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H2A and Ca-L-hipposin gene: Characteristic analysis and expression responses to Aeromonas hydrophila infection in Carassius aurutus
Fish & Shellfish Immunology 63 (2017) 344e352
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H2A and Ca-L-hipposin gene: Characteristic analysis and expression responses to Aeromonas hydrophila infection in Carassius aurutus Xianghui Kong*, Xiangmin Wu, Chao Pei, Jie Zhang, Xianliang Zhao, Li Li, Guoxing Nie, Xuejun Li College of Fisheries, Henan Normal University, Xinxiang 453007, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 November 2016 Received in revised form 13 February 2017 Accepted 17 February 2017 Available online 20 February 2017
Antimicrobial peptide is an important component of the host innate immune system and thus serves a crucial function in host defense against microbial invasion. In this study, H2A and derived antimicrobial peptide Ca-L-hipposin were cloned and characterized in Carassius aurutus. The gene H2A full-length cDNA is 908 bp and includes a 50 -terminal untranslated region (UTR) of 55 bp and a 30 -terminal UTR of 466 bp with a canonical polyadenylation signal sequence AATAA, as well as an open reading frame (ORF) of 387 bp encoding a polypeptide of 128 amino acids, with a molecular weight of 13.7 kDa, an isoelectric point of 10.7, and 94% homology with Danio rerio H2A. The secondary structure of H2A includes the a-spiral with 51 amino acids with a composition ratio of 39.8%, as well as a b-corner with 15 amino acids in a composition ratio of 11.7%. The online software ExPaSy predicted that a peptide sequence with 51 amino acids from the 2nd to 52nd amino acids in histone H2A can be produced through hydrolization by protease chymotrypsin, which indicates a difference of only three amino acids, compared with the antimicrobial peptide hipposin in Hippoglossus hippoglossus with a homology of 94%. Ca-L-hipposin includes 51 amino acids with a molecular weight of 5.4 kDa and an isoelectric point of 12.0, the secondary structure of which contains an a-helix of 17 amino acids accounting for 33.3% and a b-corner of 8 amino acids accounting for 15.7%. H2A was extensively expressed in the mRNA levels of various tissues, with higher expression levels in kidney and spleen. After C. aurutus was challenged with Aeromonas hydrophila, the mRNA expression levels of H2A were upregulated in the kidney, spleen, and liver. H2A serves an important function in the defense against the invasion of A. hydrophila. In addition, sequence characteristics reveal that Ca-L-hipposin could be a potential antimicrobial peptide for use in killing pathogenic bacteria in aquaculture. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Histone H2A Hipposin-like antimicrobial peptide Aeromonas hydrophila Carassius aurutus
1. Introduction Histone, being highly conservative and thermally stable, serves an important function in the assembly of DNA molecules. Histone can prevent DNA digestion induced by nuclease and change in topological structure. Moreover, histones can regulate gene expression, transfer, posttranslation modification, cell apoptosis, and tumor lesions [1e3]. In aquatic animals, histone-derived antimicrobial peptides are a type of cationic polypeptide that can kill pathogenic bacteria; these peptides are mainly derived from histones H1, H2A, and H2B [4e7]. Histone-derived antimicrobial
* Corresponding author. No. 46, Jianshe Road, College of Fisheries, Henan Normal University, Xinxiang 453007, PR China. E-mail address: [email protected] (X. Kong). http://dx.doi.org/10.1016/j.fsi.2017.02.028 1050-4648/© 2017 Elsevier Ltd. All rights reserved.
