Molecular cloning and characterisation of the rock bream, Oplegnathus fasciatus, Fas (CD95/APO-1), and its expression analysis in response to bacterial or viral infection

Abstract

Fas belongs to the tumour necrosis factor (TNF) receptor superfamily and can transmit a death signal leading to apoptosis. In the present study, we isolated the full-length cDNA for rock bream (Oplegnathus fasciatus) Fas (RbFas). The full-length RbFas cDNA was 1770 bp long and contained an open reading frame of 957 bp that encoded 319 amino acid residues with a predicted molecular mass of 35.1 kDa. The 319 amino-acid predicted RbFas sequence is homologous to other Fas sequences, contains three cysteine-rich domains and a death domain (DD) and two potential N-glycosylation sites. Expression of RbFas mRNA was detected in nine different tissues from healthy rock bream and was the highest in red blood cells. In analyses of mitogen-stimulated RbFas expression in peripheral blood leucocytes, expression of RbFas mRNA was observed between 1 and 36 h after stimulation with LPS, and 1 and 3 h stimulation with poly I:C. In the case of bacterial injection, the RbFas transcript peaked 6 h after injection in both the kidney and the spleen. Otherwise, the RbFas transcript peaked after 1 h in spleen and 6 h in kidney following injection with RSIV.

1. Introduction

Apoptosis, or programmed cell death, is the most common form of eukaryotic cell death, and it occurs during embryogenesis, metamorphosis, tissue atrophy and normal cell turnover [1]. Chemical agents and pathogenic infections accelerate apoptosis as it acts as an immune response in the host defence system [2]. The cytotoxicity of cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells is mediated by apoptosis [3]. Apoptosis is characterised morphologically by cell shrinkage with nuclear fragmentation and biochemically by chromatin cleavage into nucleosomal oligomers [4]. Cell components and chromatin form apoptotic bodies and are removed efficiently by neighbouring macrophages and granulocytes [1,5,6]. Thus, apoptosis is regulated to maintain immunological homoeostasis.

During the selection of immature T cells in the thymus, CTLs induce apoptosis through the Fas ligand (FasL) system against cells that react as self-antigens or are not able to recognise self-MHC molecules [7–9]. The cells that react to self-antigens attack host tissues and cause autoimmune diseases [10]. Additionally, the affinity of the T-cell receptor for the MHC molecule is essential to recognise the presentation of antigens [11].

Fas belongs to the tumour necrosis factor (TNF) receptor superfamily and can transmit a death signal leading to apoptosis [12]. The interaction between Fas and FasL has been investigated in a variety of cell lines in vitro, and the findings of these studies suggest that the binding of FasL to Fas on the target cell induces a death signal that initiates apoptosis [13]. The intracellular portion of Fas contains a protein-interaction motif termed the death domain. Death domains are found in other pro-apoptotic receptors such as TNF receptor I and DR 3–5, as well as in their adaptor proteins such as FADD, TRADD, RIP and RAIDD [12]. During Fas-mediated apoptosis, FADD binds to the Fas death domain and recruits procaspase 8, which is an apical protease for inducing apoptosis [14,15].

Fas is constitutively expressed by a broad range of normal epithelial cells and various haematopoietic cells. Notably, some tumour cells such as those of adult T-cell leukaemia, acute myelogenous leukaemia, chronic lymphocytic leukaemia, hepatocellular carcinoma and colon carcinoma abnormally over- and under-express Fas [16–19] and some of these virus-infected cells are sensitive to Fas-mediated apoptosis. The expression of Fas appears to be induced by interferon γ and CD40, which indicates that the expression of Fas is controlled by certain mechanisms [20,21].

When CTLs recognise antigens derived from the virus by the MHC molecule, they express the FasL and induce apoptosis [22]. Thus, the FasL plays an important role in maintaining homoeostasis in the immune system by inducing apoptosis. Several members of the TNF superfamily and TNF superreceptors family have been cloned in fish [23–38]. Although a Fas molecule has been reported in zebrafish and medaka [29,30], expression analyses in rock bream has not been reported. Molecular cloning and characterisation of the Fas should contribute to elucidating the mechanism of innate immunity in rock bream. Here we report the molecular cloning and sequence analysis of a Fas gene from rock bream and its expression in relation to infection by Streptococcus iniae or iridovirus.

