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. 2007 Dec 10;76(2):571–577. doi: 10.1128/IAI.01088-07

Interferons Increase Cell Resistance to Staphylococcal Alpha-Toxin▿

Timur O Yarovinsky 1,*, Martha M Monick 2, Matthias Husmann 3, Gary W Hunninghake 2
PMCID: PMC2223481  PMID: 18070901

Abstract

Many bacterial pathogens, including Staphylococcus aureus, use a variety of pore-forming toxins as important virulence factors. Staphylococcal alpha-toxin, a prototype β-barrel pore-forming toxin, triggers the release of proinflammatory mediators and induces primarily necrotic death in susceptible cells. However, whether host factors released in response to staphylococcal infections may increase cell resistance to alpha-toxin is not known. Here we show that prior exposure to interferons (IFNs) prevents alpha-toxin-induced membrane permeabilization, the depletion of ATP, and cell death. Moreover, pretreatment with IFN-α decreases alpha-toxin-induced secretion of interleukin 1β (IL-1β). IFN-α, IFN-β, and IFN-γ specifically protect cells from alpha-toxin, whereas tumor necrosis factor alpha (TNF-α), IL-6, and IL-4 have no effects. Furthermore, we show that IFN-α-induced protection from alpha-toxin is not dependent on caspase-1 or mitogen-activated protein kinases, but requires protein synthesis and fatty acid synthase activity. Our results demonstrate that IFNs may increase cell resistance to staphylococcal alpha-toxin via the regulation of lipid metabolism and suggest that interferons play a protective role during staphylococcal infections.

Staphylococcus aureus is a ubiquitous bacterial pathogen, causing a wide spectrum of severe infections, such as abscesses, pneumonia, endocarditis, and sepsis (18). Many clinical isolates of S. aureus produce a variety of pore-forming toxins (alpha-toxin, γ-toxin, binary leukocidins LukF and LukS, and Panton-Valentine leukocidin) among other virulence factors (28). Staphylococcal pore-forming toxins contribute to the pathogenesis of staphylococcal infections by inducing death in susceptible cells and triggering the release of proinflammatory mediators (16). Staphylococcal alpha-toxin is a prototype β-barrel pore-forming toxin and an important virulence factor of S. aureus (3, 28). Staphylococcal alpha-toxin induces primarily necrotic cell death via a mechanism that involves binding to the cell plasma membrane, oligomerization of the toxin, and formation of pores (3, 8). The efflux of intracellular potassium and the influx of sodium are followed by the loss of transmembrane potential, shutdown of ATP production, and predominantly necrotic cell death (3). These processes are accompanied by the release of proinflammatory and vasoactive mediators, contributing to the pathogenesis of staphylococcal infections (18).

Host cells have varying degrees of sensitivity to the cytolytic effects of alpha-toxin (3). Platelets and red blood cells are generally very sensitive to alpha-toxin-induced cytolysis (2). Among nucleated cells, epithelial and endothelial cells are more sensitive than are lymphocytes, mononuclear phagocytes, or fibroblasts, whereas neutrophils show a high degree of resistance to alpha-toxin-induced cytolysis (39). Recent studies demonstrated that epithelial cells cope with the subcytolytic concentrations of alpha-toxin and related pore-forming toxins by activating mitogen-activated protein kinases (MAPKs) or caspase-1-mediated mechanisms of plasma membrane repair (9, 11, 12). It has been reported that the production of interleukin-1β (IL-1β) or tumor necrosis factor alpha (TNF-α) may contribute to alpha-toxin-induced cell death (1, 10, 17, 30). However, whether the cell responses to alpha-toxin are regulated during staphylococcal infections is not known.

We tested a number of cytokines known to be released during staphylococcal infections to identify factors that protect cells from alpha-toxin. We found that pretreatment with alpha interferon (IFN-α), IFN-β, or IFN-γ decreased alpha-toxin-induced cell death, whereas pretreatment with TNF-α, IL-6, or IL-4 had no effect. The protective effects of IFNs were evident in human cell lines of epithelial, monocytic, and fibroblast origin and in primary mouse splenocytes. Furthermore, we found that IFN-α-induced protection from alpha-toxin was dependent on protein synthesis prior to alpha-toxin exposure, suggesting that IFN-stimulated genes mediate the protection. In contrast to the result for control cells, IFN-α-pretreated cells showed minimal depletion of ATP and maintained their plasma membrane integrity following exposure to alpha-toxin. We also found that the inhibition of MAPKs, caspase-1, or other caspases had no significant effect on IFN-α-induced protection. However, the inhibition of fatty acid synthase abolished the effects of IFN-α, suggesting that IFNs protect cells from alpha-toxin via the regulation of lipid metabolism.

MATERIALS AND METHODS

Reagents and cells.

