Effects of 4-Br-A23187 on Bacillus subtilis cells and unilamellar vesicles reveal it to be a potent copper ionophore

Abstract

Ionophores are small molecules or peptides that transport metal ions across biological membranes. Their transport capabilities are typically characterized in vitro using vesicles and single ion species. It is difficult to infer from these data which effects ionophores have on living cells in a complex environment (e.g., culture medium), since net ion movement is influenced by many factors including ion composition of the medium, concentration gradients, pH gradient, and protein-mediated transport processes across the membrane. To gain insights into the antibacterial mechanism of action of the semisynthetic polyether ionophore 4-Br-A23187, known to efficiently transport zinc and manganese in vitro, we investigated its effects on the gram-positive model organism Bacillus subtilis. In addition to monitoring cellular ion concentrations, the physiological impact of treatment was assessed on the proteome level. 4-Br-A23187 treatment resulted in an increase in intracellular copper levels, the extent of which depended on the copper concentration of the medium. Effects of copper accumulation mirrored by the proteomic response included oxidative stress, disturbance of proteostasis, metal and sulfur homeostasis. The antibiotic effect of 4-Br-A23187 is further aggravated by a decrease in intracellular manganese and magnesium. A liposome model confirmed that 4-Br-A23187 acts as copper ionophore in vitro.

Keywords: 4-Br-A23187; Bacillus subtilis; antibiotic; copper; ionophore.

FIGURE 1

Effects of the ionophore 4-Br-A23187 on B. subtilis. (A) Growth curve of B. subtilis treated with 1 μg/ml 4-Br-A23187. A representative curve is shown, for biological replicates see Supporting Information Figure S1. (B) Relative net changes in intracellular metal concentrations after 15 min treatment with 1 μg/ml 4-Br-A23187 are displayed in relation to untreated controls and normalized to cell density. Ion concentrations were determined using ICP-OES (Ca, Zn, Mg, Mn), CAS (Fe), and AAS (Cu). For absolute values see Supporting Information Table S1. (C) 2D-PAGE-based proteomic response profile of B. subtilis treated with 1 μg/ml 4-Br-A23187. The false-color overlay of the 2D gel images shows proteins synthesized during pulse labeling from min 10 to min 15 after 4-Br-A23187 addition (red) and those synthesized at the same time in the untreated control (green). Upregulated marker proteins were identified by mass spectrometry. For regulation factors see Table 1 and for information on protein identification see Supporting Information Table S2. (D) Marker proteins upregulated in response to 4-Br-A23187 and calcimycin exposure. Data for calcimycin were taken from Raatschen et al. [12]. Marker protein categorization is based on function and regulation. Representative data of three biological replicatesare shown in (A) and (C), averages of three biological replicates with standard deviations are shown in (B).

FIGURE 2

4-Br-A23187 mediated copper transport. (A) Net changes in intracellular copper concentrations after 15 min treatment of B. subtilis with 1 μg/ml 4-Br-A23187 in medium with different Cu concentrations are set in relation to Cu concentrations of untreated cells in medium without Cu supplement. Cellular Cu concentrations were determined using ICP-OES. (B) In vitro transport of copper was investigated using calcein-filled liposomes. Upon copper influx, calcein fluorescence is quenched by copper. Fluorescence intensity was normalized by the fluorescence before additives were added. (C) Leakage potential of 4-Br-A23187 evaluated with liposomes loaded with self-quenching calcein levels (750 μM). The initial fluorescence was set to 1%. Addition of Triton X-100 caused complete lysis and the resulting fluorescence signal was set to 100% (y-axis log scale). Averages of three biological replicates with standard deviations are shown in (A), representative data of duplicate experiments are shown in (B) and (C) (for the technical replicates see Supporting Information Figure S3)