List of plasmids used in this study.

AB5-type toxins are critical virulence factors in bacterial pathogenesis. Despite the identification of various B subunits in AB5 toxins across different pathogens, their assembly mechanisms and biological significance remain poorly understood. In this study, we identified and characterized a typhoid toxin-like A2B5 toxin, designated diarizonae toxin (DiaT), as a key virulence factor in Salmonella diarizonae. The DiaT genomic islet encodes two distinct B subunits, PltBd1 and PltBd2, which exhibit unique functional roles. Through genetic and functional analyses, we demonstrate that the heteropentameric assembly of PltBd1 and PltBd2 is essential for cytotoxicity, with our data suggesting PltBd1 facilitates toxin secretion and PltBd2 mediates host cell targeting. Cryo-EM structural analysis of endogenously expressed DiaT reveals a heteropentameric holotoxin with a 3:2 stoichiometry of PltBd1 to PltBd2, potentially stabilized by the PltA subunit. These findings uncover a novel assembly mechanism and synergistic functionality between distinct B subunits, advancing our understanding of the evolutionary diversity and functional complexity of AB5 toxins. This work provides new insights into bacterial pathogenesis and highlights potential targets for therapeutic intervention.