Antibacterial Peptide BSN-37 Kills Extra- and Intra-Cellular Salmonella enterica Serovar Typhimurium by a Nonlytic Mode of Action

Abstract

The increasing rates of resistance to traditional anti-Salmonella agents have made the treatment of invasive salmonellosis more problematic, which necessitates the search for new antimicrobial compounds. In this study, the action mode of BSN-37, a novel antibacterial peptide (AMP) from bovine spleen neutrophils, was investigated against Salmonella enterica serovar Typhimurium (S. Typhimurium). Minimum inhibitory concentrations (MICs) and time-kill kinetics of BSN-37 were determined. The cell membrane changes of S. Typhimurium CVCC541 (ST) treated with BSN-37 were investigated by testing the fluorescence intensity of membrane probes and the release of cytoplasmic β-galactosidase activity. Likewise, cell morphological and ultrastructural changes were also observed using scanning and transmission electron microscopes. Furthermore, the cytotoxicity of BSN-37 was detected by a CCK-8 kit and real-time cell assay. The proliferation inhibition of BSN-37 against intracellular S. Typhimurium was performed in Madin-Darby canine kidney (MDCK) cells. The results demonstrated that BSN-37 exhibited strong antibacterial activity against ST (MICs, 16.67 μg/ml), which was not remarkably affected by the serum salts at a physiological concentration. However, the presence of CaCl₂ led to an increase in MIC of BSN-37 by about 4-fold compared to that of ST. BSN-37 at the concentration of 100 μg/ml could completely kill ST after co-incubation for 6 h. Likewise, BSN-37 at different concentrations (50, 100, and 200 μg/ml) could increase the outer membrane permeability of ST but not impair its inner membrane integrity. Moreover, no broken and ruptured cells were found in the figures of scanning and transmission electron microscopes. These results demonstrate that BSN-37 exerts its antibacterial activity against S. Typhimurium by a non-lytic mode of action. Importantly, BSN-37 had no toxicity to the tested eukaryotic cells, even at a concentration of 800 μg/ml. BSN-37 could significantly inhibit the proliferation of intracellular S. Typhimurium.

Keywords: Salmonella enterica serovar Typhimurium; action mode; antibacterial activity; antibacterial peptide BSN-37; cytotoxicity; eukaryotic cells.

FIGURE 1

Construction of recombined plasmid pKP302mdtK:lacZ PmdtK was amplified from mdtK locating in the chromosome of S. Typhimurium and purified. The products of PmdtK digested by EcoRI and BamHI were ligated to the fragments of plasmid pKP302 digested with the same enzymes. The recombined plasmids were transformed into DH5α and then, identified by PCR. lacZ, a gene encoding β-galactosidase; aadA, a gene encoding resistance to streptomycin and spectinomycin; PmdtK, the promoter region of mdtK.

FIGURE 2

Time-dependent killing curves of ST incubated with BSN-37 at different concentrations of BSN-37 (0, 50, 100, and 200 μg/ml) in MH broth medium for 24 h. Aliquots were collected at 0, 1, 2, 4, 6, 8, and 24 h to count the bacteria. Error bars represent means ± SD. Assays were performed in triplicate.

FIGURE 3

The effect of BSN-37 at different concentrations (50, 100, and 200 μg/ml) and pexiganan at the concentration of 100 μg/ml on the outer membrane permeabilization and inner membrane integrity of ST. (A) Time–response curve of the outer membrane permeation of ST cells to NPN. (B) Time–response curve of the inner membrane Integrity of ST cells to DiS-C₃-(5) (C) The β-galactosidase activity in the culture medium of STpKP302mdtk:lacZ cells treated with BSN-37 and permeabilization mixture. STpKP302mdtk:lacZ cells treated with permeabilization mixture containing Z-buffer, chloroform and sodium dodecyl sulfate and PBS buffer was used as positive and negative control, respectively.

FIGURE 4

Scanning electron microscropy images of ST treated with BSN-37 and PG for 1 h. (A) ST treated with 50 μg/ml BSN-37; (B) ST treated with 100 μg/ml BSN-37; (C) ST treated with 200 μg/ml BSN-37; (D) ST treated with 10 mM PBS buffer; (E) ST treated with 50 μg/ml pexiganan.

FIGURE 5

Transmission electron microscropy images of ST treated with BSN-37 and PG for 1 h. (A) ST treated with 10 mM PBS buffer; (B) ST treated with 200 μg/ml BSN-37; (C) ST treated with 50 μg/ml pexiganan.

FIGURE 6

The survival rates of IPEC-J2 cells treated with BSN-37 and PG at a different concentrations by CCK-8 assay. Error bar represents mean ± SEM, n = 3. **** represent extremely significance (P < 0.0001 by two-way ANOVA).

FIGURE 7

Real-time measurement of the cytotoxicity of BSN-37 and pexiganan against IPEC-J2 cells and Vero cells and the survival rates of IPEC-J2 cells and Vero cells incubated with 100 μg/ml BSN-37 and 100 μg/ml pexiganan for different times. (A,C) Real-time monitoring of the cytotoxicity of IPEC-J2 cells and Vero cells, respectively, treated with BSN-37 at a different concentrations (50, 100, 200, 400, and 800 μg/ml) and pexiganan at the concentration of 100 μg/ml for 60 h using the xCELLigence system. The black arrows indicate the time points of adding the peptides. The cells without antimicrobial peptide were used as control. (B,D) parallel comparisons of the survival rates of IPEC-J2 cells and Vero cells, respectively, treated with 100 μg/ml BSN-37 and 100 μg/ml pexiganan at specific four time points. Error bars represent means ± SEM, n = 3. *** and **** represents very significance and extremely significance, respectively. (p < 0.001 and p < 0.0001 by Two-way ANOVA).

FIGURE 8

Intracellular replication of ST and SB217 in the presence and absence of BSN-37 in MDCK cells. The concentration of BSN-37 is 400 μg/ml. Error bars represent means ± SEM, n = 3. Statistical significance was defined as follows:^∗P < 0.05 and **P < 0.01 (Student’s t-test).