Single molecule resolution of the antimicrobial action of quantum dot-labeled sushi peptide on live bacteria.
BACKGROUND
Antimicrobial peptides are found in all kingdoms of life. During the evolution of multicellular organisms, antimicrobial peptides were established as key elements of innate immunity. Most antimicrobial peptides are thought to work by disrupting the integrity of cell membranes, causing pathogen death. As antimicrobial peptides target the membrane structure, pathogens can only acquire resistance by a fundamental change in membrane composition. Hence, the evolution of pathogen resistance has been a slow process. Therefore antimicrobial peptides are valuable alternatives to classical antibiotics against which multiple drug-resistant bacteria have emerged. For potential therapeutic applications as antibiotics a thorough knowledge of their mechanism of action is essential. Despite the increasingly comprehensive understanding of the biochemical properties of these peptides, the actual mechanism by which antimicrobial peptides lyse microbes is controversial.


RESULTS
Here we investigate how Sushi 1, an antimicrobial peptide derived from the horseshoe crab (Carcinoscorpius rotundicauda), induces lysis of Gram-negative bacteria. To follow the entire process of antimicrobial action, we performed a variety of experiments including transmission electron microscopy and fluorescence correlation spectroscopy as well as single molecule tracking of quantum dot-labeled antimicrobial peptides on live bacteria. Since in vitro measurements do not necessarily correlate with the in vivo action of a peptide we developed a novel fluorescent live bacteria lysis assay. Using fully functional nanoparticle-labeled Sushi 1, we observed the process of antimicrobial action at the single-molecule level.


CONCLUSIONS
Recently the hypothesis that many antimicrobial peptides act on internal targets to kill the bacterium has been discussed. Here, we demonstrate that the target sites of Sushi 1 are outer and inner membranes and are not cytosolic. Further, our findings suggest four successive steps of the bactericidal process: 1) Binding, mediated mainly by charged residues in the peptide; 2) Peptide association, as peptide concentration increases evidenced by a change in diffusive behavior; 3) Membrane disruption, during which lipopolysaccharide is not released; and 4) Lysis, by leakage of cytosolic content through large membrane defects.
Singapore-MIT Alliance, Singapore. sebastian.leptihn@chem.ox.ac.uk
Sebastian
S
Leptihn
Jia Yi
JY
Har
Jianzhu
J
Chen
Bow
B
Ho
Thorsten
T
Wohland
Jeak Ling
JL
Ding
D000595:Amino Acid Sequence; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000494:pharmacology*; D001433:Bacteriolysis / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004058:Diffusion / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects* / Q000648:ultrastructure; D006046:Gold; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D015335:Molecular Probes / Q000378:metabolism; D008969:Molecular Sequence Data; D053758:Nanoparticles / Q000648:ultrastructure; D045663:Quantum Dots; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling
D023181:Antimicrobial Cationic Peptides; D008070:Lipopolysaccharides; D015335:Molecular Probes; D049452:Green Fluorescent Proteins; D006046:Gold