5 research outputs found
Localized Permeabilization of E. coli Membranes by the Antimicrobial Peptide Cecropin A
Fluorescence microscopy enables detailed
observation of the effects
of the antimicrobial peptide Cecropin A on the outer membrane (OM)
and cytoplasmic membrane (CM) of single E. coli cells with subsecond time resolution. Fluorescence from periplasmic
GFP decays and cell growth halts when the OM is permeabilized. Fluorescence
from the DNA stain Sytox Green rises when the CM is permeabilized
and the stain enters the cytoplasm. The initial membrane disruptions
are localized and stable. Septating cells are attacked earlier than
nonseptating cells, and curved membrane surfaces are attacked in preference
to cylindrical surfaces. Below a threshold bulk Cecropin A concentration,
permeabilization is not observed over 30 min. Above this threshold,
we observe a lag time of several minutes between Cecropin A addition
and OM permeabilization and ∼30 s between OM and CM permeabilization.
The long lag times and the existence of a threshold concentration
for permeabilization suggest a nucleation mechanism. However, the
roughly linear dependence of mean lag time on bulk peptide concentration
is not easily reconciled with a nucleation step involving simultaneous
insertion of multiple peptides into the bilayer. Monte Carlo simulations
suggest that within seconds, the OM permeability becomes comparable
to that of a pore of 100 nm diameter or of numerous small pores distributed
over a similarly large area
Localized Permeabilization of E. coli Membranes by the Antimicrobial Peptide Cecropin A
Fluorescence microscopy enables detailed
observation of the effects
of the antimicrobial peptide Cecropin A on the outer membrane (OM)
and cytoplasmic membrane (CM) of single E. coli cells with subsecond time resolution. Fluorescence from periplasmic
GFP decays and cell growth halts when the OM is permeabilized. Fluorescence
from the DNA stain Sytox Green rises when the CM is permeabilized
and the stain enters the cytoplasm. The initial membrane disruptions
are localized and stable. Septating cells are attacked earlier than
nonseptating cells, and curved membrane surfaces are attacked in preference
to cylindrical surfaces. Below a threshold bulk Cecropin A concentration,
permeabilization is not observed over 30 min. Above this threshold,
we observe a lag time of several minutes between Cecropin A addition
and OM permeabilization and ∼30 s between OM and CM permeabilization.
The long lag times and the existence of a threshold concentration
for permeabilization suggest a nucleation mechanism. However, the
roughly linear dependence of mean lag time on bulk peptide concentration
is not easily reconciled with a nucleation step involving simultaneous
insertion of multiple peptides into the bilayer. Monte Carlo simulations
suggest that within seconds, the OM permeability becomes comparable
to that of a pore of 100 nm diameter or of numerous small pores distributed
over a similarly large area