Biochemical Journal

Research article

Copper redistribution in murine macrophages in response to Salmonella infection

Maud E. S. Achard, Sian L. Stafford, Nilesh J. Bokil, Jy Chartres, Paul V. Bernhardt, Mark A. Schembri, Matthew J. Sweet, Alastair G. McEwan

Abstract

The movement of key transition metal ions is recognized to be of critical importance in the interaction between macrophages and intracellular pathogens. The present study investigated the role of copper in mouse macrophage responses to Salmonella enterica sv. Typhimurium. The copper chelator BCS (bathocuproinedisulfonic acid, disodium salt) increased intracellular survival of S. Typhimurium within primary mouse BMM (bone-marrow-derived macrophages) at 24 h post-infection, implying that copper contributed to effective host defence against this pathogen. Infection of BMM with S. Typhimurium or treatment with the TLR (Toll-like receptor) 4 ligand LPS (lipopolysaccharide) induced the expression of several genes encoding proteins involved in copper transport [Ctr (copper transporter) 1, Ctr2 and Atp7a (copper-transporting ATPase 1)], as well as the multi-copper oxidase Cp (caeruloplasmin). Both LPS and infection with S. Typhimurium triggered copper accumulation within punctate intracellular vesicles (copper ‘hot spots’) in BMM as indicated by the fluorescent reporter CS1 (copper sensor 1). These copper hot spots peaked in their accumulation at approximately 18 h post-stimulation and were dependent on copper uptake into cells. Localization studies indicated that the copper hot spots were in discrete vesicles distinct from Salmonella containing vacuoles and lysosomes. We propose that copper hot spot formation contributes to antimicrobial responses against professional intracellular bacterial pathogens.

  • copper redistribution
  • copper sensor 1 (CS1)
  • innate immunity
  • macrophage
  • Salmonella

INTRODUCTION

The antimicrobial activity of copper has long been appreciated, and indeed, copper has been used in a medical context from antiquity to the present day [1]. The toxicity of copper is usually considered to arise from its redox activity; the Cu+ ion can react with hydrogen peroxide to generate hydroxyl radicals (Fenton chemistry) that can damage proteins, membrane lipids and nucleic acids. In addition, the Cu2+ ion can interact with S-nitrosothiols to generate nitric oxide [2] and drive the oxidation of cellular thiols to form disulfides [3]. Cu+ can also trigger microbial destruction by targeting iron–sulfur cluster enzymes that are essential for survival [4].

In view of the biocidal action of copper, it is not surprising that systems that confer tolerance towards copper ions have evolved in bacteria [5]. Such bacteria include intracellular pathogens such as Salmonella enterica sv. Typhimurium and Mycobacterium tuberculosis. In the case of S. Typhimurium, we have observed that a cueO mutant, which lacks a multi-copper oxidase the function of which is to oxidize the Cu+ ion to the less toxic Cu2+ ion, had reduced virulence in a mouse model of systemic infection [6]. In addition, Cavet and co-workers [7] have shown that a copA (copper-exporting ATPase)/golT double mutant, in which the two ATPases that remove copper ions from the bacterial cytoplasm have been inactivated, showed reduced survival compared with wild-type S. Typhimurium in murine macrophages. Recently, an analysis of the effect of mutation at a locus encoding an outer membrane channel (Rv1698) in M. tuberculosis showed that the loss of this channel resulted in increased sensitivity to copper ions and reduced virulence in a murine infection model [8]. These studies suggest that copper ions may play a more prominent role in the innate immune system than was hitherto recognized, although it should be noted that the link between copper deficiency and increased susceptibility to microbial infection has been known for some time [9,10].

