Cloning and characterization of the virulence factor Hop from Vibrio splendidus

Abstract

BackgroundVibrio splendidus is an aquaculture pathogen that can cause skin ulcer syndrome (SUS) in Apostichopus japonicus. HopPmaJ is a type III system effector (T3SE) that has been reported to be an important virulence factor. In this study, a gene named hop, which encodes HopPmaJ in V. splendidus was cloned and its cytotoxicity to coelomocytes and its effects on the expression of immune-related genes in A. japonicus were characterized. MethodsReal time reverse transcription PCR (RT-PCR) was used to determine the expression of the hop gene under various conditions. To obtain the purified Hop, hop gene was conditionally expressed in Escherichia coli BL21(DE3) and was purified by GST tag. The cytotoxicity of Hop to coelomocyte was determined using MTT method, and the effect of Hop on the expression of immune-related genes was determined using real time RT-PCR. ResultsThe deduced amino acid sequence of Hop from V. splendidus shared 84%–96% homology with those of Hops from other Vibrio spp. The expression of hop gene was induced not only by host-pathogen contact but also by high cell density. Purified recombinant Hop (rHop) showed cytotoxicity to the coelomocyte of A. japonicus. The cell viability decreased to approximately 42%, 26%, 32%, 30% and 20%, when 30, 50, 60, 80 and 100 μL of purified rHop was added, respectively. After being injected with rHop, the expression levels of immune-related genes that encode complement component (C1q) and caspase were significantly increased, and the production of reactive oxygen species were also increased in A. japonicus. ConclusionOur results indicated that Hop not only contributed to the cytotoxicity to coelomocyte, but also caused immune response in A. japonicus.