Identification and characterization of caspases genes in rainbow trout (Oncorhynchus mykiss) and their expression profiles after Aeromonas salmonicida and Vibrio anguillarum infection

Highlights

• Eighteen caspase genes including multiple copies were characterized in rainbow trout.

• A. salmonicida infection induced the CASPs-regulated intrinsic apoptosis.

• The casp8 exerted the greatest effect after V. anguillarum infection.

Abstract

Caspases are highly conserved cysteine-dependent aspartyl-specific proteases that play an important role in regulating cell death and inflammation. However, the caspase genes have not been systematically studied in rainbow trout (Oncorhynchus mykiss). Rainbow trout experienced 4 rounds (4R) of genome duplication in the evolutionary history. Thereby an increased numbers of paralogs are observed in trout, probably with more complicated gene functions. We identified 18 caspase genes in rainbow trout, including two inflammatory caspases (casp1a, casp1b), six apoptosis executioner caspases (casp3, casp3a1, casp3a2, casp3b, casp6, and casp7), nine apoptosis initiator caspases (casp2a, casp2b, casp8, casp9a, casp9b, casp10a, casp10b, casp20a, and casp20b) and one uncategorized caspase gene (casp17). To investigate the potentially physiological functions of caspase genes, we challenged the rainbow trout with Aeromonas salmonicida (A. salmonicida) and Vibrio anguillarum (V. anguillarum). Results showed that the CASP3-regulated intrinsic apoptosis was activated after A. salmonicida infection, while the CASP8 and CASP6-regulated extrinsic apoptosis exerted the greatest effect on trout challenged with V. anguillarum. In response to V. anguillarum infection, the data of RNA-Seq further showed the casp8 was tightly integrated with the significantly enriched Gene Ontology terms and functional pathways, including apoptosis regulation, pathogen detection and immunomodulation. Our study provides a foundation for the physiological functions and regulatory network of the caspase genes in teleosts.

Introduction

The caspases (CASPs), an evolutionarily conserved family of cysteine-dependent aspartate-directed proteases, play an important role in regulating programmed cell death and inflammatory responses (McIlwain et al., 2013; Shalini et al., 2015). The word “caspase” embodies two typically catalytic characteristics, in which the initial “c” indicates Cys protease and the following “aspase” refers to the ability to cleave after Asp (Kaushal et al., 1998). In 1989, the first caspase was described as CASP1, with the original name of interleukin 1-beta-converting enzyme (ICE) (Yuan et al., 1993). Subsequently, the Nedd2, a cysteine protease which is similar to ICE, was renamed as CASP2 (Butt et al., 1998; Kumar et al., 1994). Totally, 18 caspase genes have been identified in mammals with species-dependent expression patterns (Shalini et al., 2015). For example, the human (Homo sapiens) caspases gene family includes casp1 to casp10, casp12, casp14 and casp16, while casp11 and casp13 are identified as murine and bovine orthologues of human casp4, respectively (Shalini et al., 2015). The newly identified casp15, casp17 and casp18 are absent in human and other placental mammals (Shalini et al., 2015).

Based on physiological functions, mammalian caspases have been classified as apoptosis initiators (casp2, casp8, casp9 and casp10), apoptosis executioners (casp3, casp6 and casp7) and inflammatory caspases (casp1, casp4, casp5, casp11 and casp12) (Spead et al., 2018). The initiator caspases could be triggered by either the apoptosome-regulated intrinsic apoptosis or the death receptor-regulated extrinsic apoptosis (Shalini et al., 2015). During intrinsic apoptosis, the death-inducing stimuli, such as cellular stresses, results in apoptosome activation, further activating apoptosis initiators such as CASP9 (Czabotar et al., 2014). Death receptor-regulated extrinsic apoptosis is initiated by tumor necrosis factor receptor (TNFR) signaling. Activation of the TNFR signaling eventually forms a complicated complex, named TNFR associated death domain protein (TRADD)-dependent complex IIa, and promotes the recruitment and activation of apoptosis initiators, such as CASP8 and/or 10 (Gaur and Aggarwal 2003; Li et al., 1998). These apoptosis initiators subsequently promote apoptosis by stimulating the apoptosis executioners including CASP3, 6 and 7 (Shalini et al., 2015). The CASPs are also involved in the regulation of inflammation. Previous studies showed that CASP1 cleaves the prodomain of interleukin 1-β (IL-1β) and converts the immature IL-1β into the bioactive state (Lawson et al., 2013; Thornberry et al., 1992). The following studies further revealed that CASP8 also induces the active IL-1β (Maelfait et al., 2008). In contrast to CASP1, researches related to mice showed CASP12 inhibits the CASP1 functions, thus abrogating the inflammatory response (Scott and Saleh 2007).

