Cloning and characterisation of natural resistance associated macrophage protein (Nramp) cDNA from red sea bream (Pagrus major)

Fish & Shellfish Immunology 17 (2004) 305e313 www.elsevier.com/locate/fsi

Cloning and characterisation of natural resistance associated macrophage protein (Nramp) cDNA from red sea bream (Pagrus major) Song-Lin Chen), Mei-Yu Xu, Xiang-Shan Ji, Guo-Cai Yu Yellow Sea Fisheries Research Institute, Chinese Academy of Fisheries, Sciences, Nanjing Road 106, Qingdao 266071, PR China Received 12 January 2003; accepted 31 March 2004

Abstract Nramp (natural resistance associated macrophage protein) controls aspects of innate resistance to intracellular parasites. Its function is to enhance the ability of macrophages to kill pathogens. However, little is known about the structure and function of Nramp in lower vertebrates such as teleosts. We have recently isolated a cDNA encoding Nramp from spleen of red sea bream (Pagrus major). The full-length cDNA of the Nramp is 4709 bp in length, including 197 bp 5#-terminal untranslated region (UTR), 1662 bp encoding region and 2850 bp 3#-terminal UTR (GenBank accession number: AY485311). The 1662 nt open reading frame was found to code for a protein with 554 amino acid residues. Comparison of the amino acid sequence indicated that red sea bream Nramp consists of 12 transmembrane region (TM) domains. A consensus transport motif (CTM) containing 20 residues was observed between transmembrane domains 8 and 9. The deduced amino acid sequence of red sea bream Nramp had 77.8%, 83.0%, 82.3%, 80.0%, 81.1%, 60.4%, 70.3%, 58.5% and 69.5% identity with that of rainbow trout Nramp a and b, channel catfish Nramp, fathead minnow Nramp, common carp Nramp, mouse Nramp 1 and 2, and human Nramp1 and 2, respectively. Reverse transcriptionepolymerase chain reaction indicated that levels of Nramp expression were similar among head kidney, spleen, intestine and liver in non-challenged red sea bream, and that challenge of red sea bream with the pathogenic bacterium, Vibrio anguillarum, significantly elevated Nramp mRNA levels in liver and spleen in a time-dependent fashion. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Red sea bream; Pagrus major; Nramp; cDNA; Expression

1. Introduction Antimicrobial peptides constitute important components of the innate immune system in many species, including plants, invertebrates and vertebrates [1e4]. Natural resistance associated macrophage protein ) Corresponding author. Tel.: +86-532-584-4606; fax: +86-532-581-1514. E-mail address: [email protected] (S.-L. Chen). 1050-4648/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2004.04.003

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(Nramp) mediates antimicrobial activity of macrophages against intracellular parasites during the early stages of infection [5]. In mice, Nramp1 was identified as one of the major factors controlling early phases of natural resistance to Mycobacterium bovis [6,7]. The genetics of resistance/susceptibility to M. bovis also demonstrated that a gene on murine chromosome (MMU) 1-the Bcg/lsh/Ity gene controls the early phases of natural resistance to these intracellular pathogens. Nramp1 gene was considered to encode a protein that consists of 12 transmembrane segments with one hydrophilic N-terminal region. Homologues of murine Nramp gene have been isolated in farmed animals [8,9]. In bovine, the Nramp1 was expressed primarily in macrophages, lung and spleen [8,9]. Expression of the Nramp enhances the ability of macrophages to kill intracellular pathogens, thus playing an important role in protecting these organisms against microbial invasion. So far, Nramp genes have been cloned in mouse [10], human [11], bovine [8] and pig [12]. However, few reports on the structure and function of Nramp in teleosts are available [13e15]. In this paper, we report isolation and structural analysis of the Nramp gene from red sea bream (Pagrus major) and its expression in various tissues in response to infection with pathogenic bacteria.

