Actifensin Evolution in the Human Oral Cavity over the Past 100,000 Years.

Bacterially produced antimicrobial peptides (AMPs), or bacteriocins, play key roles in shaping microbial communities via interspecies competition. Unlike the more temporally dynamic gut microbiome, the oral microbiome exhibits long-term stability and is preserved into deep time in dental calculus, enabling evolutionary analysis across time. Here, we combine metagenomics, structural modeling, and experimental validation to investigate AMP diversity in ancient and modern dental biofilms from humans, Neanderthals, and nonhuman primates spanning 100,000 years. Using our newly developed platform, AMPcombi, we uncover evolutionary trajectories of bacteriocins and elucidate their ecological functions. Among these, we identify a conserved family of Actinomyces-derived defensin-like peptides, termed actifensins, present across all time periods. Phylogenetic, structural, and functional analyses revealed shared ancestry and adaptive diversification between ancient (paleo-) and modern actifensins, with evidence of positive selection and maintained antimicrobial activity. Our findings position the oral microbiome as a valuable reservoir for natural product discovery. In the face of rising antimicrobial resistance, evolutionary insights into AMP function open a door to next-generation therapeutics. AMPcombi streamlines this process, linking ancient biomolecules with biotechnology.