In macrophages, copper is known to induce the expression of ferroportin, the transporter associated with the efflux of ferrous iron from the cell [11]. In addition, the multi-copper oxidase Cp (caeruloplasmin) operates on the outer face of the plasma membrane and catalyses the oxidation of Fe2+, exported by Fpn1 (ferroportin 1), to Fe3+ [12]. The coupling between Fpn1 and Cp extends beyond catalysis, since loss of Cp promotes rapid turnover of Fpn1 [11,13]. Thus there are established links between copper and iron metabolism in macrophages [14]. This link can be exploited in the response of macrophages to bacterial infection. Weiss and co-workers [15] have shown that increased intracellular iron availability is associated with enhanced bacterial survival and that efflux of iron from the macrophage reduced intracellular loads of S. Typhimurium. In this context, copper plays an auxiliary role as a component of the iron efflux system. However, the potential for a more direct role for copper in macrophage defence against bacterial pathogens has been suggested by Petris and co-workers [16] who showed that exogenous copper enhanced the killing of Escherichia coli K12 by RAW264.7 mouse macrophages. This group also observed that macrophage activation by IFNγ (interferon γ) and LPS (lipopolysaccharide) induced the expression of ATP7A (copper-transporting ATPase 1) and also altered its trafficking within RAW264.7 cells. These results suggest that copper is a micronutrient that may have an important role in the antimicrobial response of macrophages.

Copper is toxic towards eukaryotic cells as well as to bacteria, and the complex system of transporters and chaperones involved in homoeostasis and trafficking of this ion is well understood [17]. Recently, the use of techniques to monitor the movement of copper ions in the eukaryotic cell has enhanced our ability to investigate cellular copper movement. Chang and co-workers [1820] have developed fluorescent reporters to directly visualize intracellular copper pools, and recently a modified version of this reporter was used to monitor copper within the mitochondrion [21]. In the present study we investigated responses of primary murine macrophages to S. Typhimurium infection in relation to changes in expression of genes involved in copper homoeostasis and to the movement of copper using the copper-responsive fluorescent reporter CS1 (copper sensor 1).

EXPERIMENTAL

Material

CS1 and the copper chelator TEMEA {tris[(ethylthio)ethyl]amine} were synthesized as described previously [18,22]. The fluorescence response of 2 μM CS1 to Cu+ was monitored in vitro at pH 7.0 as described previously [23] and at pH 5.0, 4.0 and 3.6 using 20 mM sodium acetate instead of 20 mM Hepes as buffer. Copper sulfate and the copper chelator BCS (bathocuproinedisulfonic acid, disodium salt) were purchased from Sigma–Aldrich.

Macrophages, bacterial strains and culture conditions

BMM (bone-marrow-derived macrophages) were generated from adult male C57BL/6 mice as described previously [24], and were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS (fetal bovine serum), 20 units/ml penicillin, 20 μg/ml streptomycin, 2 mM L-glutamine and 104 units/ml recombinant human CSF-1 (colony-stimulating factor-1) (a gift from Chiron, Emeryville, CA, U.S.A.). For live cell imaging, RPMI 1640 medium was replaced with CO2-independent medium (Life Technologies). S. Typhimurium strain SL1344 [25] was grown in LB (Luria–Bertani) broth and harvested for BMM infection as described previously [24].

Infection of BMM

Infection assays were performed as described previously [24] with the following modifications. S. Typhimurium was allowed to infect BMM for 1 h prior to removing any extracellular bacteria by incubation in 200 μg/ml gentamicin for 1 h. Subsequently, BMM were cultured in medium containing 5 μg/ml gentamicin (intramacrophage survival and gene expression analysis) or no antibiotic (confocal microscopy imaging) for up to 24 h. For confocal microscopy imaging, additional washes with medium supplemented with 20 μg/ml gentamicin were performed prior to any staining to remove potential extracellular bacteria.

