Engineering a multifunctional LL37-TP5 hybrid peptide with potent antimicrobial endotoxin-neutralizing and anti-inflammatory properties

The increasing prevalence of antimicrobial resistance and endotoxin-mediated inflammation highlights the need for alternative therapeutic strategies that combine antibacterial and immunomodulatory functions. Although cathelicidin LL-37 exhibits potent antimicrobial and lipopolysaccharide (LPS)-neutralizing activity, its clinical application is limited by cytotoxicity and high production costs. A hybrid peptide, LL37-TP5, was designed by fusing the active LPS-binding region of LL-37 (residues 13–36) with the immunomodulatory penta peptide thymopentin (TP5) to enhance endotoxin-neutralizing activity while reducing host cytotoxicity. Bioinformatics analyses predicted that LL37-TP5 adopt amphipathic α-helical structure with optimized hydrophobicity and a strong net positive (+9) charge. The hybrid peptide was successfully expressed recombinantly in Pichia pastoris (P. pastoris) and purified by Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC), yielding approximately 20.5 mg/L with 95.2% purity, consistent with in silico predictions. Considering the increasing prevalence of antimicrobial resistance in poultry production, the antibacterial activity of LL37-TP5 was evaluated against major poultry-associated bacterial pathogens. LL37-TP5 exhibited broad-spectrum antibacterial activity against Gram-negative and Gram-positive bacteria, with low minimum inhibitory and bactericidal concentrations. Furthermore, the hybrid peptide showed enhanced LPS-neutralizing activity and significantly suppressed nitric oxide and pro-inflammatory cytokine (IL-6, TNF-α, and IL-1β) production in LPS-stimulated RAW264.7 macrophages. Importantly, LL37-TP5 demonstrated markedly reduced cytotoxicity and negligible hemolytic activity compared with parental LL-37. Collectively, our findings indicate that hybridization of LL-37 with TP5 generated a peptide with favorable antimicrobial, endotoxin-neutralizing, and anti-inflammatory properties together with an improved safety profile compared with native LL-37.