peptides have been isolated from mollusks Chlamys farreri [8], Crassostrea gigas [9, 10], arthropod Macrobrachium rosenbergii [11], amphibians Rhacophorus schlegelii [12], and Bufo bufo gargarizans [13]. In fishes, H2A-derived antimicrobial peptides have been isolated in Hippoglossus hippoglossus [14], Parasilurus asotus [15], and Ictalurus punctatus [16]. These peptides are widely expressed in various tissues [17] and especially abundant in the surface mucus of fish [14,15,18]. H2A-derived antimicrobial peptides possess strong antimicrobial activity, which can protect against Gram-positive and Gram-negative bacteria [14,17], as well as kill fungi without hemolytic activity [15,19]. The antimicrobial activity of these peptides is 12e100 times higher in fish than in amphibians, which shows a minimum bacteriostatic concentration of 0.3 mM (1.6 mg/mL) [15], and achieves effective bacteriostatic activity in concentrations
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ranging from 0.25 mg/mL to 4 mg/mL. They can serve as the first barrier of natural immunity to the invasion of foreign pathogens [15,20,21] and are believed to be the components of innate defenses [22e28]. A novel 51-residue cationic antimicrobial peptide isolated from the skin mucus of Atlantic halibut, H. hippoglossus, was called hipposin, which is rich in Arg and Trp residues but lacks acidic amino acids [20]. A total of 50 of the 51 residues in hipposin were identical to those in the N-terminal region of H2A in rainbow trout. The fragment containing 1 to 19 amino acid residues in the N-terminal exhibits no remarkable antimicrobial activity, whereas the fragment consisting of 16e39 amino acids (similar to buforin II) indicates the antimicrobial activity of hipposin; such activity was found to be enhanced by the presence of a fragment of 40e51 amino acid residues [21]. Almost all previous reports have shown that H2A-derived antimicrobial peptides are similar to the hipposin sequence, indicating that antimicrobial activity is mainly attributable to the action of this fragment. With respect to the production of antimicrobial peptide derived from histone, two suggestions have been proposed as the following. One is that antimicrobial peptide is produced from histone, which is hydrolyzed into the smaller peptide by specific proteases to better serve its antimicrobial function [29]. For example, in Asian toad, B. bufo gargarizans, buforin I is produced by the action of pepsin isozymes called pepsin Ca and Cb, which can cleave the Tyr39eAla40 bond in histone H2A [30]. In catfish, the peptide bond between Ser19 and Arg20 in histone H2A is cleaved by cathepsin D to produce antimicrobial peptide parasin I [15,31,32]. Another viewpoint is that histone-derived antimicrobial peptide is expressed in an independent pattern (communications), the expression of which is not limited to the expression of histone, i.e., the expressions of histone and its derivative antimicrobial peptide are independent. However, up to now, the process of histonederived antimicrobial peptide production remains unclear. Therefore, more evidence should be sought to elucidate the production of histone-derived antimicrobial peptide. In fish, not only the N-terminal segment of H2A, but also the complete H2A protein has antimicrobial activity [18]. Antimicrobial peptide can be induced in the mucus of Oncorhynchus kisutch after exposure to infectious agents [23]. In fish infected with different bacteria, the expression patterns of antimicrobial peptides differ [24,25]. Among a number of aquatic pathogens, the Gram-negative Aeromonas hydrophila has been discovered in a variety of freshwater fish and found to be capable of growing in both aerobic and anaerobic conditions. This bacteria can result in the outbreak of many fish diseases, such as ulcers, fin rot, tail rot, and hemorrhagic septicemia, indicating rapid outburst, high mortality, and difficulty to control, and such outbreak could finally result in serious economic losses in fisheries [26,27]. Research on Carassius aurutus infected with A. hydrophila has been previously conducted [28]. However, the responses of H2A and its derived antimicrobial peptide to bacteria remain unclear. The Qihe crucial carp C. aurutus is an important commercial fish that is widely cultivated in the north of Henan Province, China. In this study, the complete H2A cDNA sequence in C. aurutus was cloned through reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Meanwhile, H2A-derived antimicrobial peptide Ca-L-hipposin was also achieved. The expression responses of H2A and Ca-L-hipposin to A. hydrophila challenge were studied. The aims of this research are as follows: (1) to determine the gene characteristics of H2A and CaL-hipposin in C. aurutus, (2) to analyze the gene variation and molecular evolution of H2A and Ca-L-hipposin, (3) to understand the expression responses of H2A and Ca-L-hipposin to A. hydrophila challenge.
