Fig. 3: Mechanism of action of antimicrobials from venoms.

To assess whether VEPs act on bacterial membranes, all active peptides against P. aeruginosa PAO1 were subjected to outer membrane permeabilization, and peptides active against P. aeruginosa PAO1 and S. aureus ATCC 12600 were tested in cytoplasmic membrane depolarization assays. The fluorescent probe 1-(N-phenylamino)naphthalene (NPN) was used to assess membrane permeabilization (a) induced by the tested VEPs in P. aeruginosa PAO1 cells. The fluorescent probe 3,3′-dipropylthiadicarbocyanine iodide (DiSC₃-5) was used to evaluate membrane depolarization of b P. aeruginosa PAO1 or c S. aureus ATCC 12600 caused by VEPs. The values displayed represent the relative fluorescence of both probes, with nonlinear fitting compared to the baseline of the untreated control (buffer + bacteria + fluorescent dye) and benchmarked against the antibiotics polymyxin B and levofloxacin. All experiments were performed in three independent replicates. The relative fluorescence values in (a–c) were calculated as the percentage difference between the sample and the untreated control using Eq. 3 (Methods). The untreated control (buffer + bacteria + fluorescent dye) served as the baseline, and polymyxin B was used as a positive control for benchmarking. The protein and peptide structures depicted in the figure were created with PyMOL Molecular Graphics System, version 3.1, Schrödinger, LLC. Panel (a) created in BioRender. De La Fuente-Nunez, C. (2025) https://BioRender.com/1bfl5da.