Time-Resolved, Single-Cell Study of the Attack of the Antimicrobial Peptide Ll-37 on Live E. Coli Cells

Abstract

Human LL-37 is an antimicrobial peptide whose amphiphilic helices selectively degrade bacterial membranes by a mechanism that is poorly understood. We are using single-cell, two-color fluorescence microscopy to directly observe the attack of rhodamine-LL-37 on live E. coli cells in real time. The cells express either periplasmic or cytoplasmic GFP. This enables quantitative correlation of the extent of LL-37 adsorption with leakage or lysing of GFP from the two different compartments, while simultaneously monitoring cell growth. At 15 uM, LL-37 lyses the periplasm to GFP and halts growth in 2-4 min, long before the cytoplasm lyses to GFP at 20-30 min. At 6 uM, rhodamine-LL-37 binding occurs in three distinct waves, with Wave 2 correlating in time with the halting of cell growth (t = 7-10 min). Wave 1 coats the cell periphery uniformly, but Wave 2 preferentially attacks the septal region and slowly spreads outward towards the poles. This suggests that the cell division machinery may be a target of LL-37-induced cell death. We will use FRET to discern the penetration depth of LL-37 during the different waves of attack and a variety of mutant strains to correlate the LL-37 attack with formation of the Z-ring and additional parts of the divisome. These methods will enable quantitative comparison of antimicrobial attack on real bacterial membranes with studies of lysing of synthetic lipid bilayers. They will be applicable to a wide variety of antimicrobial agents and bacterial species.