Structural and mechanistic insights into the VP14460-VP14465 effector-immunity module of the Vibrio parahaemolyticus type VI secretion system.

Vibrio parahaemolyticus, a halophilic pathogen, contaminates seafood and high-salt foods, posing significant health risks such as gastroenteritis and fatalities. With increasing seafood consumption, developing effective control strategies is imperative. The type VI secretion system, a common molecular weapon in Vibrio that mediates cross-domain interactions, is a contractile nanomachine that deploys antibacterial effectors, each paired with a cognate immunity protein to form effector-immunity (E-I) modules. Here, we present the first structural and functional characterization of this complex. The crystal structure of the VP14460-VP14465 complex unveils the molecular basis for specific E-I recognition, defining a set of critical interfacial residues. Structure-guided mutagenesis confirmed that these residues are essential for complex integrity, their disruption liberates the VP14465 toxin, unleashing potent bactericidal activity. Furthermore, we identified the active sites responsible for the DNase activity of VP14465. Notably, while VP14460 and VP14465 form a stable monomeric complex in the bound state, each isolated component exhibits distinct oligomeric behavior in vitro. This suggests a dynamic assembly-disassembly mechanism that may regulate effector delivery and toxin activation during interbacterial competition. Collectively, our findings provide mechanistic insights into type VI secretion system E-I module architecture and function, and establish a structural framework for the development of novel antibacterial therapeutics targeting DNase family effectors.