Outer membrane vesicles (OMVs) are nano-sized proteoliposomes shed from the cell envelope of all Gram-negative bacteria. OMVs are now recognized as a generalized secretion pathway which provides a means to transfer cargo to other bacterial, and eukaryotic cells. OMVs play an important role in pathogenesis, delivering virulence factors to the host cells, including toxins, adhesins, and immunomodulatory molecules. Moritella viscosa is a Gram-negative pathogen and the causative agent of winter ulcer disease in several marine fish species. M. viscosa OMVs have not been characterized and their role in the infection process and utilization as potential vaccine is unknown. Here, we develop a M. viscosa infection model in lumpfish, characterized the M. viscosa OMVs, and evaluate its toxicity and immunogenicity in in vivo lumpfish model.
We determined that the lethal dose (LD50) of M. viscosa in lumpfish was 8.1 x107 colony-forming units (CFU) fish-1. Lumpfish were intraperitoneally (i.p.) injected with 3.1 x 108 CFUfish-1 showed a rapid mortality and typical clinical signs of winter ulcer disease after 5 days post-infection. Iron is an essential nutrient for microbes and influence pathogenesis. To characterize M. viscosa OMVs, the bacterial strain was cultivated under iron-rich and iron-limited conditions. Purified M. viscosa OMVs were characterized by transmission electron microscopy and protein analysis. M. viscosa OMVs from both growing conditions showed spheres of 39.8–370 nm diameter that contains RNA and DNA. The most abundant proteins in M. viscosa OMVs have molecular sizes of 45, 30 and 20 kDa. OMVs isolated from iron-limited condition harbor an additional protein of approximately 60 kDa which is absent in OMVs isolated from bacteria grown under iron-rich conditions. Protein identification analyses of the 60 kDa protein band indicated the presence of enzyme of Metal-dependent carboxypeptidase, Glucose-6-phosphate isomerase, Glucose-6-phosphate isomerase, and transport systems including the peptide ABC transporter, extracellular solute-binding protein, and oligopeptide transport system, and permease protein B.
The results strongly suggested that M. viscosa could induce the ulcer disease in lumpfish. M. viscosa OMVs contain various bacterial components, including proteins, DNA/RNA, and did not show toxicity in lumpfish. These OMV products may partially explain that they play an important role in the pathogenesis of M. viscosa.