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. 2017 Nov 4;22(11):1898.
doi: 10.3390/molecules22111898.

Antibacterial Activity of the Non-Cytotoxic Peptide (p-BthTX-I)₂ and Its Serum Degradation Product against Multidrug-Resistant Bacteria

Norival A Santos-Filho  1 , Rafaela S Fernandes  2 , Bruna F Sgardioli  3 , Matheus A S Ramos  4 , Julia P Piccoli  5 , Ilana L B C Camargo  6 , Tais M Bauab  7 , Eduardo M Cilli  8
Affiliations

Antibacterial Activity of the Non-Cytotoxic Peptide (p-BthTX-I)₂ and Its Serum Degradation Product against Multidrug-Resistant Bacteria

Norival A Santos-Filho et al. Molecules. .

Abstract

Antimicrobial peptides can be used systemically, however, their susceptibility to proteases is a major obstacle in peptide-based therapeutic development. In the present study, the serum stability of p-BthTX-I (KKYRYHLKPFCKK) and (p-BthTX-I)₂, a p-BthTX-I disulfide-linked dimer, were analyzed by mass spectrometry and analytical high-performance liquid chromatography (HPLC). Antimicrobial activities were assessed by determining their minimum inhibitory concentrations (MIC) using cation-adjusted Mueller-Hinton broth. Furthermore, biofilm eradication and time-kill kinetics were performed. Our results showed that p-BthTX-I and (p-BthTX-I)₂ were completely degraded after 25 min. Mass spectrometry showed that the primary degradation product was a peptide that had lost four lysine residues on its C-terminus region (des-Lys12/Lys13-(p-BthTX-I)₂), which was stable after 24 h of incubation. The antibacterial activities of the peptides p-BthTX-I, (p-BthTX-I)₂, and des-Lys12/Lys13-(p-BthTX-I)₂ were evaluated against a variety of bacteria, including multidrug-resistant strains. Des-Lys12/Lys13-(p-BthTX-I)₂ and (p-BthTX-I)₂ degraded Staphylococcus epidermidis biofilms. Additionally, both the peptides exhibited bactericidal activities against planktonic S. epidermidis in time-kill assays. The emergence of bacterial resistance to a variety of antibiotics used in clinics is the ultimate challenge for microbial infection control. Therefore, our results demonstrated that both peptides analyzed and the product of proteolysis obtained from (p-BthTX-I)₂ are promising prototypes as novel drugs to treat multidrug-resistant bacterial infections.

Keywords: (p-BthTX-I)2; antimicrobial peptides; biofilm; multidrug-resistant bacteria.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Serum stability assays by analytical high-performance liquid chromatography (HPLC). (A) p-BthTX-I and (B) (p-BthTX-I)2 were incubated with human serum for the indicated times. Analytical HPLC was performed on a Shimadzu (Kyoto, Japan) system using a C18 column (4.6 × 150 mm, Phenomenex) with a linear gradient of solvent B (5–95%) at 1 mL/min for 15 min (Solvent A: 0.045% trifluoroacetic acid (TFA) in water. Solvent B: 0.036% TFA in acetonitrile). Marked boxes in the figures show the same degradation product produced by both the peptides.
Figure 2
Figure 2
Serum stability analyses by mass spectrometry. Analyses of peptides and degradation products were performed after (A) 5 min, (B) 20 min, (C) 30 min, and (D) 120 min of incubation in human serum. Molecular ions: intact (p-BthTX-I)2, black; (p-BthTX-I)2 minus one Lys residue, blue; (p-BthTX-I)2 minus two Lys residues, red; (p-BthTX-I)2 minus three Lys residues, green; and (p-BthTX-I)2 without four Lys residues, purple.
Figure 3
Figure 3
Analytical HPLC comparing the peaks of the peptides (p-BthTX-I)2, des-Lys12/Lys13-(p-BthTX-I)2, and des-Lys1/Lys2-(p-BthTX-I)2 to the RT of peaks of the stable serum degradation product of (p-BthTX-I)2. Samples were eluted at 5 min and 3 h. Analytical HPLC was performed on a Shimadzu system using a C18 column (4.6 × 150 mm, Phenomenex) with a linear gradient of solvent B (5–95%) at 1 mL/min for 30 min (Solvent A: 0.045% trifluoroacetic acid (TFA) in water. Solvent B: 0.036% TFA in acetonitrile). Marked box indicates the peak of des-Lys12/Lys13-(p-BthTX-I)2 and the stable degradation peptide produced.
Figure 4
Figure 4
Biofilm eradication activities of (p-BthTX-I)2 and des-Lys12/Lys13-(p-BthTX-I)2. S. epidermidis ATCC 35984 growth observed after treatment with the peptides at 512 µM (** p < 0.01). The columns represent mean ± standard deviation (SD) of 16 replicates for each treatment.
Figure 5
Figure 5
Time-kill curves of S. epidermidis ATCC 35984 incubated in the presence of (p-BthTX-I)2 and des-Lys12/Lys13-(p-BthTX-I)2. The bacteria were cultured in cation-adjusted BBL Mueller–Hinton II broth (CAMHB) containing 1×, 2×, or 4× MIC concentrations of either (A) (p-BthTX-I)2 or (B) des-Lys12/Lys13-(p-BthTX-I)2 as indicated. All data points represent the mean of two independent experiments. CFU: Colony Forming Units.
Figure 5
Figure 5
Time-kill curves of S. epidermidis ATCC 35984 incubated in the presence of (p-BthTX-I)2 and des-Lys12/Lys13-(p-BthTX-I)2. The bacteria were cultured in cation-adjusted BBL Mueller–Hinton II broth (CAMHB) containing 1×, 2×, or 4× MIC concentrations of either (A) (p-BthTX-I)2 or (B) des-Lys12/Lys13-(p-BthTX-I)2 as indicated. All data points represent the mean of two independent experiments. CFU: Colony Forming Units.
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