Host factors contributing to Yersinia ruckeri´s invasiveness

Content of research project: The ability to invade and multiply inside non-professional phagocytic cells, as well as the related ability to survive phagocytosis are important features of the disease process for many bacterial pathogens, including fish pathogens. Studies conducted in human have shown that most facultative intracellular bacteria hijack various mechanisms of their host cells to gain intracellular entrance, however, virtually nothing is known about fish pathogens. Hypothesis: We selected 20 genes that are likely to be involved in the intracellular survival and multiplication of other bacterial pathogens. Our hypothesis is that several of these are also targeted by the pathogen of interest and that, by inhibiting the function of these genes, we will reduce the ability of the bacterium to invade and multiply inside the cells. Methods: In this project, we are planning on using cell cultures exposed first to chemical inhibitors, known to block broad cellular functions that are often used by bacterial pathogens to gain entrance into their host. Then, small inhibitory RNA will be used to inhibit the selected genes within the host genome. We will measure the effect of both of these inhibitory methods on the susceptibility of the cells to be infected by the bacterial fish pathogen Yersinia ruckeri. Furthermore, we will apply proteomics to investigate how the bacterium can change the profile of the proteins expressed by the infected cells. New and unique aspect of the project: This project is the first investigating which genes are being hijacked by fish bacterial pathogens to gain entry into the host cell. This subject has not yet been investigated in bacterial pathogens of fish. Furthermore, the use of proteomics to investigate how bacterial pathogens can alter the protein profile of the host cells is new and will rely on the use of a third generation proteomic apparatus, a recent development.

Yersinia ruckeri is an important bacterial fish pathogen that can gain refuge inside the cells of the fish it infects. This location protect the bacteria from both the immune system and some therapeutants. The mechanisms through which this infection occur have still not been clarified and while it is likely that it makes use of mechanisms similar to that of some of its relatives, Yersiniaceae harbour multiple such mechanisms. The present study was designed to improve our understanding regarding how Y. ruckeri gains entrance into the host cells and in particular how the bacterium takes advantage of mechanisms within the host`s cells. In the first step of the project, the suitability of the infection procedure was investigated for several fish cell lines in cell cultures using a gentamycin assay. Moreover, the effect of six different chemical blockers was investigated. Because each of these chemicals target a specific aspect of the host cells` physiology, this allowed us to determine some of the general mechanisms of entry: In particular, it was shown that addition of N-acetylcysteine to a final concentration of 5 mM completely inhibited the invasion process. N-acetylcysteine is an inhibitor of the molecule Rac1 that in turn is known to act as a regulator of both the actin and the microtubule cytoskeleton. Because inhibition of either of these host factor separately only had a limited effect on the bacterial invasion, these findings suggest that Y. ruckeri is able to make use of both the actin and cytoskeleton to gain entrance into the host`s cells. The next step was to investigate individual host genes. To do so, we designed small inhibitory RNA (siRNA) specific for each of twenty pre-selected host genes. Each time, Real Time PCR (RT-qPCR) was performed to confirm that the siRNAs were efficient at reducing the expression of their target. Afterwards, the effect of each silencing was investigated. Moreover, a trypan Blue assay was applied to confirm that this silencing had no toxic side effect on the cells. The findings of these tests showed that 18 out of 20 genes played a role in the cellular invasion. Once again, silencing of Rac1 had some of the strengest effect, alongside silencing of the gene encoding the Laminin subunit alpha-2 (Lama 2). Lama 2 is an adhesion molecule, expressed on the surface of the host cells and allowing them to attach to the basement membrane. In the past, it has been shown that Lama 2 was a site of attachment for Yersinia enterocolitica, a relative of Y. ruckeri as well as the Plasminogen Activator (Pla) of Y. pestis. Finally, the effect of bacterial infection on the protein profile of the fish cell culture was also investigated. This final section of the project allowed to identify 1614 different fish proteins, of which, 76 were found at significantly differently level in the presence of bacteria. Almost as many proteins were found at lower levels than at higher level and most of these proteins played a role in cell adhesion or cell metabolism while no protein involved in cellular immunity was found to be differentially expressed. This showed that the bacteria were able to affect the physiology of the host cells to a significant degree.