2. Materials and methods

Fas cDNA was identified by analysing expressed sequence tags in the cold shock-stimulated rock bream erythrocytes library. The full-length cDNA of RbFas was obtained by 5′ RACE. The 5′ and 3′ ends of the RbFas cDNA were identified by the RACE cDNA amplification kit (Clontech, CA, USA) according to sequence information from the obtained fragment. A specific primer set (RbFas-5R and RbFas-3F) was designed according to the known EST sequences of RbFas (Table 1). Briefly, total RNA was extracted from red blood cells using Trizol Reagent (Invitrogen, USA) and first strand cDNA was synthesised using the protocol recommended by the SMART RACE cDNA Amplification Kit. The primer set of lRbFas-3F and a nested universal primer (supplied by Clontech) was used for 3′ RACE, and 5′ RACE was carried out with RbFas-5R and the nested universal primer. The generated PCR products were purified, cloned into pGEM T-easy vector system (Promega, USA) and subsequently sequenced. Thus the complete cDNA of RbFas was compiled by the 5′ and 3′ RACE DNA sequences.

Table 1.

Primers used in this study.

Primer name	Sequence (5′–3′)
For RACE PCR
RbFas-5R	CCA TCC AAT CAC TTC TGC AAT
RbFas-3F	CAG CTG ATC CGG AGA GAC AG
For RSIV quantification
MCPL sense	CCC TAT CAA AAC AGA CTG GC
MCPL anti-sense	TCA TTG TAC GGC AGA GAC AC
MCPS sense	CTG CGT GTT AAG ATC CCC TCC A
MCPS anti-sense	GAC ACC GAC ACC TCC TCA ACT A
For qRT-PCR amplification
RbFas-F	GTT TCG TGC GTC GTT TAT CA
RbFas-R	CAA ACC TGC AGC ACA CAG ACA
β-actin F	GGA CAC GGA AAG GAT TGA CA
β-actin R	CGG AAT TAA CCA GAC AAA TC

The determined nucleotide and deduced amino acid sequences and multiple sequence alignments were analysed with GENETYX ver. 8.0 (SDC Software Development Co. Ltd., Tokyo, Japan). The signal peptide was predicted using the online SignalP 3.0 programme (http://www.cbs.dtu.dk/services/SignalP/) [31] and domain identification was analysed with the PROSITE and SMART programmes (http://smart.embl-heidelberg.de/). The phylogeny was inferred using the Mega 4 programme and distance analysis by the neighbour-joining (NJ) method [32]. The values supporting each node were derived from 2000 re-samplings.

The RbFas mRNA expression levels were analysed by quantitative real-time PCR using gene-specific primers (Fig. 1). β-Actin was amplified as a control using β-actin F and β-actin R primers [33]. Tissue-specific mRNA expression was analysed in healthy rock bream gill, intestine, head kidney, trunk kidney, liver, peripheral blood leucocytes (PBLs), erythrocytes and spleen. More specifically, RNA isolated from these tissues was reverse transcribed into cDNA using a First-Strand cDNA Synthesis Kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK). For analyses of expression in mitogen-stimulated PBLs, PBLs were isolated from five fish and pooled. The PBLs were prepared as described previously [26]. Total RNA was purified from PBLs stimulated with LPS (500 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA) or poly I:C (5 μg/ml) (Sigma-Aldrich). For the bacterial challenge experiment, S. iniae (FP5228) was obtained from the Fish Pathology Division, National Fisheries Research & Development Institute (Pusan, Republic of Korea). For bacterial infection with S. iniae (3×10⁸ cells/fish) by intraperitoneal injection, sublethal doses were suspended in phosphate-buffered saline buffer. For viral infection, iridovirus was isolated from rock bream farmed in the Republic of Korea and propagated and titrated as previously described [34]. Experimental challenges were conducted on 100 fish (approximately 11–13 cm in body length) with a dose of 1×10⁶ copies/fish iridovirus administered by intraperitoneal injection. Kidneys and spleens were taken from five fish at 1, 3, 6, 12, 24 and 36 h postinfection (pi) and frozen at –80 °C for RNA extraction.

Fig. 1.

Nucleotide and deduced amino acid sequences of the rock bream Fas (RbFas). The active cysteine and other two cysteine residues, Cys⁷¹ and Cys⁷⁵, are in bold and the predicted signal peptide is underlined. The death domain predicted by the SMART programme is shaded and the polyadenylation signal AAUAAA is italicised.

