Endolysins and membrane-active peptides: innovative engineering strategies against gram-negative bacteria

Abstract

Endolysins, bacteriophage-encoded peptidoglycan hydrolases, offer promising potential in antibacterial therapy, including treatments targeting gram-negative bacteria. While these enzymes naturally act primarily on gram-positive bacteria, their application against gram-negative pathogens is more challenging due to the presence of a dual-layer cell membrane, which acts as a protective barrier. However, innovative approaches, such as fusing endolysins with antimicrobial peptides (AMPs), have demonstrated increased efficacy against gram-negative bacteria. Modifying endolysins by introducing hydrophobic properties or positive charges or combining them with agents that disrupt the outer membrane enhances their bactericidal activity. Moreover, phage endolysins that exhibit activity against gram-negative bacteria are a promising source of membrane-active peptides. Identifying new peptide sequences derived from endolysins capable of penetrating the bacterial cell membrane represents a novel and increasingly explored research direction. Studying these innovative strategies had yielded promising results, though the field remains under active investigation and development. Ongoing efforts aim to optimize these approaches to improve their effectiveness against antibiotic-resistant gram-negative bacterial strains, which are particularly difficult to treat with conventional antibiotics. This review summarizes the latest advancements and solutions in the field, highlighting the potential of endolysins and membrane-active peptides as next-generation antibacterial agents.

Keywords: antibiotics; antimicrobial agents; antimicrobial peptides; bacteriophage; endolysins; gram-negative bacteria; peptides.

Figure 1

Schematic representation of the gram-negative cell wall and cleavage sites of bacteriophage endolysins in the peptidoglycan layer: MurNac, N-acetylmuramic acid; GlcNac, N-acetylglucosamine; m-DAP, diamino acid; L-Ala, L-alanine; D-Glu, D-glutamic acid; L-Lys; L-lysine; D-Ala, D-alanine; LPS, lipopolysaccharides.

Figure 2

(A) Three-dimensional structures of the LysT84 (PDB code: 7RUM, Love et al., 2022) and PlyC (PDB code: 4F88, McGowan et al., 2012) endolysins. Enzymatically active domains are shown in green, while cell wall binding domains are depicted in blue. (B) Representation of the cell wall binding surface of PlyC endolysin, divided into eight domains. The proteins are displayed in a cartoon representation, using VMD (Humphrey et al., 1996).

Figure 3

Endolysins derived from bacteriophages active against gram-negative bacteria are classified into two groups. An example of an endolysin belonging to the first group is PHAb11, which contains a single EAD domain with lysozyme activity (PDB code: 9KBS, Hu, 2024). An example of an endolysin capable of crossing bacterial membranes is PlyF307 (its structure was predicted using the I-TASSER program) (Yang and Zhang, 2015). The helical fragment isolated as an independent antibacterial peptide is marked in red.

Figure 4

Schematic representation of selected outer membrane permeabilizers used to enhance the efficacy of endolysins against gram-negative bacteria.

Figure 5

Structures of representative peptides fused with endolysins to enhance their antibacterial activity against gram-negative bacteria. The models of cecropin A (fragment 1-8) and SMAP-29 peptides were visualized based on the solution NMR structure with PDB codes: 1DYJ (Oh et al., 1999) and 1FRY (Tack et al., 2002), respectively. Peptides form helices marked as green ribbon, with the Lys side-chains shown in blue, Trp in red, Leu in gray, Phe in orange, Ile in yellow, Ala in pink, Arg in cyan, Gly in purple, His in lime, Pro in ochre, Thr in iceblue, Tyr in black, and Val in white. The figures were prepared with VMD (Humphrey et al., 1996).

Figure 6

Schematic representation of an example of endolysin fusion with a peptide: LNT113 (Cho et al., 2024). The artilysin is composed of the Cecropin A peptide fused to the mtEC340 endolysin. The model of the mtEC340 endolysin was visualized based on the X-ray crystallography structure, PDB code: 8HP8 (Wang et al., 2023). The figure was prepared using VMD software (Humphrey et al., 1996).