Rapid Purification and Molecular Characterization of VTc Toxin from Pathogenic Vibrio parahaemolyticus

Highly lethal Vibrio disease (HLVD) is a newly emerging shrimp disease caused by VTc toxin-producing Vibrio strains, designated VHLVD. VTc toxin, composed of VTcA, VTcB, and VTcC subunits, is a novel member of the Tc toxin family. Structural and functional studies of VTc toxin have been hindered by the lack of efficient purification protocols. Here, we established a simple method for purification of milligram-scale bioactive VTc toxin via anion-exchange chromatography. The procedures of cell lysis, sample pretreatment, and anion-exchange chromatography were optimized. This protocol yielded approximately 1.5 mg of high-purity toxin from 40 mL of bacterial culture (VHLVD strain vp-HL-202005). The composition of the product was confirmed by SDS-PAGE and LC-MS/MS. Purified VTc toxin was bioactive, inducing typical HLVD mortality in shrimp post-larvae. Transmission Electron Microscopy (TEM) and native PAGE analysis revealed two distinct structural populations, which may represent the club-shaped holotoxin (VTcA2-VTcB2-VTcC2) and the bell-shaped VTcA2 homopentamer. This simple and efficient method will greatly facilitate research on the fine structure and functional mechanism of VTc toxin and thus expand our knowledge of the Tc toxin family.