Molecular Characterization and Expression Pattern of a Liver-Expressed Antimicrobial Peptide 2 (LEAP-2) Gene in Yellow Catfish (Pelteobagrus fulvidraco)

Antimicrobial peptides (AMPs) are essential components of the host nonspecific defense system in organisms, which act as antimicrobial agents by disrupting the membrane integrity of invading pathogens [14]. Because of scare drug-resistance to microbes, AMPs are considered as promising therapeutics against antibiotic-resistant bacterial strains [5,6]. Liver-expressed antimicrobial peptide 2 (LEAP-2) is the second blood-derived peptide from human that is predominantly expressed in the liver and exhibits antimicrobial activity [7]. All the members of the LEAP-2 family have a highly conserved motif containing four cysteine residues to form two disulphide bonds which makes it to exhibit distinct amino acid sequence, secondary structure and expression pattern from other peptides [7]. The characteristics of LEAP-2 have been reported in human, monkey, pig, mouse, cow, and chicken [8-12]. To date, LEAP-2 genes have been reported only in limited fish species including channel catfish [10], grass carp [11], rainbow trout [12], and blunt snout bream [13]. However, the molecular characteristics of LEAP-2 and its functions in innate host defense in fish are still unclear.


Introduction
Antimicrobial peptides (AMPs) are essential components of the host nonspecific defense system in organisms, which act as antimicrobial agents by disrupting the membrane integrity of invading pathogens [1][2][3][4]. Because of scare drug-resistance to microbes, AMPs are considered as promising therapeutics against antibiotic-resistant bacterial strains [5,6]. Liver-expressed antimicrobial peptide 2 (LEAP-2) is the second blood-derived peptide from human that is predominantly expressed in the liver and exhibits antimicrobial activity [7]. All the members of the LEAP-2 family have a highly conserved motif containing four cysteine residues to form two disulphide bonds which makes it to exhibit distinct amino acid sequence, secondary structure and expression pattern from other peptides [7]. The characteristics of LEAP-2 have been reported in human, monkey, pig, mouse, cow, and chicken [8][9][10][11][12]. To date, LEAP-2 genes have been reported only in limited fish species including channel catfish [10], grass carp [11], rainbow trout [12], and blunt snout bream [13]. However, the molecular characteristics of LEAP-2 and its functions in innate host defense in fish are still unclear.
Yellow catfish Pelteobagrus fulvidraco is an important cultured fish species in China. Despite of the importance in aquaculture, there is little information on the immunological mechanism in this fish. Actually, the problem of diseases mainly caused by pathogenic bacteria such as Edw. ictaluri and Edw. tarda is more and more serious in yellow catfish and causes serious losses [14]. In present study, we isolated and characterized a LEAP-2 gene designated PfLEAP-2 from yellow catfish P. fulvidraco. Tissue-specific expression pattern of PfLEAP-2 was tested by quantitative real-time PCR. Expression profile of PfLEAP-2 in liver after bacterial challenge was also analyzed. In addition, antimicrobial activity of recombinant PfLEAP-2 mature peptides was discussed.

Fish cultivation and sample collection
Yellow catfish with body mass about 200 g were obtained from a commercial fish farm in Shaoxing City, Zhejiang Province, China. All fish were healthy and acclimatized for 14 days in a circulating water system with temperature at 22-23°C and dissolved oxygen above 5 mg/L. The experimental fishes were euthanized with tricaine methane sulfonate (MS222) at a concentration of 100 mg per liter before sample collection. Samples were frozen in liquid nitrogen and stored at -80°C until analysis.