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2. Materials and methods 2.1. Fish The experimental fish C. aurutus with a body weight of 28 ± 2 g, obtained from aquaculture farms in Henan Normal University, were grouped and cultivated in different tanks (in triplicate), with 60 fish each tank. The fish were fed with commercial pellets twice a day (10:00 a.m. and 4:00 p.m.), and sufficient dissolved oxygen was supplied by an aerator. The water temperature was maintained at 23 ± 2 C. After acclimation for one week, the experiment was conducted. 2.2. RNA extraction and cDNA preparation After C. aurutus was dissected, the gill, liver, spleen, intestine, kidney, heart, brain, and muscle were immediately taken and placed in liquid nitrogen for RNA extraction. Total RNA was extracted using TRIzol Reagent (from TaKaRa Com) according to manufacturer's instructions. The quality of RNA was assessed by electrophoresis in a 1.0% agarose gel. The ratio of OD260/OD280 of total RNA was determined to be 1.8 to 2.0, and the concentration was determined to be 400 ng/mL to 500 ng/mL, using a microscale Nanodrop ND2000 spectrophotometer. The cDNA was synthesized using a first-strand cDNA synthesis kit (TaKaRa, No. DRR037A). 2.3. Cloning of H2A cDNA in C. aurutus The first strand of cDNA was used as a template to amplify the central fragments of histone H2A using the specific primers (PF and PR, as listed in Table 1) designed on the basis of conserved domains. PCR reactions were performed in a total volume of 20 mL, including 2 Taq Master Mix 10 mL (Taq DNA Polymerase, PCR Buffer, Mg2þ, dNTPs), 1.0 mL of each primer, 1.0 mL of cDNA, and the addition of double-distilled water (ddH2O) for a total of 20 mL. The PCR conditions were set as follows: 5 min at 95 C for predenaturation, 35 cycles consisting of 30 s at 95 C for denaturation, 30 s at 55 C for annealing, and 45 s at 72 C for extension, and 7 min at 72 C for final extension. 30 -RACE and 50 -RACE were used to obtain the 30 -terminal and 50 terminal sequences of H2A cDNA, respectively. The 30 -terminal, 50 terminal, and central sequences assembled the full-length cDNA sequences of H2A. Eight specific primers are listed in Table 1. These primers were designed on the basis of the amplified central sequences of H2A cDNA. 30 -RACE and 50 -RACE kits were purchased
Table 1 List of primers used in this study. Primer name
Primer sequences
PF PR 30 P-OUT-H2A 50 P-OUT-H2A 30 P-IN-H2A 50 P-IN-H2A 30 RACE Olig(T)-Adaptor 30 RACE Adaptor 50 RACE Olig(T)-Adaptor 50 RACE Adaptor FO-Ca-L-hipposin RE-Ca-L-hipposin FO-H2A RE-H2A FO-b-Actin RE-b-Actin
50 -ATGWGYGGRMGWGGCAAGAC-30 50 -CVGTCTTCTTKGGSAGCAG-30 50 -GGGAACGCAGCCCGAGACAACA-30 50 -TCTTGTTGTCTCGGGCTGCGTTCC-30 50 -CAGTGCTGCTGCCCAAGAAGACGG-30 50 -CCCCGGTTTTACCACTCCCAGACAT-30 50 -CTGATCTAGAGGTACCGGATCCTTTTTTTTTTTTTT-30 50 -CTGATCTAGAGGTACCGGATCC-30 50 -GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-30 50 -GACTCGAGTCGACATCG-30 50 -TCTGGGAGTGGTAAAAC-30 50 -CGCCAGATACACAGGGGCT-30 50 -TGAGTCCAAGTAATGTTATGTTCCCTA-30 50 -TTAAGTGGTGGTGTAGCTCTGTT-30 50 -CATTGACTCAGGATGCGGAAACT-30 50 -CTGTGAGGGCAGAGTGGTAGACG-30
