T6SS-Mediated Molecular Interaction Mechanism of Host Immune Response to Rahnella aquatilis Infection in Fish

Rahnella aquatilis is an emerging pathogen in fish that poses a potential risk to human and public health. However, its pathogenicity and molecular interaction mechanism with the fish host are still poorly understood. For this study, we conducted analyses into the artificial infection, bacterial load, histopathological observation, and molecular characterization of T6SS, as well as its mediated host immune response to R. aquatilis infection. The results showed that the R. aquatilis KCL-5 strain had high pathogenicity in teleosts, such as the cyprinid fish crucian carp Carassius auratus and the zebrafish Danio rerio, as well as a macrophage infection model that was successfully established, both in vivo and in vitro. A significant time-dependent increase in bacterial distribution in the infected tissues of crucian carp was examined using real-time qPCR and immunohistochemical analysis. The recombinant plasmid pET32a-hcp of T6SS was constructed and the fusion protein was of the expected size of 35.9 kD, as shown by SDS-PAGE and Western blot analysis. Moreover, the single-cell identification of kidney-derived Mφ/Mo cells was achieved, defined with the potential cellular marker gene expression in each cell and the genes’ expression of bacterial chemotaxis and flagellar assembly, inflammation, and PRRs, as well as the T6SS-mediated interaction between fish host cells and KCL-5, which was verified by multi-omics analysis. To our knowledge, this is the first report of T6SS/PAMPs-PRRs pathways related to the emerging R. aquatilis pathogen–host interaction mechanism in fish.