Actin rearrangements accompanying Aeromonas hydrophila entry into cultured fish cells

Aeromonas hydrophila can enter fish cells and exist as intracellular parasites. Phase-contrast and confocal microscopy were used to examine morphological changes and various cytoskeletal components of infected fish cells. Four fish cell lines were included in this study: (1) AS, (2) BF2, (3) CHSE-214, and (4) EPC cells. Virulent but not avirulent strains of A. hydrophila PPD 134/91 invaded fish cells, causing morphological changes, and inducing microfilament (F-actin) rearrangement. Morphological changes were observed in all infected fish cell lines and could be classified into three different stages. In stage I, the cells became detached from each other and pointed ends were observed. In stage II, tubular cytoplasmic extensions formed at contact points connecting neighbouring cells. The monolayers formed a satellite-like organization and became less confluent. Finally (stage III), cells were heavily infected with bacteria, and bacteria containing vacuoles occupied most of the cells. They eventually detached and lysed. Rearrangement of F-actin was observed as local polymerization (actin clouds) in stage I and massive reorganization in stage III of infection. Actin clouds could have been induced by A. hydrophila for 'assisted' uptake into the cells. The massive reorganization of actin in stage III may be due to products released by the bacteria and the growth of vacuoles. Pretreatment of fish cells with the microfilament inhibitors such as cytochalasins induced a similar effect. There were little if any rearrangements in intermediate and microtubule filaments during bacterial entry (stages I and II). These results suggest that A. hydrophila may bind to the surface and trigger a signal to the microfilament which then generates the force necessary for bacterial uptake.