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from TaKaRa, Japan. The amplified PCR product was verified by electrophoresis on a 1.0% agarose gel, which was then purified using an E.Z.N.A.™ gel extraction kit (TaKaRa, Japan) and ligated into PMD19-T vector (TaKaRa, Japan). Then, 10 mL of PMD19-T vector-connected objective cDNA was transferred into 100 mL of competent cells and then cultured on a solid tablet for 10 h. Eight single bacterial colonies were selected and cultured in LB liquid culture. The target sequence was verified by PCR. Two selected colonies were sequenced by the Sangon Biotech Company (Shanghai, China). 2.4. Expression of H2A and Ca-L-hipposin in mRNA levels in C. aurutus The temporal and spatial expressions of H2A and Ca-L-hipposin mRNA were studied using real-time fluorescence quantitative PCR (RT-qRT). Total RNA was extracted from the liver, spleen, heart, kidney, gill, muscle, brain, and intestine using Trizol reagent as described above. To avoid genomic DNA interference on RT-qPCR, the total RNA was incubated with RNase-free DNaseI (TaKaRa, Japan). Based on the cloned H2A cDNA sequence, the specific primers (FO-H2A, RE-H2A, FO-Ca-L-hipposin, and RE-Ca-Lhipposin) were designed and are listed in Table 1. The primer FOCa-L-hipposin and RE-Ca-L-hipposin were designed within Ca-Lhipposin cDNA to detect the expression of Ca-L-hipposin, and the primer FO-H2A and RE-H2A were designed within H2A cDNA and out of Ca-L-hipposin cDNA to detect the expression of H2A. RTqPCR was performed on a 7500 Real-time Fluorescent Quantitative Instruments. The reaction mixture of the PCR consisted of 10 mL of SYBR Premix Ex Taq TMII (TaKaRa, Japan), 0.8 mL of specific primer (10 mM), and 2.0 mL of cDNA template for a total volume of 20 mL. Real-time PCR was performed using a three-step method, which was held at 95 C for 30 s, followed by 40 cycles (at 95 C for 5 s, 60 C for 42 s, and 72 C for 30 s), and the melting curve temperature was finally maintained at 95 C for 15 s, 60 C for 15 s, and 95 C for 15 s. At the end of each PCR reaction, melting curve analysis of amplification products was performed to verify whether the PCR product was single. The b-actin gene (GenBank Accession No. AY690421.1) was used as an internal control. The relative expression levels of the target genes were expressed as the copy number ratio of the target gene relative to b-actin gene. The relative expressions of H2A and Ca-L-hipposin were presented using the 2DDct analysis method [33], with triplication for each PCR. 2.5. Responses of H2A and Ca-L-hipposin to bacterial challenge The experimental fish were randomly divided into two groups. One group was intraperitoneally injected with 100 mL of A. hydrophila (2 108 CFU/mL), and the other group was injected with 0.65% physiological saline at 100 mL. After 3, 6, 9, 12, 15, and 18 h, respectively, the gill, kidney, spleen, and liver of fish were taken out. The same tissues from three fish were mixed as one sample. The experiments were carried out in triplicate. Total RNA was extracted, and cDNA was synthesized as previously described. RT-qPCR was performed using the same protocol as described above. 2.6. Analysis of sequence characteristics and phylogeny Nucleic acid and amino acid sequence alignments were implemented in DNAman software. A phylogenetic tree was constructed using MEGA 6.0 software with a neighbor joining method. The
