A global deep-sea small protein atlas reveals a reservoir of noncanonical antimicrobial peptides

Abstract

Small proteins encoded by small open reading frames (smORFs; [≤] 100 aa) represent a largely unexplored dimension of microbial diversity, especially in the deep sea. By analyzing 708 metagenomes from five major deep-sea biomes (hadal trenches, cold seeps, hydrothermal vents, abyssal plains, and seamounts), we constructed the Deep-Sea Small Protein Atlas, comprising 88.7 million smORFs with exceptional novelty and strong habitat specificity. Deep-learning predictions identified 5.47 million candidate antimicrobial peptides (c_AMPs), revealing a peptide space far larger and structurally distinct from known AMPs. Deep-sea c_AMPs are longer, enriched in nonpolar and acidic residues, and exhibit low charge and high intrinsic disorder, suggesting non-membranolytic modes of action. We synthesized 131 representative peptides, of which 87% were antimicrobial, with MICs as low as 1.25 M, broad-spectrum antibacterial and even antifungal efficacy, and minimal mammalian cytotoxicity. Transcriptomics, TEM imaging, and peptide-protein modeling showed that representative peptides preserve membrane integrity while disrupting intracellular processes such as translation and metabolism, supporting intracellular, non-lytic mechanisms. This work uncovers a vast reservoir of previously unrecognized deep-sea small proteins and structurally unconventional AMPs, providing a foundational resource for discovering next-generation peptide therapeutics.