Journal: Nature Communications
Article Title: In situ captured antibacterial action of membrane-incising peptide lamellae
doi: 10.1038/s41467-024-47708-4
Figure Lengend Snippet: Bacterial cells were treated with 5 μM 3K in water for 120 min, and negative-stained with uranyl acetate. a NS-TEM image of a single bacterial cell highlighting the supramolecular structures formed within the cell. Scale bar: 200 nm (i–ii) The striped lamellae are observed protruding up to 100−200 nm deep into the cytoplasm from the bacterial cell wall. The inner and outer membranes (IM and OM) are also marked. Disrupted and diffuse OM and IM regions, and leakage of cell content can be clearly observed at positions where the lamellae enter the cell. Scale bar: 50 nm (for more details, see Supplementary Fig. and Fig. ). Measurements were repeated three times and similar results were obtained. b Cryo-EM image of a single bacterial cell with the supramolecular structures formed. Lamellar morphologies are highlighted by arrows. Scale bar: 200 nm. Measurements were repeated three times and similar results were obtained. c Normalized changes in outer membrane permeability induced by 3K. Normalized initial velocities of nitrocefin degradation following a 20 min incubation of bacterial cells with 3K in PBS at 1, 5, 10, 20, and 50 μM concentrations. Lysozyme was used as a positive control. Data points are the average of four biological replicates. Error bars indicate standard deviation. Normalization was done to the 0 μM nitrocefin concentration. For more details, see Supplementary Fig. .
Article Snippet: Following incubation of the cells with the peptide, nitrocefin (1 mg/mL solution freshly diluted with water from a 10 mg/mL DMSO stock solution) was added to a 50 μg/mL final concentration, and the chromogenic hydrolysis of nitrocefin was monitored by reading absorbance at 490 nm every 15 s for 100 cycles in a CLARIOstar plus plate reader (BMG Labtech).
Techniques: Staining, Cryo-EM Sample Prep, Membrane, Permeability, Incubation, Positive Control, Standard Deviation, Concentration Assay
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