Determination of the number of intracellular bacteria as a function of extracellular copper levels

Copper sulfate (100 μM) and BCS (200 μM) were used to modulate the level of available copper in the culture medium of BMM. Treatments were administered at 2 h post-infection. The concentration of copper sulfate and BCS were shown not to affect the viability of BMM by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] reduction assay (results not shown), using previously described methodology [26]. To determine the number of intracellular bacteria, BMM were lysed with 0.01% Triton X-100 in PBS at 2 or 24 h post-infection, and the lysate was plated on to LB medium supplemented with 1.5% (w/v) agar. The numbers of intracellular bacteria were calculated by colony counts after an overnight incubation of the LB agar plates at 37°C.

RNA extraction and qRT-PCR (quantitative real-time PCR) analysis of gene expression in BMM

For analysis of gene expression, macrophages were infected with S. Typhimurium, stimulated with 10 ng/ml LPS from Salmonella minnesota Re595 (Sigma–Aldrich), or left untreated. At each time point (2, 4, 8, 14 and 24 h post-challenge), RNA was extracted, and RT (reverse transcription) and RT–PCR were performed as described previously [24]. mRNA corresponding to the murine Hprt (hypoxanthine-guanine phosphoribosyl transferase) gene was used to normalize mRNA levels for genes of interest using the ΔCT method, and fold induction relative to untreated control cells was determined at each time point. Primers used for qRT-PCR are listed in Supplementary Table S1 at http://www.BiochemJ.org/bj/444/bj4440051add.htm.

CS1 staining and fluorescence microscopy

BMM were allowed to adhere to coverslips overnight and were then stimulated with 20 ng/ml LPS, infected with S. Typhimurium or left untreated. At 18 h post-challenge, cells were washed with PBS, fixed [3.7% (w/v) paraformaldehyde, 45 min], washed again with PBS and stained with CS1 (2 μM, 5–10 min). Cells were then washed with PBS and, for some experiments, incubated with TEMEA (1 mM, 5 min). For filipin staining, cells were incubated with 50 μg/ml filipin complex (Sigma–Aldrich) in PBS/10% (v/v) FBS for 2 h.

For immunofluorescence co-localization experiments, CS1-stained cells were permeabilized (0.5% Triton X-100, 5 min), washed with PBS and incubated for 2 h in blocking buffer [1% (v/v) FBS, 2% (w/v) BSA and 0.1% Triton X-100 in PBS). Cells were then incubated with either a rat anti-(mouse CD107a) antibody [Lamp-1 (lysosome-associated membrane protein-1), BD Biosciences; 1:200 dilution] or a purified mouse anti-EEA1 (early endosome antigen 1) antibody (BD Biosciences; 1:100 dilution) for 2 h, washed with 0.1% Triton X-100 in PBS and incubated with a goat anti-(rat IgG)–Alexa Fluor® 657 (Molecular Probes; 1:500 dilution) or a goat anti-(mouse IgG)–Alexa Fluor® 657 (Molecular Probes; 1:400 dilution) for 1 h. BMM and Salmonella DNA were stained using DAPI (4′,6-diamidino-2-phenylindole). Cells were imaged with a Zeiss LSM 510 fluorescence microscope or a DeltaVision personal DV imaging system.

For live imaging, BMM were stimulated with 20 ng/ml LPS for 18 h and incubated with 100 nM LysoTracker Blue (Invitrogen) during the last 1 h. CS1 (1 μM) was added to the culture medium 1 min prior to image acquisition. Each experiment was performed at least three times, and representative results are shown.

Determination of the percentage of cells harbouring copper hot spots

Where mentioned, 200 μM BCS was added to the BMM culture media 4 h prior to any LPS stimulation. BMM were challenged with 20 ng/ml LPS or left untreated for 20 h. Cells were then fixed and stained with CS1 and DAPI. The percentage of cells harbouring copper hot spots was calculated by visualizing a minimum of 600 cells per coverslip (two coverslips per treatment). Fields were randomly selected and the slides were subject to ‘blind’ analysis. The experiment was repeated three times.

