Abstract
Piscirickettsia salmonis is the causative agent of Piscirickettsiosis, a systemic infection of salmonid fish species. P. salmonis infects and survives in its host cell, a process that correlates with the expression of virulence factors including components of the type IVB secretion system. To gain further insights into the cellular and molecular mechanism behind the adaptive response of P. salmonis during host infection, we established an in vitro model of infection using the SHK-1 cell line from Atlantic salmon head kidney. The results indicated that in comparison to uninfected SHK-1 cells, infection significantly decreased cell viability after 10 days along with a significant increment of P. salmonis genome equivalents. At that time, the intracellular bacteria were localized within a spacious cytoplasmic vacuole. By using a whole-genome microarray of P. salmonis LF-89, the transcriptome of this bacterium was examined during intracellular growth in the SHK-1 cell line and exponential growth in broth. Transcriptome analysis revealed a global shutdown of translation during P. salmonis intracellular growth and suggested an induction of the stringent response. Accordingly, key genes of the stringent response pathway were up-regulated during intracellular growth as well as at stationary phase bacteria, suggesting a role of the stringent response on bacterial virulence. Our results also reinforce the participation of the Dot/Icm type IVB secretion system during P. salmonis infection and reveals many unexplored genes with potential roles in the adaptation to intracellular growth. Finally, we proposed that intracellular P. salmonis alternates between a replicative phase and a stationary phase in which the stringent response is activated.
Highlights
Piscirickettsia salmonis, the causative agent of Piscirickettsiosis, known as Salmon Rickettsial Septicemia (SRS) was first identified as a pathogenic agent in disease outbreaks among farmed Coho salmon (Oncorhynchus kisutch)
The SHK-1 cell line derived from Atlantic salmon (S. salar) macrophages (Dannevig et al, 1997) was selected because it represents a cell type that is infected by this bacterium in vivo (McCarthy et al, 2008; Rojas et al, 2009)
Given that cytopathogenicity of stationary phase P. salmonis to SHK-1 cells was significantly higher (>60%) when compared to exponential phase bacteria (Supplementary Figure 2), bacteria grown for 4 days were used for the infection assays
Summary
Piscirickettsia salmonis, the causative agent of Piscirickettsiosis, known as Salmon Rickettsial Septicemia (SRS) was first identified as a pathogenic agent in disease outbreaks among farmed Coho salmon (Oncorhynchus kisutch). P. salmonis produces a systemic infection characterized by the colonization of several organs including kidney, liver, spleen, intestine, brain, ovary, and gills (Fryer et al, 1992) This bacterium was initially isolated in 1989 from a moribund coho salmon, during an epizootic event that took place in the south of Chile (Fryer et al, 1990; Branson and Nieto Diaz-Munoz, 1991; Cvitanich et al, 1991). P. salmonis virulence factors are poorly characterized the expression of four components of the type IVB secretion system during bacterial infection has been reported by Gómez et al (2013) This is a major secretion system that can translocate virulence factors (effectors) into the host cell to subvert the host signaling pathways (Chandran Darbari and Waksman, 2015). Other reports have addressed different aspects of P. salmonis interaction with host immune cells (Isla et al, 2014; Ramírez et al, 2015; Salazar et al, 2015); further studies are required for unraveling the pathogenic mechanisms of P. salmonis
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