Strand Length-Dependent Antimicrobial Activity and Membrane-Active Mechanism of Arginine- and Valine-Rich β-Hairpin-Like Antimicrobial Peptides

Abstract

bAntimicrobial peptides with amphipathic ␤-hairpin-like structures have potent antimicrobial properties and low cytotoxicity. The effect of VR or RV motifs on ␤-hairpin-like antimicrobial peptides has not been investigated. In this study, a series of ␤-hairpin-like peptides, Ac-C(VR) n D PG (RV) n C-NH 2 (n ‫؍‬ 1, 2, 3, 4, or 5), were synthesized, and the effect of chain length on antimicrobial activity was evaluated. The antimicrobial activity of the peptides initially increased and then decreased with chain length. Longer peptides stimulated the toxicity to mammalian cells. VR3, a 16-mer peptide with seven amino acids in the strand, displayed the highest therapeutic index and represents the optimal chain length. VR3 reduced bacterial counts in the mouse peritoneum and increased the survival rate of mice at 7 days after Salmonella enterica serovar Typhimurium infection in vivo. The circular dichroism (CD) spectra demonstrated that the secondary structure of the peptides was a ␤-hairpin or ␤-sheet in the presence of an aqueous and membrane-mimicking environment. VR3 had the same degree of penetration into the outer and inner membranes as melittin. Experiments simulating the membrane environment showed that Trp-containing VRW3 (a VR3 analog) tends to interact preferentially with negatively charged vesicles in comparison to zwitterionic vesicles, which supports the biological activity data. Additionally, VR3 resulted in greater membrane damage than melittin as determined using a flow cytometry-based membrane integrity assay. Collectively, the data for synthetic lipid vesicles and whole bacteria demonstrated that the VR3 peptide killed bacteria via targeting the cell membrane. This assay could be an effective pathway to screen novel candidates for antibiotic development.

Antimicrobial activity and membrane-active mechanism of tryptophan zipper-like β-hairpin antimicrobial peptides

“…Based on these lines of evidence, the relationship between peptide length and antimicrobial potency is not linear, and there seems to be a threshold effect. As observed in this study, the antibacterial activity of the trpzip peptides initially increased and then decreased with increasing strand length, which was in agreement with our previous studies on β-sheet peptides (Dong et al 2012). The observed increase in antimicrobial activity with chain length may be attributable to increases in the hydrophobicity and cationicity (Table 1), which are features required for killing bacteria.…”

“…The Trp and Lys residues were located at every other position in the strands, resulting in all hydrophobic residues falling on the same face in the folded hairpin, while positively charged residues were located on the opposite face, forming an amphipathic structure. The two strands were connected by a rigid D Pro-Gly turn sequence that stabilizes the type II β-turn and promotes the formation of β-hairpin conformations (Dong et al 2012;Hilario et al 2003). Finally, the peptides were amidated at the C-terminal to improve peptide stabilization and increase net positive charge for enhanced antimicrobial activities (Nguyen et al 2010).…”

“…Considerable efforts have been devoted to the design and optimization of α-helical peptide sequences that possess enhanced antimicrobial activities coupled with low toxicities to host cells; however, systematic studies on β-sheet-forming peptides are acutely lacking in comparison, largely because it is difficult for short peptides to form robust β-structures (Dong et al 2006;Ong et al 2013). In particular, most existing β-sheet AMPs require one or more internal disulfide bridges as conformational constraints to stabilize their bioactive conformations, which dramatically increases the cost for large-scale manufacturing of the peptides (Dong et al 2012). Furthermore, disulfide bridges have disadvantages as structural stabilizers of bioactive peptides because they can be cleaved in vivo by reaction with free thiol groups, leading to the inevitable loss of fold stability and biological activity (Obrecht et al 2012).…”

Strategies employed in the design and optimization of synthetic antimicrobial peptide amphiphiles with enhanced therapeutic potentials

“…Other studies have also found that the appropriate positioning of Trp residue(s) at the hydrophobic-hydrophilic interface of the helical wheel projection could provide an effective strategy to enhance helical stability and also increase the partitioning or penetration of designed AMPs into microbial membrane lipid bilayers with minimal effects on mammalian cell membranes or their mimics at concentrations required for antimicrobial activities [56,58,59]. Although α-helical AMPs represent the most widely investigated class of AMPs, other types of secondary structures such as β-sheets [43,60] and β-hairpin-like AMPs [61,62] have also been designed and evaluated. For instance, our group has recently designed short synthetic linear β-sheet forming peptide amphiphiles comprised of short recurring (X 1 Y 1 X 2 Y 2 ) n -NH 2 sequences, where X 1 and X 2 : hydrophobic residues (Val, Ile, Phe or Trp), Y 1 and Y 2 : cationic residues (Arg or Lys), and n: number of repeat units based on the common occurrence of amphipathic dyads in membrane spanning β-sheets [43,60].…”

“…In another recent example, evaluation of a series of cyclic β-hairpin-like AMPs with a general sequence of Ac-C(VR) n D PG(RV) n C-NH 2 (n = 1-5), which comprise of two antiparallel Val-and Arg-containing β-sheet strands connected by a type II′ β-turn and further stabilized by a disulfide linkage between the N-and C-terminal Cys residues revealed that a 16-mer peptide with the sequence Ac-C(VR) 3 D PG(RV) 3 C-NH 2 (VR3) had the highest selectivity index and that the intraperitoneal (i.p.) administration of 1.25 to 5 mg/kg of VR3 increased the 7 day survival rate of mice intraperitoneally infected with Salmonella enteric serovar Typhimurium [62].…”