RESULTS

Role of copper in regulating S. Typhimurium survival within macrophages

In view of the potential role of copper ions in the bactericidal activity of macrophages, we tested whether addition of exogenous copper ions, at a concentration not influencing the viability of the mouse BMM, affected bacterial loads during infection with S. Typhimurium. Figure 1 shows that addition of Cu2+ ions to a concentration of 100 μM only very modestly reduced intramacrophage levels of viable S. Typhimurium at 24 h post-infection, and this effect was not statistically significant. However, the extracellular copper chelator BCS greatly enhanced S. Typhimurium survival within BMM (Figure 1), and a similar effect was also observed with RAW264.7, a commonly used macrophage cell line (results not shown). These results imply that the endogenous levels of copper present intracellularly and/or within the extracellular medium are sufficient to contribute to macrophage antimicrobial responses against S. Typhimurium.

Figure 1 Copper chelation promotes intracellular S. Typhimurium loads in mouse macrophages

BMM were infected with S. Typhimurium at an MOI (multiplicity of infection) of 10 and the infection was allowed to progress for 24 h using culture medium supplemented with 100 μM copper sulfate (black bars), 100 μM copper sulfate+200 μM BCS (grey bars), or left untreated (white bars). Intracellular bacterial loads were determined at 2 and 24 h post-infection. Results, displayed as relative survival compared with the infected control at 2 h, represent the means±S.E.M. for three independent experiments. *P<0.05.

Regulated expression of genes encoding copper transporters and metalloproteins in macrophages during infection with S. Typhimurium

The above observation, as well as studies on E. coli described by Petris and co-workers [16], indicated that copper trafficking might be important in the response of macrophages to bacterial infection. In view of this, we monitored the expression of genes encoding copper transporters and metalloproteins following LPS stimulation or Salmonella infection. At the cellular level, copper homoeostasis is tightly controlled by several transport proteins including Ctr (copper transporter) 1, which promotes copper uptake into the cell, and Ctr2, which is reportedly associated with late endosomes and lysosomes [27,28]. In addition, Atp7a and Atp7b (copper-transporting ATPase 2) regulate both the intracellular trafficking and the efflux of copper [2931]. We used qRT-PCR to measure Atp7a, Atp7b, Ctr1 and Ctr2 mRNA levels, as well as mRNA levels of Cp. Figure 2 shows that copper transport genes were dramatically up-regulated in BMM in response to either infection with S. Typhimurium or treatment with LPS: Ctr1 ~10–40-fold at 14 h (Figure 2A), Ctr2 ~30-fold at 14 h (Figure 2B), Atpa7 ~10–15-fold at 14 h (Figure 2C), and transcripts for the soluble and GPI (glycosylphosphatidylinositol)-anchored forms of Cp ~500-fold at 14 h (Figures 2D and 2E). In contrast, Atp7b and Heph, encoding the multi-copper oxidase hephaestin, were very slightly expressed at the mRNA level in BMM and were unaffected by S. Typhimurium or LPS (results not shown). We also observed an increase in levels of mRNA transcripts for copper chaperones; Atox1, Ccs [copper chaperone for SOD1 (superoxide dismutase 1)] and Cox17 (cytochrome c oxidase copper chaperone) were maximally up-regulated between and 4 and 8 h post-infection (results not shown). Taken together, these data showed that upon bacterial infection there is a co-ordinate induction of expression of genes encoding transporters that move copper into the cell across the plasma membrane (Ctr1), into the Golgi and ER (endoplasmic reticulum) (Atp7a) and into and out of intracellular vesicles (Ctr2). These observations implied that bacterial infection might trigger copper transport in macrophages.

Figure 2 Regulated expression of copper transporters in macrophages in response to infection with S. Typhimurium or treatment with LPS

BMM were infected with S. Typhimurium at an MOI (multiplicity of infection) of 100 (■), treated with LPS (10 ng/ml) (▲) or were left untreated (●), and RNA was extracted at 2, 4, 8, 14 and 24 h. (A) Ctr1, (B) Ctr2, (C) Atp7a, (D) soluble Cp and (E) GPI-anchored Cp mRNA levels were calculated relative to Hprt. Results, displayed as fold change compared with untreated cells, are combined from three independent experiments (means±S.E.M.).

