS50 MM + Zn(II)-saturated RDS50 MM + Zn(II)-free RDS51 MM + Zn(II)-saturated RDS51

8.0 ± 1.2 × 104 8.7 ± 0.9 × 104 1.0 ± 0.7 × 105 7.6 ± 1.0 × 104 5.1 ± 0.5 × 103⁎⁎⁎ 5.2 ± 0.7 × 103⁎⁎⁎,•••

Statistical significance was evaluated with respect to MM with or without added Zn(II). ⁎⁎⁎ P b 0.005 with respect to MM. ••• P b 0.005 with respect to MM+ Zn (II).


rich loop (PDB code: 2XH8). The superimpositions yield root mean square deviations in a range between 0.111 and 0.35 Å for the whole protein length (Fig. 3A). The only difference concerns residues 120– 136 belonging to the His-rich loop, that are not visible in the complex, and the small 51–65 loop, that assumes a different conformation (Fig. 3B). In the complex, the metal binding site is fully occupied by Zn(II) and in addition contains one RDS51 molecule. Zn(II) is hexa-coordinated and the overall geometry adopted by the metal ion and its coordinating ligands may be considered as a distorted square-based bi-pyramid. The vertices of the square base are occupied by the two hydroxamate oxygen atoms and the nitrogen atoms of His140 and His147 placed in the visible part of the His-rich loop, while the two apical ligands are the nitrogen atoms of the canonical His211 and His139 residues, likewise belonging to the His-rich loop (Fig. 2B). The distances between the coordinating ligands and Zn(II) range between 2.0 and 2.3 Å. The superimposition between the RDS51-Zn(II)-SeZnuA complex and Zn(II)-SeZnuA shows that RDS51 binding gives rise to a limited conformational change that involves the loops surrounding the active site. RDS51 causes the residues in the 55–61 region to shift toward the solvent. In particular, the bi-dentate Zn(II) ligand Glu59 moves ~4.1 Å (Cα–Cα distance), while its side chain rotates ~90° toward the solvent to allow accommodation of RDS51 inside the metal binding cavity (Fig. 4A). In addition, His139 of the His-rich loop becomes one of the Zn(II) ligands and hence is clearly visible whereas it is not in the other known SeZnuA structures. The cavity hosting the RDS51 molecule is located at the interface between the two SeZnuA subdomains and displays an area of 758.4 Å2 and a volume of 1511 Å3 calculated with CASTp [35]. It is lined by

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Leu285, Leu32, Lys33, Leu53, Asp55, Gly56, Ala57, Ser58, Glu59, His60, and Trp149 (Fig. 4B). The RDS51 compound forms hydrophobic interactions with the Leu285, Leu32, and Trp149 residues, coordinates Zn(II) by means of the two oxygen atoms of the hydroxamate moiety, and interacts electrostatically by means of the chloride atom located at the entrance of the cavity only with a partially protonated aspartic acid (O-Asp283 − RDS51 chloride = 2.9 Å).

4. Discussion The present study reports the successful search of compounds able to inhibit Salmonella growth by interfering with the activity of ZnuABC, the major transporter of Zn(II) under conditions of deficiency of this essential metal, such as those encountered in the infected host. This finding highlights the importance of Zn(II) in bacterial infections and in addition is of value in connection with the worrisome problem of resistance to antibiotics and the consequent need to find new compounds with antibacterial activity. The identified compounds able to affect Salmonella vitality and replication belong to the class of di-aryl pyrrole hydroxamic acids and target specifically the soluble component of the ZnuABC transporter, ZnuA (SeZnuA). This molecule was chosen as target for two reasons. Firstly, its inactivation reduces virulence and host colonization by Salmonella as well as by other bacteria responsible for nosocomial infections [13–19]. Secondly, the SeZnuA high resolution structure [26] is available; in principle, therefore, X-ray quality crystals of SeZnuA in complex with an inhibitory molecule could be obtained and shed light on the structural basis of the biological action.