The Aeromonas salmonicida (A. salmonicida) and Vibrio anguillarum (V. anguillarum) are two Gram-negative bacterial pathogens associated with severe fatal diseases in aquaculture (Frans et al., 2011; Rebl et al., 2014). A. salmonicida was firstly discovered in 1896 in brown trout hatchery (Liu et al., 2016). Subsequently, multiple studies reported that A. salmonicida results in severe furunculosis in economically important fishes including salmonid and non-salmonid species (Bricknell et al., 1999; Lago et al., 2012; Magarinos et al., 2011). V. anguillarum is the causative agent of vibriosis with the first discovery around 1900 (Bergman 1909; Canestrini 1893) (Frans et al., 2011). Up to now, more than 50 teleost species have been reported to be susceptive to V. anguillarum infection (Frans et al., 2011). Due to the high morbidity, mortality, and the resulting economic loss in aquaculture and larviculture (Frans et al., 2011; Long et al., 2015), substantial studies have already focused on genetics, physiology and pathophysiology of A. salmonicida or V. anguillarum (Altmann et al., 2016; Cao et al., 2019; Gao et al., 2016; Miest et al., 2012; Soto-Davila et al., 2019; Torrecillas et al., 2017).

Rainbow trout (Oncorhynchus mykiss) is one of the globally cultured salmonid fishes with total production over 800,000 tons in 2016 (FAO, 1940). However, bacterial diseases resulting from A. salmonicida or V. anguillarum cause high mortalities and great economic losses in both aquaculture and recreational angling industries worldwide (Frans et al., 2011; Menanteau-Ledouble et al., 2016; Wiklund and Dalsgaard 1998). A great number of these studies showed A. salmonicida or V. anguillarum infection results in apoptosis and inflammatory responses in teleosts (Altmann et al., 2016; Cao et al., 2019; Gao et al., 2016; Miest et al., 2012; Soto-Davila et al., 2019; Torrecillas et al., 2017). The caspases gene family plays an important role in the regulation of pathogen-induced apoptosis and inflammation (Shalini et al., 2015). However, most previous studies focused on mammalian caspase genes and studies regarding identification, characterization and physiology of teleost caspase genes are limited. A recent study reported that 19 different caspase genes are identified in zebrafish (Danio rerio) including the novel casp19a, casp19b, casp20, casp21, casp22, casp23 (Spead et al., 2018). Different from zebrafish, rainbow trout has experienced 4 rounds (4R) of whole-genome duplication in the evolutionary history and the 4R event results in duplications of the functional paralogs probably with novel functions (Alzaid et al., 2016; Macqueen et al., 2010). Therefore, rainbow trout has long been a model for the studies of novel gene identification and functions. One representative example is the expanded paralogs of insulin-like growth factor binding protein (IGFBP) in trout (Alzaid et al., 2016; Macqueen et al., 2010). In this study, our primary purpose is to identify the complete repertoire of caspases gene family in 4R-derived rainbow trout. Because the CASPs are closely associated with the pathogen-induced defense mechanisms and immune competences, the second purpose is to evaluate the expression profiles of caspases gene family in trout infected with A. salmonicida or V. anguillarum.