Detection of copper pools in macrophages using the fluorophore CS1

CS1 has previously been used to visualize copper pools in mammalian cells [18]. Our aim was to use this probe to visualize copper in macrophages in response to pathogen challenge. Prior to experiments with cells, we performed preliminary in vitro characterization of this probe (Figure 3A). In 20 mM Hepes, pH 7.0, the fluorescence of 2 μM CS1 increased up to 13 times upon addition of 10 μM Cu+ as described previously [23]. Addition of 200 μM TEMEA to a Cu+–CS1 complex abrogated this increase in fluorescence (results not shown). Macrophage phagosomes acidify as they mature and fuse with lysosomes, so we also monitored the fluorescence response of CS1 to Cu+ in acidic environments using 20 mM sodium acetate buffers (Figure 3A). In the absence of copper, minimal fluorescence was detected with the apo probe between pH 3.6 and 7.0. In contrast, copper increased the CS1 fluorescence signal across the pH range tested. The signal elicited by Cu+ at pH 5.0 was similar to that obtained at pH 7.0, and was comparable with previously published work [23], whereas the effect was somewhat reduced at pH 3.6 and 4.0. These results confirmed that CS1 could be used to monitor copper ions and that, although very acidic environments would be predicted to modestly reduce the strength of the signal, detection of copper by CS1 was largely independent of pH.

Figure 3 CS1 can be used to detect labile pools of copper in macrophages

(A) The fluorescence emission spectra of 2 μM CS1 in the presence (■) or absence (□) of 10 μM Cu+ were measured at neutral (Hepes buffer) and acidic (sodium acetate buffers) pH. The maximum emission recorded is presented as a function of pH. Rel., relative. (B) BMM were challenged with LPS from Salmonella (20 ng/ml) for 18 h (b and d) or left untreated (a and c). Cells were fixed with paraformaldehyde and washed with PBS. Cell were then stained with CS1 in the presence (c and d) or absence (a and b) of the membrane-permeant copper chelator TEMEA. All images were acquired with identical microscope settings and are representative of six randomly selected fields. Scale bar, 50 μm. (C) BMM were challenged with 20 ng/ml LPS from Salmonella for up to 24 h. Cells were fixed and stained with CS1 and DAPI. At each time point, the percentage of cells containing copper hot spots was determined on a minimum cell population of 600 cells per coverslip (two coverslips per time point). Fields on coverslips were randomly selected and the results represent the means±S.D. for three independent experiments. (D) BMM were cultured in the presence or absence of 200 μM BCS. After 4 h of incubation with BCS, BMM were stimulated with LPS from Salmonella (20 ng/ml) for 20 h. Cells were then fixed and stained with CS1 and DAPI. The percentage of BMM harbouring copper hot spots was determined on a minimum cell population of 600 cells per coverslip (two coverslips per time point and treatment). Results are combined from three independent experiments (means±S.E.M.) **P<0.005.

To determine if signals associated with bacterial infection could affect copper mobilization and distribution within macrophages, we stimulated BMM with Salmonella LPS for 18 h and stained the cells with CS1. Unstimulated cells presented a diffuse and faint staining of their cytoplasm (Figure 3B, panel a) that was absent when the cells had not been incubated with CS1 (results not shown). In response to LPS, we observed punctate staining (copper hot spots) inside the cells (Figure 3B, panel b). Copper hot spots were also observed in RAW264.7 macrophages following stimulation with IFNγ and LPS (results not shown). To confirm that CS1 was specifically reporting copper pools, we incubated labelled cells with the membrane-permeant copper chelator TEMEA. After addition of the chelator, the copper hot spots were not observed in LPS-stimulated macrophages (Figure 3B, panel d), and the fluorescence was similar to that of untreated cells (Figure 3B, panels a and c).

