Antibacterial Marinopyrroles and Pseudilins Act as Protonophores

Abstract

Elucidating the mechanism of action (MoA) of antibacterial natural products is crucial to evaluating their potential as novel antibiotics. Marinopyrroles, pentachloropseudilin, and pentabromopseudilin are densely halogenated, hybrid pyrrole-phenol natural products with potent activity against Gram-positive bacterial pathogens like Staphylococcus aureus. However, the exact way they exert this antibacterial activity has not been established. In this study, we explore their structure-activity relationship, determine their spatial location in bacterial cells, and investigate their MoA. We show that the natural products share a common MoA based on membrane depolarization and dissipation of the proton motive force (PMF) that is essential for cell viability. The compounds show potent protonophore activity but do not appear to destroy the integrity of the cytoplasmic membrane via the formation of larger pores or interfere with the stability of the peptidoglycan sacculus. Thus, our current model for the antibacterial MoA of marinopyrrole, pentachloropseudilin, and pentabromopseudilin stipulates that the acidic compounds insert into the membrane and transport protons inside the cell. This MoA may explain many of the deleterious biological effects in mammalian cells, plants, phytoplankton, viruses, and protozoans that have been reported for these compounds.

Figure 1

Structures of marinopyrrole A (1), pentabromopseudilin (2), and pentachloropseudilin (3), including the original bacterial producer strain, reported biological activities and reported cellular targets. Mcl-1 = myeloid cell leukemia 1; IspD = 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase.

Figure 2

Antibacterial and cytotoxic activities of compounds 1–10. (A) MICs of 1–10 against S. aureus NCTC8325 and B. subtilis 168 and half maximal inhibitory concentrations (IC₅₀s) against HeLa, A549, and MRC5 eukaryotic cells. Control = vancomycin (MIC) for the antibacterial assay and mitoxanthrone (IC₅₀) for the cytotoxicity assay. The section in gray highlights the potent antibacterial activity of the natural products (1–3). (B) Structures of 4–10. MarA = marinopyrrole A; PBP = pentabromopseudilin; PCP = pentachloropseudilin; and NT = not tested.

Figure 3

Cellular localization of coumarin probes, BODIPY probes, and membrane dye FM 5–95. (A) Structures of 1C–7C, 11, 1B, and 12. (B) B. subtilis 168 cells incubated with 1C (1 μg mL^–1, 1× MIC), 2C (0.25 μg mL^–1, 1× MIC), 3C (0.25 μg mL^–1, 1× MIC) or 1B (0.5 μg mL^–1, 2× MIC) for 10 min. Coumarin 11 (8 μg mL^–1, MIC > 64 μg mL^–1) and BODIPY 12 (8 μg mL^–1, MIC > 64 μg mL^–1) were used as negative controls. The top row shows the fluorescence channel, and the bottom row shows phase-contrast images. The micrographs display representative images of three biological replicates. Images for the coumarin probes were adjusted to the same microscopy settings for direct qualitative comparison; images for the BODIPY probes were also adjusted to the same microscopy settings, but these were different than the settings for coumarin imaging. Scale bar: 5 μm. (C) B. subtilis 168 cells incubated with 1 (0.125 μg mL^–1, 1× MIC), 2 (0.016 μg mL^–1, 1× MIC) and 3 (0.004 μg mL^–1, 1× MIC) for 15 min and then stained with the membrane dye FM5–95 (20 μg mL^–1). DMSO (1%) was used as a negative control and CCCP (100 μM) as a positive control. The top row shows the fluorescence channel, and the bottom row shows the phase-contrast images. The images displayed are representative of two biological replicates and are all adjusted to the same setting for qualitative comparison. Scale bar, 5 μm. CCCP = carbonyl cyanide m-chlorophenyl hydrazone.

Figure 4

Compounds 1–3 act as protonophores. (A) Time-resolved effect of 1–3 on the membrane potential in S. aureus NCTC8325 cells based on 3,3′-diethyloxacarbocyanine iodide [DiOC₂(3)] staining at concentrations around the respective MICs. Cells were treated with compounds 1–3 at 4×, 2×, 1×, 0.5×, and 0.25× MIC in the presence of DiOC₂(3). DMSO (1%) was used as a negative control and CCCP (5 μM) as a positive control. Error bars indicate the standard deviation (SD) of two biological replicates. The arrow shows the time point of the addition of test compounds. (B) Time-resolved effect of 1–3 on the intracellular pH in S. aureus NCTC8325 cells based on 2′,7′-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF AM) staining in relation to the MICs. Cells were treated with compounds 1–3 at 4×, 2×, 1×, 0.5×, and 0.25× MIC in the presence of BCECF AM. DMSO (1%) was used as a negative control and CCCP (50 μM) as a positive control. Error bars indicate the standard deviation (SD) of the three biological replicates. The first arrow shows the time point of test compound addition; the second arrow shows the time point for adding nigericin (20 μM), an H⁺/K⁺ antiporter further reducing the intracellular proton concentration and equilibrating it to the external environment. (C) GFP-MinD distribution in growing B. subtilis 168 cells after compound exposure. Cells were incubated for 10 min with compounds 1–3 at a 1× MIC. DMSO (1%) was used as a negative control, and CCCP (100 μM) as a positive control. The micrographs (top row) show representative images of the fluorescence channel which were all adjusted to the same microscopy settings for direct qualitative comparison. The fluorescence heatmaps (bottom row) display the spatial distribution of the median fluorescent GFP-MinD signal quantified from at least N ≥ 100 cells per experiment from three independent biological replicates. Warmer colors indicate stronger localization in this position. (D) Membrane integrity of S. aureus NCTC8325 after 35 min treatment with compounds 1–3 at 4× MIC visualized by SYTO9 (green) and propidium iodide (PI, red) costaining. DMSO (1%) and CCCP (50 μM) were used as negative controls and nisin (100 μg mL^–1) as a positive control. The images shown are an overlay of the brightfield, SYTO9, and PI channels. (E) Brightfield visualization of the formation of blebs in B. subtilis 168 cells after 10 min of treatment with compounds 1–3 at 4× MIC and fixation by acetic acid/methanol. Blebs are formed by the cytoplasmic membrane bulging through breaches in the peptidoglycan. DMSO (1%) was used as a negative control, and vancomycin (25 μg mL^–1) was used as a positive control. CCCP = carbonyl cyanide m-chlorophenyl hydrazone. Scale bars, 5 μm.