Characterization and pathogenic role of outer membrane vesicles produced by the fish pathogen Piscirickettsia salmonis under in vitro conditions Cristian Oliver a,d,e , Karla Valenzuela a,b , Mauricio Hernández c , Rodrigo Sandoval a , Ronie E. Haro a , Ruben Avendaño-Herrera d,e , Juan G. Cárcamo a,c , Maite T. Villar f , Antonio Artigues f , Rafael Garduño b , Alejandro J. Yáñez a,c,e, * a Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile b Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada c Austral-OMICS, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile d Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Santiago, Chile e Interdisciplinary Center for Aquaculture Research (INCAR), Concepción, Chile f Department of Biochemistry and Molecular Biology, School of Medicine, University of Kansas Medical Center, Kansas City, KS, United States A R T I C L E I N F O Article history: Received 8 May 2015 Received in revised form 10 September 2015 Accepted 16 September 2015 Keywords: Piscirickettsia salmonis Outer membrane vesicles (OMV) Piscirickettsiosis Hsp60 Virulence A B S T R A C T Piscirickettsia salmonis is one of the major fish pathogens affecting Chilean aquaculture. This Gram- negative bacterium is highly infectious and is the etiological agent of Piscirickettsiosis. Little is currently known about how the virulence factors expressed by P. salmonis are delivered to host cells. However, it is known that several Gram-negative microorganisms constitutively release outer membrane vesicles (OMVs), which have been implicated in the delivery of virulence factors to host cells. In this study, OMVs production by P. salmonis was observed during infection in CHSE-214 cells and during normal growth in liquid media. The OMVs were spherical vesicles ranging in size between 25 and 145 nm. SDS-PAGE analysis demonstrated that the protein profile of the OMVs was similar to the outer membrane protein profile of P. salmonis. Importantly, the bacterial chaperonin Hsp60 was found in the OMVs of P. salmonis by Western-blot and LC–MS/MS analyses. Finally, in vitro infection assays showed that purified OMVs generated a cytopathic effect on CHSE-214 cells, suggesting a role in pathogenesis. Therefore, OMVs might be an important vehicle for delivering effector molecules to host cells during P. salmonis infection. ã 2016 Published by Elsevier B.V. 1. Introduction Piscirickettsia salmonis is a facultative intracellular Gram- negative bacterium that causes the systemic disease Piscirick- ettsiosis in farmed salmonids, resulting in significant economic losses for the Chilean salmon industry. This pathogen was initially isolated from Coho salmon (Oncorhynchus kisutch) in Chile and has since then also been reported in Canada, Ireland, Norway, and Scotland (Fryer and Hedrick, 2003). Piscirickettsiosis is an infection characterized by the bacterial colonization of several organs, including kidney, liver, spleen, intestine, and gills (Fryer and Hedrick, 2003). Despite that the route of entry of this pathogen is not completely understood, it is known that P. salmonis primarily infects and multiplies within macrophages (Rojas et al., 2009). Furthermore, it has been reported that clathrin is involved in the uptake of P. salmonis by SHK-1 cells, and, following internalization, this bacterium induces disruption of the actin cytoskeleton (Ramírez et al., 2015) and replicates inside vacuoles (Smith et al., 2010). Once cell apoptosis is induced, the bacteria are released and infect other cells (Rojas et al., 2010). However, the molecular mechanisms underlying the virulence of P. salmonis are not fully understood. Secretion systems are well-characterized virulence factors that bacteria exploit to deliver soluble effector proteins into the environment or directly into the host cell cytoplasm. In Gram- negative bacteria, secreted proteins are exported across the inner and outer membranes in a single step via the type I, type III, Type IV or type VI systems (Tsai-Tien et al., 2009). Recent research on the partial genome of a virulent P. salmonis strain identified genes from * Corresponding author at: Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile. E-mail address: ayanez@uach.cl (A.J. Yáñez). http://dx.doi.org/10.1016/j.vetmic.2015.09.012 0378-1135/ ã 2016 Published by Elsevier B.V. Veterinary Microbiology 184 (2016) 94–101 Contents lists available at ScienceDirect Veterinary Microbiology journa l homepage: www.e lsevier.com/loca te/vetmic