Project PXD000181



Short cationic antimicrobial peptide RWRWRW-NH2 inhibits cell wall biosynthesis and bacterial respiration by delocalizing essential membrane proteins


Antimicrobial peptides are potent antibacterial agents. The subclass of short cationic RW-rich peptides is known to target the bacterial membrane. We employed the synthetic hexapeptide RWRWRW-NH2 to characterize in-depth the impact of short cationic RW-rich peptides on bacterial physiology and the bacterial response. Proteomics, ionomics, and amino acid analysis provided insights into both the mechanism of action and bacterial response. Peptide integration into the membrane depends on phospholipid composition and occurs in DPPG/DPPE mixed membranes but not in DPPG or DPPE membranes. Integration of the peptide changes lipid bilayer architecture resulting in delocalization of membrane-associated proteins involved in cell wall biosynthesis, respiration, and cell division. Consequently, cells suffer substantial energy limitation and cell wall integrity is corrupted. Bacteria react to this peptide stress by adjusting the membrane lipid composition andby modifying the cell wall. They further release osmoprotective amino acids, a novel strategy to survive exposure to bacteriolytic peptides. M&M Proteomics Treatment of B. subtilis 168, metabolic labeling of newly synthesized proteins with [35S]-L-methionine, and subsequent protein separation by 2D PAGE were performed as described previously [1]. For gel-free proteome analysis of membrane proteins B. subtilis 168 was grown aerobically at 37 °C in Belitzky minimal medium (BMM) supplemented with 14N-ammonium sulfate and 14N-L-tryptophane. Cells were stressed at an OD500 of 0.4 with 22.5 µg/mL MP196 for 65 minutes or left untreated as control. Untreated cultures grown on 15N-ammonium sulfate and 15N-L-tryptophane were used for relative quantification. Mixing of cell extracts prior to subcellular fractionation steps for relative quantification was carried out according to Otto et al [2].The enriched membrane protein fraction was prepared according to the workflow published by Eymann et al. [3] omitting the n-dodecyl-beta-D-maltoside treatment step. The preparation of the integral membrane peptides was carried out as described by Wolff et al.[4]. Sample preparation, mass spectrometric measurement, and subsequent data analysis were carried out as described by Otto et al.[2][1] Wenzel, M. et al.Proteomic response of Bacillus subtilis to lantibiotics reflects differences in interaction with the cytoplasmic membrane. Antimicrob. Agents Chemother. Doi:10.1128/AAC.01380-12 (2012) [2] Otto, a. et al. Systems-wide temporal proteomic profiling in glucose-starved Bacillus subtilis. Nat Commun. 2010;1:137. doi: 10.1038/ncomms1137 [3] Eymann, C. et al. A comprehensive proteome map of growing Bacillus subtilis cells. Proteomics. 2004 Oct;4(10):2849-76 [4] Wolff, S. et al. Complementary analysis of the vegetative membrane proteome of the human pathogen Staphylococcus aureus.Mol Cell Proteomics. 2008 Aug;7(8):1460-8. doi: 10.1074/mcp.M700554-MCP200

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Andreas Otto, Institute for Microbiology

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LTQ Orbitrap


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Experiment Type

Bottom-up proteomics


Publication pending