Staphylococcal alpha-toxin was purchased from Toxin Technology (Sarasota, FL) or purified as described previously (26). Streptolysin O was purchased from Sigma-Aldrich (St. Louis, MO). IFNs and other cytokines were purchased from PBL InterferonSource (Piscataway, NJ), R&D Systems (Minneapolis, MN), or PeproTech (Rocky Hill, NJ). Caspase inhibitors were purchased from BD Biosciences (San Jose, CA); other inhibitors were from EMD Biosciences (San Diego, CA). The human lung epithelial cell line A549 and monocytic cell line THP-1 were purchased from ATCC (Manassas, VA). Mouse splenocytes were isolated from C57BL/6 mice (8- to 10-week-old females; Harlan, Indianapolis, IN) following intraperitoneal treatment with mouse IFN-α (10,000 U per mouse) as approved by the University of Iowa Institutional Animal Care and Use Committee.

Assessment of cell death and relative ATP levels.

Cell death was measured using Guava ViaCount reagent and a Guava EasyCyte Mini flow cytometer (Guava Technologies, Hayward, CA). Fluorescence microscopy images were acquired using 450-nm to 490-nm band-pass excitation and 520-nm long-pass emission filters on an inverted fluorescence microscope (Leitz DM IRB; Leica Microsystems, Bannockburn, IL) and a cooled charge-coupled device color camera (Retiga EX 1350; QImaging, Surrey, British Columbia, Canada) after in situ staining with 1 mM calcein acetoxymethyl (AM) and 8 μM ethidium homodimer (Invitrogen, Carlsbad, CA).

Relative ATP levels in cell culture wells were measured using CellTiter-Glo assay (Promega, Madison, WI) and a Wallac Victor2 microplate reader (EG&G Wallac, Gaithersburg, MD) in the chemiluminescence mode. The linear range of the assay correlated with the concentration of ATP standard, which was established for all examined cell lines in pilot experiments.

Detection of alpha-toxin binding and oligomerization.

Internally 35S-labeled alpha-toxin was made by coupled in vitro transcription/translation with the pT7-NPH8S-K21C plasmid (kindly provided by H. Bayley) (42). Control or IFN-α-pretreated THP-1 cells (5 × 106) were loaded with 1 μg/ml of alpha-toxin for 40 min at 4°C and washed twice with ice-cold phosphate-buffered saline. One-half of each sample was incubated for 2 h at 37°C. Subsequently, cells were lysed in 500 μl radioimmunoprecipitation assay buffer and proteins were precipitated with 4 volumes of acetone, dissolved in sample buffer plus β-mercaptoethanol, and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%). Toxin monomers and oligomers were detected by fluorography using Amplify, FUJI super RX films, and a Kodak amplifier screen.

Immunoblotting.

Cell lysates (30 μg protein per lane) were separated in a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). Antibodies purchased were STAT1, pro-caspase-1, and ATP citrate lyase from Cell Signaling Technology (Danvers, MA); β-actin from Sigma-Aldrich; heat shock protein 90 from Stressgen (Victoria, British Columbia, Canada); and the fatty acid synthase and horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies were used with enhanced chemiluminescent substrates (ECL or ECL Plus; Amersham, Arlington Heights, IL). Following the exposure of Kodak BioMax MR films (Eastman Kodak, Rochester, NY) to the membranes, the intensity of immunoblotting signal was measured, using ImageJ software (National Institutes of Health, Bethesda, MD), as integrated density of the specific bands. The signal was normalized to β-actin and heat shock protein 90 and expressed as percent relative to the nontreated control group.

Statistical analyses.

The difference between the experimental groups was evaluated using a one-way analysis of variance test and a post hoc Bonferroni test. A two-way analysis of variance test was applied to evaluate the effects of serum deprivation or inhibitors on IFN-α-induced protection. Since cycloheximide (CHEX) and cerulenin dramatically decreased baseline ATP levels, their effects on IFN-α-induced protection from alpha-toxin were analyzed by comparing alpha-toxin-induced decreases of ATP levels in the groups treated with CHEX or the carrier solution (0.1% vol/vol dimethyl sulfoxide [DMSO] for CHEX or 0.1% vol/vol ethanol for cerulenin). All calculations were performed with GraphPad Prism software, version 4 (GraphPad Software, San Diego, CA).

RESULTS

IFNs protect cells from alpha-toxin.

In agreement with previous studies (17, 30), massive cell death was observed after the exposure of human lung epithelial cell line A549 to 0.5 μg/ml of alpha-toxin (Fig. 1A). Cell death was also noticeable for A549 cells treated with the high dose of IFN-α alone (1,000 U/ml), but was negligible at lower doses of IFN-α. Remarkably, pretreatment of A549 cells with 100 U/ml or 1,000 U/ml of IFN-α for 24 h significantly decreased alpha-toxin-induced cell death. In addition, IFN-α-pretreated A549 cells retained more ATP than did control cells following exposure to alpha-toxin (Fig. 1B). However, the pretreatment of A549 cells with IL-6, IL-4, or TNF-α had no effect on alpha-toxin-induced loss of ATP (data not shown). IFN-α-induced protection from alpha-toxin was also evident after staining with calcein AM (for live cells) and ethidium homodimer (for dead cells) (Fig. 1C). In addition, the protective properties of IFN-α were shared by IFN-β and IFN-γ (data not shown), indicating that type I and type II IFNs increase cell resistance to alpha-toxin.

FIG. 1.

FIG. 1.