The cDNAs were synthesised for real-time PCR from stimulated and non-stimulated leucocytes. The threshold cycle (Ct) values were automatically calculated based on the cycle when the fluorescence of the sample exceeded a threshold level that corresponded to 10 standard deviations from the mean of the baseline fluorescence. Amplification was performed as follows: 1 cycle at 94 °C for 2 min and 30 cycles at 94 °C for 30 s, 59 °C for 30 s and 72 °C for 1 min, with a final extension step at 72 °C for 5 min. Thermal cycling and fluorescence detection were conducted using the Thermal Cycler DICE Real Time System (TaKaRa, Tokyo, Japan). All data are given in terms of the amount of RbFas mRNA relative to that of β-actin mRNA, expressed as the mean±standard error of the mean (SEM). The results were subjected to t-test analysis, and P-values less than 0.05 were considered to be statistically significant.

3. Results and discussion

The full-length Fas sequence was isolated from a rock bream RBC cDNA library, and the sequence was named RbFas. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession number AB619804. The full-length RbFas cDNA was 1770 bp long and contained an open reading frame of 957 bp that encoded 319 amino acid residues with a predicted molecular mass of 35.1 kDa. Two potential N-glycosylation sites, ⁶⁵NLT and ¹⁶⁵NHS, which are present in many Fas genes from other species [21], were identified in RbFas (Fig. 1). The in silico analyses of RbFas revealed hydrophobic amino acids at the N-terminus, which likely represent the protein's signal peptide, one transmembrane domain signature in the middle portion and a death domain in the cytoplasmic region (Fig. 2). Death receptors exhibit an intracellular death domain (DD), which is essential for transduction of the apoptotic signal. When the sequence of the intracellular component of human Fas was compared with that of TNF-R1, a homologous region of 68 amino acids was identified. Moreover, using deletion and point mutagenesis, Tartaglia et al. [35] defined a region of TNF-R1 that was essential for the cytotoxicity mediated by the receptor. This stretch was 80 amino acids long and comprised the domain described by Itoh and Nagata [36], the DD. Several years later, additional DD-containing receptors were isolated (TRAMP, TRAIL-R1, TRAIL-R2 and DR6). Additionally, further intracellular DD-containing proteins were found, two of which bind to Fas: FADD/Mort1 [37,38] and RIP [39]. The TNFR superfamily contains several CRDS, 30–40-amino-acid regions, each containing approximately six cysteine residues [40]. The region of RbFas encoding the putative extracellular domain contained three cysteine-rich domains, which is in accordance with the corresponding region in Atlantic salmon, Japanese medaka and the zebrafish Fas genes [29,30]. Members of this family are characterised by two–five copies of the cysteine-rich extracellular repeats domain.

Fig. 2.

Amino acid sequence alignment of RbFas (predicted), Atlantic salmon Fas, Japanese medaka Fas, and zebrafish Fas. Identical (*) and similar (•) residues identified by Clustal W are indicated. The three cysteine-rich domains (CRDs) and transmembrane (TM) are boxed. Extracellular cysteines are shown in bold face. The death domains are shown in bold and italicised.

The accession numbers of the template sequences used to construct the phylogenetic tree are provided in Fig. 3. A NJ tree was constructed using the ClustalW and MEGA 4 programmes based on the amino acid sequences. The TNFR superfamily members from human and Fas genes from animals were obtained from GenBank and subjected to phylogenetic analysis. RbFas fell in the vertebrate cluster, within which RbFas was further grouped into a subcluster distinct from the sub-cluster formed by Fas of higher vertebrates, while RbFH formed another teleost Fas group. This grouping was well supported by bootstrapping.

Fig. 3.

Neighbour-joining tree of Fas and TNFR superfamily members constructed with Mega 4. The bootstrap confidence values shown at the nodes of the tree are based on 2000 bootstrap replications. GenBank accession numbers of selected genes are indicated within brackets.

Real-time PCR analysis was used to investigate the mRNA expression of RbFas in different tissues with β-actin as an internal control. The RbFas transcripts were constitutively expressed in the tissues of red blood cells (RBCs), muscle, gill and liver, and to a lesser degree in the tissue of kidney and PBLs. The expression levels in RBCs, muscle, gill and liver were 98.1-, 8.7-, 5.4- and 4.3-fold higher, respectively, than that in the head kidney (Fig. 4). mRNA of the Fas is expressed in a variety of tissues, including thymus, liver, lung, heart and ovary of the adult mouse [41], and is observed in some human cell lines [42]. In birds, erythrocytes can perish rapidly via lysis or pyknosis [43], the latter being the most characteristic expression of programmed cell death or apoptosis [43–45]. Most recent experiments suggest that injured (anucleated) erythrocytes display phosphatidylserine on their surface [46–48], a key feature of apoptosis in nucleated cells. Under different pathological conditions, oxidative stress results in the activation of Fas and initiates the extracellular pathway of apoptosis in nucleated cells [49]. The possibility exists, given that teleost erythrocytes are nucleated, that these cells can undergo apoptosis or programmed cell death.

