Enhancement of Antimicrobial Function by L/D-Lysine Substitution on a Novel Broad-Spectrum Antimicrobial Peptide, Phylloseptin-TO2: A Structure-Related Activity Research Study

Abstract

Antibiotic resistance poses a serious threat to public health globally, reducing the effectiveness of conventional antibiotics in treating bacterial infections. ESKAPE pathogens are a group of highly transmissible bacteria that mainly contribute to the spread of antibiotic resistance and cause significant morbidity and mortality in humans. Phylloseptins, a class of antimicrobial peptides (AMPs) derived from Phyllomedusidae frogs, have been proven to have antimicrobial activity via membrane interaction. However, their relatively high cytotoxicity and low stability limit the clinical development of these AMPs. This project aims to study the antimicrobial activity and mechanisms of a phylloseptin-like peptide, phylloseptin-TO2 (PSTO2), following rational amino acid modification. Here, PSTO2 (FLSLIPHAISAVSALAKHL-NH₂), identified from the skin secretion of Phyllomedusa tomopterna, was used as the template for modification to enhance antimicrobial activity. Adding positive charges to PSTO2 through substitution with L-lysines enhanced the interaction of the peptides with cell membranes and improved their antimicrobial efficacy. The analogues SRD7 and SR2D10, which incorporated D-lysines, demonstrated significant antimicrobial effects against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) while also showing reduced haemolytic activity and cytotoxicity, resulting in a higher therapeutic index. Additionally, SRD7, modified with D-lysines, exhibited notable anti-proliferative properties against human lung cancer cell lines, including H838 and H460. This study thus provides a potential development model for new antibacterial and anti-cancer drugs combating antibiotic resistance.

Keywords: D-lysine substitution; anti-cancer activity; antimicrobial peptide; membrane interaction; phylloseptin.

Figure 1

Nucleotide sequence of cDNA encoding precursor and corresponding amino acid residues of PSTO2. The signal peptide is labelled with double underlines, the amino acid composition of PSTO2 is marked with a bold line, and the termination codon is indicated by an asterisk.

Figure 2

Alignment of the peptide encoding precursor of PSTO2 and two highly similar mature peptides (phylloseptin-B2 and phylloseptin-PT) sequences of the phylloseptin family. The signal peptide sequences are highlighted in the blue box, the acid spacer regions are highlighted in the green box, and the mature peptides are highlighted in the red box. The conserved motif ‘FLSLIP’ in phylloseptins is highlighted in yellow. The asterisks (*) represent identical amino acid sequences, the colons (:) indicate conservation between groups with very similar properties, and the full stops indicate conservation between groups with weakly similar properties.

Figure 3

Predicted helical wheels of PSTO2 and its analogues (a–i). Each arrow indicates the hydrophobic face of the peptide. Different colours represent the various properties of the amino acids (yellow: hydrophobic residues; grey: non-polar residue; other colours: corresponding amino acids).

Figure 4

CD spectra of PSTO2 and its analogues in (a) 50% TFE/NH₄Ac solution and (b) 10 mM of NH₄Ac. CD spectra data are displayed as measured ellipticity in mdeg units.

Figure 5

The kinetic time–killing curves of PSTO2 and its analogues against MRSA and E. coli at a concentration of 1/2× MIC. Bacteria treated with only a culture medium were used as the negative control. The error bar indicates the SEM of the nine replicates from three repeated tests.

Figure 6

The permeability of bacterial membranes affected by PSTO2 and its selected analogues against (a) S. aureus and (b) MRSA. For the positive control, 16 μM of melittin was used. The error bar indicates the SEM of the nine replicates from three individual tests.

Figure 7

The outer membrane permeability ratio of PSTO2 and its analogues against E. coli (ATCC 8739). Bacteria incubated with 16 μM of melittin were used as the positive control. The error bar indicates the SEM of the nine replicates from three repeated tests.

Figure 8

LPS-binding capacity of PSTO2 and its four analogues at different concentrations. Melittin was used as the positive control. The error bar indicates the SEM of the nine replicates from three individual tests.

Figure 9

The survival rates of MRSA-infected Galleria mellonella larvae after treatment with PSTO2 (a), SRD7 (b), and SR2D10 (c). The infected larvae treated with corresponding vancomycin concentrations were set up as the positive control. The infected larvae treated with PBS were regarded as the negative control.

Figure 9

The survival rates of MRSA-infected Galleria mellonella larvae after treatment with PSTO2 (a), SRD7 (b), and SR2D10 (c). The infected larvae treated with corresponding vancomycin concentrations were set up as the positive control. The infected larvae treated with PBS were regarded as the negative control.

Figure 10

The cytotoxicity of different concentrations of PSTO2 and its analogues expressed by trypan blue targeting H838 human lung cancer cells. Each peptide was prepared separately for 5 μM, 10 μM, and 25 μM for 2, 6, and 24 h. The extent of cytotoxicity was indicated by the ratio of dead cells and total cells. The error bar indicates the SEM of the nine replicates from three individual tests.

Figure 11

The haemolytic activities of PSTO2 and its analogues at concentrations from 1 to 128 μM. For the positive control, 1% triton X-100 was used, and the haemolytic percentage was calculated based on this. Treatment with PBS was used as the negative control. The error bar indicates the SEM of the nine replicates from three individual tests.

Figure 12

The mode of action of PSTO2 and lysine-substituted peptide with bacteria cell membrane.