Abstract

Introduction Piscirickettsia salmonis is a Gram-negative bacterium that causes Piscirickettsiosis in all farmed salmonids in Chile. Recently, it has been reported that P. salmonis produce exotoxins that play a role in the bacterial pathogenesis. OMVs are 10-300 nm spherical-bilayer structures discharged from the surface of many Gramnegative bacteria, which are able to deliver virulence factors into the host cell. Thus, the aim of this study was to investigate if P. salmonis is able to produce OMVs and to evaluate its role in pathogenesis in vitro. Methodology OMVs isolation: P. salmonis LF-89 (ATCC VR-1361) was grown in 400 mL of a minimal broth at 18°C until early stationary phase. Bacteria were removed by centrifugation and the resultant supernatant was filtered through a 0.2-mm/pore filter. Finally, OMVs were isolated by ultracentrifugation (125,000 x g, 2h at 4°C) and stored at -80°C until use. Electron microscopy: infected cells and isolated OMVs were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 2h at RT and postfixed with 1% osmium tetroxide in 0.1 M CB. All samples were visualized with a JEOL JEM-1320 transmission electron microscope. Cytotoxicity assay: CHSE-214 cells were incubated in 1 ml of OMVs-containing medium for 4 days at 18°C. Results and conclusions P. salmonis produces OMVs during infection in CHSE-214 cells and during normal growth in liquid media. Protein analysis demonstrates the pattern of OMVs were similar to outer membrane protein profile. Finally, infection assays indicate that purified OMVs induce cytotoxic effect in a salmonids cell line.FONDAP-INCAR 15110027.