2. Materials and methods 2.1. Red sea bream and isolation of nucleic acids Red sea bream weighing 200e650 g were purchased from the Marine Fish Market (Qingdao, China). Total RNA was extracted using Trizol reagent (Qiagen) from tissues of red sea bream. Poly(A)+ RNAs were isolated from the total RNA using OligotexÔ mRNA midi kit (Qiagen). cDNA synthesis was carried out using a random primer as described [16]. 2.2. Primer design A pair of degenerate primers, Nramp D1 (5#-CTNTGGGCSTTYACNGGACC-3#) and Nramp D2 (5#-TGYAGGACGTACAYDCTRCC-3#), were designed according to conserved sequences of Nramp gene in other vertebrates (AF048760, AF048761, AF029758, D50403) and used to amplify a Nramp cDNA fragment of about 300 bp from red sea bream spleen. To isolate full-length Nramp cDNA, rapid amplification of the cDNA ends (5#-RACE and 3#-RACE) were carried out. Two specific primers (GSP5# primer and GSP3# primer) for red sea bream Nramp were designed according to the above amplified partial Nramp cDNA sequence. GSP5# primer (5#-TGTGACGACCCGACGGCGTGCAGCTAA-3#) was for amplification of the 5# end, and GSP3# primer (5#-GGGCCTTCACTGGACCAGGCTTTTTGATG-3#) was for the 3# end of red sea bream Nramp cDNA. The universal primers used for 5#-RACE and 3#-RACE were Long primer (5#-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3#) and Short primer (5#-CTAATACGACTCACTATAGGG-3#) (CloneTech). 2.3. Rapid amplification of cDNA ends Both 5#-RACE and 3#-RACE were carried out using a Smart RACE cDNA amplification kit (ClonTech) according to the manufacturer’s instructions. Touchdown polymerase chain reaction (PCR) was used for RACE amplification: 94 (C for 2 min, 94 (C for 5 s, 72 (C for 3 min, for 5 cycles; 94 (C for 5 s, 70 (C for 10 s, 72 (C for 3 min, for 5 cycles; 94 (C for 5 s, 66 (C for 10 s, 72 (C for 3 min, for 20 cycles; and 72 (C for 10 min for elongation. The amplified fragments were separated and purified with a NucleoTrap gel extraction kit (ClonTech). The purified fragments were then cloned into pMD18-T vector (Takara), propagated in Escherichia coli DH5a, and sequenced.

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2.4. Sequence analysis and alignment The DNA sequence data were analysed using DNASTAR 5.0 [17]. The alignment of the amino acid sequence of Nramp proteins was performed using ClustalX [18]. Sequences used for comparison and their GenBank accession numbers were as follows: trout Nramp a (AAD20721), trout Nramp b (AAD20722), channel catfish Nramp (AF400108), fathead minnow Nramp (AAF01778), common carp Nramp (CAB60196), mouse Nramp 1 (AAA39838), mouse Nramp 2 (AAC42051), human Nramp 1 (D50403), and human Nramp 2 (NP-000608). A phylogenetic tree was constructed using the neighbour-joining method of Saitou and Nei [19] and analysed using Mega 2 [20]. 2.5. Challenge and sampling Red sea bream weighing about 650 g were reared in tanks of 1000 l. The water temperature was kept at 23 (C. The bacterium, Vibrio anguillarum, was shown to be pathogenic to red sea bream [21]. The strain ATCC 19019 of V. anguillarum was used for infection experiments in the present study. The bacteria was cultured at 28 (C to mid-logarithmic growth in medium 2216E consisting of peptone 5.0 g, yeast extract 1.0 g, FePO4
4H2O 0.1 g, sea water 1000 ml, pH 7.6. The absorbance at 600 nm of the bacterial suspension was determined and the bacteria were resuspended to approximately 4.6!109 colony-forming units (cfu) ml ÿ1 in phosphate-buffered saline (PBS). Eighteen red sea bream were anaesthetised by immersion in MS 222 and injected intraperitoneally with 0.5 ml bacteria suspension. Uninfected fish were maintained in separate tanks as control. Two infected fish were killed at 5, 24, 48, 72 and 96 h after infection, respectively. Tissues (liver, spleen, head kidney and intestine) were removed and kept at ÿ80 (C until use. 2.6. Reverse transcription (RT)ePCR analysis of Nramp expression in different tissues Total RNA from head kidney, spleen, liver and intestine were extracted with Trizol Reagent (Qiagen). The reverse transcription of mRNA was performed according to the reported method [16]. A pair of gene-specific primers, RSB-NrampN1 (CCAGTATCCCACAGTTCCTCG) and RSB-NrampC1 (TCCTCAGGCCATAT TTGTCTA) were designed for amplifying an Nramp fragment of about 190 bp. The PCR (25 ml) consists of 0.5 ml of 20 mM of each primer, 0.2 ml of 25 mM of each dNTP, 0.5 U of Taq polymerase, 0.5ml dimeyhyl sulphoxide, and 1e2.5 ml of sea bream cDNA as template. The PCR was run as follows: Initial incubation at 94 (C for 2 min, followed by 35 cycles of 94 (C, 10 s; 52 (C, 30 s; and 72 (C, 1 min, with a final extension of 10 min at 72 (C. Expression of 18 S rRNA was used as internal control. The primers RSB-18SN1 (5#-GGCAGCGTCCGGGAAACCAAAGTC-3#) and RSB-18SC1 (5#-CCACCCACAGAATCGAGAAAGAGC-3#) were used for amplifying 18S rRNA. A fragment of about 200 bp could be amplified.

