HaloTag Assay Suggests Common Mechanism of E. coli Membrane Permeabilization Induced by Cationic Peptides

Abstract

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 E. coli OM to small molecules, with time resolution of 3 sec or better. When the profluorophore JF646 (702 Da) crosses the outer membrane (OM) and gains access to the periplasm, it binds to 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: the Gellman random β-peptide copolymer MM63:CHx37, the human AMP LL-37, and the synthetic hybrid AMP CM15. These results combined with 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 re-sealing 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 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.

Rigidification of the Escherichia coli cytoplasm by the human antimicrobial peptide LL-37 revealed by superresolution fluorescence microscopy

“…The Wimley and Stella laboratories have devised methods to measure the absolute uptake of AMP per cell at cell-killing concentrations (44, 45). We recently demonstrated a HaloTag-based, single-cell fluorescence assay that detects passage of a small dye molecule, and presumably concomitant passage of a small peptide, across the OM of E. coli (74). Strong binding of AMPs to dsDNA and to ribosomes, as reported here, may enhance the potency of many antimicrobial agents.…”

Resistance of early stationary phase E. coli to membrane permeabilization by the antimicrobial peptide Cecropin A

“…In those cases studied with good time resolution (LL-37, Cecropin A, and melittin), OM and CM permeabilization initiate locally, at the septal region or one endcap. A periplasmic HaloTag assay revealed a sequence of OM and CM permeabilization and re-sealing events to GFP common to LL-37, CM15, and melittin [36].…”

Application of Biophysical Techniques to Investigate the Interaction of Antimicrobial Peptides With Bacterial Cells

“…Using labeled peptides, it is possible to correlate permeabilization and internalization kinetics. Time-resolved FCAs performed on Rhodamine B labeled Ctn and Ctn (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34) show that binding and internalization processes occur until an equilibrium is reached; Ctn(15-34) uptake is faster and precedes bacterial membrane damage, suggesting that permeabilization occurs only upon achievement of a threshold surface concentration (33). In line with this, single cell images show that both peptides are mostly localized on the E. coli surface, but a small percentage co-localizes with the dye SYTOX green, suggesting a partial internalization.…”

“…In a different study, OM permeabilization has been detected by the profluorophore JF646, a small molecule (702 Da) emitting weak fluorescence in aqueous solution. When the AMP permeabilizes the outer membrane, JF646 enters the cytoplasm and covalently binds to the HaloTag protein emitting red fluorescence; permeabilization onset is detected with a 3s resolution ( 15 , 16 ). When peptides are labeled, information on the localization and distribution of peptides may be obtained ( 17 ).…”

“…Some techniques are suitable to investigate changes occurring both to peptides and cells; as an example, Nuclear Magnetic Resonance (NMR), that looks at changes in the chemical shift of magnetically active nuclei such as 1 H, 13 C, and 31 P, is exploited to determine the structure of peptides that interact with cells, but is also used to detect the binding of peptides to cells (8)(9)(10)(11). Similarly, fluorescence microscopy can be used to follow labeled peptides inside cells or to monitor the permeabilization of bacterial cells expressing fluorescent proteins able to cross damaged membranes (12)(13)(14)(15)(16)(17)(18). Other techniques, such as scattering techniques (DLS, SANS, SAXS) or AFM or TEM, are employed to monitor only changes at bacterial cells (19,20).…”