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 cell membrane, causing erythrocyte lysis and exhibiting cytotoxicity, cardiotoxicity, enterotoxicity, hepatotoxicity, and lethality in mice. Recombinant Gh-TDH-FITC binds to hepatocyte membranes and translocates to the nucleus. This study reveals that Gh-TDH cleaves DNA with 3'–5' nuclease activity to generate a 3-mer fragment, while cleaving RNA with ~ 70% lower efficiency. The co-crystal structure of Gh-TDH bound to ssDNA unveils a unique DNA-binding configuration, where eight ssDNA strands form four duplexes, each connected to a Gh-TDH tetramer at one end and to protomers from four different tetramers at the other. Notably, the cleavage site (Tyr87-Lys88-Asp89) deviates from the canonical 3'–5' exonuclease motif (Asp-Glu-Asp-Asp). Mutagenesis identified Lys88 as a general base essential for nuclease activity. This study provides structural and functional insights of Gh-TDH on DNA-binding and cleavage mechanism. In summary, Gh-TDH is the first pore-forming toxin identified in the Vibrio genus with dual functions of hemolytic and nuclease activities.