Mechanistic Understanding Enables the Rational Design of Salicylanilide Combination Therapies for Gram-Negative Infections

Abstract

One avenue to combat multidrug-resistant Gram-negative bacteria is the coadministration of multiple drugs (combination therapy), which can be particularly promising if drugs synergize. The identification of synergistic drug combinations, however, is challenging. Detailed understanding of antibiotic mechanisms can address this issue by facilitating the rational design of improved combination therapies. Here, using diverse biochemical and genetic assays, we examine the molecular mechanisms of niclosamide, a clinically approved salicylanilide compound, and demonstrate its potential for Gram-negative combination therapies. We discovered that Gram-negative bacteria possess two innate resistance mechanisms that reduce their niclosamide susceptibility: a primary mechanism mediated by multidrug efflux pumps and a secondary mechanism of nitroreduction. When efflux was compromised, niclosamide became a potent antibiotic, dissipating the proton motive force (PMF), increasing oxidative stress, and reducing ATP production to cause cell death. These insights guided the identification of diverse compounds that synergized with salicylanilides when coadministered (efflux inhibitors, membrane permeabilizers, and antibiotics that are expelled by PMF-dependent efflux), thus suggesting that salicylanilide compounds may have broad utility in combination therapies. We validate these findings in vivo using a murine abscess model, where we show that niclosamide synergizes with the membrane permeabilizing antibiotic colistin against high-density infections of multidrug-resistant Gram-negative clinical isolates. We further demonstrate that enhanced nitroreductase activity is a potential route to adaptive niclosamide resistance but show that this causes collateral susceptibility to clinical nitro-prodrug antibiotics. Thus, we highlight how mechanistic understanding of mode of action, innate/adaptive resistance, and synergy can rationally guide the discovery, development, and stewardship of novel combination therapies.IMPORTANCE There is a critical need for more-effective treatments to combat multidrug-resistant Gram-negative infections. Combination therapies are a promising strategy, especially when these enable existing clinical drugs to be repurposed as antibiotics. We examined the mechanisms of action and basis of innate Gram-negative resistance for the anthelmintic drug niclosamide and subsequently exploited this information to demonstrate that niclosamide and analogs kill Gram-negative bacteria when combined with antibiotics that inhibit drug efflux or permeabilize membranes. We confirm the synergistic potential of niclosamide in vitro against a diverse range of recalcitrant Gram-negative clinical isolates and in vivo in a mouse abscess model. We also demonstrate that nitroreductases can confer resistance to niclosamide but show that evolution of these enzymes for enhanced niclosamide resistance confers a collateral sensitivity to other clinical antibiotics. Our results highlight how detailed mechanistic understanding can accelerate the evaluation and implementation of new combination therapies.

Keywords: antibiotic resistance; colistin; drug efflux; efflux; niclosamide; nitroreductase; proton motive force; repurposing; resistance; synergy.

FIG 1

Niclosamide resistance mechanisms. (a) Structure of niclosamide. (b) MIC of E. coli wild-type (WT), ΔtolC, Δ7NR, and Δ7NRtolC strains. Asterisks (*) indicate >32 μg · ml⁻¹, which is the solubility limit of niclosamide in growth media. (c) IC₅₀ analysis of Δ7NRtolC strains individually overexpressing candidate E. coli nitroreductases or a vector-only control following niclosamide administration. Error bars indicate standard errors of the means (SEM). (d and e) Covariance plots displaying the interrelated profiles of niclosamide, metronidazole, and nitrofurantoin resistance. A total of 90 colonies of NfsA variants were picked from agar plates without niclosamide (red) or with 0.2 μg · ml⁻¹ niclosamide (orange) or 2 μg · ml⁻¹ niclosamide (yellow). Variants were grown overnight and then screened for niclosamide resistance (growth at 2 μg · ml⁻¹) and (d) nitrofurantoin resistance or (e) metronidazole resistance (growth at 5 or 10 μg · ml⁻¹, respectively). Variant distribution is shown as gray histograms that are overlaid on the x and y axes. R² values (linear regression analysis) are displayed; P < 0.01. E. coli NfsA and vector-only controls are displayed in cyan and gray, respectively. All panels are constructed from pooled data from at least three independent biological replicates.

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.

