A novel truncated CHAP modular endolysin, CHAPSAP26-161, that lysesStaphylococcus aureus,Acinetobacter baumannii,andClostridioides difficile

Abstract

AbstractBackgroundDevelopment of novel antimicrobial agents is imperative due to the increasing threat of antibiotic-resistant pathogens. This study aimed to validate the enhanced antibacterial activity andin vivoefficacy of a novel truncated endolysin, CHAPSAP26-161, derived from the CHAP domain of LysSAP26, against multidrug-resistant bacteria.MethodsTwo deletion mutants, CHAPSAP26-139 and CHAPSAP26-161, were constructed by deleting the C-terminal portion of LysSAP26. These were cloned and expressed, and their antibacterial activities, together with protein purification efficiency, were evaluated against 12 bacterial species under various environmental conditions. To test the temperature and pH stability of the three recombinant proteins, the antibacterial effects of the proteins at various temperatures (4°C–60°C) and pH values (3–10) were measured. Time-kill assay measured the optical density (600 nm) and colony-forming units after incubation for 0, 2, 4, 6, 8, and 24 h. We verified this throughin vivoexperiments using mouse models to evaluate the therapeutic potential of CHAPSAP26-161 againstAcinetobacter baumannii.ResultsCHAPSAP26-161 exhibited higher protein purification efficiency and antibacterial activity than LysSAP26. Moreover, CHAPSAP26-161 showed the highest lytic activity againstA. baumanniiwith a minimal bactericidal concentration (MBC) of 5–10 µg/mL, followed byStaphylococcus aureuswith an MBC of 10–25 µg/mL. Interestingly, CHAPSAP26-161 could lyse anaerobic bacteria, such asC. difficile, with an MBC of 25–50 µg/mL. At pH 4–8 and temperatures of 4°C–45°C, CHAPSAP26-161 exhibited optimal hydrolase activity. The lytic activity of CHAPSAP26-161 was dependent on divalent metal ions, especially Zn2+, and increased in the presence of ethylenediamine tetraacetic acid. CHAPSAP26-161 demonstrated superior protein purification efficiency and antibacterial activity than LysSAP26. It showed high lytic activity against gram-positive, gram-negative, and anaerobic bacteria, includingS. aureusandClostridioides difficile.Enhanced stability under varied temperatures and pH conditions.In vivo,tests demonstrated promising therapeutic effects of CHAPSAP26-161 in murine systemicA. baumanniiinfection models.ConclusionsCHAPSAP26-161, a truncated modular endolysin containing only the CHAP domain of LysSAP26, demonstrated higher protein purification efficiency and antibacterial activity than LysSAP26. It also exhibited extended-spectrum antibacterial activity against gram-positive, gram-negative, and anaerobic bacteria, such asS. aureus,A. baumannii, andC. difficile. Its successfulin vivoapplication in murine models highlights its potential as an alternative therapeutic agent in combating antibiotic resistance.