Chemiosmotic mechanism of antimicrobial activity of Ag(+) in Vibrio cholerae

Abstract

Although the antimicrobial effects of silver salts were noticed long ago, the molecular mechanism of the bactericidal action of Ag(+) in low concentrations has not been elucidated. Here, we show that low concentrations of Ag(+) induce a massive proton leakage through the Vibrio cholerae membrane, which results in complete deenergization and, with a high degree of probability, cell death.

FIG. 1.

Effects of Ag⁺ on the H⁺ permeability of the membrane measured in the inside-out membrane vesicles prepared from V. cholerae. Respiration-dependent formation of ΔpH was initiated by the addition of 10 mM Tris-succinate (down arrows). Where indicated, 2.0 μM AgNO₃ was added to the vesicles (up arrows). (A) Addition of Ag⁺ after succinate collapses ΔpH generated in wild-type (WT) NQR (upper trek) as well as ΔNQR (lower trek) membranes. (B) Ag⁺ prevents the formation of the respiration-dependent ΔpH. (C) Ag⁺-induced ΔpH collapse is very fast in the presence of 0.2 μM valinomycin. In each case, typical treks from five independent experiments are shown.

FIG. 2.

Effects of Ag⁺ on Δψ in subbacterial vesicles of V. cholerae. The Δψ-sensitive dye Oxonol V (1.0 μM) was used instead of AO. Ag⁺ (4.0 μM) completely dissipates Δψ generated by respiration in vesicles derived from the strain possessing the wild-type (WT) NQR (upper trek). Nearly identical results were obtained with ΔNQR vesicles (data not shown). In the control experiment (lower trek), 0.2 μM valinomycin was added instead of AgNO₃. Shown are typical treks from five independent experiments.

FIG. 3.

The initial rate of ΔpH dissipation in the wild-type and ΔNQR membrane vesicles as a function of the AgNO₃ added. A transmembrane pH gradient was generated by the addition of 10 mM Tris-succinate to the vesicles at pH 7.5, and varying concentrations of AgNO₃ were added after a steady-state ΔpH was reached. In each case, changes in AO fluorescence were monitored for 20 s after Ag⁺ addition. Initial dequenching rates are expressed in arbitrary fluorescence units per minute.