Cloning of PfLEAP-2 cDNA
Total RNA was extracted from the liver of yellow catfish using TRIzol Reagent (Invitrogen, Carlsbad, USA) according to the manufacturer protocol, and quantified by measuring absorbance at 260 nm with a Nano Drop 2000 spectrophotometer (Thermo Scientific, Wilmington, USA). Gene-specific primers for RT-PCR, LEAP-2F and LEAP-2R, were designed based on PfLEAP-2 sequences of channel catfish Ictalurus punctatus (GenBank accession no. AY845141) and blue catfish I. furcatus (GenBank accession no. AY845142). The firststrand cDNA was synthesized with the M-MLV Reverse Transcriptase Rnase H Minus, Point Mutant (Promega, Madison, USA) in a reaction mixture of 20 µl containing 4 µl×5 reaction mix, 200 ng total RNA, 1 µl primer of Oligo(dT)12-18 (20 µm), 2 µl SuperScript III RT/Platinum Taq polymerase mix, 1 µl dNTP. The reactions were completed in a thermocycler at 42°C for 60 min. The first strand of cDNA was used as the template to amplify the fragment using the gene-specific primers LEAP-2F (5′-TCTAGAAAAGAGCAACATCATTTCAGCAG-3′) and LEAP-2R (5′-GAATTCTTAGGAAATTATAGGTTGGCTAAAGG-3′). The amplification conditions were: 94°C for 2 min, then 30 cycles of 94°C for 45 s, 55°C for 45 s, 72°C for 60 s, followed by a final extension step of 10 min at 72°C. Amplified products were analyzed on a 1.2% agarose gel stained. PCR products were isolated from gel by using a Gel Extraction Kit (Promega) before they were ligated with pGEM-T vector and transformed into DH5α competent cell of Escherichia coli (TaKaRa, Dalian, China). Ten positive clones were selected randomly and sequenced.

Bioinformatics analysis
Determinations of the gene and amino acid sequence were performed using the Expert Protein Analysis System (ExPASy; http://au.expasy.org). The signal peptide was predicted using the SignalP program (http://www.cbs.dtu.dk/services/SignalP/). Domain prediction was performed using the simple modular architecture research tool (SMART; http://smart.embl-heidelberg.de/). Needle program (http://www.ebi.ac.uk/Tools/emboss/align/) was used to calculate identities among nucleotide sequences of fish LEAP-2. The deduced amino acid sequence of PfLEAP-2 was aligned with these of the other fish LEAP-2 using Cluster W 1.81 [15] (Table 1). Phylogenetic tree was constructed with MEGA 5 program [16] by the Neighbor-Joining method [17]. The evolutionary distance was measured as the proportion of difference (p-distance). The confidence in the phylogenetic tree branch topology was accessed with bootstrap using 10, 000 replicates.

Tissue-specific expression pattern of PfLEAP-2 by quantitative real-time PCR
Tissue-specific expression pattern of PfLEAP-2 in 11 tissues of five yellow catfish including the brain, liver, hind kidney, head kidney, spleen, intestine, heart, gill, muscle, skin, and stomach were analyzed by quantitative real-time PCR (qRT-PCR) using the iQTM5 apparatus (BioRad, Hercules, USA). The expression levels of PfLEAP-2 mRNA in liver tissues of infected yellow catfish at different stages were also analyzed by qRT-PCR. cDNA from total RNA was synthesized as described in section 2.2. The gene-specific primers (RT-HcAmyF: 5′-TCGGCATACCATCACCCAT-3′; RT-HcAmyR: 5′-GATTGATTACCAGATCAGGGAAGAT-3′) for qRT-PCR were designed basing on the cDNA sequence of PfLEAP-2. β-actin of yellow catfish (GenBank accession no. EU161066) was selected as the endogenous reference gene for calibration and amplified with primers actin-F (5′-CCGCACTGGTTGTTGACAACG-3′) and actin-R (5′-TGCCAATGGTGATGACCTGTCC-3′). qRT-PCR was performed on an iQTM5 Real Time PCR Detection System (Bio-Rad, Hercules, USA) with the SYBR® Premix Ex Taq TM PCR kit (TaKaRa) according to the manufacturer protocol. The reaction was performed in the following program: denaturation at 95°C for 10 s, 40 cycles of 95°C for 5 s, 60°C for 30 s, and 72°C for 20 s, followed by the determination of the melting curve, which was carried out through 0.5°C increments from 55°C to 95°C. All samples were performed in triplicate with a standard curve of serially diluted synthetic RNA. The relative mRNA level was determined by the 2-(ΔΔCt) method [18] using β-actin as internal control and liver as calibrator tissue.

Expression profile of PfLEAP-2 after bacterium challenge
The bacterium Edw. tarda was cultured for 24 hours at 28°C in shaken bottles with LB medium. The culture was centrifuged at 5000 rpm for 15 minutes at 4°C. The pellet was re-suspended in sterile PBS and the cell density was correlated to a dose of 107 cells per mL count using serial dilution and spread plate techniques. Yellow catfish were divided randomly into two groups (20 fish per group) and cultured under the same conditions. Fish of treatment group was injected with Edw. tarda 0.1 mL per individual, while fish of control group with 0.1 mL sterile PBS. The sample of liver from five fish of two groups were isolated at 4 h, 24 h, 3 d, and 6 d post-injection respectively and stored at −80°C until analysis. The expression profile of PfLEAP-2 mRNA was tested with the qRT-PCR method as described in section 2.4.

