Bioactivity and Bactericidal Mechanism of Histidine-Rich β-Hairpin Peptide Against Gram-Negative Bacteria

Abstract

Antibacterial peptides (APMs) are a new type of antibacterial substance. The relationship between their structure and function remains indistinct; in particular, there is a lack of a definitive and fixed template for designing new antimicrobial peptides. Previous studies have shown that porcine Protegrin-1 (PG-1) exhibits considerable antimicrobial activity and cytotoxicity. In this study, to reduce cytotoxicity and increase cell selectivity, we designed histidine-rich peptides based on the sequence template RR(XY)2XDPGX(YX)2RR-NH2, where X represents I, W, V, and F. The results showed that the peptides form more β-hairpin structures in a lipid-rich environment that mimics cell membranes. Among them, the antimicrobial peptide HV2 showed strong antibacterial activity against Gram-negative strains and almost no toxicity to normal cells. The results of our analysis of its antibacterial mechanism showed that peptide HV2 acts on the bacterial cell membrane to increase its permeability, resulting in cell membrane disruption and death. Furthermore, peptide HV2 inhibited bacterial movement in a concentration-dependent manner and had a more robust anti-inflammatory effect by inhibiting the production of TNF-α. In summary, peptide HV2 exhibits high bactericidal activity and cell selectivity, making it a promising candidate for future use as an antibiotic.

High Cell Selectivity and Bactericidal Mechanism of Symmetric Peptides Centered on d-Pro–Gly Pairs

“…The results of MICs indicated that the designed peptides had good antibacterial activity toward Gram-negative bacteria ( Table 2). Compared to the single membrane and thick peptidoglycan layer of Gram-positive bacteria, the double-membrane structure of Gram-negative bacteria represents a weaker barrier; thus, Gram-negative bacteria are more sensitive to AMPs [17,21,24]. The antibacterial activity of AMPs can be affected by many factors.…”

“…Previous studies demonstrated that cationic AMPs combine with anions on the surface of bacteria via electrostatic action, and their hydrophobic amino acids are inserted into the bacterial cell membrane. Once the critical concentration is exceeded, the membrane breaks, and this ultimately leads to death of the bacteria [21,22]. LR pG firstly bound to the LPS of the outer membrane of E. coli ATCC25922 through electrostatic action (Figure 8), and then destroyed the cell membrane.…”

“…Since the outer membrane of the cell is negatively charged at physiological pH, the permeability of the cationic AMPs to the outer membrane is increased [21]. The permeability of peptides to the Gram-negative bacterial outer membrane can be reflected by N-phenyl-1-naphthylamine (NPN) uptake [15,17,21]. NPN is normally excluded from the outer membrane; however, once the outer membrane is permeabilized, NPN is taken up intracellularly and shows increased fluorescence.…”

“…The outer membrane permeability was examined using fluorescent dye NPN according to the method mentioned previously [17,21,41]. Briefly, E. coli ATCC25922 cells were washed three times and diluted to 10 5 CFU/mL in 5 mM HEPES buffer (pH 7.4, containing 5 mM glucose).…”

“…The positive charge and hydrophobicity were consistent with LR pG . Finally, by amidating the C-terminus of the peptides, the charge of the AMPs was increased, and the stability of the structure was improved [21][22][23]. Using this method of designing and optimizing AMPs, we obtained LR pG (X: L, zz: pG) with high cell selectivity and good conditional stability.…”