De novo origin and evolution of an antimicrobial protein function

Antimicrobial peptides (AMPs) constitute key components of innate immunity across the tree of life. Canonical AMPs are typically translated as small proteins and secreted from host cells to act against microbes. However, cryptic AMP-like domains are also embedded within diverse proteins not classically associated with antimicrobial function. How such embedded AMPs first emerge and diversify remains unclear. Here we retrace the origin and evolution of the abundant mammalian protein lactoferrin and its embedded AMP, lactoferricin. By resurrecting extinct lactoferrin ancestors dating back to the earliest mammals, we identify a gradual enrichment of cationic and hydrophobic amino acids in the lactoferricin domain over time. These changes enabled ancient lactoferricin to first rupture bacterial membranes, an activity that was later enhanced in extant mammals conferring potent bactericidal activity against diverse bacteria. In addition, we find that recent natural selection within the lactoferricin domain has continued to modulate antimicrobial activity on recent evolutionary timescales. In particular, we pinpoint a single rapidly evolving site in lactoferricin among great apes that significantly enhances antimicrobial potency against major pathogenic bacteria. Together our study illustrates how novel immune protein functions can arise, evolve, and diversify to strengthen host defense against diverse microbial pathogens. Significance StatementCryptic antimicrobial peptide (AMP) domains have been increasingly identified within larger proteins, yet how these embedded AMPs first emerge and diversify remains unclear. Here we retrace the origin and evolution of the abundant mammalian immunity protein lactoferrin and its embedded AMP, lactoferricin. By resurrecting extinct mammalian lactoferrin ancestors, we identify molecular alterations that first enabled lactoferricin to rupture bacterial membranes and later acquire bacterial-killing activity. Our findings illustrate how new protein functions can quickly arise and evolve to confer immune defense against deadly pathogens. More broadly, this work highlights the proteome as a reservoir of latent antimicrobial defenses, suggesting new strategies for discovering AMP-based therapeutics to combat the escalating threat of antibiotic-resistance.