peptidase cutting locus was analyzed online at http://web.expasy. org/peptide cutter/. The phosphorylation site was analyzed online at http://www.cbs.dtu.dk/services/NetPhos/. The spatial structure was predicted online at http://www.sbg.bio.ic.ac.uk/phyre2/. 2.7. Statistical analysis The significant difference in gene expression between the experimental and control groups was statistically analyzed using one-way analysis of variance, which was implemented in EXCEL 2010 software. The significance level was set at P < 0.05. 3. Results 3.1. Molecular characterization of H2A and Ca-L-hipposin in C. aurutus The sequences of the H2A cDNA conserved region, 30 -untranslated region (UTR), and 50 -UTR were respectively achieved through RT-PCR, 30 -RACE, and 50 -RACE in C. aurutus. The complete cDNA of H2A, registered in GenBank with Accession No. KJ704981, was 908 bp (Fig. 1) and contained an ORF of 387 bp encoding a peptide of 128 amino acids, a 50 -UTR of 55 bp, and a 30 -UTR of 466 bp. The polyadenylation signal (AATAA) was located at the upstream position of 71 bp ahead of the poly A tail. ExPaSy software was used to predict that the molecular weight of H2A is 13.7 kDa, isoelectric point is 10.7, and instability index is 48.6. The estimated half-life of H2A is 30 h in mammalian reticulocytes in vitro, >20 h in yeast in vivo, and >10 h in Escherichia coli in vivo. As regards the secondary structure, an a-helix composed of 51 amino acids accounts for 39.8% of the total length, whereas the b-corner involved in 15 amino acids accounts for 11.7%. H2A-derived antimicrobial peptide Ca-L-hipposin was predicted online by ExPaSy software. The results indicated that the molecular weight is 5.4 kDa, isoelectric point is 12.0, and instability index is 42.5. The secondary structure a-helix of Ca-L-hipposin, which is composed of 17 amino acids, accounts for 33.3%, and the b-corner containing eight amino acids accounts for 15.7%. The estimated half-life is 1.9 h in mammalian reticulocytes in vitro, >20 h in yeast in vivo, and >10 h in E. coli in vivo. Therefore, H2A and its derived Ca-L-hipposin belong to the instability proteins. H2A contains alkaline amino acid residues (13 Lys, 12 Arg, and 2 His), acidic amino acid residues (7 Glu and 2 Asp), and 6 Pro without Cys. Ca-L-hipposin contains alkaline amino acid residue (5 Lys, 8 Arg, and 1 His), acidic amino acid residues (1 Glu), and 2 Pro without Cys. NetPhos2.0 Server online analysis showed that histone H2A phosphorylation sites were located at positions Ser17/19/20/127, Thr7/121, and Tyr40. Ca-L-hipposin phosphorylation sites were located at positions Ser16/18/19, Thr6, and Tyr39. 3.2. Spatial structure of H2A and Ca-L-hipposin in C. aurutus The spatial structures of histone H2A and Ca-L-hipposin were predicted using online Phyre2 software. The spatial structure prediction of H2A was based on the model d1kx5c, and the prediction of Ca-L-hipposin was based on the model d1eqza. The model adopted for the prediction has a coverage of 100%, with a credibility of more than 99%. The predicted spatial structures of H2A and Ca-Lhipposin are shown in Fig. 2. The spatial structure of H2A contains a-helix and b-corner, without a b-sheet, and that of Ca-L-hipposin was similar but with a reduction in quantity. It was postulated that
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Fig. 1. Histone H2A gene and deduced amino acid sequence in C. aurutus. The H2A-derived antimicrobial peptides Ca-L-hipposin are shown in the gray background.
