Engineering a multifunctional LL37-TP5 hybrid peptide with potent antimicrobial endotoxin-neutralizing and anti-inflammatory properties
Abstract
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.
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BibTeX
@article{ahmad2026engineering,
title = {{Engineering a multifunctional LL37-TP5 hybrid peptide with potent antimicrobial endotoxin-neutralizing and anti-inflammatory properties}},
author = {Baseer Ahmad and Kiran Khurshid and Wei Xubiao and Kashif Hussain and Muhammad Asif Raza and Haizhou Gong and Christina Hölzel and Si Dayong},
journal = {Frontiers in Pharmacology},
year = {2026},
month = nov,
doi = {10.3389/fphar.2026.1886263},
url = {https://www.frontiersin.org/articles/10.3389/fphar.2026.1886263},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Engineering a multifunctional LL37-TP5 hybrid peptide with potent antimicrobial endotoxin-neutralizing and anti-inflammatory properties
AU - Baseer Ahmad
AU - Kiran Khurshid
AU - Wei Xubiao
AU - Kashif Hussain
AU - Muhammad Asif Raza
AU - Haizhou Gong
AU - Christina Hölzel
AU - Si Dayong
PY - 2026
DA - 2026/11/04
JO - Frontiers in Pharmacology
DO - 10.3389/fphar.2026.1886263
UR - https://www.frontiersin.org/articles/10.3389/fphar.2026.1886263
AB - 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.
SN - 1663-9812
ER -