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Antimicrobial Agents and Chemotherapy, December 2009, p. 5236-5244, Vol. 53, No. 12
0066-4804/09/$08.00+0     doi:10.1128/AAC.00555-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Eradication of Intracellular Salmonella enterica Serovar Typhimurium with a Small-Molecule, Host Cell-Directed Agent{triangledown}

Hao-Chieh Chiu,1 Samuel K. Kulp,1 Shilpa Soni,2,3 Dasheng Wang,1 John S. Gunn,2,3,4 Larry S. Schlesinger,2,3,4 and Ching-Shih Chen1*

Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy,1 Center for Microbial Interface Biology,2 Department of Molecular Virology, Immunology and Medical Genetics,3 Division of Infectious Diseases, Department of Internal Medicine, The Ohio State University, Columbus, Ohio 432104

Received 24 April 2009/ Returned for modification 6 July 2009/ Accepted 28 September 2009


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ABSTRACT
 
Eradication of intracellular pathogenic bacteria with host-directed chemical agents has been an anticipated innovation in the treatment of antibiotic-resistant bacteria. We previously synthesized and characterized a novel small-molecule agent, AR-12, that induces autophagy and inhibits the Akt kinase in cancer cells. As both autophagy and the Akt kinase have been shown recently to play roles in the intracellular survival of several intracellular bacteria, including Salmonella enterica serovar Typhimurium, we investigated the effect of AR-12 on the intracellular survival of Salmonella serovar Typhimurium in macrophages. Our results show that AR-12 induces autophagy in macrophages, as indicated by increased autophagosome formation, and potently inhibits the survival of serovar Typhimurium in macrophages in association with increased colocalization of intracellular bacteria with autophagosomes. Intracellular bacterial growth was partially rescued in the presence of AR-12 by the short hairpin RNA-mediated knockdown of Beclin-1 or Atg7 in macrophages. Moreover, AR-12 inhibits Akt kinase activity in infected macrophages, which we show to be important for its antibacterial effect as the enforced expression of constitutively activated Akt1 in these cells reverses the AR-12-induced inhibition of intracellular serovar Typhimurium survival. Finally, oral administration of AR-12 at 2.5 mg/kg/day to serovar Typhimurium-infected mice reduced hepatic and splenic bacterial burdens and significantly prolonged survival. These findings show that AR-12 represents a proof of principle that the survival of intracellular bacteria can be suppressed by small-molecule agents that target both innate immunity and host cell factors modulated by bacteria.


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INTRODUCTION
 
Salmonella enterica, a gram-negative facultative intracellular pathogen, causes up to 1.3 billion cases of disease worldwide per year (7). Among over 2,500 serovars identified within the six subspecies of S. enterica (11, 27), the serovar Typhimurium infects both humans and animals, and leads to a typhoid-like systemic illness in mice (2), making it a useful model of infection with S. enterica serovar Typhi, the causative agent of typhoid fever in humans (32, 47). Salmonella serovar Typhimurium is capable of invading both phagocytic and nonphagocytic cells through Salmonella pathogenicity island 1 (SPI-1)-induced endocytosis (16, 48, 53). After entering host cells, Salmonella serovar Typhimurium is enclosed in an endosome-like vacuole, called the Salmonella-containing vacuole (SCV), which gradually migrates to the trans-Golgi network, where bacteria proliferate inside the vacuole by exploiting cellular nutrition through an alteration in host cell vesicle trafficking (14, 21, 37). The maturation and migration of SCV rely on the activation of host cell Akt1 kinase, which is mediated by the Salmonella effector, SopB (22, 43). Inhibition of Akt1 activity with small interfering RNA or a small-molecule compound was shown to interfere with the intracellular survival of Salmonella serovar Typhimurium in macrophages (22). On the host side, the macrophage uses various antimicrobial mechanisms to defend against intracellular invasion, including the generation of reactive nitrogen and oxygen species, and the ubiquitin-proteasome system (31, 34, 41). More recently, there is evidence that macroautophagy also plays an important role in the cellular innate defense against intracellular pathogens, including Salmonella serovar Typhimurium. Bacteria that escape from damaged SCVs were shown to be engulfed and lysed by the autophagosome (4, 5).

Macroautophagy (called autophagy hereafter) was initially identified as a cellular energy scavenging process. Under conditions of starvation, mammalian cells acquire energy from the self-digestion of long-lived proteins, and organelles accumulated inside autophagosomes (24). In addition to serving as a response to nutrition deprivation, autophagy has been reported to be involved in the clearance of damaged organelles and unfolded proteins (55), homeostasis of the endoplasmic reticulum (9, 28, 56), and innate defense against intracellular pathogens (8, 29), including Mycobacterium tuberculosis (15, 42), group A Streptococcus pyogenes (26), Rickettsia spp. (10, 49), and Salmonella serovar Typhimurium (4, 5). Thus, from a clinical perspective, targeting the induction of autophagy represents a therapeutically relevant strategy for the treatment of infectious diseases caused by intracellular pathogens.

In our efforts to develop new anticancer drugs, we identified a celecoxib derivative, AR-12 (formerly OSU-03012; Arno Therapeutics, Inc., Fairfield, NJ) (Fig. 1A) (58), which suppresses tumor cell viability through multiple mechanisms, including the inhibition of PDK-1/Akt signaling, activation of endoplasmic reticulum stress, and more recently, the induction of autophagy (13, 20, 30, 38, 45, 46, 50, 51, 54, 58). In light of the role of autophagy and Akt in the intracellular survival of Salmonella serovar Typhimurium, we hypothesized that AR-12 could inhibit intracellular survival of Salmonella serovar Typhimurium in macrophages via effects on host cells. Here, our results show that AR-12 has no direct antibacterial effects on Salmonella serovar Typhimurium but exhibits the ability to effectively kill Salmonella serovar Typhimurium in infected macrophages in vitro at submicromolar concentrations via both autophagy- and Akt-dependent mechanisms. These findings were extended to Salmonella serovar Typhimurium-infected mice, in which oral treatment with AR-12 at 2.5 mg/kg per day significantly reduced hepatic and splenic bacterial burdens and prolonged survival of the infected mice. Together, these findings indicate that the intracellular survival of bacteria can be effectively suppressed by chemical agents targeting both cellular innate immunity and the host factors modulated by bacteria.