FIG 3

Analyses of salicylanilide synergy interactions. (a to c) The combined inhibitory effects of 0 to 8 μg · ml⁻¹ niclosamide and (a) 0 to 128 μg · ml⁻¹ PAβN or (b) 0 to 250 ng · ml⁻¹ colistin or (c) 0 to 500 ng · ml⁻¹ polymyxin B were tested against E. coli using checkerboard analysis. ZIP synergy scores (δ) are presented. Bacterial growth is depicted as a heat plot. (d and e) The combined inhibitory effects of 0 to 250 ng · ml⁻¹ niclosamide and either (d) 0 to 250 ng · ml⁻¹ colistin or (e) 0 to 500 ng · ml⁻¹ polymyxin B were tested against E. coli ΔtolC in checkerboard analyses. Bacterial growth is depicted as a heat plot. (f) A bar graph of ZIP scores (δ) depicting the synergism of oxyclozanide (OXY), rafoxanide (RAF), or closantel (CTL) in combination with PAβN or colistin against E. coli. Error bars indicate SEM. (g and h) Analysis of oxyclozanide synergy with nitrofurantoin (NIT), metronidazole (MTZ), cefotaxime (CEF), rifampin (RIF), tetracycline (TET), gentamicin (GEN), ciprofloxacin (CIP), chloramphenicol (CAM), trimethoprim (TMP), fosfomycin (FOS), meropenem, (MER) or vancomycin (VAN). (g) A covariance plot of antibiotic ZIP scores from checkerboard assays conducted in minimal media with oxyclozanide against E. coli and E. coli ΔtolC. (h) A bar chart displaying fold changes of IC₅₀ values in the ΔtolC strain compared to the E. coli WT strain; uncertainty is indicated by error bars. (i) Fold change in the rate of Hoechst 33342 fluorescence (compared to a DMSO control) in ΔtolC cells or WT E. coli following administration of 28 μg · ml⁻¹ PAβN, 5 μg · ml⁻¹ CCCP, or 11.2 to 128 μg · ml⁻¹ oxyclozanide. Error bars indicate SEM. All panels were constructed from pooled data from at least three independent biological replicates.

FIG 4

Proposed salicylanilide mechanisms of action. (a) Salicylanilide crosses the outer membrane but is expelled from the cell via (PMF-dependent) TolC-mediated efflux; electron transport, membrane polarization, oxygen consumption, and ATP synthesis are not affected. (b) When TolC is inhibited by compounds such as PAβN, salicylanilides uncouple the electron transport chain, dissipate the PMF, increase oxygen consumption, and decrease ATP production. (c) When the outer membrane is disrupted via compounds such as colistin, salicylanilides rapidly enter the cell, overwhelming TolC-mediated efflux, uncoupling the electron transport chain, dissipating the PMF (inhibiting PMF-dependent efflux), increasing oxygen consumption, and decreasing ATP production.

FIG 5

Niclosamide/colistin combination therapy was effective against recalcitrant MDR Gram-negative strains. (a) Bar graph depicting in vitro ZIP scores (δ) of niclosamide and colistin coadministration against the following clinical MDR Gram-negative strains: P. aeruginosa LESB58, P. aeruginosa NZRM4034, K. pneumoniae KPLN649, K. pneumoniae NZRM4387, A. baumannii Ab5075, A. baumannii NZRM3289, A. baumannii C4, E. coli E38, E. coli NZRM4403, E. coli NCTC 13846, and E. cloacae 218R1. The ZIP synergy score (δ) represents the average of interaction data from an 8-by-8 dose-response matrix. Data were averaged from at least three independent experiments, and error bars indicate SEM. (b) Diagram of abscess model procedure and analysis. inj., injection. (c and d) Dot plots of (c) colistin-resistant P. aeruginosa LESB58 and (d) K. pneumoniae KPLN649 survival, represented as CFU recovered per abscess after administration of 10 mg · kg⁻¹ niclosamide ethanolamine salt and 0.15 mg · kg⁻¹ (P. aeruginosa) or 2.5 mg · kg⁻¹ (K. pneumoniae) colistin as individual or combined therapeutics. Labels indicate significant responses over the PEG control (*, P < 0.05; **, P < 0.01) or synergistic responses, i.e., significant differences measured for the combination therapy over the sum of the effects of each agent alone (^##, P < 0.01). Statistical analyses were performed using one-way analysis of variance (ANOVA) and the Kruskal-Wallis test with Dunn’s correction (two sided).