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IFN-α protects A549 and THP-1 cells from alpha-toxin. A549 or THP-1 cells were pretreated with the indicated concentrations of IFN-α for 24 h prior to challenge with alpha-toxin for an additional 24 h. (A and D) Cell death was measured based on membrane permeability for PI in nucleated cells by using the Guava ViaCount reagent and flow cytometry. (B and E) Relative ATP levels were measured at 24 h after alpha-toxin exposure. (C) In situ analyses of dead and live cells were performed by staining A549 cells with calcein AM (green) and ethidium homodimer (red) and fluorescence microscopy. The data are means ± SD of five (B) or four (E) independent experiments, each performed at least in triplicate. The data in panels A and D are means ± SD (error bars) of triplicates and are representative of two independent experiments.

The protective effects of IFN-α were specific for alpha-toxin, since pretreatment with IFN-α sensitized A549 cells to TNF-α and had no effect on the loss of ATP triggered by exposure to hydrogen peroxide (data not shown). Moreover, pretreatment with IFN-α did not protect A549 cells from the depletion of ATP induced by streptolysin O, a pore-forming toxin from Streptococcus pyogenes, or from valinomycin, a potassium ionophore (data not shown).

Human monocytic THP-1 cells are naturally more resistant to alpha-toxin than are A549 and many other cells (7); therefore, higher concentrations of alpha-toxin were required to trigger cell death in THP-1 cells (Fig. 1D). The pretreatment of THP-1 cells with 1,000 U/ml IFN-α for 24 h significantly decreased alpha-toxin-induced cell death (Fig. 1D) and loss of ATP (Fig. 1E). These data indicate that the resistance of monocytic cells to alpha-toxin is further increased by pretreatment with IFN-α.

A number of other cells, such as monocytic cell line U937, nontransformed human fibroblast line HFL-1, and skin epithelial cell line HaCaT, could be protected from alpha-toxin by pretreatment with IFN-α (data not shown). Moreover, splenocytes isolated from mice that were pretreated with IFN-α in vivo were more resistant to alpha-toxin than were splenocytes from control mice (data not shown). Thus, pretreatment with IFNs increases the resistance of cells to the cytolytic action of alpha-toxin in various cell systems.

Previous studies demonstrated that alpha-toxin and other pore-forming toxins trigger the secretion of IL-1β, TNF-α, IL-8 and a number of other proinflammatory mediators (1, 7, 29, 30, 33-35). We found that IFN-α-pretreated THP-1 cells secreted less IL-1β than did control cells after exposure to 1 or 5 μg/ml of alpha-toxin (Fig. 2). Although the challenge with 5 μg/ml of alpha-toxin resulted in similar levels of production of TNF-α and IL-8 from control and IFN-α-pretreated cells, the latter cells secreted less TNF-α and IL-8 after stimulation with 1 μg/ml of alpha-toxin. These data suggest that treatment with IFN-α decreases proinflammatory responses to alpha-toxin by raising the threshold for cell activation.

FIG. 2.

FIG. 2.

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Effects of IFN-α pretreatment on alpha-toxin-induced secretion of cytokines by THP-1 cells. Concentrations of IL-1β, TNF-α, and IL-8 in cell culture supernatants were measured by enzyme-linked immunosorbent assay following treatment with alpha-toxin for 24 h. The data are means ± standard deviations (error bars) of triplicate cultures from a representative experiment.

IFN-α protects cells from alpha-toxin without interfering with alpha-toxin binding and oligomerization.

Next, we examined whether treatment with IFN-α affects alpha-toxin binding and oligomerization by incubating control and IFN-α-pretreated THP-1 cells with 35S-labeled alpha-toxin and subjecting the lysates of washed cells to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography revealed that alpha-toxin was present predominantly in the monomeric form (34 kDa) at the start of the incubation (Fig. 3A). In agreement with results of previous studies (40, 41), amounts of alpha-toxin were essentially equal in monomeric and heptameric forms at 2 h of incubation, although the overall amounts of membrane-bound alpha-toxin decreased, probably due to shedding. Control and THP-1 cells showed similar amounts of monomeric and heptameric forms of alpha-toxin, indicating that pretreatment with IFN-α has no effect on alpha-toxin binding or oligomerization.

FIG. 3.

FIG. 3.

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IFN-α protects cells without interfering with alpha-toxin binding and oligomerization. (A) Binding to and oligomerization on control and IFN-α-pretreated THP-1 cells of labeled alpha-toxin were assessed as described in Materials and Methods. The data are representative of two independent experiments. (B) Cell permeability for PI was measured by flow cytometry following short-term exposure of control or IFN-α-pretreated THP-1 cells. Representative dot plots are shown. (C) ATP levels in control and IFN-α-pretreated THP cells were measured following exposure to 5 μg/ml of alpha-toxin. Means ± standard deviations (error bars) of triplicate samples from a representative experiment are shown.

