D-enantiomeric β-casein 197 disrupts biofilm-associated infection of MDR Pseudomonas aeruginosa and Acinetobacter baumannii through superior LPS binding and host cell protection
- 1Shaanxi Institute for Pediatric Diseases, Xi’an Children’s Hospital, Affiliated Children’s Hospital of Xi’an Jiaotong University, Xi’an, China
- 2Department of Clinical Laboratory, Xi’an Children’s Hospital, Affiliated Children’s Hospital of Xi’an Jiaotong University, Xi’an, China
- 3School of Electrical and Information Engineering, Jiangsu University of Technology, Changzhou, China
Abstract
Biofilms formed by multidrug-resistant (MDR) Pseudomonas aeruginosa and Acinetobacter baumannii are a major cause of chronic nosocomial infections and therapeutic failure. L-form antimicrobial peptides (AMPs) are rapidly degraded by proteases, whereas D-enantiomeric peptides offer enhanced resistance to proteases. Here, we synthesized L- and D-β-casein 197, a milk-derived AMP, and systematically compared their antibiofilm activities against eight clinical isolates of P. aeruginosa and A. baumannii. The D-form reduced biofilm biomass by up to 83% during formation and by up to 88% in preformed biofilms. Mechanistic analyses revealed that the D-form caused more extensive membrane disruption, as demonstrated by flow cytometry and transmission electron microscopy. Isothermal titration calorimetry showed that the D-form bound LPS with higher affinity (KD 0.8 μM vs 3.1 μM), and molecular docking identified additional hydrogen bonds with lipid A. The D-form also more effectively inhibited bacterial motility. Both peptides exhibited low cytotoxicity toward cells (>90% viability at 100 μM), and the D-form reduced bacteria-induced cytotoxicity and bacterial internalization in infected macrophages more effectively than the L-form. In a Galleria mellonella infection model, D-β-casein 197 significantly improved survival against P. aeruginosa no. R1 and A. baumannii no. 13 challenge over 96 h compared to the L-form and untreated infected controls, while larval survival remained comparable to PBS controls, indicating negligible host toxicity. These findings identify D-β-casein 197 as a potent antibiofilm peptide against MDR Gram-negative biofilms, with enhanced activity, and LPS-neutralization assays confirmed that its antimicrobial potency is, at least in part, attributable to its stronger LPS binding affinity.
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BibTeX
@article{jiang2026denantiomeric,
title = {{D-enantiomeric β-casein 197 disrupts biofilm-associated infection of MDR Pseudomonas aeruginosa and Acinetobacter baumannii through superior LPS binding and host cell protection}},
author = {Kaichong Jiang and Wei Wang and Hong Lei and Tianhao Wang and Lei Xu and Ying Yang and Rui Zhou and Tianwei Lin and Yinong Huang},
journal = {Virulence},
year = {2026},
month = oct,
doi = {10.1080/21505594.2026.2749928},
url = {https://doi.org/10.1080/21505594.2026.2749928},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - D-enantiomeric β-casein 197 disrupts biofilm-associated infection of MDR Pseudomonas aeruginosa and Acinetobacter baumannii through superior LPS binding and host cell protection
AU - Kaichong Jiang
AU - Wei Wang
AU - Hong Lei
AU - Tianhao Wang
AU - Lei Xu
AU - Ying Yang
AU - Rui Zhou
AU - Tianwei Lin
AU - Yinong Huang
PY - 2026
DA - 2026/10/22
JO - Virulence
DO - 10.1080/21505594.2026.2749928
UR - https://doi.org/10.1080/21505594.2026.2749928
AB - Biofilms formed by multidrug-resistant (MDR) Pseudomonas aeruginosa and Acinetobacter baumannii are a major cause of chronic nosocomial infections and therapeutic failure. L-form antimicrobial peptides (AMPs) are rapidly degraded by proteases, whereas D-enantiomeric peptides offer enhanced resistance to proteases. Here, we synthesized L- and D-β-casein 197, a milk-derived AMP, and systematically compared their antibiofilm activities against eight clinical isolates of P. aeruginosa and A. baumannii. The D-form reduced biofilm biomass by up to 83% during formation and by up to 88% in preformed biofilms. Mechanistic analyses revealed that the D-form caused more extensive membrane disruption, as demonstrated by flow cytometry and transmission electron microscopy. Isothermal titration calorimetry showed that the D-form bound LPS with higher affinity (KD 0.8 μM vs 3.1 μM), and molecular docking identified additional hydrogen bonds with lipid A. The D-form also more effectively inhibited bacterial motility. Both peptides exhibited low cytotoxicity toward cells (>90% viability at 100 μM), and the D-form reduced bacteria-induced cytotoxicity and bacterial internalization in infected macrophages more effectively than the L-form. In a Galleria mellonella infection model, D-β-casein 197 significantly improved survival against P. aeruginosa no. R1 and A. baumannii no. 13 challenge over 96 h compared to the L-form and untreated infected controls, while larval survival remained comparable to PBS controls, indicating negligible host toxicity. These findings identify D-β-casein 197 as a potent antibiofilm peptide against MDR Gram-negative biofilms, with enhanced activity, and LPS-neutralization assays confirmed that its antimicrobial potency is, at least in part, attributable to its stronger LPS binding affinity.
SN - 2150-5594
SN - 2150-5608
ER -