Fig. 4. A Superimposition of the RDS51-Zn(II)-SeZnuA complex (in slate, PDB code: 4BBP) with Zn(II) bound SeZnuA (in pink, PDB code: 2XQV). The Glu59 residue, which is shifted away from the zinc binding site upon RDS51 binding, is indicated. B RDS51 binding site. The cavity identified with CASTp (http://sts.bioe.uic.edu/castp/) and the residues interacting with RDS51 and/or coordinating Zn(II) are represented as sticks.

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The RDS compounds used for screening the antibacterial activity in vitro on Salmonella have been chosen on the basis of their ability to bind Zn(II). The only two active ones among the 36 assayed (see Table S1) are RDS50 and RDS51, belonging to the class of di-aryl pyrrole hydroxamic acids. The inhibition of Salmonella growth and replication in minimal medium (MM) without added zinc manifests itself at 250 μM for RDS50 and RDS51 and is clearly evident at 500 μM (Tables 1 and 2). The data also show that RDS51 is more active than RDS50 (IC50 = 221 μM vs 233 μM). Meaningfully, the efficacy of both compounds increases significantly when their metal binding capacity is saturated upon addition of equimolar amounts of Zn(II) to MM (Tables 1 and 2). In fact, the IC50 of Zn(II)-saturated RDS50 and RDS51 becomes 186 μM and 128 μM, respectively. In general, RDS51 is more effective than RDS50, especially when saturated with Zn(II). The high resolution structure of the RDS51-Zn(II)-SeZnuA complex (Figs. 2-4) provides the molecular explanation for the ability of the pyrrole hydroxamic acids to inhibit Salmonella growth as well as for the effect of Zn(II) on their biological activity. RDS51 is harbored concomitantly with Zn(II) in the metal binding pocket created between the two SeZnuA domains and establishes a number of interactions with both the protein moiety and the metal ion. Specifically, RDS51 interacts with the Leu285, Leu32, and Trp149 residues that line the hydrophobic domain interface (Fig. 4A, B), forms a hydrogen bond with Asp283 by means of the chloride atom, and with the carboxy-amide oxygen atoms binds tightly Zn(II) which is also coordinated by four protein histidine (His139, His140, His147, His211) residues (Figs. 2B, 3). Therefore, Zn(II) enhances binding of the pyrrole hydroxamic acids to the protein active site by establishing interactions that tie the compound to the protein moiety. The structure of the RDS51-Zn(II)-SeZnuA complex also shows that the Zn(II) binding cavity is strongly hydrophobic and that both the benzyl and phenyl groups bound to the pyrrole are necessary to establish interactions with the protein moiety. Finally, the higher efficacy of RDS51 with respect to RDS50 is in nice agreement with the different position of the chloride atom in the chloro-benzylic ring (Table S1 and Fig. 2B). In fact, a hydrogen bond with Asp283 can be formed when chloride is placed in ortho, as in RDS51, but not when the chloride is in para as in RDS50. RDS51 binding is accompanied by localized conformational changes in the vicinity of the SeZnuA metal binding site. These involve the bidentate Zn(II) ligand Glu59, which rotates and shifts toward solvent jointly with the whole 55–61 region, as well as the His-rich loop where His139 takes the place of His140 as Zn(II) ligand (Fig. 2B). The interaction between RDS51 and Zn(II) disclosed by the RDS51Zn(II)-SeZnuA complex depicts only the last step of the RDS51 and RDS50 biological action. Indeed, the manifestation of its relevance is apparent in the capacity of both compounds to inhibit Salmonella vitality and replication and in the increased efficiency attendant their saturation with zinc (Tables 1 and 2). Lastly, the reduction of intracellular Salmonella in Caco-2 cells, which is significant in the case of Zn(II)-saturated RDS51 (Table 3), shows that the complex decreases Salmonella invasion ability of human Caco-2 cells. In conclusion, the present data show that in those Gram-negative bacteria that, like Salmonella, have ZnuABC as unique importer, affecting Zn(II) transport may open a new research field in the search of novel antibacterial agents. A good strategy appears to be targeting the periplasmic component ZnuA of ZnuABC. In this framework, RDS51 may be considered a lead compound that still requires optimization to increase its efficacy.

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