Structural Insights to the Heterotetrameric Interaction between the Vibrio parahaemolyticus PirA(vp) and PirB(vp) Toxins and Activation of the Cry-Like Pore-Forming Domain

Acute hepatopancreatic necrosis disease (AHPND) is a newly emergent penaeid shrimp disease which can cause 70–100% mortality in Penaeus vannamei and Penaeus monodon, and has resulted in enormous economic losses since its appearance. AHPND is caused by the specific strains of Vibrio parahaemolyticus that harbor the pVA1 plasmid and express PirA(vp) and PirB(vp) toxins. These two toxins have been reported to form a binary complex. When both are present, they lead to the death of shrimp epithelial cells in the hepatopancreas and cause the typical histological symptoms of AHPND. However, the binding mode of PirA(vp) and PirB(vp) has not yet been determined. Here, we used isothermal titration calorimetry (ITC) to measure the binding affinity of PirA(vp) and PirB(vp). Since the dissociation constant (K(d) = 7.33 ± 1.20 μM) was considered too low to form a sufficiently stable complex for X-ray crystallographic analysis, we used alternative methods to investigate PirA(vp)-PirB(vp) interaction, first by using gel filtration to evaluate the molecular weight of the PirA(vp)/PirB(vp) complex, and then by using cross-linking and hydrogen-deuterium exchange (HDX) mass spectrometry to further understand the interaction interface between PirA(vp) and PirB(vp). Based on these results, we propose a heterotetrameric interaction model of this binary toxin complex. This model provides insight of how conformational changes might activate the PirB(vp) N-terminal pore-forming domain and should be helpful for devising effective anti-AHPND strategies in the future.