Mitochondria-targeted antioxidants as highly effective antibiotics

Abstract

Mitochondria-targeted antioxidants are known to alleviate mitochondrial oxidative damage that is associated with a variety of diseases. Here, we showed that SkQ1, a decyltriphenyl phosphonium cation conjugated to a quinone moiety, exhibited strong antibacterial activity towards Gram-positive Bacillus subtilis, Mycobacterium sp. and Staphylococcus aureus and Gram-negative Photobacterium phosphoreum and Rhodobacter sphaeroides in submicromolar and micromolar concentrations. SkQ1 exhibited less antibiotic activity towards Escherichia coli due to the presence of the highly effective multidrug resistance pump AcrAB-TolC. E. coli mutants lacking AcrAB-TolC showed similar SkQ1 sensitivity, as B. subtilis. Lowering of the bacterial membrane potential by SkQ1 might be involved in the mechanism of its bactericidal action. No significant cytotoxic effect on mammalian cells was observed at bacteriotoxic concentrations of SkQ1. Therefore, SkQ1 may be effective in protection of the infected mammals by killing invading bacteria.

Figure 1

Dilution Antimicrobial Susceptibility Test. Bacterial growth was observed visually alongside the OD620 measurement.

Figure 2

Growth of E. coli strains having deletions in various transporters in the presence of 10 μM SkQ1. SkQ1 was added at “0” time to the LB medium. Growth was evaluated after 15–35 h incubation at 30 °C or 37 °C by absorbance at 620 nm. The growth of WT E. coli cells in the absence of SkQ1 is referred as a control. The data points represent mean ± SD of three experiments.

Figure 3

Effect of SkQ1 on ethidium bromide accumulation by WT (top panel) and ΔtolC (bottom panel) E. coli cells. E. coli cells were added to PBS with 20 μM ethidium bromide (black) and SkQ1 was added subsequently after 8 min of incubation (red). Osmotic shock by deionized water was used as a control (blue).

Figure 4

Effect of SkQ1 (10 µM) on the accumulation of ethidium bromide by E. coli WT and ΔtolC cells, monitored by FCS. Fluorescence intensity traces of ethidium (10 nM) were recorded with the FCS set-up in the presence or absence of bacterial cells (10⁶ per ml of PBS). Insert: Corresponding dependences of the number of peaks with the fluorescence intensity F exceeding the threshold F₀, n(F > F₀), on the value of F₀.

Figure 5

(A) Effect of SkQ1 on membrane potential in B. subtilis. Changes in the membrane potential were monitored by measuring fluorescence of DiS-C₃-(5) (10 µM) in PBS buffer. Gramicidin A concentration, 0.5 ng/ml. (B) Propidium iodide membrane permeability was tested via its fluorescence at 600 nm at 1 µM. Propidium iodide was present in all samples. Deionized water was used as a positive control of membrane permeabilization. SkQ1 concentration, 1 µM.

Figure 6

Induction of the translation inhibittion fluorescent reporter (left) or DNA damage fluorescent reporter (right) by SkQ1 on Petri plates. E. coli ΔtolC transformed with pRFPCER-TrpL2A (left) or pRFPCER-sulA (right) exhibited red fluorescence owing to RFP expression. SkQ1; erythromycin (ery) and levofloxacin (lev) were spotted on agar. Rings of cerulean fluorescence (cyan-green) were formed under the influence of the antibiotic causing ribosome stalling (left panel), and under the influence of the DNA-damaging antibiotic (right panel). Petri dishes were illuminated at UV (254-nm) and photographed by a digital camera (bottom panels) while the signal of cerulean protein fluorescence was detected in Cy2 channel by means of ChemiDoc (top panel).

Figure 7

Viability of HeLa cells after the addition of SkQ1. HeLa cells were incubated for 17 h. Cell viability was determined with Cell Titer-Blue reagent (Promega).

Figure 8

The SkQ1-mediated protonophorous cycling of fatty acids in bacterial membrane. In the absence of SkQ1-expelling transporters, SkQ1 acts as protonophore-like uncoupler with the help of endogenous free fatty acids (left). Export of SkQ1 from the bacterial cell by means of AcrA, AcrB, and TolC pump (right).