To investigate whether the formation of copper hot spots correlated with inducible mRNA expression of genes encoding copper transporters, we monitored the kinetics of formation of copper hot spots in BMM at 2, 4, 8, 14, 18 and 24 h after LPS stimulation (Figure 3C). At 2 h, only 1–3% of cells presented punctate staining. At 4 h and 8 h, the percentage of cells presenting a copper redistribution doubled. Between 8 and 14 h, this percentage increased from 9±1% to 65±7%. At 18 h, the percentage of cells with these copper pools reached 73±7%, and similar levels were apparent up to 24 h post-stimulation. Since the ability of BMM to clear intracellular Salmonella (Figure 1) was impaired by the extracellular copper chelator BCS, we next investigated whether the presence of BCS in culture medium had an impact on the formation of copper hot spots in LPS-stimulated BMM. Figure 3(D) shows that the percentage of BMM harbouring copper hot spots 20 h after stimulation with LPS was reduced by 45% when the BMM were cultured in the presence of BCS. These results suggest that the LPS-induced formation of copper hot spots is at least partially dependent on copper uptake.

Visualization of copper hot spots during infection of macrophages with S. Typhimurium

We next extended our study by investigating the formation of copper hot spots in BMM after infection with S. Typhimurium. As for LPS, S. Typhimurium triggered copper mobilization and redistribution within BMM as indicated by the fluorescence reported by CS1 (Figure 4A, middle panel). In view of the suggestion that copper ions might exert a direct antibacterial role [16], we were interested to determine whether the copper hot spots co-localized with S. Typhimurium. Figure 4(A) shows macrophages infected with S. Typhimurium and co-stained with CS1 and DAPI to visualize the bacterial cells. The copper hot spots showed no co-localization with S. Typhimurium in infected macrophages (Figure 4A). This was further confirmed by labelling the endosomal–lysosomal marker Lamp-1, which also marks the SCV (Salmonella-containing vacuole) in macrophages [32]. Figure 4(B) shows that the rod-shaped Lamp-1+ SCV membranes (green) did not contain copper hot spots (red), and that no Salmonella Lamp-1+ vesicle containing copper could be observed (Figure 4B). Similarly, copper did not accumulate in early endosomal compartments, as assessed by labelling the early endosome marker EEA1 (Supplementary Movie S1 at http://www.BiochemJ.org/bj/444/bj4440051add.htm). Live cell imaging also confirmed that the copper hot spots are distinct from acidic lysosomal vesicles, as assessed by CS1 and LysoTracker Blue staining (Figure 5A). However, filipin staining indicated that the copper hot spots are isolated from the macrophage cytoplasm by a lipid membrane (Figure 5B).

Figure 4 Copper hot spot localization in Salmonella-infected macrophages

BMM were infected with S. Typhimurium at an MOI (multiplicity of infection) of 4 for 18 h. (A) BMM were fixed and stained with DAPI (left-hand panel) and CS1 (middle panel). The right-hand panel image is a false colour overlay with Cu+–CS1 in red and BMM and Salmonella DNA (DAPI-stained) in grey. (B) Fixed BMM were stained with DAPI (left-hand panel) and CS1 (second panel from left). The late-endosomal compartments and the Salmonella-containing vacuoles were detected with an anti-CD107a (Lamp-1) primary antibody (third panel from left). The right-hand panel image is a false colour overlay (CS1, red; Lamp-1, green; DNA, grey). Scale bars, 5 μm.

Figure 5 Localization of copper hot spots in LPS-stimulated macorphages

BMM were challenged with LPS from Salmonella (20 ng/ml) for 18 h. (A) Live BMM were stained with LysoTracker Blue (middle panel) and CS1 (left-hand panel). The right-hand panel image is a false colour overlay with Cu+–CS1 in red and the acidic compartment (LysoTracker Blue-stained) in blue. (B) BMM were fixed and subsequently stained with CS1 (left-hand panel) and with filipin (middle panel) to visualize membrane-associated cholesterol. The right-hand panel image is a false colour overlay with Cu+–CS1 in red and filipin in white. Scale bars, 5 μm.

