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BMC Biology

, 7:22

First Online: 11 May 2009Received: 10 November 2008Accepted: 11 May 2009

Abstract

BackgroundAntimicrobial 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.

ResultsHere 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.

ConclusionRecently 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.

AbbreviationsACFautocorrelation function

APDavalanche photodiode

AMPantimicrobial peptide

FCSfluorescence correlation spectroscopy

GFPgreen fluorescent protein

GNBGram-negative bacteria

GPBGram-positive bacteria

LPSlipopolysaccharide

PBSphosphate-buffered saline

PMBpolymixin B

Qdotquantum dot

rFCrecombinant Factor C

SPTsingle particle tracking

S1Sushi 1

SDSsodium dodecyl sulfate

TEMtransmission electron microscopy.

Electronic supplementary materialThe online version of this article doi:10.1186-1741-7007-7-22 contains supplementary material, which is available to authorized users.

Sebastian Leptihn, Jia Yi Har, Thorsten Wohland and Jeak Ling Ding contributed equally to this work.

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Autor: Sebastian Leptihn - Jia Yi Har - Jianzhu Chen - Bow Ho - Thorsten Wohland - Jeak Ling Ding

Fuente: https://link.springer.com/article/10.1186/1741-7007-7-22



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