A Novel Dibenzoxazepine Attenuates Intracellular Salmonella Typhimurium Oxidative Stress Resistance

Abstract

Salmonella enterica serovar Typhimurium is the leading cause of invasive nontyphoidal salmonellosis. Additionally, the emergence of multidrug-resistant S. Typhimurium has further increased the difficulty of controlling its infection. Previously, we showed that an antipsychotic drug, loxapine, suppressed intracellular Salmonella in macrophages. To exploit loxapine's antibacterial activity, we simultaneously evaluated the anti-intracellular Salmonella activity and cytotoxicity of newly synthesized loxapine derivatives using an image-based high-content assay. We identified that SW14 exhibits potent suppressive effects on intramacrophagic S. Typhimurium with an 50% effective concentration (EC₅₀) of 0.5 μM. SW14 also sensitized intracellular Salmonella to ciprofloxacin and cefixime and effectively controlled intracellular multidrug- and fluoroquinolone-resistant S. Typhimurium strains. However, SW14 did not affect bacterial growth in standard microbiological broth or minimal medium that mimics the phagosomal environment. Cellular autophagy blockade by 3-methyladenine (3-MA) or shATG7 elevated the susceptibility of intracellular Salmonella to SW14. Finally, reactive oxygen species (ROS) scavengers reduced the antibacterial efficacy of SW14, but the ROS levels in SW14-treated macrophages were not elevated. SW14 decreased the resistance of outer membrane-compromised S. Typhimurium to H₂O₂. Collectively, our data indicated that the structure of loxapine can be further optimized to develop new antibacterial agents by targeting bacterial resistance to host oxidative-stress defense. IMPORTANCE The incidence of diseases caused by pathogenic bacteria with resistance to common antibiotics is consistently increasing. In addition, Gram-negative bacteria are particularly difficult to treat with antibiotics, especially those that can invade and proliferate intracellularly. In order to find a new antibacterial compound against intracellular Salmonella, we established a cell-based high-content assay and identified SW14 from the derivatives of the antipsychotic drug loxapine. Our data indicate that SW14 has no effect on free bacteria in the medium but can suppress the intracellular proliferation of multidrug-resistant (MDR) S. Typhimurium in macrophages. We also found that SW14 can suppress the resistance of outer membrane compromised Salmonella to H₂O₂, and its anti-intracellular Salmonella activity can be reversed by reactive oxygen species (ROS) scavengers. Together, the findings suggest that SW14 might act via a virulence-targeted mechanism and that its structure has the potential to be further developed as a new therapeutic against MDR Salmonella.

Keywords: ROS; antipsychotics; high-content assay; loxapine.

FIG 1

SW14 suppresses the intracellular replication of S. Typhimurium in macrophages. (A) Structures, 50% cytotoxic concentration (CC₅₀) toward macrophages, 50% effective concentration (EC₅₀) against intracellular Salmonella, and selectivity index (SI; CC₅₀/EC₅₀) of loxapine and SW14. The data are representative of two independent experiments. (B) RAW264.7 cells were treated with various doses of SW14, and cell viability was evaluated using HCA and an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell viability assay. The data are expressed as percentages relative to the untreated control and are presented as the means ± SD (n = 3/group). (C) S. Typhimurium-infected RAW264.7 cells were treated with a range of SW14 doses in the presence of gentamicin (20 mg/L) for 24 h. Intracellular bacteria survival was assessed using HCA and a CFU assay. The data are expressed as percentages relative to the untreated control and are presented as the means ± SD (n = 3/group). (D) S. Typhimurium-infected RAW264.7 cells were treated with 0.5 μM SW14 in combination with gentamicin (20 mg/L). The relative number of intracellular bacteria was determined at designed times using HCA for a total of 24 h, and the results are expressed as relative fluorescence units (RFU). The data are presented as the means ± SD (n = 3/group). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 2