3. Results 3.1. Red sea bream Nramp cDNA sequence Nramp cDNA was isolated from red sea bream spleen using PCR and 5#- and 3#-RACE. A fragment of 295 bp was first amplified by PCR by using degenerate primers D1 and D2 designed on the basis of Nramp conserved regions in mammals. On the basis of the sequence of the 295 bp fragment, two specific primers, GSP5# primer and GSP3# primer, were designed and used for 5#-RACE and 3#-RACE, respectively. A 576 bp 5#-RACE fragment and a 4319 bp 3#-RACE fragment were amplified separately (data not shown). After splicing and assembling of the two fragments, a full-length Nramp cDNA fragment of 4709 bp was

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obtained, including 197 bp 5#-terminal untranslated sequence (UTR), 1662 bp encoding region and 2850 bp 3#-terminal UTR (Fig. 1, GenBank accession number AY485311). 3.2. Structure analysis of red sea bream Nramp protein The amino acid sequence of red sea bream Nramp was deduced from the nucleotide sequence of the cDNA. The 1662 nt open reading frame was found to code for a protein with 554 amino acid residues. Comparison of amino acid sequence indicated that red sea bream Nramp consists of 12 transmembrane region (TM) domains (Fig. 1). A highly conserved transport motif (CTM) containing 20 residues was observed between transmembrane domains 8 and 9. Three N-linked glycosylation sites (N-X-S/T) were identified between TM domain 7 and TM domain 8. One potential kinase C phosphorylation site (S/T-X-R/ K) was identified just before TM domain 1. A casein kinase II site (S/T-X-X-D/E) was found to be after TM domain 12 (Fig. 1). 3.3. Sequence alignment and phylogenetic analysis The deduced amino acid sequence of red sea bream Nramp had 77.8%, 83.0%, 82.3%, 80.0%, 81.1%, 60.4%, 70.3%, 58.5% and 69.5% identity with that of rainbow trout Nrampa (AAD20721) and Nrampb (AAD20722), channel catfish Nramp (AF400108), fathead minnow Nramp (AAF01778), carp Nramp (CAB60196), mouse Nramp1 (AAA39838) and Nramp2 (AAC42051), and human Nramp1 (D50403) and Nramp2 (NP-000608), respectively (Fig. 2). It is evident that red sea bream Nramp was more similar to Nramp2 than to Nramp1 of mouse and human. Phylogenetic analysis indicated that red sea bream Nramp is the closest to that of rainbow trout Nrampb among the examined species (Fig. 3). 3.4. Nramp gene expression in red sea bream tissues Nramp mRNA expression in different tissues of red sea bream was analysed using RTePCR. Levels of expression were similar among head kidney, spleen, intestine and liver from non-challenged individuals (Fig. 4). The mRNA levels increased gradually and significantly in the liver and spleen from 5 to 96 h after challenge with pathogenic bacteria (Fig. 4). Change in Nramp expression in intestine and head kidney prior to and after challenge was not evident (Fig. 4). The expression of 18 S rRNA was not affected by challenge.

