Antimicrobial activity, membrane interaction and structural features of short arginine-rich antimicrobial peptides

Abstract

Antimicrobial activity of many AMPs can be improved by lysine-to-arginine substitution due to a more favourable interaction of arginine guanidinium moiety with bacterial membranes. In a previous work, the structural and functional characterization of an amphipathic antimicrobial peptide named RiLK1, including lysine and arginine as the positively charged amino acids in its sequence, was reported. Specifically, RiLK1 retained its β-sheet structure under a wide range of environmental conditions (temperature, pH, and ionic strength), and exhibited bactericidal activity against Gram-positive and Gram-negative bacteria and fungal pathogens with no evidence of toxicity on mammalian cells. To further elucidate the influence of a lysine-to-arginine replacement on RiLK1 conformational properties, antimicrobial activity and peptide-liposome interaction, a new RiLK1-derivative, named RiLK3, in which the lysine is replaced with an arginine residue, was projected and characterised in comparison with its parental compound. The results evidenced that lysine-to-arginine mutation not only did not assure an improvement in the antimicrobial potency of RiLK1 in terms of bactericidal, virucidal and fungicidal activities, but rather it was completely abolished against the hepatitis A virus. Therefore, RiLK1 exhibited a wide range of antimicrobial activity like other cationic peptides, although the exact mechanisms of action are not completely understood. Moreover, tryptophan fluorescence measurements confirmed that RiLK3 bound to negatively charged lipid vesicles with an affinity lower than that of RiLK1, although no substantial differences from the structural and self-assembled point of view were evidenced. Therefore, our findings imply that antimicrobial efficacy and selectivity are affected by several complex and interrelated factors related to substitution of lysine with arginine, such as their relative proportion and position. In this context, this study could provide a better rationalisation for the optimization of antimicrobial peptide sequences, paving the way for the development of novel AMPs with broad applications.

Keywords: antimicrobial compound; arginine; cationic arginine-rich peptide; membrane interaction; spectroscopy.

Figure 1

Antifungal activity of RiLK3 against two pathogenic fungi. (A) Aspergillus brasiliensis and (B) Candida albicans. CTRL: each tested pathogen without peptide treatment. The fungal cultures (1×10⁵ CFU/mL) treated or not with different peptide concentrations (25 and 50 μM) for 6 h at 37°C, were seeded on DG18 plates. The photographs are representative of three independent experiments performed in triplicate.

Figure 2

Effect of SDS concentration on the secondary structure of RiLK3 and RiLK1 monitored by circular dichroism. Far-UV CD spectra of (A) RiLK3 and (B) RiLK1. All spectra were recorded at a peptide concentration of 80 μM in 10 mM Tris-HCl. pH 7.0 and at 25°C in the absence (green lines) or presence of SDS at different concentrations.

Figure 3

Time-dependent effect of SDS on the secondary and tertiary structure of RiLK3 and RiLK1 monitored by spectroscopic techniques. Far-UV circular dichroism spectra of (A) RiLK3 and (B) RiLK1. Intrinsic fluorescence emission spectra of (C) RiLK3 and (D) RiLK1. All spectra were recorded at a peptide concentration of 80 μM in 10 mM Tris-HCl. pH 7.0 in the presence or absence (blue lines) of SDS (50 mM) during 24 h incubation at 25°C.

Figure 4

Analysis of RiLK3 and RiLK1 oligomerization in SDS micelles by electrophoresis. Tris-tricine SDS-PAGE (17%) of RiLK1 or RiLK3 at 240 μM concentration treated or not with glutaraldehyde (4% concentration) in presence of SDS (150 mM) for 24 h at 37°C. MW: molecular weight references; lane 1: RiLK1; lane 2: RiLK1 + SDS (150 mM); lane 3: RiLK1 + SDS + glutaraldehyde; lane 4: RiLK3; lane 5: RiLK3 + SDS; lane 6: RiLK3 + SDS + glutaraldehyde. A 10 μL amount of each sample was loaded onto the gel.

Figure 5

Gel filtration chromatography of the crosslinked products of RiLK3 and RiLK1 in presence of SDS micelles. Size exclusion chromatography was performed on the Superdex 30 Increase column, pre-equilibrated with 50 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl and 20% acetonitrile. (A) RiLK1 (240 μM) incubated at 37°C for 24 h with SDS (150 mM) in presence of glutaraldehyde (4%). (B) RiLK3 (240 μM) incubated at 37°C for 24 h with SDS (150 mM) in presence of glutaraldehyde (4%). (C) Glutaraldehyde (4%) incubated at 37°C for 24 h. After incubation, all the samples were analysed by gel filtration. The dashed lines represent the peptide solutions (240 μM) incubated at 37°C for 24 h without SDS and glutaraldehyde. The reported chromatograms are representative of three independent experiments.

Figure 6

FTIR absorption spectra of RILK1 and RILK3 in powder form. Main peaks are underlined. Into the boxes, the amide I bands of RILK1 and RILK3, respectively with their deconvolution data analysis, were reported.

Figure 7

Trp fluorescence analysis of binding of RiLK1 to MLVs. Binding isotherms calculated from Trp fluorescence intensity at 335 nm of RiLK1 (1 μM) with model membrane vesicles (30 μM) in HEPES buffer with NaCl 150 mM. Scatchard plot analysis for the binding data of RiLK1. Data are presented as means ± s.d. of different samples analysed in quadruplicate. n.d. the equation could not fit the data, not possible to determine.

Figure 8

Trp fluorescence analysis of binding of RiLK3 to MLVs. Binding isotherms calculated from Trp fluorescence intensity at 335 nm of RiLK3 (1 μM) with model membrane vesicles (30 μM) in HEPES buffer with NaCl 150 mM. Scatchard plot analysis for the binding data of RiLK3. Data are presented as means ± s.d of different samples analysed in quadruplicate. n.d. the equation could not fit the data, not possible to determine.

Figure 9

Dynamic light scattering and ζ-potential analysis of bacterial membrane liposomes. (A) Hydrodynamic size distribution (d) of Salmonella-like liposomes (0.2 mM). The mean d value, and the PDI are reported (n = 3). (B) Hydrodynamic size distribution of Staphylococcus-like liposomes (0.2 mM). Two main populations, whose mean sizes are denoted as d₁ and d₂, are identified as wells as the PDI is reported (n = 3). (C) Schematic representation of the interaction between liposomes (Salmonella and Staphylococcus) and peptides (RiLK1 and RiLK3). (D) ζ-potential histograms of Salmonella liposomes (blue), interacting with RiLK1 and RiLK3 peptides in liposome:peptide ratio of 20:1 (red) and 2:1 (green). ζ-potential of the peptides alone are also reported (yellow). (E) ζ-potential histograms of Staphylococcus liposomes (blue), interacting with RiLK1 and RiLK3 peptides in liposome:peptide ratio of 20:1 (red) and 2:1 (green). ζ-potential of the peptides alone are also reported (yellow). ^*Significant differences (p < 0.05) between the bacterial-like liposomes alone (light blue) and the bacterial-like liposomes in the presence of the peptides (red or green).