Internalization and cytotoxicity are important virulence mechanisms in Vibrio-fish epithelial cell interactions

Vibrio anguillarum and Vibrio damselae are Gram-negative bacteria that cause systemic infections called vibriosis in fish. They can enter fish cells and survive as intracellular parasites. The host-pathogen interactions between these Vibrio species and the fish epithelial cell lines epithelioma papillosum of carp (EPC) and grunt-fin tissue (GF) cells, were examined using phase-contrast, scanning electron and confocal microscopy. In addition, potential signal transduction pathways that precede bacterial internalization were studied by using signal transduction inhibitors. Some Vibrio species induced morphological changes in fish cells and this allowed classification into a cytopathic group and a noncytopathic group. The cytopathic group could be subdivided into two invasive groups (I and II) and a cytotoxic group. Of the invasive strains V. anguillarum 811218-5W (group I) and G/Virus/5(3) (group II), genistein, a tyrosine kinase inhibitor, only inhibited internalization of V. anguillarum G/Virus/5(3) into EPC cells, whereas staurosporine, a protein kinase C inhibitor, accelerated internalization of both strains. Cytochalasin D, an inhibitor of microfilament polymerization, prevented internalization of both strains, whilst vincristin, a microtubule inhibitor, only inhibited internalization of V. anguillarum G/Virus/5(3). For the cytotoxic strain V. damselae ATCC 33539, extracellular products (ECP) alone caused morphological changes in EPC and GF. Bacterial internalization may not be important in the pathogenesis of this group. The non-cytopathic strain V. anguillarum S2/5/93(2) did not enter cells or induce any changes in EPC and GF monolayers. This study has identified some major differences between Vibrio species in their interactions with fish cells in vitro and will thus facilitate future studies of the molecular basis of pathogenesis of vibriosis.