Helicolysins Emerge as a New Family of Metzincin Metallopeptidases in Host–pathogen Interactions

The Helicobacter pylori Hom family of outer-membrane proteins (OMPs) has been implicated in virulence-related processes. Here, we present a structural and functional characterization of HomA from H. pylori strain 26695. Comparative in vivo analyses of the wild-type strain and a homA deletion mutant demonstrate a role for HomA in focal adhesion kinase phosphorylation at Y 397 and in bacterial adhesion to human leukaemia-derived MEC-1 cells. Recombinant production of several HomA variants identified a soluble ectodomain ( ECD HomA) that harbours the conserved zinc-binding motif of metzincin metallopeptidases (MPs) and displays dimerization capacity, acid resistance, and immunomodulatory activity by triggering a pro-inflammatory response in Raji human B-lymphoblastoid cancer cells. Site-directed mutagenesis of the zinc-binding motif confirmed its contribution to zinc coordination and thermal stability. Structural analysis of ECD HomA revealed a two-domain organization comprising an N-terminal accessory β-sandwich domain (ND) and a C-terminal catalytic domain (CD), in which a non-canonical ‘Thr-turn’ replaces the hallmark metzincin ‘Met-turn’. Integrating bioinformatics with AI-based structure predictions uncovered putative HomA homologues across 44 proteomes, predominantly from Gram-negative bacteria. Structural and biochemical characterization of the closest non- Helicobacter homologue from the oral pathogen Capnocytophaga canimorsus , dubbed canilysin, revealed an architecture equivalent to ECD HomA and highly specific MP activity. Together, these findings define the Hom family and canilysin as helicolysins, a previously uncharacterized metzincin subfamily distinguished by a conserved Thr-turn and an accessory ND, and implicates these proteins in host–pathogen interactions, adhesion, and immunomodulation.