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

Abstract

<jats:title>Abstract</jats:title><jats:p>Bacterial pathogens are known to co-opt mechanisms of the host cells’ physiology to gain intracellular entrance. Among the facultative intracellular bacteria is <jats:italic>Yersinia ruckeri</jats:italic>, an enterobacterium mostly known as the causative agent of enteric redmouth disease in salmonid fish. In the present study, we applied RNA inhibition to silence twenty pre-selected genes on the genome of a fish cell line (CHSE-214) followed by a gentamycin assay to quantify the effect of this silencing on the susceptibility of the cells to infection. It was found that silencing of 16 out of 20 genes significantly reduced the number of <jats:italic>Y. ruckeri</jats:italic> recovered at the end of the gentamycin assay. Among the genes with the strongest impact were Rab1A, actin and Rac1, which is consistent with our previous findings that N-acetylcysteine, a chemical inhibitor of Rac1, completely prevented invasion of cells by <jats:italic>Y. ruckeri</jats:italic>. Conversely, silencing of the Rho GTPase activating protein had no statistically significant effect, possibly because <jats:italic>Y. ruckeri</jats:italic>, like some other members of the <jats:italic>Yersinia</jats:italic> genus is able to activate Rho GTPase directly. Similarly, the effect of silencing E-cadherin was not statistically significant, suggesting that this might not be a target for the adhesion molecules of <jats:italic>Y. ruckeri</jats:italic>. Taken together, these findings improve our understanding of the infection process by <jats:italic>Y. ruckeri</jats:italic> and of the interactions between this bacterial pathogen and host cells.</jats:p><jats:sec><jats:title>Importance</jats:title><jats:p>Intracellular invasiveness is a mean for bacterial pathogen to gain shelter from the immune system as well as access nutrients. The enterobacterium <jats:italic>Y. ruckeri</jats:italic> is well characterised as a facultative intracellular pathogen. However, the mechanisms of invasion scrutiny. Investigations have mostly focused on the bacterial virulence rather than on the host’s mechanisms hicjacked during invasion. The present findings therefore allow us to better understand the interaction between this important potentially zoonotic pathogen of fish and host cells in vitro.</jats:p></jats:sec>

Effects of Yersinia ruckeri invasion on the proteome of the Chinook salmon cell line CHSE-214

“…However, like Y. enterocolitica and Y. pseudotuberculosis, it is a known facultative intracellular pathogen 3,4 . Recently, it was shown that silencing of the integrin β-1 precursor in an in-vitro culture of Chinook Salmon embryo cell culture (CHSE) resulted in a significant reduction of the cells' suceptibility to invasion by Y. ruckeri 44 and it is therefore very plausible that Y. ruckeri can make use of the zipper mechanism to invade host cells. In this context, the fact that several adhesion molecules, and in particular integrin molecules were detected at a lower level was unexpected.…”

Principle, application and challenges of development siRNA-based therapeutics against bacterial and viral infections: a comprehensive review

“…Some bacterial pathogens like Mycobacterium tuberculosis (MTB), Listeria monocytogenes, Mycobacterium fortuitum, S. typhimurium, and Yersiniaceae need host cell facilities for entry and invasion (Agaisse et al, 2005). Inactivating the invasion related genes like SEC22A, Rab1B, and VPS33B by siRNA can prevent bacterial cell entrance to the host cells (Menanteau-Ledouble et al, 2020). In our recent in vitro study, we designed specific siRNA against urease B subunit (ureB) and cytotoxinassociated gene A (CagA) genes from Helicobacter pylori.…”

“…Menanteau-Ledouble et al, reported that siRNA targets and silence zipper and trigger processes related to genes in Yersinia ruckeri, the causative agent of enteric red mouth in fish. They reported that Rab1A, myotubularin, Lama2, and Rac1 were more strongly silenced by siRNA (Criss and Casanova, 2003;Menanteau-Ledouble et al, 2020). Silencing of Caveolin-2 as a host gene involved in the invasion of Pseudomonas aeruginosa by siRNAs leads to the reduction of bacterial pathogenesis (Zaas et al, 2005).…”