Structural and biochemical characterization of Grimontia hollisae thermostable direct hemolysin with DNA reveals first Vibrio hemolysin with nuclease activity.

Grimontia hollisae thermostable direct hemolysin (Gh-TDH) is a pore-forming toxin that disrupts the cell membrane, leading to erythrocyte lysis and cytotoxicity. Previous studies showed that fluorescently labeled Gh-TDH-FITC binds to hepatocyte membranes and subsequently translocates to the nucleus. Here, we demonstrate for the first time that Gh-TDH cleaves DNA with 3'-5' nuclease activity. The crystal structure of Gh-TDH in complex with ssDNA unveils a unique DNA-binding configuration. Notably, the putative cleavage site (Tyr87-Lys88-Asp89) deviates from the canonical 3'-5' exonuclease motif (Asp-Glu-Asp-Asp). Site-directed mutagenesis and binding kinetics assays demonstrate that Lys88 is essential for nuclease activity, supporting its central role in catalysis. TDH homologues are widespread in Vibrio, with DNA-binding and catalytic residues highly conserved. Consistently, V. parahaemolyticus TDH also exhibits 3'-5' exonuclease activity, indicating this nuclease function is an intrinsic, conserved feature of the TDH family rather than incidental. These findings provide the structural basis and mechanism of DNA binding and cleavage by Gh-TDH. Gh-TDH is thus the first Vibrio pore-forming toxin shown to have dual hemolytic and nuclease activities. This work opens new avenues to explore why Vibrio pore-forming toxins have evolved nuclease activity and what physiological or pathogenic roles it may play in vivo.