Development of Broad-Spectrum Antimicrobial Peptides through the Conjugation of FtsZ-Binding and Cell-Penetrating Peptides

Abstract

The rapid increase in bacterial resistance to conventional antibiotics has led to a great demand for novel antibacterial agents. Antimicrobial peptides (AMPs) are emerging as next-generation antimicrobial alternative drugs to conventional antibiotics because of their broad-spectrum antimicrobial activities and minimal potential for drug resistance induction. This work describes novel antimicrobial peptides (FtsZpcpp) synthesized through the conjugation of a cell penetration peptide ((RXR)₄XB) to nonantimicrobial peptides (FtsZp). The FtsZp peptides were previously identified to bind FtsZ (flaming-temperature-sensitive protein Z), a crucial protein in regulating bacterial cell divisions. Newly designed FtsZpcpp peptides have broad antimicrobial activities against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains. Besides, these new peptides exert minimal hemolytic activity toward human red blood cells and low cytotoxicity toward human skin cells. Comprehensive studies on the antimicrobial mechanism of FtsZpcpp peptides revealed that they exert antimicrobial activities through multiple mechanisms, including membrane disruption and intracellular actions (e.g., interference with cell divisions, DNA binding, and reactive oxygen species (ROS) generation). Our results have shown that FtsZpcpp peptides have the potential to serve as future antimicrobial drugs in combating the increasing global problem of antibiotic resistance.

Keywords: antibiofilm; antimicrobial peptides; antimicrobial resistance; cell division; cell membrane; cell penetrating peptide.

1

Antibiofilm potential of peptides was assessed using the crystal violet assay. ATCC 25922 and ATCC 27853 were cultured in a 96-well polystyrene and polypropylene plate for 24 h at 37 °C, respectively. Images of (A) and (C) biofilm stained with crystal violet. Quantification of (B) and (D) biofilm formation in response to different concentrations of peptides by measuring the absorbance at 570 nm. Experimental data are presented as the mean ± SD obtained from three biological replicates. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns: not significant. The p values were determined using one-way ANOVA (Dunnett’s test).

2

CD spectra of peptides in different environments. Peptide (100 μM) was dissolved in 10 mM SDS (green), 100% TFE (red), or PBS (blue) at room temperature. Each data point represents the mean for three replicates (n = 3) and CD spectra of the corresponding buffer were subtracted.

3

Effects of peptides on the cell membrane. (A) Alternation of membrane permeabilization detected by the uptake of SYTOX Green. was treated with peptides (μM) and antibiotics (μg/mL) at various concentrations for 10, 30, and 4 h (shorter incubation time corresponding to lower saturation color). Fluorescence intensity was measured with an excitation/emission wavelength of 485/525 nm. (B) Plasma-membrane permeability of peptides against by measuring the uptake of propidium iodide. All experiments were performed with three biological replications. Data are presented as the mean ± SD obtained from three biological replicates. The p values were determined using one-way ANOVA (Dunnett’s test). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns: not significant. (C) Fluorescence microscopy images of stained with SYTOX green (green, nucleic acid), DAPI (blue, nucleic acid), and FM 4–64 (red, membrane). was treated with FtsZp1acpp (12 μM), FtsZp2acpp (12 μM,), FtsZp3acpp (8 μM) and (RXR)₄XB (96 μM) in MHB for 4 h. Images of each fluorescent dye were acquired separately and kept at the same exposure time and intensity for each channel and overlaid into a single image. The scale bar is 5 μm. Red arrows mark the spherical cellular morphologies of . SYTOX Green uptake in cells is annotated with white arrows. In cells without peptide treatment, no spherical morphologies and SYTOX Green uptake are observed.

4

Peptides induced membrane permeabilization and depolarization. (A) Outer membrane permeabilization induced by peptides was detected by NPN (N-phenyl-1-naphthylamine) uptake in . The percentage of outer membrane permeabilization was plotted to compare with 10 μg/mL polymyxin B. (B) Inner membrane permeabilization by peptides at 24 and 48 μM using DiSC₃(5). All experiments were performed with three biological replications. Data are presented as the mean ± SD obtained from three biological replicates. The p values were determined using one-way ANOVA (Dunnett’s test). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns: not significant. (C) Observations on cell morphology using scanning electron microscopy (SEM). after 2 h incubation in MHB in absence of peptides (1) or exposure to 1 × MIC FtsZp1acpp (2), 2 × MIC FtsZp1acpp (3), 1 × MIC FtsZp2acpp (4), 2 × MIC FtsZp2acpp (5), 1 × MIC FtsZp3acpp (6), or 2 × MIC FtsZp3acpp (7). The scale bar represents 1 μm.

5

ITC of LPS from O55:B5 with peptides. (A) FtsZp1acpp binding to LPS. (B) FtsZp2acpp binding to LPS. (C) FtsZp3acpp binding to LPS. (D) (RXR)₄XB binding to LPS. The top panel of each graph is the raw ITC data and the bottom panel of each graph is the integrated heat peaks against the molar ratio of peptides.

6

Effects of the peptides on FtsZ. (A) Fluorescence microscopy for FtsZ-GFP localizations in stained with membrane dye. BW 25113 harboring a plasmid containing IPTG inducible FtsZ-GFP was cultured in MHB containing 30 μg/mL chloramphenicol at 37 °C and FtsZ-GFP was overexpressed by 5 μM IPTG before being treated with 12 μM FtsZp1acpp, FtsZp2acpp, or FtsZp3acpp. Cell membranes were stained by FM 4–64 (red) before being imaged. The scale bar is 5 μm. (B) Effects of peptides on the GTPase activity of FtsZ. FtsZ (3 μM) in 50 mM MOPS buffer (pH 6.5) was incubated with peptides (1 mM) at room temperature for 30 min. GTPase activity was measured after the addition of 5 mM MgCl₂, 200 mM KCl, and 0.25 μM GTP. Data are presented as the mean ± SD obtained from three biological replicates. The p values were determined using one-way ANOVA (Dunnett’s test). **p ≤ 0.01, and ***p ≤ 0.001.

7

Intracellular mechanisms. (A) DNA binding assay. The interaction of peptides with ATCC 25922 genomic DNA was assessed by measuring the DNA migration through a 1% agarose gel. Peptide concentration was indicated at the top of each lane. DNA genomics without treatment was used as a control in the first lane of each gel for comparison. (B) ROS level affected by peptides. ROS level in bacteria was determined using a DCFH-DA after exposure to peptides for 1 h at room temperature (ex/em: 488 nm/535 nm). Experiments were carried out with three biological replicates, and all data were given as mean ± SD. One-way ANOVA was used to determine the statistical significance (***p ≤ 0.001 and ****p ≤ 0.0001).

8

Hemolytic potency and cytotoxicity of peptides. (A) Hemolytic potency of peptides against mammalian red blood cells (RBCs). 1% Triton X-100 was used as a positive control, and the concentration of tested peptides was 1 mM. Each data point stands for the mean ± SD obtained from three biological replicates, and one-way ANOVA was used to determine the statistical significance (***p ≤ 0.001). (B) Human BJ cells viabilities after treatment with various concentrations of peptides. The dashed black line indicates 50% cell viability. Each data point stands for the mean ± SD obtained from three biological replicates.

9

Serial passage resistance induction of peptides against ATCC 25922. was challenged with sub-MIC of FtsZp1acpp (blue), FtsZp2acpp (red), FtsZp3acpp (green), or Cirfloxacin (black) for 12 continuous passages.