Fig. 4.

Expression of RbFas mRNA in various tissues of healthy rock bream. PBLs, RBC, head kidney, trunk kidney, spleen, liver, intestine, gill and muscle were examined. The data are presented as the mean relative ratio of RbFas/β-actin mRNA levels. Errors bars represent SEM (n=3).

During mitogen-stimulated PBL expression analyses, we observed the induction of RbFas expression between 1 and 36 h after the PBLs were stimulated with lipopolysaccharide (LPS) and from 1 and 3 h after the PBLs were stimulated with poly I:C (Fig. 5). Additionally, the mRNA expressions of the RbFas in the kidney and the spleen were examined under bacterial and viral challenge via real-time RT-PCR analysis. Symptoms of the disease were first apparent on approximately day 4 postinjection, and each pathogen was reconfirmed via PCR (red sea bream iridovirus, RSIV) and cell culture (bacteria). The experimental challenge of the rock bream with S. iniae or RSIV resulted in significant increases in the RbFas mRNA in the kidney and the spleen. In the case of bacterial injection, the RbFas transcript peaked 6 h after injection in both the kidney and the spleen (Fig. 6A). Otherwise, the RbFas transcript peaked after 1 h in spleen and 6 h in kidney following the injection with RSIV (Fig. 6B). LPS stimulates the polyclonal proliferation of salmonid lymphocytes, the respiratory burst and phagocytic activity of macrophages [50] and, in addition, it has adjuvant properties. It has been shown to elicit the expression of cytokines like IL-1 in channel catfish [51] and TNF α in rainbow trout [52]. Cell death pathway can be initiated by a variety of cytotoxic agents, such as LPS, which induce activation of pro-inflammatory cytokines, caspases and other signalling pathways that ultimately lead to apoptosis and cell death [53]. Poly I:C is a synthetic analogue of dsRNAs that are generally produced during virus reproduction and triggers antiviral responses in host cells [54]. Robert et al. reported that a single poly I:C challenge is sufficient to induce an acute increase in apoptosis [55]. The LPS activation systems have previously been used in the analysis of molecular determinants of fish leucocyte proliferation. In Paralichthys olivaceus PBLs, the expression of the FasL and TNFR increased after stimulation with LPS or poly I:C [26,28]. The typical histopathological features of acute RSIV infections are splenomegaly and the presence of enlarged basophilic cells in the spleen, gill, kidney, heart and liver [56]. Pathologically, the Fas and FasL system is involved in eliminating autoreactive immune cells, malignant cells or virally infected cells [12,57,58]. FasL-induced apoptosis is important in the elimination of virus-infected cells and cancer cells by NK cells and cytotoxic T lymphocytes [12,59]. The FasL and Fas system has been implicated in the nonspecific cytotoxic response of teleost fish. Jaso-Friedmann et al. demonstrated that tilapia nonspecific cytotoxic cells contain a cytosolic soluble FasL that is released after stimulation [60].

Fig. 5.

Quantitative real-time PCR analysis of RbFas expression in rockbream PBLs stimulated with LPS or poly I:C at 1, 3, 6, 12, 24 and 36 h. Error bars represent the SEM (n=3). Asterisk indicates a statistically significant difference (P<0.05) compared with the control (0 h).

Fig. 6.

Expression of RbFas in rock bream tissues in response to experimental challenges with Streptococcus iniae (A) and RSIV (B) modulation were determined by quantitative real time reverse transcriptase PCR at various times post-challenge. Error bars represent the SEM (n=3). Asterisk indicates a statistically significant difference (P<0.05) compared with the control (0 h).

We conclude that the RbFas cDNA encodes for a novel 319-amino-acid protein that is homologous with members of the TNFR superfamily and that it contains CRDs and a DD. Domain organisations and phylogenetic analysis strongly suggest that the rock bream gene identified in this study is a true orthologue for Fas. After bacterial and viral stimulation, the expression of RbFas was upregulated in kidney and spleen. The RbTRx1 expression profile after infection indicates that it is inducible and may be involved in rock bream immune response. Further studies on rock bream FasL and Fas would contribute to a better understanding of the fish immune system and might elucidate the fish apoptosis induction pathway.