In the next set of experiments, we evaluated the effects of IFN-α on alpha-toxin-induced plasma membrane permeabilization in THP-1 cells. Although the initial β-barrel pores formed by heptamerized alpha-toxin are permeable to monovalent cations only (38, 40, 41), the exposure of the cells to microgram concentrations of alpha-toxin results in the formation of pores permeable to larger molecules, such as propidium iodide (PI) (8). Therefore, to evaluate alpha-toxin-induced membrane permeabilization, we stained control and IFN-α-pretreated THP-1 cells with PI prior to, and at early time points after, exposure to alpha-toxin (Fig. 3B). IFN-α alone had no effect on the number of PI-positive cells. A considerable fraction of control cells became permeable to PI as early as 2 h after alpha-toxin exposure, whereas very few IFN-α-pretreated cells became permeable to PI (18.0% ± 2.2% cells were PI positive in the control group versus 3.8% ± 0.5% PI-positive cells in the IFN-α-pretreated group; P < 0.01; data are means ± standard deviations [SD] of triplicate samples from a representative experiment). By 4 h of alpha-toxin exposure, 36.1% ± 3.4% cells in the control group became permeable to PI, whereas only 6.7% ± 0.3% PI-positive cells were found in the IFN-α-pretreated group (P < 0.01). Remarkably, a fraction of IFN-α-pretreated cells showed a decrease in forward scatter without membrane permeabilization, which may indicate transient ion fluxes and cell volume decrease.

Since the detection of alpha-toxin-induced permeabilization using flow cytometry was problematic in A549 cells, which are cultured under adherent conditions, we replicated our experiments using primary splenocytes from control and IFN-α-pretreated mice. Similar numbers of PI-positive cells were found in splenocytes isolated from control (13.5% ± 1.3%, n = 3) and IFN-α-pretreated mice (13.7% ± 1.2%) following ex vivo culture for 4 h without additional stimulation. The number of PI-positive splenocytes in the control group almost doubled to 27.5% ± 1.5% after treatment with alpha-toxin for 4 h, whereas little increase was found for the splenocytes from IFN-α-pretreated mice (18.3% ± 0.2%, significantly different from control; P < 0.01). Thus, our data suggest that IFN-α protects cells from alpha-toxin by interfering with the formation of alpha-toxin pores.

It is known that alpha-toxin triggers rapid depletion of ATP and that alpha-toxin-resistant cells quickly repair membrane lesions and recover their pools of ATP (38, 41). To determine the effects of IFN-α on the early alpha-toxin-triggered loss of ATP, we measured changes in ATP levels for up to 6 h after exposure to alpha-toxin. The levels of ATP in control and IFN-α-pretreated groups increased without alpha-toxin treatment, which may reflect ongoing cell proliferation (Fig. 3C). Treatment of control THP-1 cells with 5 μg/ml of alpha-toxin resulted in dramatic depletion of ATP within the first 2 to 3 h. However, IFN-α-pretreated cells retained high levels of ATP within 6 h after exposure to alpha-toxin. Thus, our data suggest that pretreatment with IFN-α prevents early loss of ATP following exposure to alpha-toxin.

IFN-α-induced protection from alpha-toxin is dependent on protein synthesis and activity of fatty acid synthase.

Recent studies implicated MAPKs in the protection of cells from subcytolytic concentrations of alpha-toxin and other structurally related pore-forming toxins (11, 12). Extracellular signal-regulated kinase and p38 MAPKs are activated by type I IFNs and participate in the regulation of cell survival (15). However, we found that well-characterized inhibitors of p38 (SB203580) and extracellular signal-regulated kinase (UO126) had no significant effect on IFN-α-induced protection of THP-1 or A549 cells from alpha-toxin (data not shown).

Most of the biological effects of IFNs are realized via expression of IFN-stimulated genes, many of which are involved in the regulation of cell death and survival (6). During our initial screening experiments, we found that IFN-α showed maximal protection if used 16 to 24 h prior to alpha-toxin exposure, was less efficient if used 4 h prior to alpha-toxin, and showed no protection if used simultaneously with alpha-toxin (data not shown). These kinetics suggest that IFN-stimulated genes mediate protection from alpha-toxin. Therefore, we examined the effects of CHEX on the responses of control and IFN-α-pretreated cells to alpha-toxin. CHEX alone decreased ATP levels by 30 to 40% relative to ATP levels in the cells exposed to 0.1% DMSO, which was used as a carrier. However, both groups responded to alpha-toxin by similar reductions of the ATP levels (Fig. 4A and B) and the presence of CHEX had little effect on cell viability within the frame of the experiments (data not shown). When CHEX was added 24 h prior to alpha-toxin, ATP levels decreased to levels similar to those in cells treated with CHEX alone or IFN-α plus CHEX (Fig. 4A). There was no significant difference between the CHEX and IFN-α plus CHEX groups in ATP levels after treatment with 1 or 5 μg/ml alpha-toxin. These data indicate that CHEX abolishes the protective effects of IFN-α if added simultaneously with IFN-α. However, CHEX did not abolish the protective effects of IFN-α if added 23 h later, since cells treated with IFN-α plus CHEX retained significantly higher ATP levels than did cells treated with CHEX alone after alpha-toxin exposure (Fig. 4B). In composite, these data suggest that the protective effects of IFN-α are dependent on protein synthesis prior to alpha-toxin exposure.

