Outer membrane vesicles from Piscirickettsia salmonis induce the expression of inflammatory genes and production of IgM in Atlantic salmon Salmo salar.

Piscirickettsiosis outbreaks due to Piscirickettsia salmonis occur globally in the Chilean salmon aquaculture generating significant monetary losses in the industry. P. salmonis secretes outer membrane vesicles (OMVs) which are naturally non-replicating and highly immunogenic spherical nanoparticles. P. salmonis OMVs has been shown to induce immune response in zebrafish; however, the immune response induced by these vesicles in salmonids has not been evaluated. In this study, we inoculated Atlantic salmon with 10 and 30 µg doses of P. salmonis OMVs and took samples for 12 days. qPCR analysis indicated an inflammatory response. Thus, the inflammatory genes evaluated were up- or down-regulated at several times in liver, head kidney and spleen. In addition, the liver was the organ most immune-induced, mainly in the 30 µg-dose. Interestingly, co-expression of pro- and anti-inflammatory cytokines was evidenced by the prominent expression of il-10 at day 1 in spleen and also in head kidney on days 3, 6 and 12, while il-10 and tgf-ß were up-regulated on days 3, 6 and 12 in liver. Importantly, we detected the production of IgM against proteins of P. salmonis in the serum collected from immunized fish after 14 days. Thus, 40 and 400 µg OMVs induced the production of highest IgM levels; however, no statistical difference in the immunoglobulin levels produced by these OMVs doses were detected. The current study provides evidence that OMVs released by P. salmonis induced a pro-inflammatory responses and IgM production in S. salar, while regulatory genes were induced in order to regulate their effects and achieve the balance of the inflammatory response.

The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins.

Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs) released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain.