An antisense oligomer conjugate with unpredicted bactericidal activity against <i>Fusobacterium nucleatum</i>

Fusobacteria are commensal members of the oral microbiome that can spread from their primary niche and colonize distal sites in the human body. Their enrichment in colorectal and breast cancer tissue has been associated with tumor growth, metastasis, and chemotherapeutic resistance. The use of non-selective antibiotics to remove fusobacteria impairs tumor progression, but prolonged application causes side effects, such as gastrointestinal problems and dysbiosis. Species-specific antisense antibiotics based on peptide nucleic acid (PNA) have shown efficacy in many gram-negative species, suggesting that antisense PNAs may also enable a tailored depletion of fusobacteria. Here, we have investigated the antibacterial potential of cell-penetrating peptide (CPP)-PNA conjugates targeting the mRNA of putative essential genes in <i>Fusobacterium nucleatum</i>. Unexpectedly, we observed no growth inhibition with any of the target-specific PNAs but identified a non-targeting control CPP-PNA [FUS79, (RXR)₄XB-GACATAATTGT] as a potent growth inhibitor of <i>F. nucleatum</i>. Our data suggest that the CPP and specific sequence features of FUS79 are responsible for its activity, rather than an antisense effect. Interestingly, FUS79 also inhibits the growth of five additional fusobacterial strains but not of <i>F. nucleatum</i> subsp. <i>vincentii</i> (FNV). RNA-seq analysis indicates that FUS79 induces a membrane stress response in a vulnerable <i>F. nucleatum</i> strain but not in FNV. Collectively, our attempt at developing antisense antibiotics for fusobacteria discovers a potent growth inhibitor, whose bactericidal effect appears independent of target-specific mRNA inhibition.IMPORTANCEEnrichment of <i>F. nucleatum</i> at cancer sites is associated with increased tumor growth and metastasis. Antibiotic treatment to remove the bacteria was shown to change the course of cancer progression. Here, we explore first steps to establish peptide nucleic acids (PNAs) as specific antisense antibiotics, thereby laying the foundation for further development of antisense technology in fusobacteria. Although the CPP-PNA FUS79 was initially designed as a control, we observed that the compound was bactericidal for specific fusobacterial strains. Our results suggest that FUS79 might be able to selectively deplete fusobacterial strains from bacterial communities, offering a new perspective on fusobacterial removal at the tumor site.