View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 584 (2010) 1543–1548 journal homepage: www.FEBSLetters.org Functional interaction of human neutrophil peptide-1 with the cell wall precursor lipid II Erik de Leeuw a,*, Changqing Li a, Pengyun Zeng b, Chong Li b, Marlies Diepeveen-de Buin c, Wei-Yue Lu b, Eefjan Breukink c, Wuyuan Lu a,** a University of Maryland Baltimore School of Medicine, Institute of Human Virology and Department of Biochemistry and Molecular Biology, 725 West Lombard Street, Baltimore, MD 21201, USA b Fudan University School of Pharmacy, Shanghai, China c Utrecht University, Department of Biochemistry of Membranes, Bijvoet Center for Biomolecular Research, Padualaan 8, 3585 CH, Utrecht, The Netherlands article info abstract Article history: Defensins constitute a major class of cationic antimicrobial peptides in mammals and vertebrates, Received 11 January 2010 acting as effectors of innate immunity against infectious microorganisms. It is generally accepted Revised 1 March 2010 that defensins are bactericidal by disrupting the anionic microbial membrane. Here, we provide evi- Accepted 2 March 2010 dence that membrane activity of human a-defensins does not correlate with antibacterial killing. Available online 7 March 2010 We further show that the a-defensin human neutrophil peptide-1 (HNP1) binds to the cell wall pre- Edited by Renee Tsolis cursor lipid II and that reduction of lipid II levels in the bacterial membrane significantly reduces bacterial killing. The interaction between defensins and lipid II suggests the inhibition of cell wall synthesis as a novel antibacterial mechanism of this important class of host defense peptides. Keywords: Human neutrophil peptide-1 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. Defensin Lipid II 1. Introduction Defensins are widely accepted to kill bacteria through pore for- mation in the microbial membrane, causing leakage of intracellular Defensins form a large subfamily of cationic antimicrobial pep- contents and cell lysis [17,18]. The specific disruption of the bacte- tides that kill a broad range of microorganisms [1–4]. Human rial membrane by defensins is believed to be driven by electro- defensins are cysteine-rich, cationic peptides with molecular static attractions between these cationic peptides and the masses ranging from 3 to 5 kDa. Based on the connectivity of the negatively charged membrane. However, alternative mechanisms six conserved cysteine residues and sequence homology, human for bacterial killing have been proposed, including membrane- defensins are classified into a and b families. Both families of independent mechanisms and targeting of intracellular com- defensins have similar three-dimensional structures as determined pounds by defensins [19–21]. by X-ray crystallography and NMR studies [5–9], sharing a com- Recent observations on the bacterial killing by human defensins mon fold of three-stranded anti-parallel b-sheets constrained by could not fully be explained by the membrane-disruption model, three intra-molecular disulfide bonds. Human defensins were dis- suggesting a more nuanced mode of action. First, a-defensins were covered originally as natural peptide antibiotics in neutrophils. shown to preferentially kill Gram-positive bacteria, whereas b- These defensins were named human neutrophil peptides (HNP) defensins kill Gram-negative strains more effectively [22,23]. How- 1–3 of the a-defensin family [10]. Subsequently, a fourth a-defen- ever, human b-defensins carry more positive charges, indicating sin was discovered in neutrophils, termed HNP-4 [11–13]. More re- that cationicity of defensins alone does not explain this strain- cently, two additional a-defensins were described, termed human specificity. Second, disruption of the membrane via stable pore for- defensin 5 and 6 [14,15]. HD-5 and HD-6 are stored in the granules mation is believed to require peptide structure. However, we and of Paneth cells, specialized epithelial cells in the small intestine others have shown that bacterial killing by defensins can be [16]. structure-independent [24,25]. Third, we recently observed that a-defensins composed entirely of D-amino acids show greatly re- duced antibacterial activity against Staphylococcus aureus com- pared to the L-peptide, suggesting that the microbial membrane * Corresponding author. Fax: +1 410 706 7583. is not the sole target [26]. Here, we further examine the bacterial ** Corresponding author. Fax: +1 410 706 7583. E-mail addresses: [email protected] (E. de Leeuw), wlu@ihv. killing by a-defensins and provide evidence for an interaction with umaryland.edu (W. Lu). the bacterial target lipid II. 0014-5793/$36.00 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. doi:10.1016/j.febslet.2010.03.004 1544 E. de Leeuw et al. / FEBS Letters 584 (2010) 1543–1548 2. Materials and methods [31]. Typically, M. flavus vesicles (120 lmol lipid-Pi) were incu- bated