FIG. 4.

FIG. 4.

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The protective effects of IFN-α are dependent on protein synthesis prior to exposure to alpha-toxin. THP-1 cells were treated with 1,000 U/ml IFN-α for 24 h prior to exposure to alpha-toxin. CHEX (100 μg/ml) was added simultaneously with IFN-α (A) or 23 h later (B). The timelines for treatment are shown in the diagrams above the corresponding bar graphs. Asterisks denote significant decreases of ATP levels 24 h after challenge with the indicated concentration of alpha-toxin (*, P < 0.05; **, P < 0.001). A significant difference in ATP levels was observed between IFN-α and IFN-α plus CHEX groups only when CHEX was added 23 h after IFN-α (#, P < 0.01). The data are from a representative experiment performed in quadruplicate. (C) The expression of the indicated proteins in THP-1 cells treated with 10 to 1,000 U/ml of IFN-α was analyzed by immunoblotting. The left panel shows representative scans, and the right panel shows relative intensity of the specific bands normalized to β-actin and heat shock protein. Error bars indicate standard deviations.

A recent study showed that the activation of caspase-1 and the induction of lipogenic genes facilitate epithelial cell recovery from the attack by alpha-toxin and a structurally related pore-forming toxin, aerolysin (9). It is well known that type I IFNs modify lipid metabolism and increase the activity of fatty acid synthase, a key enzyme involved in the biosynthesis of membrane phospholipids (22). We found that the treatment of THP-1 cells with IFN-α increased the abundance of fatty acid synthase, but slightly decreased the abundance of ATP citrate lyase, which is involved in the regulation of lipid biosynthesis by generating cytosolic acetyl-coenzymeA (Fig. 4C). This result coincided with the induction of pro-caspase-1 and STAT1.

To determine the role of fatty acid synthase in IFN-α-induced protection from alpha-toxin, we used cerulenin, a well-characterized inhibitor of this enzyme (23). We found that cerulenin decreased baseline ATP levels, sensitized cells to alpha-toxin, and reversed the protective effects of pretreatment with IFN-α (Fig. 5A). Similar effects of cerulenin were observed in A549 cells (data not shown). To determine the role of caspase-1, we exposed control and IFN-α-pretreated THP-1 cells to alpha-toxin in the presence of the caspase-1-specific, cell-permeable peptide inhibitor Ac-YVAD-CMK. We observed that neither the loss of ATP in control cells after alpha-toxin exposure nor the IFN-α-induced protection from alpha-toxin was altered in the presence of Ac-YAVD-CMK (Fig. 5B). Similarly, a cell-permeable pan-caspase inhibitor zVAD-FMK had no effect on alpha-toxin-induced loss of ATP or IFN-α-induced protection from alpha-toxin (Fig. 5C). However, IL-1β secretion in control cells exposed to alpha-toxin decreased in the presence of Ac-YVAD-CMK (Fig. 5D) or zVAD-FMK (Fig. 5E), which confirmed the efficiency of the inhibitors, since the secretion of IL-1β is dependent on caspase-1-mediated cleavage of pro-IL-1β. Thus, our data show that IFN-induced resistance to alpha-toxin is not dependent on caspases, but requires the activity of fatty acid synthase.

FIG. 5.

FIG. 5.

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The protective effects of IFN-α are dependent on activity of fatty acid synthase, but do not require the activity of caspase-1 or other caspases. (A) Cerulenin (5 μg/ml) or an equivalent amount of ethanol was added to control and IFN-α-pretreated THP-1 cells 30 min prior to exposure to alpha-toxin. Relative ATP levels were measured 2 h later. Asterisks denote a significant decrease of ATP levels 24 h after challenge with the indicated concentration of alpha-toxin (*, P < 0.05; **, P < 0.001). (B and C) Cell-permeable caspase-1 inhibitor Ac-YVAD-CMK (100 μM), pan-caspase inhibitor zVAD-FMK (20 μM), control peptide zFA-FMK (20 μM), or an equivalent amount of DMSO was added to control and IFN-α-pretreated THP-1 cells 30 min prior to exposure to alpha-toxin. Relative ATP levels were measured 24 h later. (D and E) The effects of IFN-α and caspase inhibitors on alpha-toxin-induced secretion of IL-1β were measured by enzyme-linked immunosorbent assay. Error bars indicate standard deviations.

DISCUSSION

Our findings demonstrate that IFNs protect a broad spectrum of cells from staphylococcal alpha-toxin, which is a prototypical microbial toxin forming β-barrel pores. The protective effects are shared by type I IFNs (IFN-α and IFN-β) and type II IFN (IFN-γ), but not by other cytokines tested so far. Moreover, the protective effects are specific for alpha-toxin, as we found no protection from streptolysin O, and a previous study showed that type I IFNs actually sensitize lymphocytes to listeriolysin O, a pore-forming toxin from Listeria monocytogenes (4). The variations in the effects of IFNs are likely due to differences in specificity of binding at the molecular level and the mechanisms of pore formation between the pore-forming toxins (37).