Figure 1
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  		FIG. 1. Induction of autophagy in macrophages by AR-12. (A) Structure of AR-12. (B) AR-12 induced autophagosome formation in uninfected RAW264.7 cells. The arrows indicate LC3-positive puncta with diameters of ≥1 µm (giant LC3 puncta). Scale bar, 10 µm. (C) Pattern of LC3 punctum formation in AR-12-treated RAW264.7 cells. The numbers of giant LC3 puncta in AR-12-treated macrophages were counted at different time points during treatment. Points represent the average number of puncta/cell (50 cells counted/time point). (D) Dose-dependent effect of AR-12 on the viability of uninfected RAW264.7 cells. Cells were treated with the indicated doses of AR-12 for 8 h, and cell viability was assessed by the MTT assay. Each data point represents the mean; error bars indicate the SD (n = 6).


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MATERIALS AND METHODS
 
Bacterial strain and phagocytic cell lines. Wild-type Salmonella enterica serovar Typhimurium (14028) was obtained from the American Type Culture Collection (Manassas, VA) and cultured in Luria-Bertani (LB) broth (Difco, Detroit, MI) at 37°C. RAW264.7 and J774.1 murine macrophage cell lines were maintained in Dulbecco's modified Eagle's medium (GIBCO-BRL, Invitrogen Corp., Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; GIBCO-BRL). The THP-1 human monocytic leukemia cell line was maintained in RPMI 1640 containing 10% FBS. THP-1 cells were differentiated with 20 nM 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma-Aldrich, St. Louis, MO) for 48 h. All of the murine and human cells were cultured at 37°C in a humidified incubator containing 5% CO2, and they were seeded into six-well tissue culture plates at 5.0 x 105 cells/well for 12 to 16 h prior to experimentation.

Reagents and antibodies. AR-12 (NSC D728209) was synthesized in-house as described previously (58), with purity exceeding 99% as shown by nuclear magnetic resonance spectroscopy (300 MHz). Bafilomycin A1 and rapamycin were purchased from LC Laboratories (Woburn, MA). Stock solutions were prepared in dimethyl sulfoxide (DMSO) and diluted in culture medium for treatment of cells (final concentration of DMSO, 0.1%). The following antibodies were used in this study: anti-LC3 II (MBL, Woburn, MA); anti-Salmonella group B (BD Biosciences, Franklin Lakes, NJ); anti-ATG7 (Abgent, San Diego, CA); anti-Beclin-1, anti-Akt, anti-phospho-473Ser-Akt, anti-S6 ribosomal protein, and anti-phospho-235/236Ser-S6 ribosomal protein (Cell Signaling Technology, Danvers, MA); anti-phospho-308Thr-Akt (Santa Cruz Biotechnology, Santa Cruz, CA); anti-β-actin (MP Biomedicals, Solon, OH); horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (IgG) and anti-rabbit IgG (Sigma-Aldrich); and Alexa Red-conjugated goat anti-rabbit IgG and fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Invitrogen).

Analysis of bacterial growth in macrophages. Overnight cultures of Salmonella serovar Typhimurium were prepared for infection of macrophages by subculture (1:33) in fresh LB broth and incubation for 3 h at 37°C. Bacteria were then collected by centrifugation at 3,000 x g for 10 min and suspended in phosphate-buffered saline (PBS; pH 7.2) to an optical density of 0.6 at 600 nm, which was equivalent to 5 x 108 CFU/ml. Cultured macrophages were infected by the addition of Salmonella serovar Typhimurium at a multiplicity of infection of 25. Thirty minutes later, the infected cells were exposed to 100 µg/ml gentamicin in culture medium for 1 h and then thoroughly washed with prewarmed PBS three times to remove extracellular bacteria. The infected cells were then treated with test agent(s) at the concentrations and durations indicated in the figures in fresh culture medium containing 10% FBS and 10 µg/ml of gentamicin, which was added to eliminate potential contamination by extracellular bacteria. After treatment, the infected cells were washed three times with PBS, and the surviving intracellular bacteria were harvested by lysis with 0.1% Triton X-100 (Calbiochem, San Diego, CA) in PBS for 10 min at 37°C. The supernatants were immediately serially diluted with PBS and spread onto LB agar plates. After incubation for 16 h at 37°C, the numbers of bacterial colonies for each sample were counted and expressed as CFU. To assess the effect of AR-12 on the entry of Salmonella serovar Typhimurium into macrophages, bacteria were exposed to the vehicle (DMSO) or AR-12 at a concentration of 1 µM in culture medium for 30 min and then used for infection of RAW264.7 cells. After removal of extracellular bacteria, numbers of intracellular bacteria were determined 1 h later.

Cell viability analysis. The effect of AR-12 on the viability of uninfected and infected RAW264.7 cells was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay in six replicates per treatment group. Cells were seeded into 96-well, flat-bottomed plates at 2.5 x 104 cells/well, cultured for 24 h, and then exposed to the concentrations of AR-12 indicated in the figures for 8 h. Controls received DMSO vehicle at a concentration equal to that in drug-treated cells. At the end of treatments, the medium was replaced by 200 µl of 0.5 mg/ml of MTT in culture medium, and cells were incubated in the CO2 incubator at 37°C for an additional 1 h. Supernatants were removed from the wells, and the reduced MTT dye was solubilized in 200 µl/well of DMSO. Absorbance at 570 nm was determined on a microplate reader.

