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. 2008 Jul;52(7):2599-607.
doi: 10.1128/AAC.00028-08. Epub 2008 Apr 28.

The human CXC chemokine granulocyte chemotactic protein 2 (GCP-2)/CXCL6 possesses membrane-disrupting properties and is antibacterial

Helena M Linge  1 , Mattias Collin, Pontus Nordenfelt, Matthias Mörgelin, Martin Malmsten, Arne Egesten
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V体育平台登录 - The human CXC chemokine granulocyte chemotactic protein 2 (GCP-2)/CXCL6 possesses membrane-disrupting properties and is antibacterial

Helena M Linge et al. Antimicrob Agents Chemother. 2008 Jul.

Abstract

Granulocyte chemotactic protein 2 (GCP-2)/CXCL6 is a CXC chemokine expressed by macrophages and epithelial and mesenchymal cells during inflammation. Through binding and activation of its receptors (CXCR1 and CXCR2), it exerts neutrophil-activating and angiogenic activities. Here we show that GCP-2/CXCL6 itself is antibacterial. Antibacterial activity against gram-positive and gram-negative pathogenic bacteria of relevance to mucosal infections was seen at submicromolar concentrations (minimal bactericidal concentration at which 50% of strains tested were killed, 0 VSports手机版. 063 +/- 0. 01 to 0. 37 +/- 0. 03 muM). In killed bacteria, GCP-2/CXCL6 associated with bacterial surfaces, which showed membrane disruption and leakage. A structural prediction indicated the presence of three antiparallel NH(2)-terminal beta-sheets and a short amphipathic COOH-terminal alpha-helix; the latter feature is typical of antimicrobial peptides. However, when the synthetic derivatives corresponding to the NH(2)-terminal (50 amino acids) and COOH-terminal (19 amino acids, corresponding to the putative alpha-helix) regions were compared, higher antibacterial activity was observed for the NH(2)-terminus-derived peptide, indicating that the holopeptide is necessary for full antibacterial activity. An artificial model of bacterial membranes confirmed these findings. The helical content of GCP-2/CXCL6 in the presence or absence of lipopolysaccharide or negatively charged membranes was studied by circular dichroism. As with many antibacterial peptides, membrane disruption by GCP-2/CXCL6 was dose-dependently reduced in the presence of NaCl, which, we here demonstrate, inhibited the binding of the peptide to the bacterial surface. Compared with CXC chemokines ENA-78/CXCL5 and NAP-2/CXCL7, GCP-2/CXCL6 showed a 90-fold-higher antibacterial activity. Taken together, GCP/CXCL6, in addition to its chemotactic and angiogenic properties, is likely to contribute to direct antibacterial activity during localized infection. .

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Figures

FIG. 1.
FIG. 1.
GCP-2/CXCL6 associates with the bacterial surface and disrupts bacterial membranes. (A to D) E. coli was exposed to GCP-2/CXCL6 (1 μM) for 1 h at 37°C. After fixation, the bacteria were labeled and immobilized on glass slides. (A) Differential interference contrast image of the stained bacteria. (B) Bacterial DNA is visualized by binding of the fluorescent dye DAPI. (C) Immunolabeled GCP-2/CXCL6. (D) Merge composite of panels B and C, showing DNA in blue and immunolabeled GCP-2/CXCL6 in red. Results are representative of three separate experiments. Scale bar, 10 μm. (E) The stained bacteria were enumerated, and results are displayed in a bar graph. (F and G) Electron micrographs showing bacteria incubated in buffer alone (F) or with GCP-2/CXCL6 (1 μM) (G) for 1 h at 37°C, followed by negative staining. Bacteria exposed to GCP-2/CXCL6, but not control samples, show protrusions and leakage of intracellular contents. Scale bars, 0.5 μm.
FIG. 2.
FIG. 2.
Predicted structure of GCP-2/CXCL6 in relation to antibacterial activity. (A) Predictive model based on known structures of other members of the chemokine family. The NH2 terminus, most likely, is an unstructured region followed by three antiparallel β-sheets that are held together by disulfide bonds involving the cysteines of the CXC motif (yellow). The COOH terminus is presumably an α-helix (purple). (B) The putative COOH-terminal α-helix (amino acids 56 to 77) has an amphipathic structure where hydrophobic (blue) and hydrophilic (white/yellow) amino acids are arranged in a polarized fashion. (C) The amphipathic character of the COOH terminus is further elucidated by depicting it as a helical wheel, giving a view of a helix from a protein sequence, looking down the axis of the helix.
FIG. 3.
FIG. 3.
GCP-2/CXCL6 and its derivatives GPV-50 and APF-19 cause liposome leakage by membrane disruption. (A and B) CF-loaded liposomes were incubated with the holopeptide or one of its derivatives at the indicated concentrations in buffer alone (A) or buffer containing 150 mM NaCl (B). Leakage was determined by monitoring the fluorescence emitted at 520 nm and was expressed as a percentage of the leakage induced by Triton X-100, taken as 100%. Measurements were performed at 37°C. (C) The kinetics of leakage induction by the peptides was assessed, and results are shown.
FIG. 4.
FIG. 4.
Extent of helix formation of GCP-2/CXCL6 and its derivatives as studied by CD spectroscopy. Measurements were performed using a peptide concentration of 10 μM in buffer with or without the addition of liposomes or LPS as indicated. The helix content was quantified at a wavelength of 222 to 225 nm and 37°C. (A) GCP-2/CXCL6; (B) GPV-50; (C) APF-19.
FIG. 5.
FIG. 5.
Effects of plasma and NaCl on the antibacterial activity of GCP-2/CXCL6, investigated by using a bactericidal assay (peptide concentration, 1 μM for 1 h at 37°C). (A) The presence of plasma at low concentrations strongly inhibited the killing of S. aureus, while plasma concentrations of 10% or higher were required to decrease the killing of E. coli. (B) S. aureus was chosen for examination of the effect of NaCl on the antibacterial activity of GCP-2/CXCL6. The antibacterial activity in this setup was greatly reduced in the presence of 100 and 150 mM NaCl. Data are means from three separate experiments. Error bars, standard errors of the means. (C) The ability of GCP-2/CXCL6 to interact with the bacterial membrane in the absence (lane 1) and presence (lane 2) of 150 mM NaCl was investigated. GCP-2/CXCL6 or IgG was adsorbed to S. aureus in the presence or absence of NaCl as indicated, followed by SDS-PAGE separation and Western blot analysis using anti-GCP-2 antibodies or protein G, respectively. Molecular mass is indicated on the left.
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