FIG 2.

Antibiotic mechanisms of niclosamide. (a) Fold change in DiSC₃(5) fluorescence. E. coli was grown in MHB with 10 mM EDTA to an OD₆₀₀ of 1. Cells were incubated with DiSC₃(5) for 20 min prior to administration of 0.5 μg/ml valinomycin (VAL; a ΔΨ-dissipating ionophore), 0.5 μg/ml nigericin (NIG; a ΔpH-dissipating ionophore), or 0.03 to 1 μg/ml niclosamide. KCl (100 mM) was added to cells prior to valinomycin treatment. RFU, relative fluorescence units. (b and c) The combined inhibitory effects of 0 to 250 ng · ml⁻¹ niclosamide and either (b) 0 to 4 μg · ml⁻¹ kanamycin or (c) 0 to 500 ng · ml⁻¹ tetracycline were tested against the Δ7NRtolC strain in a checkerboard format. Bacterial growth is shown as a heat plot. (d) Oxygen consumption was measured using the MitoXpress oxygen probe in Δ7NRtolC cells (mid-log phase; OD₆₀₀ = 0.15) overlaid with mineral oil for 20 min. a.u., arbitrary units. (e) Relative cellular ATP levels were estimated by luciferase activity and compared to an unchallenged (DMSO-only) control. (f and g) Intracellular oxidation levels were measured in (f) WT E. coli and (g) ΔtolC strains constitutively expressing redox-sensitive GFP (roGFP) following administration of 5 mM H₂O₂ (oxidized control), 10 mM DTT (reduced control), or niclosamide. (h) Representative high-throughput fluorescence microscopy images of Δ7NR and Δ7NRtolC cells 120 min after administration of DTT, H₂O₂, or niclosamide. Images on the right are pseudocolored ratio images after analysis with ImageJ. Panels a to g were constructed from pooled data from at least three independent biological replicates. Labels indicate significant responses over the control (* = P < 0.05; ** = P < 0.01). Statistical analyses were performed using one-way analysis of variance (ANOVA) and the Kruskal-Wallis test. Error bars indicate SEM.