Assembly of a multivalent aptamer for efficient inhibition of thermostable direct hemolysin toxicity induced by Vibrio parahaemolyticus

Thermostable direct hemolysin (TDH) is a key virulence factor of Vibrio parahaemolyticus, capable of causing seafood-mediated outbreaks of gastroenteritis, posing a threat to the aquatic environment and global public health. In the present study, we explored a multivalent aptamer-mediated inhibition strategy to mitigate TDH toxicity. Based on the characteristic structure of TDH, a stable multivalent aptamer, Ap3–5, was rationally designed by truncation, key fragment evolution, and end fixation. Ap3–5 exhibited strong affinity (Kd=39.24 nM), and thermal (Tm=57.6 °C) and enzymatic stability. In silico studies also revealed that Ap3–5 occupied more active sites of TDH and covered its central pore, indicating its potential as a blocking agent for inhibiting TDH toxicity. In the hemolysis assay, Ap3–5 significantly suppressed the hemolytic effect of TDH. A cellular study revealed a substantial (∼80 %) reduction in TDH cytotoxicity. Supporting these findings, in vivo trials confirmed the inhibitory action of Ap3–5 on both the acute and intestinal toxicity of TDH. Overall, benefiting from the strong binding affinity, high stability, and multisite occupation of the multivalent aptamer with TDH, Ap3–5 displayed robust potential against TDH toxicity by inhibiting membrane pore formation, providing a new approach for alleviating bacterial infections.