DISCUSSION

Although the antibacterial properties of copper have long been recognized, consideration of specific roles for copper ions as part of the innate immune system is relatively recent. It is the case that many bacterial pathogens possess systems that provide for tolerance towards copper ions [5]. In the case of S. Typhimurium, it has been observed that a copA/golT mutant, which lacks the ability to export copper from the cytoplasm, is less competitive compared with the isogenic wild-type strain in a macrophage survival assay [7]. This difference in survival capability between mutant and wild type was only observed after 24 h. Similarly, we observed that copper chelation increased Salmonella loads within macrophages at 24 h, but not 2 h, post-infection (Figure 1). Thus it may be concluded that bactericidal effects of copper, within the context of the innate immune system, manifest themselves at a late stage in the Salmonella infection. In contrast, Petris and co-workers [16] have shown that addition of copper ions to macrophages enhanced killing of E. coli K12 after only 1 h. The production of reactive oxygen species via the NADPH oxidase system is a rapid response, which peaks 30 min after phagocytosis [33]. Thus the findings of Petris and co-workers [16] are consistent with the toxic effect of copper ions arising through enhancement of the effects of hydrogen peroxide via the Fenton reaction. Our results would suggest that Salmonella are resistant to such acute antimicrobial effects of copper, consistent with the known ability of this pathogen to subvert trafficking of the NADPH oxidase system [34]. In contrast, however, we did find evidence for bactericidal effects of copper at the later (24 h) time point in the Salmonella model. In this regard, it is possible that copper ions might exert a more slow-acting effect via their interaction with S-nitrosothiols, formed during the nitrosative burst [2]. Alternatively, a recent study demonstrated that TLR (Toll-like receptor) ligands induced delayed production of mitochondrial reactive oxygen species in macrophages and that this contributed to host defence against Salmonella [35]. It therefore remains possible that copper does act as a Fenton catalyst in this second wave of reactive oxygen species production. Finally, it is also possible that copper limits Salmonella growth by promoting iron export from macrophages [12].

In view of the effects of copper chelation on Salmonella survival, it was of interest to determine whether there was a change in expression of genes encoding copper transporters and metalloproteins. Petris and co-workers [16] have shown that IFNγ increases Ctr1 expression in RAW264.7 mouse macrophages, and we observed a similar effect upon challenge of primary macrophages with either LPS or Salmonella. However, we also observed that Ctr2 and Atp7a mRNA levels were similarly increased (Figures 2B and 2C), consistent with a change in expression of genes encoding all of the key transporters associated with copper trafficking within the cell. The most dramatic change in gene expression was associated with the induction of Cp (Figures 2D and 2E). Cp is a multi-copper oxidase whose ferroxidase activity is coupled to the Fe2+-export activity of the ferroportin iron exporter [12,13]. Thus the changes in gene expression observed upon LPS or Salmonella challenge indicate that the entire suite of genes encoding copper transporters and metalloproteins is induced upon infection. These observations suggest that bacterial infection is likely to have a major impact on the movement of copper within macrophages. The changes in gene expression that we observed coincided with the appearance of copper hot spots that increased between 8 and 14 h post-LPS stimulation and persisted inside the macrophage (Figure 3C). The formation of these copper hot spots was at least partly dependent on cellular uptake, since the cell-impermeant copper chelator BCS reduced their accumulation (Figure 3D). However, it remains possible that copper redistribution within the cell also contributes to their formation and to the antimicrobial effects of copper in infected macrophages.

