A Novel β-Hairpin Peptide Z-d14CFR Enhances Multidrug-Resistant Bacterial Clearance in a Murine Model of Mastitis

Abstract

The widespread prevalence of antimicrobial resistance has spawned the development of novel antimicrobial agents. Antimicrobial peptides (AMPs) have gained comprehensive attention as one of the major alternatives to antibiotics. However, low antibacterial activity and high-cost production have limited the applications of natural AMPs. In this study, we successfully expressed recombinant Zophobas atratus (Z. atratus) defensin for the first time. In order to increase the antimicrobial activity of peptide, we designed 5 analogues derived from Z. atratus defensin, Z-d13, Z-d14C, Z-d14CF, Z-d14CR and Z-d14CFR. Our results showed that Z-d14CFR (RGCRCNSKSFCVCR-NH₂) exhibited a broad-spectrum antimicrobial activity to both Gram-positive bacteria and Gram-negative bacteria, including multidrug-resistant bacteria. It possessed less than 5% hemolysis and 10% cytotoxicity, even at a high concentration of 1 mg/mL. Antimicrobial mechanism studies indicated that Z-d14CFR performed antimicrobial effect via inhibiting biofilm formation, disrupting bacterial membrane integrity and inducing cellular contents release. Furthermore, Z-d14CFR showed a great therapeutic effect on the treatment of multidrug-resistant Escherichia coli (E. coli) infection by enhancing bacterial clearance, decreasing neutrophils infiltration and the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) in a murine model of mastitis. Our findings suggest that Z-d14CFR could be a promising candidate against multidrug-resistant bacteria.

Keywords: Zophobas atratus; antimicrobial peptide; defensin; mastitis; multidrug-resistant bacteria; β-hairpin.

Figure 1

Expression and verification of recombinant Z. atratus defensin. (A) Construction of pGEX 6P-ZA-defensin. The gene codes mature Z. atratus defensin was cloned into pGEX 6P-1 with BamH I and Not I to construct recombinant plasmid pGEX 6P-ZA-defensin. (B) SDS-PAGE of recombinant ZA-defensin (rZA-defensin) expression. M: marker (10–180 kDa); 1: expression product of rZA-defensin induced by IPTG; 2: purified rZA-defensin; 3: GST-tag cut from rZA-defensin. (C) Western blotting analysis of purified rZA-defensin. 1: GST-tag cut from rZA-defensin defensin; M: marker (10–180 kDa); 2: purified rZA-defensin. (D) Purification of rZA-defensin by Superdex 30 after cutting off GST-tag shows a sharp peak at the elution volume of 26 mL. (E) rZA-defensin exhibits favorable antibacterial activity against E. coli ATCC25922 and S. aureus ATCC29213.

Figure 2

Design and structural analysis of Z. atratus defensin analogues. (A) Sequence alignment between Z. atratus defensin analogues and Protegrin-1 (PG-1, PDB ID: 1PG1). Rose lines represent the β-sheet region of peptides. The conserved cysteines are shadowed in yellow, the mutated residues are colored in red, the black arrows indicate the disulfide bonds of peptides. (B) Structural model (ribbon diagram) of Z. atratus defensin, Z-d13 and Z-d14C in accordance with (A). The structure of Z. atratus defensin was predicted according to Sapecin (PDB ID: 1L4V) by SWISS-MODEL (http://swissmodel.expasy.org/) (Accessed: 20 March 2022). Z. atratus defensin consists of N-terminal random coil followed by a α-helical and antiparallel β-sheet. Z-d14C forms a new disulfide bond between Cys1–Cys4 after mutating Gly into Cys at the 3rd position. (C) Clustering of hydrophilic residues (green), hydrophobic residues (blue) and positive residues (rose red) in Z-d14C and Z-d14CFR. (D) Circular dichroism (CD) spectra of Z. atratus defensin analogues in 10 mM PBS (green line), 50% trifluoroethanol (TFE) (red line) and 30 mM SDS (blue line).

