Green fluorescent protein-tagged Vibrio splendidus for monitoring bacterial infection in the sea cucumber Apostichopus japonicus

Abstract

Vibrio splendidus is an important opportunistic pathogen that infects many important marine animals; for example, it can cause skin ulcer syndrome in the sea cucumber Apostichopus japonicus. In this study, a green fluorescent protein (GFP)-tagged V. splendidus was constructed by ligating GFP into the plasmid pET28a. The growth of GFP-tagged V. splendidus showed no difference from that of the wild type strain. The distribution of V. splendidus in various tissues after artificial infection was determined by colony counting. When V. splendidus infected A. japonicus at a concentration of 5.0 × 107 CFU·mL−1, V. splendidus could be detected in the tissues of respiratory tree, intestine, body wall, tentacle, and muscle, with a level over 1.0 × 106 CFU·g−1 at 48 h post-infection and with V. splendidus remaining in coelomic fluid at a level of 2.1 × 109 CFU·mL−1. Green fluorescence was only observed in the respiratory tree and intestine in a paraffin section at 48 h of infection, which indicated that V. splendidus could adhere to these two tissues. Moreover, V. splendidus could adhere to coelomocytes of A. japonicus but it could poorly internalize coelomocytes in vitro. The adherence of V. splendidus to coelomocyte was dependent on the β-integrin on the surface of coelomocytes.