Immunofluorescence microscopy. Immunofluorescence microscopy was used to visualize intracellular Salmonella serovar Typhimurium and autophagosomes in RAW264.7 cells. Treated cells were washed three times with cold PBS, fixed with 4% formaldehyde (Sigma-Aldrich) in PBS for 20 min at 25°C, permeabilized with 0.5% Triton X-100 in PBS for 20 min, and then blocked with 3% bovine serum albumin in PBS overnight at 4°C. After three washes with PBS, the cells were incubated with primary antibody in PBS containing 1% bovine serum albumin for 1 h at 25°C and then with Alexa Red- or fluorescein isothiocyanate-conjugated secondary antibody for 1 h at 25°C. Macrophage nuclei were stained with DAPI (4',6-diamidino-2-phenylindole) contained in the Vectashield mounting medium (Vector Laboratories, Burlingame, CA). The slides were examined using a Nikon TE300 wide-field fluorescent microscope equipped with a digital camera (CoolSnap HQ; Roper Scientific) or a Zeiss LSM 510 confocal laser scanning microscope system. To assess the colocalization of bacteria with autophagosomes, three-dimensional images acquired by confocal microscopy were examined to ensure the inclusion of data from only those bacteria in direct contact with autophagosomes.

Extracellular bacterial growth assay. A late-log-phase Salmonella serovar Typhimurium culture (optical density, 0.6) was diluted 1:200 in fresh LB broth or magnesium minimal medium [100 mM Tris-Cl (pH 5.0), 5 mM KCl, 7.5 mM (NH4)2SO4, 0.5 mM K2SO4, 1 mM KH2PO4, 8 µM MgCl2, 38 mM glycerol, and 0.1% Casamino Acids] (3) and then dispensed into tubes containing different concentrations of AR-12. After thorough mixing, the bacteria suspension was dispensed onto a 96-well plate (six replicates per treatment group). Bacterial growth in each well was monitored spectrophotometrically in a microplate reader (Molecular Devices, Sunnyvale, CA) at 37°C and a wavelength of 600 nm, with readings taken every 30 min for 8 h.

Knockdown of Beclin-1 and Atg7 expression in macrophages. Knockdowns of Beclin-1 and Atg7 expression were achieved by transfection of macrophages with plasmids expressing short hairpin RNA (shRNA) against the target genes (OriGene Technologies, Rockville, MD). Four plasmids coding for different shRNA sequences for each of the two target genes were obtained, amplified in the K-12 strain of Escherichia coli, and then purified using the EndoFree plasmid purification kit (Qiagen, Valencia, CA). Cells were transfected by nucleofection by the use of the Amaxa Nucleofector system (Amaxa Biosystems, Gaithersburg, MD) according to the manufacturer's instructions. Briefly, RAW264.7 cells (2 x 106) were gently suspended in 100 µl of Nucleofector solution V and then mixed with 2 µg of plasmid DNA. The mixture was transferred to a sterile cuvette and nucleofected using program D-32 of the Nucleofector system. The transfected cells were immediately transferred into prewarmed medium for culture. Macrophages transfected with shRNA for green fluorescent protein (GFP) served as controls.

Cells were harvested at 48, 72, and 96 h after transfection, and the knockdown efficiency of each plasmid was assessed by immunoblotting. The plasmid that caused maximal repression for each target was chosen for subsequent experimentation. Stably transfected clones were selected by using medium supplemented with puromycin (2 µg/ml), and immunoblotting was used to screen for knockdown of target gene expression. The selected clones were maintained in the presence of 1 µg/ml of puromycin to retain clonal homogeneity.

Ectopic expression of constitutively active Akt1 in macrophages. The ectopic expression of constitutively active Akt1 (CA-Akt1) was achieved by transfection of RAW264.7 cells with the plasmid encoding hemagglutinin (HA)-tagged CA-Akt1, which was kindly provided by James R. Woodgett (University of Toronto) (39). Constitutive activation of Akt1 was accomplished by replacing the threonine-308 and serine-473 residues with aspartic acid to mimic the phosphorylation at these sites that is required for activation of Akt. The HA tag has been reported to have no effect on Akt1 kinase activity (1). Plasmids were amplified in the K-12 strain of E. coli and then purified using the EndoFree plasmid purification kit (Qiagen, Valencia, CA). Cells were transfected by nucleofection by the use of the Amaxa Nucleofector system (Amaxa Biosystems, Gaithersburg, MD) as described above. Macrophages transfected with empty pcDNA3.1 plasmids served as controls. Stably transfected clones were selected by using medium containing G418 (600 µg/ml), and immunoblotting was used to screen for CA-Akt1 expression. The selected clones were maintained in the presence of 300 µg/ml of G418 to retain clonal homogeneity.

Immunoblotting. Cells were washed with cold PBS, suspended in the M-PER protein extraction reagent (Pierce, Rockford, IL), and then incubated on ice for 10 min. After centrifugation at 11,000 x g for 10 min at 4°C, equivalent amounts of total protein from the lysate supernatants were mixed with 4x Laemmli buffer, incubated at 95°C for 10 min, resolved on a sodium dodecyl sulfate-acrylamide gel (25 µg/lane), and transferred to 0.2 µm nitrocellulose membranes (Gelman, Pall Corp., East Hills, NY). The membranes were blocked with 3% skim milk in Tris-buffered saline (TBS) for 1 h and then washed twice with 0.5% Tween 20 in TBS (TBST). The membrane was incubated with primary antibody at the appropriate dilution in TBST for 12 h at 4°C, washed three times with TBST, incubated with horseradish peroxidase-conjugated goat IgG secondary antibody in TBST containing 1% skim milk, and then washed three times with TBST. The immunopositive bands were visualized by enhanced chemiluminescence (GE Amersham, Piscataway, NJ) followed by exposure to X-ray film (Hyperfilm; GE Amersham).

