Dataset Information

Daptomycin Pore Formation and Stoichiometry Depend on Membrane Potential of Target Membrane.

ABSTRACT: Daptomycin is a calcium-dependent lipodepsipeptide antibiotic clinically used to treat serious infections caused by Gram-positive pathogens. Its precise mode of action is somewhat controversial; the biggest issue is daptomycin pore formation, which we directly investigated here. We first performed a screening experiment using propidium iodide (PI) entry to Bacillus subtilis cells and chose the optimum and therapeutically relevant conditions (10?µg/ml daptomycin and 1.25?mM CaCl₂) for the subsequent analyses. Using conductance measurements on planar lipid bilayers, we show that daptomycin forms nonuniform oligomeric pores with conductance ranging from 120 pS to 14 nS. The smallest conductance unit is probably a dimer; however, tetramers and pentamers occur in the membrane most frequently. Moreover, daptomycin pore-forming activity is exponentially dependent on the applied membrane voltage. We further analyzed the membrane-permeabilizing activity in B. subtilis cells using fluorescence methods [PI and DiSC₃(5)]. Daptomycin most rapidly permeabilizes cells with high initial membrane potential and dissipates it within a few minutes. Low initial membrane potential hinders daptomycin pore formation.

SUBMITTER: Seydlova G

PROVIDER: S-EPMC6325215 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Daptomycin Pore Formation and Stoichiometry Depend on Membrane Potential of Target Membrane.

Seydlová Gabriela G Sokol Albert A Lišková Petra P Konopásek Ivo I Fišer Radovan R

Antimicrobial agents and chemotherapy 20181221 1

Daptomycin is a calcium-dependent lipodepsipeptide antibiotic clinically used to treat serious infections caused by Gram-positive pathogens. Its precise mode of action is somewhat controversial; the biggest issue is daptomycin pore formation, which we directly investigated here. We first performed a screening experiment using propidium iodide (PI) entry to <i>Bacillus subtilis</i> cells and chose the optimum and therapeutically relevant conditions (10 µg/ml daptomycin and 1.25 mM CaCl<sub>2</sub ...[more]

PMID: 30323037

Similar Datasets

Project description:Medin, a 50-amino-acid cleavage product of the milk fat globule-EGF factor 8 protein, is one of the most common forms of localized amyloid found in the vasculature of individuals older than 50 years. Medin induces endothelial dysfunction and vascular inflammation, yet despite its prevalence in the human aorta and multiple arterial beds, little is known about the nature of its pathology. Medin oligomers have been implicated in the pathology of aortic aneurysm, aortic dissection, and more recently, vascular dementia. Recent in vitro biomechanical measurements found increased oligomer levels in aneurysm patients with altered aortic wall integrity. Our results suggest an oligomer-mediated toxicity mechanism for medin pathology. Using lipid bilayer electrophysiology, we show that medin oligomers induce ionic membrane permeability by pore formation. Pore activity was primarily observed for preaggregated medin species from the growth-phase and rarely for lag-phase species. Atomic force microscopy (AFM) imaging of medin aggregates at different stages of aggregation revealed the gradual formation of flat domains resembling the morphology of supported lipid bilayers. Transmission electron microscopy images showed the coexistence of compact oligomers, largely consistent with the AFM data, and larger protofibrillar structures. Circular dichroism spectroscopy revealed the presence of largely disordered species and suggested the presence of ?-sheets. This observation and the significantly lower thioflavin T fluorescence emitted by medin aggregates compared to amyloid-? fibrils, along with the absence of amyloid fibers in the AFM and transmission electron microscopy images, suggest that medin aggregation into pores follows a nonamyloidogenic pathway. In silico modeling by molecular dynamics simulations provides atomic-level structural detail of medin pores with the CNpNC barrel topology and diameters comparable to values estimated from experimental pore conductances.

Dataset Information

Daptomycin Pore Formation and Stoichiometry Depend on Membrane Potential of Target Membrane.

Publications

Daptomycin Pore Formation and Stoichiometry Depend on Membrane Potential of Target Membrane.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets