Cloning and characterization of Vshppd, a gene inducing haemolysis and immune response of Apostichopus japonicus

Abstract

Vibrio splendidus is one of the most important opportunistic pathogens that are ubiquitously presented in the marine environment. It is the main pathogen of “skin ulcer syndrome” which occurs in Apostichopus japonicus; however, little is known about its virulence factors and pathogenic mechanism. In this study, V. splendidus isolated from diseased A. japonicus was determined to possess α-haemolytic activity, and a gene named Vshppd, encoding a 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) homologue from pathogenic V. splendidus, was cloned, and conditionally expressed in Escherichia coli. Haemolytic activity analyses of both purified protein and recombinant BL21(DE3)/pET258h revealed that Vshppd was responsible for the haemolysis of V. splendidus. What's more, expressions of Vshppd under various conditions were also determined using real-time reverse transcriptase PCR. Transcription of Vshppd was up-regulated by both iron chelator and coelomic fluid of A. japonicus, while it was down-regulated by glucose. Furthermore, DH5α/pBATh, an E. coli strain displaying Vshppd on the cell surface was genetically constructed and showed significantly stimulatory effects on the expression of immune-related genes encoding LBP and caspase of A. japonicus. Our results indicated that haemolysin Vshppd was not only involved in the interaction between V. splendidus and A. japonicus but also contributed to the stimulatory effect on immune response. Statement of relevanceV. splendidus is an important opportunistic pathogen that can infect a broad range of aquatic animals including A. japonicus, but little was known about its pathogenic mechanism. Our study firstly showed that Vshppd conferred the haemolytic activity of V. splendidus, involved in the interaction between V. splendidus and host, and contributed to the stimulatory effect on immune response.