HaloTag Assay Suggests Common Mechanism of <i>E. coli</i> Membrane Permeabilization Induced by Cationic Peptides

Permeabilization of the Gram-negative bacterial outer membrane (OM) by antimicrobial peptides (AMPs) is the initial step enabling access of the AMP to the cytoplasmic membrane. We present a new single-cell, time-resolved fluorescence microscopy assay that reports on the permeabilization of the <i>E. coli</i> OM to small molecules with a time resolution of 3 s or better. When profluorophore JF₆₄₆ (702 Da) crosses the outer membrane (OM) and gains access to the periplasm, it binds to the localized HaloTag protein (34 kDa) and fluoresces in a characteristic hollow spatial pattern. Previous work used the much larger periplasmic GFP (27 kDa) probe, which reports on OM permeabilization to globular proteins. We test the assay on three cationic agents: Gellman random β–peptide copolymer MM₆₃:CHx₃₇, human AMP LL-37, and synthetic hybrid AMP CM15. These results combined with the previous work suggest a unifying sequence of OM and cytoplasmic membrane (CM) events that may prove commonplace in the attack of cationic peptides on Gram-negative bacteria. The peptide initially induces gradual OM permeabilization to small molecules, likely including the peptide itself. After a lag time, abrupt permeabilization of the OM, abrupt resealing of the OM, and abrupt permeabilization of the CM (all to globular proteins) occur in rapid sequence. We propose a mechanism based on membrane curvature stress induced by the time-dependent differential binding of peptide to the outer leaflet of the OM and CM. The results provide fresh insight into the critical OM-permeabilization step leading to a variety of damaging downstream events