In vivo studies. Female BALB/c mice (8 to 10 weeks of age) were purchased from Harlan (Indianapolis, IN). Mice were housed as groups under conditions of constant photoperiod (12 h light, 12 h dark) with ad libitum access to sterilized food and water. All experimental procedures with these mice were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of The Ohio State University. Mice were infected by intragastric administration of an overnight culture of Salmonella serovar Typhimurium (105 organisms in 0.1 ml PBS; 50% lethal dose) through a gavage tube. The same overnight culture was plated onto LB agar to confirm the number of organisms inoculated. At 24 h postinfection, mice were assigned to treatment groups and began receiving vehicle (0.5% methylcellulose-0.1% Tween 80 in sterile water) or AR-12 administered intragastrically by gavage once daily. Observations of general health and measurements of body weight were recorded daily.

Assessment of hepatic and splenic bacterial burdens in AR-12-treated, infected mice. Mice were treated with vehicle or AR-12 at 1, 2.5, or 5 mg/kg (seven to nine mice) once daily for 4 days. On the fifth day postinfection, mice were sacrificed and livers and spleens harvested for the determination of bacterial content. Each organ was mechanically homogenized in 10 ml of cold PBS. The homogenates were then serially diluted with PBS and spread onto LB agar plates. After incubation for 16 h at 37°C, the numbers of bacterial colonies for each sample were counted and expressed as CFU.

Assessment of survival in AR-12-treated, infected mice. Mice were treated with vehicle or AR-12 at 2.5 mg/kg (n = 8) once daily for the duration of the study. The survival time of each mouse was recorded and was defined as the time in days from the start of the study to when mice were sacrificed upon exhibiting signs of significant morbidity, which included, though were not limited to, weight loss of ≥20% of initial body weight.

Statistical analysis. Data are expressed as means ± the standard deviation (SD). Group means were compared using a two-tailed t test for independent samples. Differences were considered significant at a P value of <0.05. Statistical analyses were performed using SPSS for Windows (version 16.0; SPSS, Inc., Chicago, IL).


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RESULTS
 
Evidence that AR-12 induces autophagy in RAW264.7 cells without causing cytotoxicity. Recent evidence indicates that exposure of tumor cells to AR-12 in the range of 1 to 5 µM led to cellular responses characteristic of autophagy (13, 30), followed by those characteristic of apoptosis, in a dose-dependent manner. In light of the role of autophagy in cellular innate immunity, we assessed the time-dependent effect of AR-12 (1 µM) on the induction of autophagosome formation (18) in the murine macrophage cell line RAW264.7. During autophagy, the cytoplasmic form LC3-I is processed and recruited to the autophagosomes, where LC3-II is generated via site-specific lipidation. Images indicate that AR-12 induced a transient increase in the formation of LC3-positive puncta, as observed by immunofluorescence microscopy (Fig. 1B). Moreover, AR-12 treatment resulted in the appearance of LC3-positive puncta with diameters greater than 1 µm (giant LC3 puncta) in the cytoplasm. Enumeration of these giant LC3 puncta in AR-12-treated macrophages at different time points revealed a transient increase in their formation (Fig. 1C). Although autophagy can result in cell death due to excessive self-digestion (23), AR-12 at 1 µM had no significant cytotoxic effect on macrophages even after prolonged treatment, as the 50% inhibitory concentration (IC50) for suppressing macrophage cell viability was greater than 10 µM (Fig. 1D). Together, these findings indicated that low doses of AR-12 are capable of inducing autophagy in macrophages without causing cytotoxicity.

AR-12 inhibits intracellular survival of Salmonella serovar Typhimurium in macrophages. To examine whether AR-12-induced autophagy is capable of inhibiting the growth of intracellular bacteria in macrophages, we first assessed the effect of AR-12 on the colocalization of Salmonella serovar Typhimurium with autophagosomes in infected RAW264.7 cells by use of immunofluorescence microscopy. The data show that treatment with 1 µM of AR-12 for 1 h, which induced peak numbers of LC3-positive puncta in macrophages (Fig. 1C), resulted in a 21.2% colocalization of Salmonella serovar Typhimurium with LC3-positive puncta, compared to 3.0% in cells treated with vehicle alone. Next, we determined that treatment of Salmonella serovar Typhimurium-infected RAW264.7 cells with the same concentration of AR-12 (1 µM) for 2 and 8 h reduced bacterial survival by approximately 25% and 90%, respectively, relative to that of DMSO vehicle-treated cells (Fig. 2A, left panel). This reduction in the bacterial load of AR-12-treated RAW264.7 cells was confirmed by immunofluorescent staining, which revealed an 82% decrease in the number of infected macrophages after exposure to 1 µM of AR-12 for 8 h (data not shown). These findings demonstrate the ability of AR-12, at a concentration that induces autophagy and increases the association of autophagosomes with bacteria, to effectively kill intracellular Salmonella serovar Typhimurium in macrophages.


Figure 2
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  		FIG. 2. Inhibition of intracellular survival of Salmonella serovar Typhimurium in macrophages by AR-12. (A) Time-dependent effect of AR-12 on the intracellular survival of Salmonella serovar Typhimurium in RAW264.7 cells. Left panel, comparison of two autophagy-inducing stimuli (1 µM AR-12 and HBSS) on intracellular bacterial survival. Data are expressed as percentages of the mean numbers of CFU in the DMSO-treated control groups (100%) at the end of the respective treatment periods. Error bars represent the SD (n = 3). **, P < 0.01; ***, P < 0.001, for the difference between treated groups and their respective control groups. Right panel, time course of AR-12's effect on intracellular Salmonella serovar Typhimurium survival in CFU. Error bars represent the SD (n = 3). (B) Left panel, dose-dependent effect of AR-12 (8-h treatment) on the intracellular Salmonella serovar Typhimurium survival in RAW264.7 cells. Posttreatment CFU values are expressed as a percentage of those in the control group. Error bars represent the SD (n = 3). Right panel, effect of AR-12 on the viability of infected RAW264.7 cells. Infected RAW264.7 cells were treated with 1 µM AR-12 for 8 h, and the effects on intracellular bacterial survival and macrophage viability were determined. Data represent the mean; error bars represent the SD (n = 3). ***, P < 0.001, for the difference between the drug-treated group and the respective control group.

