1. Nazarov P.A1*., Kotova Е.А.1, Antonenko Y.N.1
The mitochondria-targeted antioxidant SkQ1 demonstrates severe antimicrobial action
Nazarov P.A1*., Kotova Е.А.1, Antonenko Y.N.1
Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia
* ASM Young Ambassador to Russia,
Mitochondria-targeted antioxidants are known to alleviate mitochondrial oxidative damage that causes a variety of diseases. By monitoring time courses of optical density at 620 nm after 2-fold broth microdilution, we found that SkQ1, a decyltriphenyl phosphonium cation conjugated to a quinone moiety, produced strong antibacterial effect on Gram-positive Bacillus subtilis, Mycobacterium sp. and Staphylococcus aureus and Gram-negative Photobacterium phosphoreum and Rhodobacter sphaeroides at submicromolar and micromolar concentrations SkQ1 exhibited much lower antibiotic activity towards Escherichia coli obviously due to the presence of the highly effective multidrug resistance pump AcrAB-TolC. E. coli mutants lacking any of AcrAB-TolC transporter proteins showed similar SkQ1 sensitivity, as B. subtilis. To confirm that SkQ1 cations compete with ethidium for AcrAB-TolC as a transporter, we applied fluorescence correlation spectroscopy (FCS) for measuring ethidium accumulation in E. coli cells. By measuring fluorescence of the potential-sensitive dye DiS-C3-(5), we examined the effect of SkQ1 on the membrane potential of B. subtilis. Submicromolar concentrations of SkQ1 caused a decrease in the membrane potential of B. subtilis in the minute time scale, whereas 5 µM SkQ1 caused a rapid drop of membrane potential to the level observed with the channel-forming antibiotic gramicidin A. SkQ1 did not cause an increase in fluorescence of the cell membrane-impermeant probe propidium iodide, therefore the action of SkQ1 on the membrane potential of B. subtilis was not due to a detergent effect on bacterial membrane. The mechanism of SkQ1 toxicity towards bacterial cells was neither associated with DNA damage, nor with inhibition of translation. Lowering of the bacterial membrane potential by SkQ1 might be involved in the mechanism of its bactericidal action. No significant cytotoxic effect on mammalian HeLa cells was observed at bacteriotoxic concentrations of SkQ1. Therefore, SkQ1 may be effective in protection of the infected mammals by killing invading bacteria. B A C D E F H G
Figure 1. (A) Chemical structures of reduced SkQ1 and MitoQ; (В) Effects of 1 µM SkQ1 and 1 µM MitoQ on the growth of B. subtilis. Effect of SkQ1 on the growth of Mycobacterium sp. (C), S. aureus (D), R.sphaeroedes (E) and P. phosphoreum (F); (G), effects of 1-50 µM SkQ1 on the growth of WT E. coli. (H), effects of 1 µM SkQ1 on the growth of E. coli WT and tolC. Open triangles and circles show control growth of the bacteria without inhibitors. The data points represent mean SD of three experiments. A G B
TolC interaction created by STRING v10.
Figure 2. Influence of SkQ1 on ethidium bromide accumulation by WT (A) and tolC (B) 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); (C) Effect of SkQ1 on membrane potential in B. subtilis. Measurement of the membrane potential performed via fluorescence of the potential-sensitive dye DiS-C3-(5) (10 µM) in PBS buffer. (D) Propidium iodide membrane permeability test via its fluorescence at 600 nm at 1 µM. Deionized water was used as a positive control of membrane permeabilization. SkQ1 concentration, 1 µM. (E) Growth of E.coli strains having deletions in various transporters in the presence of 10 M SkQ1. (F) Viability of HeLa cells after addition of SkQ1. (G) Scheme of proton-transport cycling of SkQ1 and fatty acids in bacterial membrane. In the absence of SkQ1-expelling transporters, SkQ1 acts as protonophore-like uncoupler in assistance with endogenous free fatty acids. [1, 2] E C
Figure 3. 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 pRFPCERsulA (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). [2] F D
REFERENCES
ACKNOWLEGMENTS
This work was supported by grants from the Russian Science Foundation and Attendance at ASM Microbe was sponsored by American Society for Microbiology.
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