Fusion expression, purification of mature peptide
The cDNA fragment of PfLEAP-2 mature peptide was amplified using the primers of Mp-LEAP2F (5′-GAATTCATGGCCCGTATGA-3′) and Mp-LEAP2R (5′-CTACTCGAG TTAGGAAATTAT-3′) designed on the sequence of PfLEAP-2 cDNA sequence, and were ligated to the vector pGEX-4T-1 (TransGen Biotech, Beijing, China). The recombinant plasmids, pGEX-m-leap was transformed into DH5α competent cells of E. coli (TaKaRa). The forward positive clones were screened by PCR with vector primer M13F primer (5′-CACGACGTTGTAAAACGAC-3′) and Mp-LEAP2R, and further confirmed by sequencing. The positive recombinant plasmid, pGEXm-leap was isolated by Axy Prep TM Plasmid Miniprep Kit (Axygen Biosciences, Union City, USA), transferred into E. coli BL21 (DE3) plysS Competent Cells (Promega), and subsequently incubated in LB medium. The E. coli BL21 (DE3) plysS inserted empty pGEX-4T-1 vector was also cultured in LB medium as negative control. The expression of recombinant protein was induced for 5 h in LB medium with 1.0 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) at 28°C. Recombinant protein was purified by affinity chromatography using a Glutathione Sepharose 4B column (GE Healthcare, Little Chalfont, UK). Purified products were assessed on 15% SDS-PAGE gel. Half of the fusion proteins were digested with thrombin on column before elution with PBS, whereas the remaining proteins were eluted from the column with glutathione. Purification and elution were performed according to the instruction manual. Purified products were assessed with western-blotting performed as described in [19].

Antimicrobial activity assay
The antimicrobial activity was determined by microdilution susceptibility assays as described in [20]. The bacteria Edw. tarda were cultured overnight in LB medium at 28°C. The suspension used for each assay was adjusted to 1×10 5 CFU/mL in LB medium. Recombinant mature peptide of LEAP-2 was added into bacteria suspension with final concentrations of (0, 20, 40, 60 μg/mL). All assays were performed in triplicate. The suspensions were incubated at 28°C in a shaker at 200 rpm. The concentrations of bacteria suspension were determined with ATB expression densimat (Merieux, France) every 2 hours until the absorption values of control group (0 μL/mL) at OD 630 reached 0. 6 Suggested location in line 97 of manuscript

Statistical analysis
The differences of mRNA expression levels in tissues and at different stages after challenge with Edw. tarda were analyzed with oneway ANOVA, and further comparisons were performed with Tukey's HSD test using the SPSS software (version 19.0). Significance level was set at P<0.05.

Results cDNA and protein sequences of PfLEAP-2
The 285-bp cDNA sequence cloned in present study contained the full open reading frame (ORF) of yellow fish PfLEAP-2, which was deposited in GenBank with accession no. GQ228478. The deduced PfLEAP-2 protein comprised of 94 amino acids and contained a putative signal peptide of 28 residues, a prodomain peptide of 21 residues, and a mature peptide of 45 residues. The mature peptide had 4 conserved cysteine residues, namely Cys70, Cys76, Cys81, and Cys86 ( Figure 1). The conserved cleavage site of RMAR was also identified in the mature peptide. Sequence identities of PfLEAP-2 to LEAP-2 from other fishes ranged between 42.4-89.4% and 63.2-97.8% for the entire proteins and mature peptides, respectively (Table1 and Figure 1).

Phylogenetic analysis
The neighbor-joining tree based on the amino acid sequences of LEAP-2 showed that the PfLEAP-2 had closest relationship with Ictalurus punctatus which are other species from Siluriformes ( Figure  1). Some LEAP-2 from same species, such as Oryzias latipes, Maylandia zebra and Oncorhynchus mykiss were clustered into different clades ( Figure 2)

Tissue distribution of PfLEAP-2 mRNA in yellow catfish
The tissue-specific expression pattern of PfLEAP-2 in yellow catfish was examined by qRT-PCR using β-actin as an internal control. PfLEAP-2 transcripts were abundant in the gill, detectable in the skin, stomach, and head kidney, but low in the muscle, spleen, liver, hind kidney, intestine, heart and brain (Figure 3).

