Avendaño-Herrera, Rubén

Oliver, Cristian

Sanchez, Fabián

Sanchez, Patricio

Sanhueza, Constanza

Valenzuela, Karla

Yáñez, Alejandro

English

University of Maine

 DETERMINATION OF PRO-IMMFLAMATORY CYTOKINES EXPRESSION IN SALMONIDS MACROPHAGES INDUCED BY OUTER MEMBRANE VESICLES PURIFIED FROM PISCIRICKETTSIA SALMONIS. Cristian Oliver 1,2,5, Karla Valenzuela 2,3, Constanza Sanhueza 2 Fabián Sánchez 2, Patricio Sánchez 2, Alejandro Yáñez 2,4,5, Rubén Avendaño-Herrera 1,5 1 Departament of Biological Sciences, Universidad Andrés Bello, Santiago, Chile. 2 Department of Biochemistry and Microbiology, Universidad Austral de Chile, Valdivia, Chile. 3Microbiology and Immunology Department, Dalhousie University, Halifax, Nova Scotia, Canada. 4Austral-OMICS, Faculty of Sciences, Universidad Austral de Chile, Valdivia, Chile 5Interdisciplinary Center for Aquaculture Research INCAR, Concepcion, Chile  ABSTRACT Piscirickettsia salmonis is a Gram-negative intracellular bacterium that causes Piscirickettsiosis in salmonids farms in Chile. It was recently reported that P. salmonis produces exotoxins that play a role in the pathogenesis. However a delivery system has not yet been identified. Outer membrane vesicles (OMVs) are 10-300 nm spherical-bilayer structures discharged from the surface of many Gram-negative bacteria, which are able to deliver toxins and virulence factors. Recently, we described the production of OMVs by P. salmonis in both, in normal growth in broth and inside the host cells after infection. However, pro-immflamatory cytokines expression induced by P. salmonis OMVs has not yet been characterized. Thus, the aim of this study was to investigate if OMVs purified from P. salmonis are able to generate an immflamatory immune response in fish macrophages.  P. salmonis was grown in basal broth supplemented with Cysteine (3.18 mM) and ferric chloride (0.05 mM) at 18°C until early stationary phase. Bacteria were removed by centrifugation (5000 x g, 10 min at 4°C) and the supernatant were filtrated through a 0.22-μm-pore-size filter. Then, vesicles present in the bacterial free supernatant were isolated by ultracentrifugation (125,000 x g, 2h at 4°C) and analyzed by SDS-PAGE. For gene expression analysis, specific primers for IL-1ß, IL-8 and TNF-α genes were designed to evaluate changes in gene expression in primary culture of macrophages isolated from head kidney. Thus, 1x106 cells were incubated with 5, 10 or 20 µg of purified OMVs and gene expression was evaluated by real time PCR. 18S and EF-1α were used as reference genes for normalization of the relative transcription levels.   P. salmonis is able to produce OMVs. The purified OMVs were able to generate a strong pro-immflamatory immune response in fish macrophages. Thus, IL-1ß, IL-8 and TNF-α genes were differentially expressed in fish macrophages after incubation with P. salmonis OMVs, suggesting a potential role in fish immune cells. Additionally, P. salmonis OMVs could be a potential candidate for vaccine development against Piscirickettsiosis.  KEYWORDS Piscirickettsia salmonis, SRS, OMVs, Proteome, fish immune response  §Corresponding author. Tel.: +56 91596891; Fax: +56 63 2221332 E-mail address: ayanez@uach.cl  

DETERMINATION OF PRO-IMMFLAMATORY CYTOKINES EXPRESSION IN SALMONIDS MACROPHAGES INDUCED BY OUTER MEMBRANE VESICLES PURIFIED FROM PISCIRICKETTSIA SALMONIS

https://digitalcommons.library.umaine.edu/cgi/viewcontent.cgi?article=1267&amp;context=isfsi

DETERMINATION OF PRO-IMMFLAMATORY CYTOKINES EXPRESSION IN SALMONIDS MACROPHAGES INDUCED BY OUTER MEMBRANE VESICLES PURIFIED FROM PISCIRICKETTSIA SALMONIS

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