The fact that IFNs are protective was surprising to some degree, given that the net effects of IFNs are usually associated with sensitization to apoptotic cell death via the induction of IFN-stimulated genes (6, 32). However, the mechanism of alpha-toxin-induced cell death is primarily necrotic (8) and there are several examples in the literature showing that IFNs may promote cell survival (14, 19, 27).

With respect to the mechanisms of IFN-induced protection from alpha-toxin, we found that IFN-α does not have any significant effect on alpha-toxin binding or oligomerization. However, we observed that pretreatment with IFN-α prevents membrane permeabilization and protects cells from the early depletion of ATP. We also found that IFN-α-mediated protection from alpha-toxin was dependent on timely protein synthesis, suggesting a role for IFN-stimulated genes. Furthermore, we found that IFN-α-mediated protection required the activity of fatty acid synthase, but was not dependent on the activity of MAPKs, caspase-1, or other caspases. Our findings are in line with those of other studies emphasizing the role of membrane lipid turnover in protection from pore-forming toxins (9, 21, 38). However, previous studies focused on cell responses to the efflux of potassium through the pores, which triggers the induction of lipogenic genes to repair the pores, whereas our study shows that IFN-α minimizes cell injury by preparing the cells to attack with alpha-toxin in advance.

Fatty acid synthase has previously been implicated in the regulation of membrane lipid turnover by IFNs (5). This enzyme is regulated at the level of gene transcription via sterol regulatory element binding proteins and at the level of enzyme activity via substrate availability (5). Further studies will be necessary to determine whether IFNs act primarily by increasing expression levels or activity of fatty acid synthase and whether other IFN-stimulated genes contribute to protection from alpha-toxin.

A number of studies showed that the ablation of alpha-toxin secretion or passive immunization against alpha-toxin decreases virulence of S. aureus (13, 20, 24, 25, 43). The recognition of S. aureus protein A by mononuclear phagocytes and/or plasmacytoid dendritic cells triggers the release of IFN-α, albeit at relatively low levels (31, 36). Our findings prompt further investigation of the role of type I IFNs in the pathogenesis of staphylococcal infections. It will be important to determine whether IFN-induced protection of the host cells from alpha-toxin may improve the outcome of staphylococcal infections and whether IFNs regulate cell resistance to other staphylococcal pore-forming toxins, such as leukocidins.

Acknowledgments

This work was supported by a VA Merit Review grant and NIH grants HL073967, HL077431, and HL007638 (G.W.H.).

We acknowledge advice and support from Sucharit Bhakdi and the skillful work of Claudia Neukirch (Institute of Medical Microbiology and Hygiene, Johannes Gutenberg-University Mainz, Mainz, Germany) as well as the technical assistance of Philip Ryan (University of Iowa Carver College of Medicine).

Editor: B. A. McCormick

Footnotes

▿

Published ahead of print on 10 December 2007.

