Antibacterial Activity of T22, a Specific Peptidic Ligand of the Tumoral Marker CXCR4

Abstract

CXCR4 is a cytokine receptor used by HIV during cell attachment and infection. Overexpressed in the cancer stem cells of more than 20 human neoplasias, CXCR4 is a convenient antitumoral drug target. T22 is a polyphemusin-derived peptide and an effective CXCR4 ligand. Its highly selective CXCR4 binding can be exploited as an agent for the cell-targeted delivery and internalization of associated antitumor drugs. Sharing chemical and structural traits with antimicrobial peptides (AMPs), the capability of T22 as an antibacterial agent remains unexplored. Here, we have detected T22-associated antimicrobial activity and biofilm formation inhibition over Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, in a spectrum broader than the reference AMP GWH1. In contrast to GWH1, T22 shows neither cytotoxicity over mammalian cells nor hemolytic activity and is active when displayed on protein-only nanoparticles through genetic fusion. Under the pushing need for novel antimicrobial agents, the discovery of T22 as an AMP is particularly appealing, not only as its mere addition to the expanding catalogue of antibacterial drugs. The recognized clinical uses of T22 might allow its combined and multivalent application in complex clinical conditions, such as colorectal cancer, that might benefit from the synchronous destruction of cancer stem cells and local bacterial biofilms.

Keywords: antimicrobial peptides; fusion proteins; inhibition of biofilm formation; multivalent drugs; nanoparticles.

Figure 1

Structure of GWH1, T22 and related AMPs. (A) Columns contain the following data: peptide name (β-hairpin-forming peptides with background colored as in (B); length in number of amino acids; peptide sequence aligned with T22 (all but GWH1), residues used for superposition in bold; hydrophobic-moment-vector magnitude; average absolute electrostatic potential on the peptide’s surface; net charge; RMSD from T22 superposition; surface hydrophobicity; hydrophobic free energy (see methods for definition); FoldX total energy after repair. (B) Superposition of all β-hairpin AMPs used in this study and their calculated HM vectors against model T22 (RMSD and colors for each peptide given in (A). (C) Superposition of the calculated HM vector for T22 and GHW1, oriented parallel to the membrane normal. (D) Representation of the surface electrostatic potential for T22 and GWH1 (scale: top red = −10, top blue = 10).

Figure 2

Impact of peptides T22 and GWH1 on bacterial growth in liquid culture. (A) Bacterial growth of E. coli, S. aureus and P. aeruginosa cultures, measured by their optical density at 620 nm, treated with T22 and control GWH1 peptides in serial 2-fold dilutions at 37 °C for 18 h. Each point represents an average of at least two different values and error bar indicates standard deviation. (B) Minimum inhibitory concentration (MIC) of the peptides for E. coli and S. aureus. The lowest concentration showing no bacterial growth (evaluated by visual inspection) in the broth microdilution method was taken as the MIC. Significant differences between groups are indicated as * p < 0.01, one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test.

Figure 3

Time- and concentration-dependent impact of the peptides on bacterial growth and biofilm formation, respectively. (A) Time-kill kinetics of E. coli (up) and S. aureus (down) exposed to T22 and GWH1 peptides at different concentrations for 0, 0.5, 1, 2, 3, 4, 5 and 24 h. Each point represents an average of at least two different values and error bars indicate standard deviation. Control represents the bacterial growth without peptide exposure. Figures indicate concentration in µmol/L. (B) Effect of peptide concentration on biofilm formation on the surface of the microtiter wells. Significant differences over bacterial control are indicated as * p < 0.05, one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test.

Figure 4

Cytotoxicity of peptides T22 and GWH1 on mammalian cells. (A) Cell viability in the presence of peptides over cultured mammalian cells, recorded 48 h after exposure. (B) Hemolytic activity associated with peptides over human erythrocytes. Significant differences over cell control are indicated as * p < 0.01, one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. Figures indicate concentration in µmol/L.

Figure 5

Antibacterial activity and possible structure of a recombinant T22 displayed on protein nanoparticles. (A) Volume size distribution of T22-PE24-H6, T22-GFP-H6 and GWH1-GFP-H6 nanoparticles and unassembled GFP-H6, as determined by dynamic light scattering. Data are represented as mean ± standard error on the mean (SEM). (B) Bacterial integrity of S. aureus measured by the optical density at 620 nm, after incubation of GWH1-GFP-H6, T22-GFP-H6 and T22-PE24-H6 nanoparticles and GFP-H6 in serial 2-fold dilutions at 37 °C for 18 h. Protein concentration at the X axis refers to monomers. Significant differences over GFP control protein are indicated as * p < 0.01, one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. (C) A proposed model for the T22-GFP-H6 nanoparticle according to a previous approach [17]. Each T22 peptide has been colored differently and its calculated HM vector has been drawn in black. (D) Left: detail of (C) for a single nanoparticle monomer. Right: Closeup of the T22 region with PM1-selected model superposed (RMSD 1.34 calculated with PyMOL’s “super” function).