Plantaricin JK from Lactiplantibacillus plantarum ZJ316 inhibits enterohemorrhagic Escherichia coli O104:H4 and alters fecal microbial communities in vitro

Abstract

Enterohemorrhagic Escherichia coli (EHEC) O104:H4 is a highly virulent foodborne pathogen for which antibiotic therapy is contraindicated, severely restricting conventional treatment options; therefore, the development of novel antimicrobial strategies is essential. This study investigated the antibacterial activity and gut microbiota-modulating effects of plantaricin JK (PlnJK), a two-peptide bacteriocin from Lactiplantibacillus plantarum. Chemically synthesized PlnJK exhibited a minimum inhibitory concentration (MIC) of 32 M against E. coli O104:H4, disrupting membrane integrity as evidenced by increased extracellular conductivity, ATP depletion, and ultrastructural damage. In an in vitro fecal fermentation model, PlnJK reduced Escherichia-Shigella abundance from 36.70% to 30.60% which contains E. coli O104:H4-related taxa, enriched Clostridium (5.70% vs 2.40% in infection group), Rikenellaceae, and Lactobacillaceae, and significantly elevated short-chain fatty acid (SCFA) levels including butyrate (p < 0.01) and isovalerate (p < 0.05). To translate this into a deliverable format, CRISPR-Cas9-engineered L. plantarum strains with plnJK knockout, complementation, and overexpression were constructed. Compared with the chemically synthesized peptide, LAB-derived PlnJK--especially from the overexpression strain--showed superior efficacy in restoring microbial diversity and enhancing SCFA production, significantly increased Shannon index and reduced dominance (p < 0.01), while promoting butyrate (p < 0.01) and isovalerate (p < 0.01) production compared to the knockout control. The engineered strains further revealed a clear dose-dependent effect, as overexpression potentiated while knockout abolished the beneficial modulation. These findings demonstrate that PlnJK acts through membrane disruption and beneficially modulates gut microecology, with LAB-mediated in situ delivery offering a promising strategy for anti-EHEC live biotherapeutic development.