the a-helix structure serves an important function in antimicrobial activity. 3.3. Molecular variations and phylogenetic analysis The 51 Aa of H2A N-terminal (52 Aa minus 1 Met) in C. aurutus corresponds to the antimicrobial peptide hipposin in H. hippoglossus with a homology of 94% and is therefore called Ca-Lhipposin (like hipposin in C. aurutus). Other H2A-derived antimicrobial peptides, such as parasin I (19 Aa), buforin I (39 Aa), buforin II (21 Aa), artificially designed buforin IIA (17 Aa), and buforin IIB (21 Aa) based on buforin II, himantura (39 Aa), abhisin (40 Aa), Rainbow Tr (51 Aa), scallop A (40 Aa), litopenaeu (38 Aa), and molluskin (25 Aa) [10,14,15,21,29,34], which were derived from other animals, were aligned as shown in Fig. 3. Most of these peptides were derived from the N-terminal region of H2A. Ca-Lhipposin showed only a three amino acid difference with hipposin in H. hippoglossus, indicating that the Arg/3rd, Thr/16th, and His/
41st in hipposin respectively correspond to Ser, Ser, and Glu in Ca-Lhipposin. Ca-L-hipposin has high similarity with N-terminal 2e40 Aa of Buforin I . Ca-L-hipposin is the same as 17e37 Aa in Buforin II with the exception of the 17th Aa. Similarly, Ca-L-hipposin, was found to be highly homologous with H2A-derived antimicrobial peptide sequences in other animals. The orthologous sequences of H2A cDNA were searched in GenBank using the searching tool Blastn on the NCBI website. Based on the aligned H2A sequence matrix, a phylogenetic relationship was constructed using the neighbor-joining method implemented in MEGA 6.0 software (Fig. 4). The confidence of the phylogenetic tree was assessed by performing 1000 bootstrap repetitions. Phylogenetic analyses indicated that fish were clustered into two clades in teleosts: one clade includes the fish species in Cypriniformes and Salmoniformes, and another clade includes the fish species of two other orders. In Cypriniformes, C. aurutus shared the highest homology of H2A with Danio rerio, indicating the closest relationship in the phylogenetic tree. The H2A sequence was highly conserved, with C. aurutus being 94% homologous with D. rerio and moderately similar to Oryzias latipes (83%), Salmo salar and Oncorhynchus mykiss (82%), and Haplochromis burtoni (81%).
3.4. H2A mRNA expression in different tissues and organs of C. aurutus
Fig. 2. Predicted protein spatial structures of H2A and Ca-L-hipposin in C. aurutus. The predicted spatial structure is based on the model d1kx5c for H2A and the model for CaL-hipposin.
The expression of histone H2A was detected through RT-qPCR in various tissues and organs (kidney, spleen, gill, liver, brain, intestine, heart, and muscle) of healthy C. aurutus (Fig. 5). The results indicated that H2A was predominantly expressed in kidney, spleen, and gill (10- to 31-fold), secondarily expressed in liver and brain (<5-fold), and less expressed in intestine, heart, and muscle (<2fold).
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Fig. 3. Comparison of H2A-derived antimicrobial peptides. Gray region is on behalf of conservative amino acids.
3.5. mRNA expressions of H2A and Ca-L-hipposin in C. aurutus injected A. hydrophila After injection of A. hydrophila, H2A mRNA expression levels were upregulated in kidney, spleen, gills, and liver of C. aurutus at most time points, as shown in Fig. 6. In the liver, the expression level of H2A mRNA gradually increased and reached the highest value at 15 h after injection, indicating significant differences compared with the controls (P < 0.01). A similar tendency was observed in the spleen and kidney, indicating that the expression level of H2A mRNA reached the highest value at 15 h after injection (P < 0.01) and then significantly decreased and returned to normal levels. However, in the gills, a significant upregulation of H2A mRNA expression was observed at 3 and 12 h after bacterial challenge
(P < 0.01). After injection of A. hydrophila, the expression profile of Ca-Lhipposin at mRNA level was similar with that of H2A in the same tissue of C. aurutus (data not shown). 4. Discussions Recent studies have reported that almost all histone H2As and their derivative antimicrobial peptides are cationic antimicrobial peptides with an amphiphilic a-helix structure [35]. H2A-derived antimicrobial peptides, with remarkable specificity to prokaryotes and low toxicity to eukaryotic cells, have been favored for application as a new antibiotic with antimicrobial activity against a broad spectrum of pathogenic organisms, capability to resist high
Fig. 4. Phylogenetic tree constructed by NJ based on histone H2A implemented in software Mega 6.0. This phylogenetic tree is the unrooted tree. + indicates the position of C. aurutus; The used H2A gene sequences are from the species Xenopus tropicalis (XM_002944612.1), Danio rerio (BX_120005.6), Oryzias latipes (XM_004065529.1), Oncorhynchus mykiss (BT_073517.1), Salmo salar (BT_047220.1), Haplochromis burtoni (XM_005928954.1), Sus scrofa (XM_001927727.3), Rattus norvegicus (XM_003749860.2), Homo sapiens (NM_002105.2), Mus musculus (NM_175662.1), Tursiops truncates (XM_004330511.1), Orcinus orca (XM_004284025.1), Equus caballus (XM_005603620.1), Capra hircus (XM_005696813.1), Microtus ochrogaster (XM_005369911.1), Cavia porcellus (XM_003468798.2), Chrysochloris asiatica (XM_006872785.1).
