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 , Δ opaR , Δ luxO, and Δ opaR Δ luxO ), 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 .