A closer examination of this time-dependent killing of intramacrophage Salmonella serovar Typhimurium by 1 µM of AR-12 revealed a biphasic mode of action over the 8-h exposure period (Fig. 2A, right panel). In the first 4 h of treatment, the drug caused a modest suppression of intracellular bacterial growth compared to that of the DMSO-treated controls, as reflected in the number of CFU isolated from the macrophages after treatment. This growth-inhibitory phase, however, was followed by a dramatic bactericidal stage, in which, over the next 4 h of treatment, more than 90% of intracellular Salmonella serovar Typhimurium was eradicated from RAW264.7 cells. Interestingly, exposure of Salmonella serovar Typhimurium-infected RAW264.7 cells to nutrient-deprived Hanks' balanced salt solution (HBSS), which has been shown to induce autophagy-mediated clearance of intracellular bacteria (15), also suppressed bacterial growth, but it did not induce the second bactericidal phase that was observed after AR-12 treatment (Fig. 2A, left panel), suggesting a divergence in the mechanisms of bacterial killing by these two treatments at the 8-h time point.

The dose-response curve for this second, bactericidal phase of AR-12's effect on intracellular bacteria indicates an IC50 of 0.2 µM (Fig. 2B, left panel). This AR-12-induced bacterial killing, however, was not attributable to the death of the infected host cells, since AR-12 at 1 µM had no appreciable effect on the viability of bacteria-infected RAW264.7 cells (Fig. 2B, right panel). The antibacterial effect of AR-12 was also evident in murine J774.1 macrophages and, to a lesser extent, in TPA-differentiated THP-1 human monocytic leukemia cells (data not shown), indicating that this AR-12-induced reduction in the intracellular survival of Salmonella serovar Typhimurium was not a cell line-specific event.

To determine if this AR-12-induced clearance of intracellular Salmonella serovar Typhimurium from macrophages involved a direct action of the drug on the salmonellae, bacteria were exposed to AR-12 during growth in LB broth (Fig. 3A); magnesium minimal medium (pH 5.0), which mimics endosomal conditions (data not shown); and a disc diffusion assay (data not shown). In all of these assays, AR-12 exhibited no appreciable antibacterial activity against Salmonella serovar Typhimurium, suggesting that drug-induced suppression of intracellular bacterial survival is mediated indirectly via effects on host cells. Moreover, pretreatment of bacteria with 1 µM of AR-12 for 30 min prior to infection of RAW264.7 cells did not cause a reduction in the number of bacteria isolated 1 h later from macrophages compared to vehicle-pretreated controls, indicating that the ability of AR-12 to inhibit intracellular survival of Salmonella serovar Typhimurium was not a consequence of impaired bacterial entry into cells (Fig. 3B). Comparison of the kinetics of AR-12-mediated antibacterial action was distinctly different from that observed after treatment with bafilomycin A1 (200 nM), an inhibitor of the vacuolar ATPase, which facilitates the acidification of SCVs (Fig. 3C). Importantly, cotreatment of infected RAW264.7 cells with AR-12 and bafilomycin A1 enhanced the inhibition of the survival of intracellular Salmonella serovar Typhimurium in comparison to treatment with either AR-12 or bafilomycin A1 alone (data not shown), thereby providing an additional indication that the pharmacological action of AR-12 is independent of vacuolar ATPase inhibition. Also, the effect of AR-12 on the antibacterial activity of gentamicin was assessed by treatment of Salmonella serovar Typhimurium in LB broth with gentamicin at 10 to 0.15 mg/liter in the presence or absence of 1 µM of AR-12. After 8 and 24 h of treatment, the MIC of gentamicin was unaltered by AR-12 (data not shown), indicating that AR-12 does not potentiate the antibacterial activity of the antibiotic.


Figure 3
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  		FIG. 3. AR-12 does not affect extracellular bacterial growth, bacterial entry into macrophages, or the role of the vacuolar ATPase in the intracellular growth of bacteria. (A) Effect of AR-12 on the growth of Salmonella serovar Typhimurium in LB broth. Data shown are the means; error bars represent the SD (n = 6). (B) Effect of pretreatment with AR-12 on entry of Salmonella serovar Typhimurium into macrophages. Data shown are the means; error bars represent the SD (n = 3). (C) Comparison of the effect of AR-12 with that of the vacuolar ATPase inhibitor bafilomycin A1 (BA) on intracellular growth of Salmonella serovar Typhimurium in RAW264.7 cells. Infected RAW264.7 cells were treated with DMSO, AR-12 (1 µM), or bafilomycin A1 (200 nM), and the numbers of surviving intracellular bacteria were determined. Data shown are the means; error bars represent the SD (n = 3).