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Based on the spatial structures of H2A and Ca-L-hipposin, the ahelix was the main structural component. Therefore, a-helical structures in Ca-L-hipposin serve an important function in antimicrobial function through interaction with the cell membrane. Previous studies have reported that a-helical structures help antimicrobial peptides facilitate to combine with and damage the cell membrane [19,36].
4.2. Function of H2A-derived antimicrobial peptides
Fig. 5. Relative H2A mRNA expression levels in various tissues and organs of C. aurutus. The different letters above the columns denote the significance between the different tissues and organs (P < 0.05), and the same letter signs no significant difference (P > 0.05).
temperatures, and widely distributed in organisms, especially in the lower animals (fish and amphibians) [7,21].
4.1. H2A and derived antimicrobial peptide Ca-L-hipposin Based on the composition and structure of H2A and Ca-Lhipposin in C. aurutus, the 51 Aa of H2A N-terminal coding region (minus 1 Met) in C. aurutus corresponds to the antimicrobial peptide hipposin in H. hippoglossus, with a homology of 94%. Therefore, it was suggested that antimicrobial peptide Ca-L-hipposin is derived from H2A. Compared with the H2A-derived antimicrobial peptides in other animals [10,14,15,21,30,34], the variations of sequences might result in differences in antimicrobial activities. Therefore, such peptides could supply the reference data for the redesign to achieve a high-activity antimicrobial peptide.
Most studies have focused on the antimicrobial activities of cationic antimicrobial peptides [37,38]. Antimicrobial peptide activity mainly depends on amino acid composition, amphipathicity, cationic charges, and structure size. These properties can cause these peptides to adhere to the surface of bacteria, insert into membrane bilayers to form pores, and finally, to break down the bacteria [37,39,40]. Some antimicrobial peptides (e.g., magainins and cecropins) preferentially act against bacteria, whereas others (e.g., melittin and gramicidins) interact favorably with both bacteria and eukaryotic cells [41]. Although the majority of antimicrobial peptides can kill bacteria through membrane disruption, some antimicrobial peptides can enter bacterial cells and interfere with physiological function. Examples are pyrrhocoricin, which inhibits ATPase actions and prevents chaperone-assisted protein folding, and indolicidin, which inhibits DNA synthesis in E. coli [42]. H2A-derived buforin II can enter cells to combine with DNA/ RNA, the binding force of which with nucleic acids is 20 times that of magainin 2 [13]. Antimicrobial peptides could inhibit the synthesis of nucleic acids, proteins, and cell walls as well as reduce enzymatic activity [43]. Buforin II can also damage the cell membrane of bacteria, causing a leakage of contents, the intensity of which is 100 times that of magainin 2 [34]. In this study, buforin II corresponds to the partial fragment of Ca-L-hipposin, which was
Fig. 6. mRNA expression levels of H2A in gill, kidney, spleen, and liver of C. aurutus after A. hydrophila infection. All values present with mean ± standard deviation (n ¼ 3). The statistically significant differences between the experimental group (gray bar) and the control (white bar) are indicated with asterisk above the gray bar (*, P < 0.05; **, P < 0.01).
