Real-time attack on single Escherichia coli cells by the human antimicrobial peptide LL-37

Abstract

Natural antimicrobial peptides (AMPs) provide prototypes for the design of unconventional antimicrobial agents. Existing bulk assays measure AMP activity but do not provide details of the growthhalting mechanism. We use fluorescence microscopy to directly observe the attack of the human antimicrobial peptide LL-37 on single Escherichia coli cells in real time. Our findings strongly suggest that disruption of the cytoplasmic membrane is not the growth-halting mechanism. At 8 μM, LL-37 binding saturates the outer membrane (OM) within 1 min. Translocation across the OM and access to the periplasmic space (5-25 min later) correlates in time with the halting of growth. Septating cells are attacked more readily than nonseptating cells. The halting of growth may occur because of LL-37 interference with cell wall biogenesis. Only well after growth halts does the peptide permeabilize the cytoplasmic membrane to GFP and the small dye Sytox Green. The assay enables dissection of antimicrobial design criteria into two parts: translocation across the OM and the subsequent halting of growth.

Single-cell, real-time detection of oxidative stress induced in Escherichia coli by the antimicrobial peptide CM15

“…Figure 1C shows the time evolution of total GFP fluorescence intensity and of tip-to-tip cell length. The inward movement of GFP implies rapid translocation of the peptide across the outer membrane (OM) without permeabilization to GFP, followed by permeabilization of the cytoplasmic membrane (CM) to GFP (16). Diffusive transfer of GFP to the cytoplasm is driven by the large cytoplasmic volume compared with the periplasmic volume.…”

“…We used previously developed single-cell, real-time imaging assays (14)(15)(16) to monitor the disruption of K12 E. coli membranes by CM15. The modified K12 cells include a plasmid to express GFP with the twin-arginine translocase signal peptide appended to the N terminus.…”

“…The MIC for CM15 was determined using a broth microdilution method as previously described (16). Twofold serial dilutions of CM15 in 1× EZRDM were performed in separate rows of a polystyrene 96-well plate with each plate containing an inoculum of E. coli MG1655.…”

Plasmonic imaging of protein interactions with single bacterial cells

“…The data generated with these bulk assays are averaged over many bacteria, which wash out important variability or heterogeneity of different bacterial cells. Various imaging techniques, such as fluorescence (Cywes--Bentley et al, 2013;Sochacki et al, 2011), AFM (Fantner et al, 2010;Lower et al, 2011) and transmission electron microscopy (TEM) (Cywes--Bentley et al, 2013), and non--imaging microfluidics techniques, such as flow cytometry (Tracy et al, 2010) and micro electrophoresis (van der Mei and Busscher, 2012), have been used to study bacterial surfaces. These techniques have contributed to the understanding of bacteria, but each has disadvantages.…”

“…The surfaces of bacteria act as the first line of defense against harmful external stimuli, including antibiotics (Delcour, 2009) and antimicrobial peptides, (Fantner et al, 2010;Sochacki et al, 2011) and also play crucial roles in interacting with other surfaces, including host tissues (Van Houdt and Michiels, 2005) and medical plastics, (Lower et al, 2011) to help bacterial cells attach and colonize. In order to survive in a changing environment, bacteria replicate and evolve quickly, (Carnes et al, 2010;van der Mei and Busscher, 2012) leading to diversity of different bacteria species, and variability within the same species.…”