AR-12 inhibits the intracellular survival of Salmonella serovar Typhimurium via both autophagy-dependent and -independent mechanisms. To shed light onto the role of autophagy in the AR-12-induced suppression of intracellular bacterial survival, we examined the effect of shRNA-mediated knockdown of two important autophagy regulatory proteins, Beclin-1 and Atg7, on the response of Salmonella serovar Typhimurium-infected RAW264.7 cells to AR-12. While Beclin-1 represents an integral component of the Beclin-1-phosphatidylinositol-3-kinase {gamma} complex (VPS34) (19, 57), Atg7 (an E1 ubiquitin ligase-like protein) facilitates the formation of Atg12-Atg5 complexes (44) and the coupling of phosphatidylethanolamine to Atg8 (LC3) in the autophagic process (17). The knockdown of Beclin-1 and Atg7 expression has been shown to impair the cellular autophagic response to nutritional deprivation in glioblastoma cells (57) and to Mycobacterium tuberculosis infection in RAW264.7 cells (42), respectively. In this study, RAW264.7 cells were stably transfected with shRNA against Beclin-1 or Atg7, which reduced the expression of the respective proteins by 83% and 43%, respectively, relative to that in cells stably transfected with GFP shRNA (Fig. 4A and B, upper panels). These stable transfectants were infected with Salmonella serovar Typhimurium and then treated with AR-12 or DMSO vehicle as indicated (Fig. 4A and B, lower panels). It is intriguing to note that, while decreased expression of Beclin-1 and Atg7 reversed the inhibitory effect of AR-12 on the survival of intracellular bacteria after 2 h of treatment, virtually all bacteria were eliminated after 8 h of treatment, irrespective of the expression level of either Beclin-1 or Atg7. This finding suggests that distinct mechanisms are involved in each of the two phases of the antibacterial action of AR-12 and that autophagy represents the major pathway in suppressing bacterial survival in the first few hours of drug exposure.


Figure 4
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  		FIG. 4. AR-12 inhibits the intracellular survival of Salmonella serovar Typhimurium in macrophages via both autophagy-dependent and -independent mechanisms. (A and B) Effect of shRNA-mediated repression of Beclin-1 (A) and Atg7 (B) expression on AR-12-induced inhibition of intracellular bacterial survival. RAW264.7 cells stably transfected to express shRNA targeting Beclin-1 (sh-Beclin-1), Atg7 (sh-Atg7), or GFP (sh-GFP) were infected with Salmonella serovar Typhimurium and then treated with AR-12 or DMSO as indicated. The numbers of surviving intracellular bacteria were determined and expressed as a percentage of those in the respective sh-GFP control group. Upper panels show the levels of Beclin-1 (A) and Atg7 (B) proteins in stable transfectants expressing the shRNA targeting Beclin-1, Atg7, or GFP, as determined by immunoblotting. Bands for Beclin-1 and Atg7 were quantified by densitometry and normalized to those of β-actin. Percentages represent the relative expression levels of Beclin-1 or Atg7 in sh-Beclin-1 or sh-Atg7 transfectants compared to those in sh-GFP transfectants. Lower panels show the intracellular bacterial survival in AR-12-treated RAW264.7 cells with shRNA-mediated knockdown Beclin-1 (A) or Atg7 (B) expression. Data shown are the means; error bars represent the SD (n = 3). **, P < 0.01; ***, P < 0.001, for the difference between drug-treated groups and their respective control groups.

Inhibition of Akt kinase by AR-12 contributes to the suppression of intracellular survival of Salmonella serovar Typhimurium. To survive and proliferate inside macrophages, Salmonella serovar Typhimurium exploits several host cell kinases, including the Akt1 kinase (22). Because AR-12 is an inhibitor of PDK-1/Akt signaling, we investigated the role of suppressed Akt activity in the killing of intracellular Salmonella serovar Typhimurium by AR-12. First, we assessed the phosphorylation status and activity of the Akt kinase in Salmonella serovar Typhimurium-infected RAW264.7 cells after 8 h of treatment with various concentrations of AR-12. As shown in Fig. 5A, AR-12 decreased the levels of both p-473Ser- and p-308Thr-Akt in infected RAW264.7 cells in a dose-dependent manner. To confirm a functional consequence of reduced phospho-Akt, we examined the phosphorylation status of the Akt substrate, glycogen synthase kinase 3β (GSK3β), which was also suppressed by AR-12 treatment at a concentration of 1 µM.


Figure 5
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  		FIG. 5. Inhibition of the Akt kinase by AR-12 contributes to the suppression of intracellular survival of Salmonella serovar Typhimurium. (A) Western blot analysis of the effect of AR-12 on the phosphorylation status of Akt and the Akt substrate, GSK3β, in Salmonella serovar Typhimurium-infected RAW264.7 cells. RAW264.7 cells were treated with the indicated concentrations of AR-12 or DMSO for 8 h, after which proteins in cell lysates were analyzed by immunoblotting. (B) Levels of the Akt1 protein in selected stably transfected RAW264.7 clones expressing HA-tagged CA-Akt1 as determined by immunoblotting. Empty vector (pcDNA3.1)-transfected cells served as the control. (C) Effects of ectopic expression of CA-Akt1 on the antibacterial activity of AR-12 against intracellular Salmonella serovar Typhimurium. Stable CA-Akt1-expressing RAW264.7 clones were infected with Salmonella serovar Typhimurium and treated with DMSO (–) or 1 µM AR-12 (+) for 2 and 8 h. After treatment, the numbers of surviving intracellular bacteria were determined and expressed as percentages of those in the respective DMSO-treated control group. Data represent the means; error bars represent the SD (n = 3). *, P < 0.05, for the difference between drug-treated groups and their respective control groups.

To validate Akt inhibition as a mechanism for AR-12-induced inhibition of intracellular bacterial survival, the effect of ectopic expression of CA-Akt1 on the ability of AR-12 to inhibit intracellular survival of Salmonella serovar Typhimurium was examined. Two stably transfected RAW264.7 clones (c3 and c4) expressing different levels of HA-tagged CA-Akt1 (Fig. 5B) were infected and then treated with 1 µM of AR-12 for both 2 and 8 h to assess effects on the biphasic activity of the drug. As shown in Fig. 5C, the ectopic expression of CA-Akt1 had no effect on the AR-12-induced suppression of intracellular bacterial survival after 2 h of treatment, in comparison to the empty vector (pcDNA3.1)-transfected control (Fig. 5C, upper panel). This finding indicates that the induction of autophagy by AR-12 is independent of its inhibitory activity against Akt1. In contrast, the marked inhibition of bacterial survival observed at 8 h of treatment was significantly reversed by the expression of CA-Akt1 (Fig. 5C, lower panel). Moreover, this effect occurred in a manner dependent upon the dose of CA-Akt1, in that AR-12 was less effective in reducing bacteria in RAW264.7 c4, which, of the two clones, expressed the higher level of CA-Akt1. Together, these findings indicate that the later, autophagy-independent phase of AR-12's antibacterial action can be attributed, at least in part, to the inhibition of the Akt kinase in infected macrophages.

