Targeted therapy of oncomicrobe F. nucleatum with bioengineered probiotic expressing guided antimicrobial peptide (gAMP)

Abstract Colorectal cancer (CRC) is a leading cause of cancer-related mortality, with Fusobacterium nucleatum ( F. nucleatum ) identified as a key contributor to its progression. This study explores a novel targeted therapy using bioengineered probiotics expressing guided antimicrobial peptides (gAMPs) to selectively inhibit F. nucleatum . We engineered Lactococcus lactis MG1363 to express gAMPs derived from Ovispirin and Cathelin-related peptide SCF, linked to a Statherin-derived guide peptide (YQPVPE) that binds specifically to the F. nucleatum membrane porin FomA. Our results demonstrate that the Statherin-derived guide peptide enhances the binding affinity to F. nucleatum , significantly increasing the preferential attachment compared to control peptides. In vitro assays revealed that both unguided and guided AMPs effectively inhibited biofilm formation in F. nucleatum , with gAMPs showing reduced toxicity against non-target bacteria ( Bacteroides fragilis and Escherichia coli ). The gAMPs were also more effective in modulating growth kinetics, exhibiting selective toxicity towards F. nucleatum at lower concentrations. Co-culture experiments in a simulated human gut microbiome demonstrated that the gAMP probiotic maintained microbial diversity while effectively reducing F. nucleatum abundance. Quantitative PCR and 16S rRNA sequencing confirmed that gAMP treatment preserved the richness of the microbiota, contrasting with significant dysbiosis observed in control samples. These findings support the potential of engineered probiotics as a targeted therapeutic approach to combat F. nucleatum -associated CRC. By leveraging the specificity of Statherin-derived peptides, this strategy not only addresses the pathogenicity of F. nucleatum but also mitigates the adverse effects of traditional antimicrobial therapies on beneficial gut microbiota. Future studies will explore the clinical applicability of this approach in CRC management and its impact on overall gut health.