Characterization of a metalloprotease involved in Vibrio splendidus infection in the sea cucumber, Apostichopus japonicus

Vibrio splendidus is an important aquatic pathogen that infects a broad range of hosts, leading to typical symptoms of "skin ulceration syndrome" in the sea cucumber Apostichopus japonicus. However, there are few reports on the virulence factors and pathogenic mechanism of V. splendidus. In the present study, V. splendidus could survive in the coelomic fluid of A. japonicus but poorly internalized into the coelomocyte under the tested conditions. It was thus postulated that V. splendidus was pathogenic to A. japonicus, mainly due to its extracellular products. The main extracellular proteins of V. splendidus were detected using MALDI-TOF-TOF/MS, and a metalloprotease was identified. The gene encoding a metalloprotease belonging to the thermolysin family, named vsm, was cloned and characterized. Furthermore, the expression of vsm was growth dependent and the supernatant of V. splendidus also showed high protease activity. The vsm gene was conditionally expressed in Escherichia coli BL21(DE3). Enzyme activity analysis showed that the optimal temperature and pH for purified recombinant Vsm were approximately 40 °C and 7.0, respectively. Furthermore, Vsm was determined to be associated with the pathogenesis of V. splendidus due to the following aspects: (1) real-time reverse transcriptase PCR showed that expression of vsm was significantly up-regulated when V. splendidus was co-incubated with the coelomic fluid of A. japonicus; and (2) purified recombinant Vsm showed obvious cytotoxicity to the coelomocyte of A. japonicus. Our results indicated that Vsm is involved in the interaction between V. splendidus and A. japonicus and also contributed to the cytotoxic effects on the coelomocyte of A. japonicus.