Figure 3

Hemolysis and cytotoxicity of rZA-defensin and its analogues. (A) Hemolysis of rZA-defensin defensin and its analogues. (B–D) Cytotoxicity of rZA-defensin defensin and its analogues to MAC-T (B), Hela (C), and Vero (D). The results are represented as mean ± SEM from three independent experiments.

Figure 4

Time-killing curves of Z-d14CFR. Bacterial count of E. coli ATCC25922 (A) and S. aureus ATCC29213 (B) in logarithmic phase incubated with 2 MIC Z-d14CFR, polymyxin B or gentamicin. Bacterial count of E. coli ATCC25922 (C) and S. aureus ATCC29213 (D) in plateau incubated with 2 MIC Z-d14CFR, polymyxin B or gentamicin. The results are represented as mean ± SEM from three independent experiments.

Figure 5

Morphological changes of bacteria treated with or without Z-d14CFR. Scanning electron microscopy (SEM) observation of E. coli ATCC25922 and S. aureus ATCC29213 treated with 10 mM PBS or 2 MIC Z-d14CFR for 3 h. E. coli ATCC25922 (A) and S. aureus ATCC29213 (B) treated with 10 mM PBS. E. coli ATCC25922 (C,E) and S. aureus ATCC29213 (D,F) treated with Z-d14CFR. The bacteria showed membrane breakage (red short arrows), cells shrinkage (red long arrows) and rupture (blue long arrows).

Figure 6

Antibacterial mechanism of Z-d14CFR. (A) Dynamic curves of NPN uptake in E. coli ATCC25922 after incubating with 2 MIC, 1 MIC, 1/2 MIC, 1/4 MIC Z-d14CFR, 10 mM PBS as the negative control. (B) Dynamic curves of DiSC₃(5) release in E. coli ATCC25922 and S. aureus ATCC29213 after incubating with 2 MIC, 1 MIC, 1/2 MIC, 1/4 MIC Z-d14CFR, 10 mM PBS as the negative control. (C) Extracellular release of β-galactosidase in E. coli ATCC25922 and S. aureus ATCC29213 induced by 2 MIC Z-d14CFR, 10 mM PBS as the negative control, ultrasonication as the positive control. (D) SYTO9/PI staining of E. coli ATCC25922 and S. aureus ATCC29213 treated with 10 mM PBS or 2 MIC Z-d14CFR for 3 h under laser confocal microscope. (E) Flow cytometry of PI-positive cells treated with 10 mM PBS or 2 MIC Z-d14CFR for 3 h. (F) Biofilm formation of E. coli CAU201919 and E. coli CAU201920 treated with 2 MIC, 1 MIC, 1/2 MIC, 1/4 MIC, 1/8 MIC and 1/16 MIC Z-d14CFR or 10 mM PBS. All the results are represented as mean ± SEM from three independent experiments, *** p < 0.001, unpaired t-test.

Figure 7

A murine model of multidrug-resistant E. coli-induced mastitis treated with or without Z-d14CFR. (A) Experimental protocol of murine mastitis model induced by multidrug-resistant E. coli CAU201919. (B) Histopathologic changes of the mammary gland. Long arrows indicate the thickening of mammary acinar walls, short arrows indicate neutrophils infiltration in acinar cavity. (C) Bacterial burden in the mammary gland of mice. (D) IL-1 β and TNF-α mRNA expression in the mammary gland of mice. The results are represented as mean ± SEM from three independent experiments, * p < 0.05, *** p < 0.001, unpaired t-test.

Figure 8

Possible mechanism of Z-d14CFR against E. coli and S. aureus. Z-d14CFR binds to bacterial membranes through electrostatic interactions, induces cytoplasmic membrane depolarization, inserts into the cell membranes and forms β-barrels, thereby disrupting the integrity of cell membranes, promoting the release of cellular contents and then killing the bacteria.