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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 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 ?-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.

SUBMITTER: Yang Z

PROVIDER: S-EPMC6097915 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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

Yang Zhilin Z Weisshaar James C JC

ACS chemical biology 20180612 8

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 a time resolution of 3 s or better. When profluorophore JF<sub>646</sub> (702 Da) crosses the outer membrane (OM) and gains access to the periplasm, it binds to the local ...[more]

PMID: 29812894

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Project description:BackgroundThe question of how the aggregation of the neuronal protein ?-synuclein contributes to neuronal toxicity in Parkinson's disease has been the subject of intensive research over the past decade. Recently, attention has shifted from the amyloid fibrils to soluble oligomeric intermediates in the ?-synuclein aggregation process. These oligomers are hypothesized to be cytotoxic and to permeabilize cellular membranes, possibly by forming pore-like complexes in the bilayer. Although the subject of ?-synuclein oligomer-membrane interactions has attracted much attention, there is only limited evidence that supports the pore formation by ?-synuclein oligomers. In addition the existing data are contradictory.Methodology/principal findingsHere we have studied the mechanism of lipid bilayer disruption by a well-characterized ?-synuclein oligomer species in detail using a number of in vitro bilayer systems and assays. Dye efflux from vesicles induced by oligomeric ?-synuclein was found to be a fast all-or-none process. Individual vesicles swiftly lose their contents but overall vesicle morphology remains unaltered. A newly developed assay based on a dextran-coupled dye showed that non-equilibrium processes dominate the disruption of the vesicles. The membrane is highly permeable to solute influx directly after oligomer addition, after which membrane integrity is partly restored. The permeabilization of the membrane is possibly related to the intrinsic instability of the bilayer. Vesicles composed of negatively charged lipids, which are generally used for measuring ?-synuclein-lipid interactions, were unstable to protein adsorption in general.Conclusions/significanceThe dye efflux from negatively charged vesicles upon addition of ?-synuclein has been hypothesized to occur through the formation of oligomeric membrane pores. However, our results show that the dye efflux characteristics are consistent with bilayer defects caused by membrane instability. These data shed new insights into potential mechanisms of toxicity of oligomeric ?-synuclein species.

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

Publications

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

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