Extracellular vesicles from Streptococcus parauberis facilitate the efficient delivery of LL37 with enhanced antibacterial activity.

Bacterial extracellular vesicles (BEVs) serve as efficient drug-delivery vehicles for bioactive molecules including antimicrobial peptides (AMPs). In the present study, human cathelicidin-derived AMP LL37 was encapsulated in Streptococcus parauberis-derived BEVs (SpEVs) using a co-incubation method, achieving an encapsulation efficiency and loading capacity of 28.5 and 57 %, respectively. The LL37 loaded SpEVs (SpEVs-LL37) retained the characteristic morphology of native SpEVs, as confirmed by field-emission electron microscopy. A slight increase in particle size was observed via nanoparticle tracking analysis (NTA), with SpEVs-LL37 measuring 190.7 ± 3.1 nm compared to 154.5 ± 2.7 nm for SpEVs. Both SpEVs and SpEVs-LL37 maintained their negative surface charge. SpEVs-LL37 exhibited significantly lower cytotoxicity against murine macrophage Raw 264.7 cells, compared with LL37 alone, while both showed similar levels of cellular internalization. SpEVs-LL37 demonstrated enhanced antibacterial activity against S. parauberis, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 5 and 10 μg/mL, respectively, compared to 10 and 25 μg/mL for LL37 alone. Time-kill kinetics and bacterial viability assays confirmed the superior antibacterial efficacy of SpEVs-LL37. Mechanistically, SpEVs-LL37 treatment induced greater alterations in bacterial membrane permeability and reactive oxygen species (ROS) generation than treatment with LL37 alone. In vivo, SpEVs-LL37 significantly upregulated immune-related genes in zebrafish compared with SpEVs and LL37 alone. Additionally, SpEVs-LL37 conferred higher disease resistance against S. parauberis infection in zebrafish. These findings highlight the potential of SpEVs-LL37 as an effective drug-delivery and therapeutic platform for combating S. parauberis and other pathogenic infections.