RNA-Seq-Based Analysis of Cortisol-Induced Differential Gene Expression Associated with Piscirickettsia salmonis Infection in Rainbow Trout (Oncorhynchus mykiss) Myotubes.

Rodrigo Zuloaga,Phillip Dettleff,Claudio Meneses,Macarena Bastias-Molina,Claudia Altamirano,Juan Antonio Valdés,Alfredo Molina

doi:10.3390/ani11082399

Abstract

Simple SummarySkeletal muscle is the most abundant tissue in fish and the main product of the Chilean salmonid aquaculture industry. Intensive farming conditions generate stress and increased susceptibility to infectious diseases, such as salmonid rickettsial septicemia (SRS), that directly affect this tissue. However, the immunocompetence of skeletal muscle during infection is poorly understood. To further explore the interplay between pathogen infection and stress on this tissue, we analyze the transcriptional profile of isolated rainbow trout (Oncorhynchus mykiss) muscle cells pretreated with 3 h of the stress hormone cortisol, and then infected with the SRS etiologic agent Piscirickettsia salmonis for 8 h, using RNA sequencing technology. For the first time, the obtained data reveals the biological processes related to programmed cell death, negative regulation of cell proliferation, and innate immune response. These results are validated by real-time qPCR. Furthermore, cortisol pretreatment significantly stimulated bacterial gene expression compared to infected cells. These data demonstrated that fish skeletal muscle can activate an intrinsic immune-like response against P. salmonis that is differentially regulated by cortisol. The information provided here will help us to understand the molecular mechanisms of fish muscle cells respond to infection, which could prevent P. salmonis outbreaks in skeletal muscle under stress conditions.Salmonid rickettsial septicemia (SRS) is the major infectious disease of the Chilean salmonid aquaculture industry caused by Piscirickettsia salmonis. Intensive farming conditions generate stress and increased susceptibility to diseases, being skeletal muscle mainly affected. However, the interplay between pathogen infection and stress in muscle is poorly understood. In this study, we perform an RNA-seq analysis on rainbow trout myotubes that are pretreated for 3 h with cortisol (100 ng/mL) and then infected with P. salmonis strain LF-89 for 8 h (MOI 50). Twelve libraries are constructed from RNA samples (n = 3 per group) and sequenced on Illumina HiSeq 4000. A total of 704,979,454 high-quality reads are obtained, with 70.25% mapped against the reference genome. In silico DETs include 175 total genes—124 are upregulated and 51 are downregulated. GO enrichment analysis reveals highly impacted biological processes related to apoptosis, negative regulation of cell proliferation, and innate immune response. These results are validated by RT-qPCR of nine candidate transcripts. Furthermore, cortisol pretreatment significantly stimulated bacterial gene expression of ahpC and 23s compared to infection. In conclusion, for the first time, we describe a transcriptomic response of trout myotubes infected with P. salmonis by inducing apoptosis, downregulating cell proliferation, and intrinsic immune-like response that is differentially regulated by cortisol.

Highlights

Piscirickettsiosis, called salmonid rickettsial septicemia (SRS), is one of the most important infectious diseases affecting the Chilean salmonid aquaculture industry [1].SRS is caused by Piscirickettsia salmonis, which is a gram-negative bacterium that is aerobic, non-encapsulated, generally non-motile, pleomorphic, but predominately coccoid, and facultative intracellular [2]
No P. salmonis was detected in the CTRL and CORT
Our results present high correlations in the group infected with P. salmonis (r = 0.808), in the group treated with cortisol (r = 0.756), and in the group pretreated with cortisol and infected with P. salmonis (r = 0.747), between the expression values of those candidate genes used in the RNA-seq and room temperature (RT)-qPCR

Summary

Introduction

Piscirickettsiosis, called salmonid rickettsial septicemia (SRS), is one of the most important infectious diseases affecting the Chilean salmonid aquaculture industry [1].SRS is caused by Piscirickettsia salmonis, which is a gram-negative bacterium that is aerobic, non-encapsulated, generally non-motile, pleomorphic, but predominately coccoid, and facultative intracellular [2]. Piscirickettsiosis, called salmonid rickettsial septicemia (SRS), is one of the most important infectious diseases affecting the Chilean salmonid aquaculture industry [1]. Among the mechanisms that P. salmonis uses to infect are secretion system type 4 deficient in organelle trafficking/intracellular multiplication (Dot/Icm), adhesion molecules (type 4 pili), and the inhibition of reactive oxygen species [5]. This septicemic disease affects a variety of salmonid species, such as Atlantic salmon (Salmo salar), Coho salmon (Oncorhynchus kisutch), and rainbow trout [6]. Rainbow trout is a salmonid of high commercial interest for the industry, with its skeletal muscle being the most important tissue in terms of aquaculture products [7]

Methods

Results

Discussion

Conclusion