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.

Understudied proteins and understudied functions in the model bacterium Bacillus subtilis—A major challenge in current research

“…For example, transcriptome or proteome profiling of changes in a mutant and a comparison to established catalogs may yield functional insights. Such an approach has already been successfully used to identify the mode of action of unknown antibacterial compounds in B. subtilis (Senges et al, 2021, 2022). Proteomic profiling may prove to be especially useful for the functional analysis of unknown ribosome‐interacting proteins, since extensive cataloged information of proteome changes under conditions of ribosome perturbation is available for B. subtilis (Senges et al, 2021).…”

“…For example, transcriptome or proteome profiling of changes in a mutant and a comparison to established catalogs may yield functional insights. Such an approach has already been successfully used to identify the mode of action of unknown antibacterial compounds in B. subtilis (Senges et al, 2021(Senges et al, , 2022.…”

Cuproptosis: mechanisms and links with cancers

“…Some antimicrobial drugs also serve as copper ionophores, which inhibit the growth of microorganisms by elevating intracellular copper ion concentration. Zinc pyrithione mediated copper influx can inhibit the growth of yeast [ 196 ], 4-Br-A23187 and Dimethyldithiocarbamate can increase cell copper level and play an antibacterial role [ 197 , 198 ]. Lipographic copper containing complexes with bis (thiosemicarbazone) ligands can also increase the concentration of copper ions in cancer cells and host cells of chlamydial [ 199 , 200 ].…”

Understudied Proteins and Understudied Functions in the Model Bacterium Bacillus subtilis -- a Major Challenge in Current Research

“…For example, transcriptome or proteome profiling of changes in a mutant and a comparison to established catalogues may yield functional insights. Such an approach has already been successfully used to identify the mode of action of unknown antibacterial compounds in B. subtilis (Senges et al, 2021;Senges et al, 2022). Proteomic profiling may prove to be especially useful for the functional analysis of unknown ribosome-interacting proteins, since extensive catalogued information of proteome changes under conditions of ribosome perturbation is available for B. subtilis (Senges et al, 2021).…”