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Salmonid rickettsial septicemia (SRS) is a critical sanitary problem in the Chilean aquaculture industry since it induces the highest mortality rate in salmonids among all infectious diseases. Piscirickettsia salmonis, a facultative intracellular bacterium, is the biological agent of SRS. In Chile, two genogroups of P. salmonis, designated as LF-89 and EM-90, have been identified. Previous studies suggested that their cohabitation triggers the expression of virulence effectors, which may be related to a higher pathogenicity in salmonids during co-infection with both P. salmonis genogroups. Therefore, we aimed to evaluate if the physical contact between two isolates from LF-89 and EM-90 is necessary to activate this effect. Through a spatially separated in vivo co-culture inside Atlantic salmon (Salmo salar) post smolts and RNA-seq analysis, we compared the differentially expressed genes (DEGs) with previous results from an in vivo mixed co-culture. The results showed that although the LF-89-like isolate and the EM-90-like isolate had a similar DEG profile under both co-culture conditions, important virulence factors observed during the mixed co-cultures (i.e., flagellar-related genes, CydD, and NCS2) were absent in the spatially separated co-cultures. Hence, the synergistic effect linked to increased pathogenicity to the host may be driven by the physical co-localization and contact between the P. salmonis LF-89-like and EM-90-like isolates. [ABSTRACT FROM AUTHOR]

The Piscirickettsia salmonis genome was screened to evaluate potential flagella-related open reading frames, as well as their genomic organization and eventual expression. A complete and organized set of flagellar genes was found for P. salmonis, although no structural flagellum has ever been reported for this bacterium. To gain further understanding, the hierarchical flagellar cascade described for Legionella pneumophila was used as a reference model for putative analysis in P. salmonis. Specifically, 5 of the most relevant genes from this cascade were chosen, including 3 regulatory genes (fleQ, triggers the cascade; fliA, regulates the σ28-coding gene; and rpoN, an RNA polymerase-dependent gene) and 2 terminal structural genes (flaA and flaB, flagellin and a flagellin-like protein, respectively). Kinetic experiments evaluated gene expressions over time, with P. salmonis assessed in 2 liquid, cell-free media and during infection of the SHK-1 fish cell line. Under all conditions, the 5 target genes were primarily expressed during early growth/infection and were differentially expressed when bacteria encountered environmental stress (i.e. a high-salt concentration). Intriguingly, the flagellin monomer was fully expressed under all growth conditions and was located near the bacterial membrane. While no structural flagellum was detected under any condition, the recombinant flagellin monomer induced a proinflammatory response in SHK-1 cells, suggesting a possible immunomodulatory function. The potential implications of these observations are discussed in the context of P. salmonis biology and pathogenic potential.