together with 500 lmol UDP-GlcNAc, 500 lmol UDP-Mur- 2.1. Materials NAC-pentapeptide and 400 lmol farnesyl phosphate in 100 mM Tris–HCl pH 8.0, 5 mM MgCl2. The incubation lasted 2 h at room Chemicals used for solid phase peptide synthesis were obtained temperature for 3-P. The synthesis of 3-lipid II was followed using as described [27]. S. aureus ATCC 29213 was obtained from RP-8 reversed phase TLC (Merck) developed in 75% methanol. For Microbiologics (St. Cloud, MN). The phospholipids palmitoyl– purification, the membranes were removed by centrifugation at oleoyl-phosphatidylcholine (POPC), palmitoyl–oleoyl-phosphati- 40 000Âg and the supernatant was collected and loaded on a C18 dylglycerol (POPG) and dipalmitoyl–phosphatidyl choline (DPPC) HPLC column and eluted with a linear gradient from 50 mM were purchased from Avanti Polar Lipids (Alabaster, AL). 8-Amino- ammonium bicarbonate to 100% methanol in 30 min. Farnesyl-li- naphthalene-1,3,6-trisulfonic acid sodium salt (ANTS) and p-xyl- pid II (3-lipid II) eluted at approximately 60% methanol. Its identity enebis(pyridinium) bromide (DPX) were from Molecular Probes was confirmed by mass spectroscopy. (Eugene, OR). Poly-L-lysine (MW = 3800) was obtained from Sigma. Bacitracin, D-cycloserine and fosfomycine were purchased from 2.6. Surface plasmon resonance Sigma, Calbiochem and LKT Laboratories respectively. Surface plasmon resonance binding experiments were carried 2.2. Solid phase peptide synthesis out on a BIAcore T100 system (BIAcore Inc., Piscataway, NY) at 25 °C. The assay buffer was 10 mM HEPES, 150 mM NaCl, 0.05% Chemical synthesis and folding of defensins was carried out as surfactant P20, pH 7.4 (±3 mM EDTA). L-HNP1 (780 RUs) or D- described [27,28]. The molecular mass of the peptides was verified HNP1 (790 RUs) were immobilized on CM5 sensor chips using by electrospray ionization mass spectrometry (ESI–MS) as de- the amine-coupling chemistry recommended by the manufacturer. scribed [27]. Peptide stock solutions prepared with water were Lipid II was introduced into the flow-cells at 30 ll/min in the run- quantified spectroscopically using molar extinction coefficients at ning buffer. Association and dissociation were assessed for 300 and 280 nm calculated according to the algorithm of Pace et al. [29]. 600 s, respectively. Resonance signals were corrected for non-spe- cific binding by subtracting the background of the control flow- 2.3. LUVs preparation cell. After each analysis, the sensor chip surfaces were regenerated with 15 mM HCl for 30 s at a flow rate 100 ll/min, and equilibrated LUVs with the low molecular weight fluorophore/quencher pair with the buffer prior to next injection. Binding isotherms were (ANTS/DPX) encapsulated were prepared using the standard extru- analyzed with manufacturer-supplied software for BIAcore T100 sion method. Specifically, phospholipids were dissolved in chloro- and/or GraphPad Prism 4.0. form at a desired molar ratio, dried as a film by solvent evaporation. After removal of residual solvent, the lipid film was 2.7. Antibacterial activity assay hydrated in the fluorescent solution containing 5 mM HEPES, 12.5 mM ANTS, 45 mM DPX, and 20 mM NaCl, pH 7.0, freeze– The antibacterial activity of HNP1 against S. aureus ATCC 29213 thawed for 10 cycles and extruded 10 times through 0.4-lm poly- was carried out in a 96-well turbidimetric assay essentially as de- carbonate membranes. LUVs were separated from unencapsulated scribed previously [22]. Lipid II levels in S. aureus were manipu- materials by gel filtration chromatography using a Sepharose CL- lated by the addition of three different inhibitors of cell wall 4B column eluted with 5 mM HEPES, 100 mM NaCl, pH 7.4 (high- synthesis: bacitracin (250 lg/ml), D-cycloserine (64 lg/ml) and salt). For leakage assays in a low-salt buffer, purified vesicles were fosfomycine (250 lg/ml). Bacterial cultures were pre-treated with further diluted with 5 mM HEPES containing 10 mM NaCl, pH 7.4. these compounds for 30 min under shaking at 37 °C. Subsequently, cells were exposed to HNP1 peptide ranging from 256 to 1 lg/ml 2.4. Leakage assay for 15 min, after which HNP1 activity was neutralized by the addi- tion of Mueller Hinton broth. Bacterial growth was monitored for Leakage of ANTS from LUVs, monitored on a LS-55 Perkin Elmer 12 h and data were analyzed as described [22]. luminescence spectrometer, was characterized by an increase in fluorescence, which was quenched by DPX when encapsulated to- gether inside liposomes [30]. 270 ll ANTS/DPX-encapsulated LUVs 3. Results (in either high-salt or low-salt buffers) were added to each well of a 96-well plate to a final lipid concentration of 600 lM. 30 llH2O 3.1. Membrane lipid interaction of a-defensins was added to the first well of each row as a blank, and 30 ll 2.5% (v/v) Triton X-100 to the last (twelfth) well as the control for 100% Defensins are believed to kill bacteria by permeabilizing the leakage.