REFERENCES

  • 1.Bhakdi, S., M. Muhly, S. Korom, and F. Hugo. 1989. Release of interleukin-1 beta associated with potent cytocidal action of staphylococcal alpha-toxin on human monocytes. Infect. Immun. 573512-3519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Bhakdi, S., M. Muhly, U. Mannhardt, F. Hugo, K. Klapettek, C. Mueller-Eckhardt, and L. Roka. 1988. Staphylococcal alpha toxin promotes blood coagulation via attack on human platelets. J. Exp. Med. 168527-542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Bhakdi, S., and J. Tranum-Jensen. 1991. Alpha-toxin of Staphylococcus aureus. Microbiol. Rev. 55733-751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Carrero, J. A., B. Calderon, and E. R. Unanue. 2004. Type I interferon sensitizes lymphocytes to apoptosis and reduces resistance to Listeria infection. J. Exp. Med. 200535-540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Castoreno, A. B., Y. Wang, W. Stockinger, L. A. Jarzylo, H. Du, J. C. Pagnon, E. C. Shieh, and A. Nohturfft. 2005. Transcriptional regulation of phagocytosis-induced membrane biogenesis by sterol regulatory element binding proteins. Proc. Natl. Acad. Sci. USA 10213129-13134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Chawla-Sarkar, M., D. J. Lindner, Y. F. Liu, B. R. Williams, G. C. Sen, R. H. Silverman, and E. C. Borden. 2003. Apoptosis and interferons: role of interferon-stimulated genes as mediators of apoptosis. Apoptosis 8237-249. [DOI] [PubMed] [Google Scholar]
  • 7.Dragneva, Y., C. D. Anuradha, A. Valeva, A. Hoffmann, S. Bhakdi, and M. Husmann. 2001. Subcytocidal attack by staphylococcal alpha-toxin activates NF-kappaB and induces interleukin-8 production. Infect. Immun. 692630-2635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Essmann, F., H. Bantel, G. Totzke, I. H. Engels, B. Sinha, K. Schulze-Osthoff, and R. U. Janicke. 2003. Staphylococcus aureus alpha-toxin-induced cell death: predominant necrosis despite apoptotic caspase activation. Cell Death Differ. 101260-1272. [DOI] [PubMed] [Google Scholar]
  • 9.Gurcel, L., L. Abrami, S. Girardin, J. Tschopp, and F. G. van der Goot. 2006. Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival. Cell 1261135-1145. [DOI] [PubMed] [Google Scholar]
  • 10.Haslinger, B., K. Strangfeld, G. Peters, K. Schulze-Osthoff, and B. Sinha. 2003. Staphylococcus aureus alpha-toxin induces apoptosis in peripheral blood mononuclear cells: role of endogenous tumour necrosis factor-alpha and the mitochondrial death pathway. Cell. Microbiol. 5729-741. [DOI] [PubMed] [Google Scholar]
  • 11.Huffman, D. L., L. Abrami, R. Sasik, J. Corbeil, F. G. van der Goot, and R. V. Aroian. 2004. Mitogen-activated protein kinase pathways defend against bacterial pore-forming toxins. Proc. Natl. Acad. Sci. USA 10110995-11000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Husmann, M., K. Dersch, W. Bobkiewicz, E. Beckmann, G. Veerachato, and S. Bhakdi. 2006. Differential role of p38 mitogen activated protein kinase for cellular recovery from attack by pore-forming S. aureus alpha-toxin or streptolysin O. Biochem. Biophys. Res. Commun. 3441128-1134. [DOI] [PubMed] [Google Scholar]
  • 13.Ji, Y., A. Marra, M. Rosenberg, and G. Woodnutt. 1999. Regulated antisense RNA eliminates alpha-toxin virulence in Staphylococcus aureus infection. J. Bacteriol. 1816585-6590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Kaneko, S., N. Suzuki, H. Koizumi, S. Yamamoto, and T. Sakane. 1997. Rescue by cytokines of apoptotic cell death induced by IL-2 deprivation of human antigen-specific T cell clones. Clin. Exp. Immunol. 109185-193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Katsoulidis, E., Y. Li, H. Mears, and L. C. Platanias. 2005. The p38 mitogen-activated protein kinase pathway in interferon signal transduction. J. Interferon Cytokine Res. 25749-756. [DOI] [PubMed] [Google Scholar]
  • 16.Labandeira-Rey, M., F. Couzon, S. Boisset, E. L. Brown, M. Bes, Y. Benito, E. M. Barbu, V. Vazquez, M. Hook, J. Etienne, F. Vandenesch, and M. G. Bowden. 2007. Staphylococcus aureus Panton-Valentine leukocidin causes necrotizing pneumonia. Science 3151130-1133. [DOI] [PubMed] [Google Scholar]
  • 17.Liang, X., and Y. Ji. 2007. Involvement of alpha5beta1-integrin and TNF-alpha in Staphylococcus aureus alpha-toxin-induced death of epithelial cells. Cell. Microbiol. 91809-1821. [DOI] [PubMed] [Google Scholar]
  • 18.Lowy, F. D. 1998. Staphylococcus aureus infections. N. Engl. J. Med. 339520-532. [DOI] [PubMed] [Google Scholar]
  • 19.Marrack, P., J. Kappler, and T. Mitchell. 1999. Type I interferons keep activated T cells alive. J. Exp. Med. 189521-530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.McElroy, M. C., H. R. Harty, G. E. Hosford, G. M. Boylan, J. F. Pittet, and T. J. Foster. 1999. Alpha-toxin damages the air-blood barrier of the lung in a rat model of Staphylococcus aureus-induced pneumonia. Infect. Immun. 675541-5544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.McNeil, P. L., K. Miyake, and S. S. Vogel. 2003. The endomembrane requirement for cell surface repair. Proc. Natl. Acad. Sci. USA 1004592-4597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Memon, R. A., K. R. Feingold, A. H. Moser, W. Doerrler, and C. Grunfeld. 1992. In vivo effects of interferon-alpha and interferon-gamma on lipolysis and ketogenesis. Endocrinology 1311695-1702. [DOI] [PubMed] [Google Scholar]
