Invasion and replication of Yersinia ruckeri in fish cell cultures

Abstract

BackgroundLike many members of the Enterobacteriaceae family, Yersinia ruckeri has the ability to invade non professional phagocytic cells. Intracellular location is advantageous for the bacterium because it shields it from the immune system and can help it cross epithelial membranes and gain entry into the host. In the present manuscript, we report on our investigation regarding the mechanisms of Y. ruckeri’s invasion of host cells.ResultsA gentamycin assay was applied to two isolates, belonging to both the biotype 1 (ATCC 29473) and biotype 2 (A7959–11) and using several cell culture types: Atlantic Salmon Kidney, Salmon Head Kidney and, Chinook salmon embryos cells at both low and high passage numbers. Varying degrees of sensitivity to Y. ruckeri infection were found between the cell types and the biotype 1 strain was found to be more invasive than the non-motile biotype 2 isolate. Furthermore, the effect of six chemical compounds (Cytochalasin D, TAE 226, vinblastine, genistein, colchicine and, N-acetylcysteine), known to interfere with bacterial invasion strategies, were investigated. All of these compounds had a significant impact on the ability of the bacterium to invade host cells. Changes in the concentration of bacterial cells over time were investigated and the results suggested that neither isolate could survive intracellularly for sustained periods.ConclusionsThese results suggest that Y. ruckeri can gain entrance into host cells through several mechanisms, and might take advantage of both the actin and microtubule cytoskeletal systems.

Effects of siRNA silencing on the susceptibility of the fish cell line CHSE-214 toYersinia ruckeri

“…This study made use of CHSE-214 cells. The identity of this cell line derived from Chinook Salmon Embryo has been confirmed by PCR and sequencing and their suitability for gentamycin assay has been confirmed before (6). CHSE-214 cells were grown in 24 well plates at 20°C supplied with 1250 µl Minimum Essential Medium (MEM-glutaMAX™, Gibco) containing 2% FBS, where they reached about 80% confluence one day after seeding.…”

“…Notably, this Rac1 suppressive activity is most likely to hinder phagocytosis mediated killing (43) while intracellular invasion with the zipper pathway relies on the CDC42-independent activation of Rac1 (44). Moreover; in a previous experiment, we investigated the effect of multiple chemical and found out that the inhibitor of Rac1 N-acetylcysteine completely inhibited entrance of Y. ruckeri into CHSE-214 cells (6).…”

“…The gentamycin assay was performed as previously described (6). Briefly, Y. ruckeri ATCC 29473 was cultivated overnight in brain heart infusion (BHI, Oxoid).…”

“…An important feature of both invasion mechanisms is that they require active uptake of the bacterium by the cell and it is possible to prevent host cells from internalising the bacteria, for example by treating them with chemical blockers (24). This is for example the case for Y. ruckeri and several chemical blockers have been shown to prevent bacterial invasion (6, 7).…”

“…The bacterium has zoonotic potential and has been associated with topical infections in humans (4). Like several other enterobacteriaceae including other members of the genus Yersinia, Y. ruckeri has demonstrated the ability to survive within macrophages (5) as well as invade non-professional phagocytic cells (6, 7). This likely allows the access to some restricted nutrients and protects it from the immune system, moreover it might play a role in the bacterium’s crossing of epithelial membranes as known from other members of the genus Yersinia (8).…”