The comprehension of the mechanism of action of antimicrobial peptides is fundamental for the design of new antibiotics. Studies performed looking at the interaction of peptides with bacterial cells offer a faithful picture of what really happens in nature. In this work we focused on the interaction of the peptide Temporin L with E. coli cells, using a variety of biochemical and biophysical techniques that include: functional proteomics, docking, optical microscopy, TEM, DLS, SANS, fluorescence. We identified bacterial proteins specifically interacting with the peptides that belong to the divisome machinery; our data suggest that the GTPase FtsZ is the specific peptide target. Docking experiments supported the FtsZ-TL interaction; binding and enzymatic assays using recombinant FtsZ confirmed this hypothesis and revealed a competitive inhibition mechanism. Optical microscopy and TEM measurements demonstrated that, upon incubation with the peptide, bacterial cells are unable to divide forming long necklace-like cell filaments. Dynamic light scattering studies and Small Angle Neutron Scattering experiments performed on treated and untreated bacterial cells, indicated a change at the nanoscale level of the bacterial membrane. The peptide temporin L acts by a non-membrane-lytic mechanism of action, inhibiting the divisome machinery. Identification of targets of antimicrobial peptides is pivotal to the tailored design of new antimicrobials.
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