Rhodamine 19 Alkyl Esters as Effective Antibacterial Agents

Abstract

Mitochondria-targeted antioxidants (MTAs) have been studied quite intensively in recent years as potential therapeutic agents and vectors for the delivery of other active substances to mitochondria and bacteria. Their most studied representatives are MitoQ and SkQ1, with its fluorescent rhodamine analog SkQR1, a decyl ester of rhodamine 19 carrying plastoquinone. In the present work, we observed a pronounced antibacterial action of SkQR1 against Gram-positive bacteria, but virtually no effect on Gram-negative bacteria. The MDR pump AcrAB-TolC, known to expel SkQ1, did not recognize and did not pump out SkQR1 and dodecyl ester of rhodamine 19 (C12R1). Rhodamine 19 butyl (C4R1) and ethyl (C2R1) esters more effectively suppressed the growth of ΔtolC Escherichia coli, but lost their potency with the wild-type E. coli pumping them out. The mechanism of the antibacterial action of SkQR1 may differ from that of SkQ1. The rhodamine derivatives also proved to be effective antibacterial agents against various Gram-positive species, including Staphylococcus aureus and Mycobacterium smegmatis. By using fluorescence correlation spectroscopy and fluorescence microscopy, SkQR1 was shown to accumulate in the bacterial membrane. Thus, the presentation of SkQR1 as a fluorescent analogue of SkQ1 and its use for visualization should be performed with caution.

Keywords: AcrAB-TolC; MDR pumps; mitochondria-targeted antioxidants; phosphonium; rhodamine.

Figure 1

Kinetic growth curves. (A) Effect of 0.12–2.8 μg/mL SkQR1 (top panel, left), C10R1 (top panel, right), C4R1 (bottom panel, left), and C2R1 (bottom panel, right) on the growth of B. subtilis. (B) Effect of 1–64 μg/mL SkQR1 (top panel), and C4R1 (bottom panel) on the growth of E. coli WT (left) and ∆TolC (right). Kinetic growth curves for SkQ1 are presented in Figure S1.

Figure 2

Growth of E. coli strains having deletions in various transporters in the presence of 6 μg/mL SkQ1 (top), 21 μg/mL SkQR1 (middle), and 10 μg/mL C4R1 (bottom). Substances were added at “0” time to the LB medium. Growth was evaluated after 15–24 h incubation at 37 °C by absorbance at 620 nm. The growth of WT E. coli cells in the absence of substances is referred to as a control, and, in the presence of substances, is referred to as WT. The data points represent mean ± SD of three experiments.

Figure 3

Accumulation of SkQR1 and C4R1 by B. subtilis and E. coli cells (black) and the effect of CCCP (10 µM) on it (purple), monitored using FCS: (A) accumulation of SkQR1 in B. subtilis cells, (B) accumulation of SkQR1 in E. coli cells, (C) accumulation of C4R1 in B. subtilis cells, (D) accumulation of C4R1 in E. coli cells. Cells E. coli or B. subtilis (red), rhodamine derivatives SkQR1 or C4R1 (blue), and cells with added CCCP uncoupler were used as controls. The fluorescence intensity traces of SkQR1 and C4R1 recorded with the FCS set-up in the presence or absence of bacterial cells (10⁶ per mL of PBS). The 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 4

Fluorescence from B. subtilis cells stained with SkQR1. The cells were incubated with 3.5 µg/mL of the dyes for 5 min, washed with LB, and imaged.

Figure 5

Chemical structures of SkQ1, SkQR1, and alkyl ester derivatives of rhodamine 19 (CnR1).