SW14 is effective against multiple antibiotic-resistant S. Typhimurium strains in macrophages. (A, B) Salmonella-infected RAW264.7 cells were treated with various concentrations of cefixime (CFM; A) and ciprofloxacin (CIP; B) alone or in combination with 0.5 μM SW14. After 24 h, the relative numbers of intracellular bacteria were determined using HCA, and the results are expressed as RFUs. The data are presented as the means ± SD (n = 3/group). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (C, D) RAW264.7 cells were infected with multidrug-resistant isolates 0911R and NL08.10 (C) or ciprofloxacin-resistant strains SA10 and SB10 (D) of S. Typhimurium followed by treatment with various doses of SW14 combined with 20 mg/mL gentamicin for 24 h. The relative numbers of intracellular bacteria were determined using HCA, and the results are expressed as relative fluorescence units (RFU). The data are presented as the means ± SD (n = 3/group). *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

FIG 3

The antibacterial activity of SW14 is not mediated by suppressing bacterial growth or cellular autophagic defense. (A) The growth of S. Typhimurium in LB medium, cell culture medium (CCM), or LPM medium containing various concentrations of SW14 was monitored by measuring the OD₆₀₀ of each bacterial culture at designated times for a total of 24 h. The data are presented as the means ± SD (n = 3/group). (B) Infected RAW264.7 cells were treated with 3-MA (10 mM) for 1 h followed by SW14 (0.5 μM) or mock (DMSO) treatment for 24 h. The viability of the intracellular bacteria was assessed using HCA, and the results are expressed as percentages relative to the mock-treated cells. The data are presented as the means ± SD (n = 3/group). ****, P < 0.0001. (C) RAW264.7 cells were stably transfected with a plasmid to express shRNA targeting Atg7 (shATG7) or with the empty vector (Vec) and then infected with S. Typhimurium followed by treatment with various concentrations of SW14 and 20 mg/mL gentamicin for 24 h. The numbers of surviving intracellular bacteria were determined by HCA, and the results are expressed as a percentage of the control group. The left panel shows the levels of the Atg7 protein in the stable transfectants as determined by immunoblotting. Bands for Atg7 were quantified by densitometry and normalized to those of β-actin. Percentages represent the relative expression levels of Atg7 in the shATG7 transfectants compared to the vector transfectants. The data are representative of two independent experiments. The right panel shows intracellular bacterial survival in SW14-treated RAW264.7 cells with shRNA-mediated knockdown of Atg7 expression. The data shown are the means; error bars represent the SD (n = 3). *, P < 0.05 for the differences between the drug-treated groups and their respective control groups. LPM, low-phosphate, low-magnesium medium; 3-MA, 3-methyladenine; ns, nonsignificant. OD₆₀₀, optical density at 600 nm.

FIG 4

SW14 sensitizes outer membrane-compromised S. Typhimurium to oxidative stress. (A) ROS scavengers reversed the suppressive effects of SW14 on intracellular S. Typhimurium. The viabilities of intracellular Salmonella in RAW264.7 cells treated with SW14 with/without the ROS scavengers N-acetylcysteine (NAC; 10 mM) and glutathione (GSH; 10 mM) were assessed using HCA. The data are presented as the means ± SD (n = 3/group). *, P < 0.05; ***, P < 0.001. (B) SW14 did not induce ROS production in macrophages. Salmonella-infected RAW264.7 cells were loaded with 25 μM H₂DCFDA for 45 min followed by treatment with SW14 or PMA at various concentrations. The data are expressed as percentages relative to the untreated control and are presented as the means ± SD (n = 3/group). ns, nonsignificant; ***, P < 0.001; ****, P < 0.0001. (C, D) S. Typhimurium ATCC 14028 was treated with H₂O₂ alone or in combination with SW14 (0.5 μM) in LB (C) or LPM (D) for 1 h followed by evaluation by the CFU assay to determine bacterial viability. The data are expressed as percentages relative to the number of bacteria in H₂O₂-free medium and are presented as the means ± SD (n = 3/group). ns, nonsignificant; *, P < 0.05.