Expression of the AHPND Toxins PirA(vp) and PirB(vp) Is Regulated by Components of the Vibrio parahaemolyticus Quorum Sensing (QS) System

Acute hepatopancreatic necrosis disease (AHPND) in shrimp is caused by Vibrio strains that harbor a pVA1-like plasmid containing the pirA and pirB genes. It is also known that the production of the PirA and PirB proteins, which are the key factors that drive the observed symptoms of AHPND, can be influenced by environmental conditions and that this leads to changes in the virulence of the bacteria. However, to our knowledge, the mechanisms involved in regulating the expression of the pirA/pirB genes have not previously been investigated. In this study, we show that in the AHPND-causing Vibrio parahaemolyticus 3HP strain, the pirA(vp) and pirB(vp) genes are highly expressed in the early log phase of the growth curve. Subsequently, the expression of the PirA(vp) and PirB(vp) proteins continues throughout the log phase. When we compared mutant strains with a deletion or substitution in two of the quorum sensing (QS) master regulators, luxO and/or opaR (luxO(D47E), ΔopaR, ΔluxO, and ΔopaRΔluxO), our results suggested that expression of the pirA(vp) and pirB(vp) genes was related to the QS system, with luxO acting as a negative regulator of pirA(vp) and pirB(vp) without any mediation by opaR(vp). In the promoter region of the pirA(vp)/pirB(vp) operon, we also identified a putative consensus binding site for the QS transcriptional regulator AphB. Real-time PCR further showed that aphB(vp) was negatively controlled by LuxO(vp), and that its expression paralleled the expression patterns of pirA(vp) and pirB(vp). An electrophoretic mobility shift assay (EMSA) showed that AphB(vp) could bind to this predicted region, even though another QS transcriptional regulator, AphA(vp), could not. Taken together, these findings suggest that the QS system may regulate pirA(vp)/pirB(vp) expression through AphB(vp).