Expression pattern of PfLEAP-2 gene in the liver after Edw. tarda challenge
The change of PfLEAP-2 transcripts after the pathogenic bacteria stimulation was performed by qRT-PCR. As shown in Figure 4, the expression of PfLEAP-2 mRNA in liver was up-regulated rapidly after infection time of 4 h (P<0.05), but then returned to the control level afterwards (Figure 4).

Expression of recombinant PfLEAP-2 mature peptide and Western blot analysis
SDS-PAGE analysis showed that the recombinant PfLEAP-2 mature peptide expressed a 31 kD fusion protein ( Figure 5). The content of recombinant protein in E. coli was increased in a timedependent manner. Western-blotting analysis indicated that the 31 kDa fusion protein were cleaved into two parts by thrombin, including GST proteins with a molecular weight of 26.4 kDa and mature peptides with a molecular weight of 4.6 kDa ( Figure 6).

Antimicrobial activity of recombinant PfLEAP-2 mature peptide
The results of antimicrobial assay showed that the purified recombinant PfLEAP-2 mature peptide exhibited no activity against Edw. tarda under the protein concentrations of 20 to 60 μg/mL ( Figure  7).

Discussion
As an important component of antimicrobial peptides in innate immune system, LEAP-2 has been identified in some fish species [21]. In this study, we reported a novel LEAP-2, named PfLEAP-2 from the      Time (hours) Racteria Concentration (x10 5 /mL) 0 µ g/mL 20 µ g/mL 40 µ g/mL 60 µ g/mL overlapping distribution of LEAP-2 in phylogenetic tree suggests that gene duplication of LEAP-2 occurred in teleosts [10].
In vertebrate, the liver produces two cysteine-containing molecules LEAP-1 (hepcidin) and LEAP-2 [22]. Beside of liver, the transcripts of LEAP-2 mRNA was also detected abundantly in the intestine of rainbow O. mykiss and trout blunt snout bream M. amblycephala [12,13]. In this study, the transcripts of PfLEAP-2 mRNA were predominantly expressed in the gill, skin and stomach but low in the liver. Similar result of tissues distribution of LEAP-2 was also detected in channel catfish [10]. Gill, stomach, and skin serve as the first barrier against infection in catfish [23]. The higher expression levels of PfLEAP-2 mRNA in these tissue indicated that PfLEAP-2 may play a role during early period of immune defense against pathogens.
Edwardsiella sp. of the family Enterobacteriaceae, such as Edw. tarda and Edw. ictaluri were the important causative agent, and have caused serious disease problems in catfish culture industries [14,[24][25][26], and reduced enteric septicaemia of catfish (ESC). To date, both Edw. tarda and Edw. ictaluri were isolated from the diseased yellow catfish, and caused "red head" disease [27] and "hole -in-the-head" disease [26]. In this study, the up-regulation of PfLEAP-2 at 4h after the challenge of Edw. tarda ( Figure 4) suggests that this gene react rapidly to the pathogen invasion and may play an important role in the early immune defense of the yellow catfish against bacterial pathogen. Similar induction pattern was also reported in the spleen of channel catfish in which LEAP-2 was significantly inducted at 4 h after challenge of Edw. ictaluri [10]. However, in another report [28], LEAP2 was not significantly different from that of control fish at all-time points after Edw. ictaluri infection.
Recombinant LEAP-2 obtained by in vitro expression has been proven to exhibit dose-dependent and selective antimicrobial activity against bacterial pathogen in human, bird, and fish [7,11,29]. In this study, the recombinant PfLEAP-2 has been successfully expressed in E. coli and purified. The PfLEAP-2 mature peptide cleaved by thrombin is verified by Western-blotting analysis. However, the purified recombinant PfLEAP-2 mature peptides displayed no antimicrobial activity against Edw. tarda even at the terminal concentration of 60 ug/ mL (Figure 7). This result indicated that the antimicrobial activity of PfLEAP-2 against pathogen may also selective. The role of PfLEAP-2 in the immune defense of yellow catfish is needed to be further studied.