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 therapy that targets this pathogen by using a bioengineered probiotic that expresses guided antimicrobial peptides (gAMPs) to selectively inhibit F. nucleatum. Lactococcus lactis MG1363 was engineered to express gAMPs derived from Ovispirin and Cathelin-related peptide SCF, linked to a Statherin-derived guide peptide that binds specifically to the F. nucleatum membrane porin FomA. The bacteria expressed the AMP/gAMP under the induction of the PNisA promoter by nisin and secreted it via the extracellular secretion signal usp45. The resultant synthetic peptides and probiotics were assayed for antimicrobial activity against the targeted F. nucleatum and other non-target bacteria. Biofilm inhibition and growth kinetic assays were performed with synthetic peptides in vitro or the probiotic in co-culture with a polymicrobial community. Statherin-derived guide peptide enhanced the binding affinity to F. nucleatum, significantly increasing 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. The gAMPs were more effective in modulating growth kinetics, exhibiting selective toxicity towards F. nucleatum at lower concentrations. Co-culture experiments in a simulated human gut microbiome showed 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 therapeutic approach that targets CRC-associated F. nucleatum.