  • 23.Menendez, J. A., I. Mehmi, V. A. Verma, P. K. Teng, and R. Lupu. 2004. Pharmacological inhibition of fatty acid synthase (FAS): a novel therapeutic approach for breast cancer chemoprevention through its ability to suppress Her-2/neu (erbB-2) oncogene-induced malignant transformation. Mol. Carcinog. 41164-178. [DOI] [PubMed] [Google Scholar]
  • 24.Menzies, B. E., and D. S. Kernodle. 1996. Passive immunization with antiserum to a nontoxic alpha-toxin mutant from Staphylococcus aureus is protective in a murine model. Infect. Immun. 641839-1841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.O'Reilly, M., J. C. de Azavedo, S. Kennedy, and T. J. Foster. 1986. Inactivation of the alpha-haemolysin gene of Staphylococcus aureus 8325-4 by site-directed mutagenesis and studies on the expression of its haemolysins. Microb. Pathog. 1125-138. [DOI] [PubMed] [Google Scholar]
  • 26.Palmer, M., R. Jursch, U. Weller, A. Valeva, K. Hilgert, M. Kehoe, and S. Bhakdi. 1993. Staphylococcus aureus alpha-toxin. Production of functionally intact, site-specifically modifiable protein by introduction of cysteine at positions 69, 130, and 186. J. Biol. Chem. 26811959-11962. [PubMed] [Google Scholar]
  • 27.Pilling, D., A. N. Akbar, J. Girdlestone, C. H. Orteu, N. J. Borthwick, N. Amft, D. Scheel-Toellner, C. D. Buckley, and M. Salmon. 1999. Interferon-beta mediates stromal cell rescue of T cells from apoptosis. Eur. J. Immunol. 291041-1050. [DOI] [PubMed] [Google Scholar]
  • 28.Prévost, G., L. Mourey, D. A. Colin, and G. Menestrina. 2001. Staphylococcal pore-forming toxins. Curr. Top. Microbiol. Immunol. 25753-83. [DOI] [PubMed] [Google Scholar]
  • 29.Ratner, A. J., K. R. Hippe, J. L. Aguilar, M. H. Bender, A. L. Nelson, and J. N. Weiser. 2006. Epithelial cells are sensitive detectors of bacterial pore-forming toxins. J. Biol. Chem. 28112994-12998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Rose, F., G. Dahlem, B. Guthmann, F. Grimminger, U. Maus, J. Hanze, N. Duemmer, U. Grandel, W. Seeger, and H. A. Ghofrani. 2002. Mediator generation and signaling events in alveolar epithelial cells attacked by S. aureus alpha-toxin. Am. J. Physiol. Lung Cell. Mol. Physiol. 282L207-L214. [DOI] [PubMed] [Google Scholar]
  • 31.Smith, E. M., H. M. Johnson, and J. E. Blalock. 1983. Staphylococcus aureus protein A induces the production of interferon-alpha in human lymphocytes and interferon-alpha/beta in mouse spleen cells. J. Immunol. 130773-776. [PubMed] [Google Scholar]
  • 32.Stetson, D. B., and R. Medzhitov. 2006. Type I interferons in host defense. Immunity 25373-381. [DOI] [PubMed] [Google Scholar]
  • 33.Suttorp, N., M. Buerke, and S. Tannert-Otto. 1992. Stimulation of PAF-synthesis in pulmonary artery endothelial cells by Staphylococcus aureus alpha-toxin. Thromb. Res. 67243-252. [DOI] [PubMed] [Google Scholar]
  • 34.Suttorp, N., M. Fuhrmann, S. Tannert-Otto, F. Grimminger, and S. Bhadki. 1993. Pore-forming bacterial toxins potently induce release of nitric oxide in porcine endothelial cells. J. Exp. Med. 178337-341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Suttorp, N., W. Seeger, J. Zucker-Reimann, L. Roka, and S. Bhakdi. 1987. Mechanism of leukotriene generation in polymorphonuclear leukocytes by staphylococcal alpha-toxin. Infect. Immun. 55104-110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Svensson, H., B. Cederblad, M. Lindahl, and G. Alm. 1996. Stimulation of natural interferon-alpha/beta-producing cells by Staphylococcus aureus. J. Interferon Cytokine Res. 167-16. [DOI] [PubMed] [Google Scholar]
  • 37.Tilley, S. J., and H. R. Saibil. 2006. The mechanism of pore formation by bacterial toxins. Curr. Opin. Struct. Biol. 16230-236. [DOI] [PubMed] [Google Scholar]
  • 38.Valeva, A., I. Walev, A. Gerber, J. Klein, M. Palmer, and S. Bhakdi. 2000. Staphylococcal alpha-toxin: repair of a calcium-impermeable pore in the target cell membrane. Mol. Microbiol. 36467-476. [DOI] [PubMed] [Google Scholar]
  • 39.Valeva, A., I. Walev, M. Pinkernell, B. Walker, H. Bayley, M. Palmer, and S. Bhakdi. 1997. Transmembrane beta-barrel of staphylococcal alpha-toxin forms in sensitive but not in resistant cells. Proc. Natl. Acad. Sci. USA 9411607-11611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Walev, I., E. Martin, D. Jonas, M. Mohamadzadeh, W. Muller-Klieser, L. Kunz, and S. Bhakdi. 1993. Staphylococcal alpha-toxin kills human keratinocytes by permeabilizing the plasma membrane for monovalent ions. Infect. Immun. 614972-4979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Walev, I., M. Palmer, E. Martin, D. Jonas, U. Weller, H. Hohn-Bentz, M. Husmann, and S. Bhakdi. 1994. Recovery of human fibroblasts from attack by the pore-forming alpha-toxin of Staphylococcus aureus. Microb. Pathog. 17187-201. [DOI] [PubMed] [Google Scholar]
  • 42.Walker, B., and H. Bayley. 1995. Key residues for membrane binding, oligomerization, and pore forming activity of staphylococcal alpha-hemolysin identified by cysteine scanning mutagenesis and targeted chemical modification. J. Biol. Chem. 27023065-23071. [DOI] [PubMed] [Google Scholar]
  • 43.Wardenburg, J. B., R. J. Patel, and O. Schneewind. 2007. Surface proteins and exotoxins are required for the pathogenesis of Staphylococcus aureus pneumonia. Infect. Immun. 751040-1044. [DOI] [PMC free article] [PubMed] [Google Scholar]

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