Eradication of intracellular Salmonella enterica serovar Typhimurium with a small-molecule, host cell-directed agent

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.

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).

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.

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).

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.

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.

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.