Oral AR-12 decreases bacterial burden and prolongs the survival of Salmonella serovar Typhimurium-infected mice. Based on the in vitro findings described above, we next determined whether AR-12 demonstrated activity in a mouse model of Salmonella infection. First, the effect of AR-12 on hepatic and splenic bacterial burdens in Salmonella serovar Typhimurium-infected mice was assessed. BALB/c mice were inoculated intragastrically by gavage with 105 CFU of Salmonella serovar Typhimurium and then, 24 h later, randomly assigned to four treatment groups (seven to nine mice each) receiving vehicle or AR-12 at 1, 2.5, or 5 mg/kg administered intragastrically by gavage once daily for 4 days. Two mice in the vehicle-treated group and one in the group treated with 1 mg/kg AR-12 died on the fourth and fifth days postinfection, respectively. All remaining mice were sacrificed on the fifth day postinfection, and the bacterial loads of the liver and spleen of each mouse were determined. Although the bacterial counts varied widely within individual groups, the data suggest that daily oral doses of AR-12 of 2.5 and 5 mg/kg provided substantial protection against bacterial infection, reducing the mean CFU counts in the liver by 99% and 96%, respectively, and the mean CFU counts in the spleen by 98% and 90%, respectively, relative to the vehicle-treated control (Fig. 6A). Pursuant to this finding, we examined whether this marked reduction in organ bacterial burdens would be reflected in prolonged survival of AR-12-treated, Salmonella serovar Typhimurium-infected mice. Twenty-four hours after infection with Salmonella serovar Typhimurium (105 organisms), BALB/c mice were treated with 2.5 mg/kg AR-12 or vehicle once daily by gavage (n = 8). As in the previous study, vehicle-treated mice rapidly developed clinical signs of infection, as evidenced primarily by precipitous weight loss, as well as dehydration, piloerection, and lethargy, which limited the mean survival time to 5.7 ± 2.1 days (mean ± SD). In contrast, oral AR-12 treatment delayed the onset of disease and significantly prolonged the survival of infected mice (mean survival time, 8.1 ± 1.2 days; P < 0.05) (Fig. 6B).


Figure 6
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  		FIG. 6. Oral AR-12 reduces the bacterial burden in organs and improves survival in Salmonella serovar Typhimurium-infected mice. (A) Effect of oral administration of AR-12 on hepatic and splenic bacterial burdens in Salmonella serovar Typhimurium-infected mice. BALB/c mice were inoculated intragastrically with 105 CFU of Salmonella serovar Typhimurium. Beginning at 24 h postinfection, mice were treated orally once daily with vehicle or AR-12 with the indicated doses. At 5 days postinfection, mice were sacrificed and livers and spleens harvested for the determination of bacterial numbers. Data points represent the number of CFU from the indicated organ of individual mice; horizontal bars indicate the mean CFU values for each group (of seven to nine mice). (B) Effect of oral administration of AR-12 on survival of Salmonella serovar Typhimurium-infected mice. Twenty-four hours after intragastric inoculation with 105 CFU of Salmonella serovar Typhimurium, BALB/c mice were treated orally with AR-12 at 2.5 mg/kg or vehicle once daily for the duration of the study. Survival data are presented as Kaplan-Meier survival curves for each treatment group (n = 8). The difference in the mean survival times of the groups was statistically significant at P values of <0.05.


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DISCUSSION
 
The high worldwide incidence of Salmonella infection and the rapid emergence of antibiotic-resistant strains as epitomized by the multidrug-resistant Salmonella serovar Typhi (25, 36) underscore the importance of this pathogen as a public health concern and the urgent need for new approaches for therapeutic intervention. Recently, the concept of eradicating intracellular pathogens through activation of host defense mechanisms and targeting host factors modulated by pathogens has received wide attention in the infectious-disease arena (12, 40). From a therapeutic perspective, targeting host immunity and factors with an orally bioavailable small-molecule agent represents a novel strategy for antimicrobial therapy. Here, we present findings that provide proof of principle of the feasibility of treating Salmonella infection by targeting both cellular innate immunity and host factors manipulated by bacterial effectors in phagocytes with a single small-molecule agent. Our results show that the novel anticancer agent AR-12 is a potent inhibitor of the intracellular survival of Salmonella serovar Typhimurium in macrophages. It induces these effects with a submicromolar IC50 and, importantly, in the absence of host cell toxicity and without direct activity against the bacteria. This lack of direct inhibitory effect on bacterial growth is particularly noteworthy, as it suggests that AR-12 is less likely to promote microbial resistance than are conventional antibiotics. Moreover, our previous in vivo evaluations of AR-12 in murine models of cancer revealed that continuous treatment with doses of up to 200 mg/kg/day was well tolerated and induced no dose-limiting toxicities (D. Wang and S. C. Weng, unpublished data). These doses are up to 80-fold greater than that which reduced organ bacterial burdens and prolonged survival in Salmonella serovar Typhimurium-infected mice in the current study (2.5 mg/kg). These findings suggest that toxicities associated with this low antibacterial dose of AR-12, if they occur, will be minimal.