Our control experiments clearly showed that the fluorescence of the CS1 probe reports the presence of copper, consistent with previous observations [18,23]. This is not the first observation of intracellular copper accumulation in discrete hot spots. Lutsenko and co-workers [36,37] have described the movement of copper in hepatocytes from patients with Wilson's disease, where large amounts of copper accumulate in the cytosol as a consequence of the lack of the active copper-efflux transporter Atp7b. Copper accumulation in deposits (hot spots) occurs in stage II of the Wilson's disease phenotype and is regarded as a protective mechanism that helps to minimize the damaging activities of copper [37]. The chemical properties of the accumulating copper hot spots in hepatocytes are not established. Lutsenko and co-workers [37] suggested that copper hot spots in hepatocytes might be associated with a time-dependent polymerization of metallothioneins. However, we did not observe co-location of copper hot spots and metallothionein using confocal immunofluorescence microscopy (results not shown) and, on the basis of our observation using filipin staining, it does appear that the copper hot spots are surrounded by lipid membrane (Figure 5B). We were not able to identify definitively the nature of the copper hot spots in the macrophage, but could rule out their association with the SCV (Figure 4B and Supplementary Movie S2 at http://www.BiochemJ.org/bj/444/bj4440051add.htm), early endosomes (Supplementary Movie S1) and lysosomes (Figure 5A).

Although copper may exert a direct antimicrobial effect in macrophages by catalysing the production of hydroxyl radicals from hydrogen peroxide immediately after bacterial phagocytosis as suggested by Petris and co-workers [16], copper hot spots are unlikely to be involved in this process since they only began to appear 8–14 h post-infection. Understanding the functional significance of intracellular copper hot spots represents an interesting new challenge in innate immunity research.

AUTHOR CONTRIBUTION

Maud Achard, Sian Stafford, Matthew Sweet, Mark Schembri and Alastair McEwan designed the research and analysed the data. Maud Achard, Sian Stafford and Nilesh Bokil conducted the research. Maud Achard, Jy Chartres and Paul Bernhardt synthesized and tested CS1 and TEMEA. Alastair McEwan, Maud Achard, Matthew Sweet, Sian Stafford and Mark Schembri wrote the paper. All of the authors participated in reviewing and editing the paper prior to submission.

FUNDING

This work was supported by the National Health and Medical Research Council (NHMRC, Australia) [grant number 519722] (to A.G.M and M.A.S). P.V.B is the recipient of an Australian Research Council (ARS) Discovery grant [grant number DP1096029]; M.A.S. is the recipient of an ARC Future Fellowship [grant number FT100100662]; M.J.S. is the recipient of an ARC Future Fellowship [grant number FT100100657] and an honorary NHMRC Senior Research Fellowship [grant number APP1003470]; S.L.S. is the recipient of a University of Queensland Postgraduate Scholarship; and N.J.B. is the recipient of an Endeavour international postgraduate research scholarship and a University of Queensland research scholarship. Confocal microscopy was performed at the Institute for Molecular Bioscience Dynamic Imaging Facility for Cancer Biology, University of Queensland, which was established with the support of the Australian Cancer Research Foundation (ACRF).

Acknowledgments

We thank Dr Timothy Barnett (School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Australia) for help with the DeltaVision personal DV imaging.

Abbreviations: ATP7A/Atp7a, copper-transporting ATPase 1; Atp7b, copper-transporting ATPase 2; BCS, bathocuproinedisulfonic acid, disodium salt; BMM, bone-marrow-derived macrophages; copA, copper-exporting ATPase; Cp, caeruloplasmin; CS1, copper sensor 1; Ctr, copper transporter; DAPI, 4′,6-diamidino-2-phenylindole; EEA1, early endosome antigen 1; FBS, fetal bovine serum; Fpn1, ferroportin 1; GPI, glycosylphosphatidylinositol; IFNγ, interferon γ; Lamp-1, lysosome-associated membrane protein-1; LB, Luria–Bertani; LPS, lipopolysaccharide; qRT-PCR, quantitative real-time PCR; RT, reverse transcription; SCV, Salmonella-containing vacuole; TEMEA, tris[(ethylthio)ethyl]amine

References

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