4. Discussion Nramp1 gene has been identified as one of the major candidate genes for controlling natural resistance and/or susceptibility to intracellular pathogens in human, mouse, bovine and pig [8,9,11,12]. However, few homologues of Nramp are available in teleosts [14,15]. To characterise the Nramp gene in fish and study the function of the Nramp in fish innate defence, an Nramp cDNA has been isolated and characterised from red sea bream spleen. The deduced amino acid sequence of encoding region of red sea bream Nramp shares 58e83% identity with the sequence of previously reported Nramps. Phylogenetic comparison of deduced Fig. 1. cDNA and predicted amino acid sequence of red sea bream Nramp. Nucleotides are indicated above and numbered to the right of each lane (upper row). The deduced amino acid sequence is shown below the nucleotide sequence. Amino acids are indicated with italic letters and numbered to the right of each lane (lower row). The transmembrane regions (TM) are underlined with and numbered 1e12. The ‘consensus transport motif’ (CTM) located between TM8 and TM9 is underlined with d. The polyA signal is represented with ]. One predicted protein kinase C phosphorylation site is underlined with e e e e, three predicted N-linked glycosylation sites are underlined with - - - -, a casein kinase II site is underlined with ee.

310 S.-L. Chen et al. / Fish & Shellfish Immunology 17 (2004) 305e313 Fig. 2. Amino acid alignment of red sea bream Nramp (AY485311), rainbow trout Nramp a (AAD20721) and b (AAD20722), channel catfish Nramp (AF400108), fathead minnow Nramp (AAF01778), carp Nramp (CAB60196), mouse Nramp 1 (AAA39838) and 2 (AAC42051), human Nramp 1 (D50403) and 2 (NP-000608). The amino acid sequence of red sea bream Nramp is given with 12 putative transmembrane regions underlined. Identity is indicated by dots (..), and gaps used to maximise the alignment are shown by dashes (- -).

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Fig. 3. Phylogenetic tree based on the genetic distances between deduced amino acid sequences of Nramps from a variety of fish. The tree was constructed using a neighbour-joining method. Gaps were completely deleted. The scale bar length is 0.05. Numbers on nodes indicate frequency with which this node was recovered per 100 bootstrap replications in a total of 500.

Fig. 4. Reverse transcriptionePCR analysis of Nramp gene expression in various tissues of control red sea bream (uninfected) and those challenged with V. anguillarum sampled at 5, 24, 48, 72 and 96 h after challenge. 18S rRNA was used as control. M, DNA markers.

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amino acid sequences demonstrated that red sea bream Nramp most closely resembles that of rainbow trout. Like other fish Nramp, red sea bream Nramp is most similar to Nramp2 of human and mouse [13e15]. The structural features of Nramp protein can be observed in red sea bream Nramp, including 12 transmembrane regions (TM), a consensus transport motif (CTM), three N-linked glycosylation sites (N-XS/T), a potential kinase C phosphorylation site (S/T-X-R/K) and a casein kinase II site (S/T-X-X-D/E). These structural features of red sea bream Nramp are very similar to those present in human [22,23], mouse [24,25], rainbow trout [14] and carp [13]. In mammals, Nramp1 expression is tissue specific. Human Nramp1 mRNA is detected in spleen, liver and lung [11], while mouse Nramp1 mRNA can be detected in spleen and in liver [10]. Bovine Nramp1 is expressed primarily in macrophages of the reticuloendothelial system [26]. However, the present study demonstrated that in red sea bream Nramp mRNA expression was similar in liver, spleen, intestine and head kidney, which is in agreement with that reported in channel catfish [15]. Challenge of red sea bream with the pathogenic bacterium, V. anguillarum, significantly up-regulated the Nramp expression in liver and spleen in a time-dependent fashion, but didn’t influence the expression level of Nramp mRNA in intestine and head kidney. A similar up-regulating effect following infection was also observed in mouse [24] and pig [27]. Lamas et al. [28] demonstrated that V. anguillarum multiplied extensively in spleen and liver and caused pathological changes of these tissues in rainbow trout, which provides evidence for the bacteriainduced increase in Nramp mRNA level in red sea bream in the present study. Similarly, Chen et al. [15] indicated that treatment of channel catfish with 3 mg/kg lipopolysaccharide (LPS) significantly elevated the level of Nramp mRNA in spleen at 5 h post-treatment, but not beyond 5 h, and no change was detected in the liver. The present study demonstrated that the levels of Nramp mRNA in spleen and liver of red sea bream challenged with pathogenic bacteria gradually and significantly increased with a maximum level at 96 h post-infection. These results implied that Nramp plays an important role in immune response of red sea bream to infection, and that spleen and liver might be major sites for Nramp function. Further studies will be needed for elucidating the precise role and mechanism of Nramp in innate defence in fish.

Acknowledgements This work was supported by grants from National Nature Science Foundation of China (40376047) and from State 863 High-Technology R&D Project of China (2002AA626010).

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