This study demonstrates a biphasic effect of AR-12 on intracellular survival of Salmonella serovar Typhimurium and provides evidence that each of these phases involves a distinct mechanism for bacterial killing. The data indicate that autophagy plays a major role in the early phase of AR-12's antibacterial effect, as the shRNA-mediated knockdown of Beclin-1 or Atg7 expression reversed the bacterial growth inhibition observed at 2 h of treatment. In contrast, the later phase of bacterial killing, observed at 8 h of treatment, was unaffected by the same shRNA-mediated disruption of the autophagy machinery, indicating an autophagy-independent process. Our data showing the effect of nutrient-deprived HBSS on Salmonella serovar Typhimurium-infected RAW264.7 cells (Fig. 2B) are supportive of this conclusion. Although HBSS, which has been shown to induce autophagy-mediated clearance of intracellular bacteria (15), suppressed Salmonella serovar Typhimurium growth, it did not exhibit the marked second bactericidal phase characteristic of AR-12, suggesting a nonautophagic mechanism for bacterial killing at the later time point. In addition, data from our laboratory indicate that the mTOR inhibitor rapamycin, a potent autophagy-inducing agent, eliminates intracellular Salmonella serovar Typhimurium (data not shown), but only at suprapharmacological concentrations (i.e., >50 µM). Together, these findings suggest that in Salmonella serovar Typhimurium-infected RAW264.7 cells, induction of autophagy results in only a moderate reduction of intracellular Salmonella serovar Typhimurium and that the bulk of bacterial killing after AR-12 treatment occurs through another mechanism.

Our assays of intracellular bacterial survival include the presence of gentamicin during treatment with AR-12, which raises the possibility that AR-12-mediated changes in cell permeability permitted the entry of gentamicin into the macrophage, resulting in antibiotic-induced bacterial killing. Since we have not directly assessed the intracellular concentrations of gentamicin in untreated phagocytes versus those in AR-12-treated phagocytes, or the effect of AR-12 on membrane permeability, an AR-12-mediated increase in intracellular gentamicin levels cannot be definitively ruled out. However, data from our laboratory show that while overall intracellular bacteria counts were much higher under gentamicin-free conditions than in the presence of the antibiotic, AR-12 still induced significant inhibition of intracellular Salmonella survival (data not shown). This result suggests that the observed anti-Salmonella activity of AR-12 was largely independent of gentamicin.

Evidence presented here implicates the inhibition of Akt as a mechanism for the later bactericidal phase of AR-12 activity in Salmonella serovar Typhimurium-infected macrophages. Ectopic expression of CA-Akt1 in infected macrophages did not affect the suppression of bacterial growth at 2 h of AR-12 treatment, but it partially protected bacteria against the marked drug-induced inhibition of survival at 8 h. This finding is consistent with recent work showing that host cell Akt1 activity is important for the intracellular growth of bacteria (22). It is notable that ectopic expression of CA-Akt1 did not completely reverse AR-12-induced inhibition of bacterial survival. It is possible that AR-12 inhibits not only Akt but also other signaling pathways involved in promoting intracellular survival of Salmonella serovar Typhimurium. For example, the p21-activated kinase 1 (PAK1) has been reported to be inhibited by AR-12 at concentrations similar to those needed for Akt inhibition (33). Since the role of PAKs in the control of actin dynamics by intracellular Salmonella has been reported (6, 22), it is possible that PAK inhibition may contribute to the suppression of intracellular bacterial survival induced by AR-12. In addition to the inhibition of kinases, we cannot exclude the possibility that AR-12 mediates the enhancement of other host defense mechanisms against intracellular Salmonella, including increased activity of the NADPH oxidase, inducible nitric oxide synthase, cationic peptides, and the ubiquitin system (31, 34, 35, 52). Our preliminary studies have revealed that the antibacterial activity of AR-12 is unaffected by pharmacological inhibition of NADPH oxidase and proteasome activity, indicating that these antimicrobial factors do not play significant roles in the AR-12-induced inhibition of intracellular bacterial survival. Further investigation of the precise antimicrobial factors involved in AR-12-mediated bacterial killing is currently under way. Understanding the complete antibacterial mechanism of AR-12 may provide further insight into the interaction between Salmonella and host cells, as well as support the continued development of this novel class of antibacterial agent.

Our previous work on AR-12 in cancer cells showed that concentrations of ≥1 µM were needed to suppress the phosphorylation of Akt (38, 58). This contrasts with the findings presented here, which indicate that AR-12 at a concentration as low as 0.25 µM is able to decrease phospho-Akt levels in infected RAW264.7 cells, which exhibit marked elevation of Akt phosphorylation (Fig. 5A). This low Akt-inhibitory concentration in infected macrophages is consistent with the observed IC50 for the inhibition of intracellular survival of Salmonella serovar Typhimurium (Fig. 2C). The mechanistic basis for this difference in the sensitivities of cancer cells and infected RAW264.7 cells to AR-12-induced suppression of Akt activity is unknown. Nonetheless, these findings suggest the use of low doses of AR-12 for the treatment of Salmonella serovar Typhimurium infection to selectively suppress the Akt kinase in infected cells without inducing potential side effects in uninfected cells.

In conclusion, AR-12 represents a promising lead compound that can be used for the development of novel agents targeting host cells for the treatment of intracellular bacterial infections, especially those caused by antibiotic-resistant facultative bacteria.


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ACKNOWLEDGMENTS
 
This work was supported by the NIH/NIAID Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research (RCE) Program and the Lucius A. Wing Endowed Chair Fund (to C.-S.C.). We acknowledge membership within and support from the Region V "Great Lakes" RCE (NIH award 1-U54-AI-057153).

We also thank Hsiao-Ching Chuang, Aaron M. Sargeant, and Shu-Chuan Weng for assistance with experiments.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Medicinal Chemistry, College of Pharmacy, 336 L. M. Parks Hall, 500 West 12th Avenue, Columbus, OH 43210. Phone: (614) 688-4008. Fax: (614) 688-8556. E-mail: chen.844{at}osu.edu Back

{triangledown} Published ahead of print on 5 October 2009. Back


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Antimicrobial Agents and Chemotherapy, December 2009, p. 5236-5244, Vol. 53, No. 12
0066-4804/09/$08.00+0     doi:10.1128/AAC.00555-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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