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highly homologous with H2A-derived antimicrobial peptide sequences in other animals, indicating Ca-L-hipposin had functions in antimicrobial activity. 4.3. Phosphorylation sites and prolines in Ca-L-hipposin The phosphorylated form of protein can strengthen its function and stability. Histone H1, which has three phosphorylation sites, can not only improve the capability to bind DNA but can also resist the hydrolysis of protein, and without phosphorylation, can be digested by protease [44]. Phosphorylation in both end residues of H1-derived antimicrobial peptide oncorhyncin II can prevent damage from bacterial proteases. In this study, Ca-L-hipposin has phosphorylation sites at Ser/16th/18th/19th, Thr/6th, and Tyr/39th. These phosphorylation sites are postulated to serve important contributions to physiological function and protection in C. aurutus. The H2A-derived antimicrobial peptide buforin II can cause cell death by translocation across membranes and interaction with nucleic acids [45]. To understand the function of proline in buforin II, the effects of changes in proline position on antimicrobial activity were further studied using four mutants obtained through proline substitution, indicating that the variation of Pro could have an important influence on antimicrobial activity [34]. In this study, the antimicrobial peptide Ca-L-hipposin in C. aurutus contains Pro, the antimicrobial activity of which could be improved by altering its position and number in the future. 4.4. Variations of acidic and alkaline amino acids in H2A-derived antimicrobial peptides The cationic antimicrobial peptides generally have charges of þ2 to þ9, whereas the amino acids are at 10 to 50 [46]. The basic amino acid is essential for antimicrobial peptides, which serve an important function in maintaining antimicrobial activity [47]. Actually, the change in a single amino acid indicates a negligible effect on antimicrobial activity. For example, defenin in rabbits, with the lack of Arg residue, also showed similar antimicrobial activity [48]. However, an increase in cationic peptides could decrease antimicrobial activity. The amino acid fragments from positions 16 to 36 in Ca-Lhipposin, corresponding to buforin II, and positions 40 to 51 are the main antimicrobial motifs. Outside of the two fragments, the mutation implies a slight effect on antimicrobial activity, for example, a mutation in the third amino acid [26]. In this study, the variations of amino acids, such as that at the 16th and 41st positions are studied. In particular, we examine that of the 41st position, which is the basic amino acid in H. hippoglossus, and is the acidic amino acid in C. aurutus. Given that the acidic amino acid reduces the number of net positive charges, we speculated that acidic amino acids could have an important influence on antimicrobial activity. 4.5. mRNA expression responses of H2A and Ca-L-hipposin to the injected A. hydrophila As regards the tissue expression of H2A mRNA, the expression levels of H2A mRNA were higher in kidney, spleen, gill, liver, and brain, which indicated that the higher expression levels of H2A mRNA were maintained in immune organs (the highest in the kidney and the second highest in the spleen). This result was in agreement with the suggestions of previous studies [49]. After aquatic animals are infected by pathogenic bacterium, antimicrobial peptide expression presents a series of responses to
prevent and kill bacteria [49]. In this study, after A. hydrophila was injected into C. aurutus, the expression levels of H2A increased in the kidney, spleen, and liver, which indicated that the expression first increased and then decreased with the extension of time. The expression responses of H2A to A. hydrophila are sensitive to C. aurutus in the early stages. These results are similar to the responses of hepcidin to the challenge of Vibrio anguillarum in common carp (Cyprinus carpio L.) in that the expression level of hepcidin was quickly upregulated in liver, spleen, head kidney, and hindgut, indicating that H2A serves an important function in innate immunity [49]. After sea bass (Dicentrarchus labrax) was acutely stressed by crowding, the expressions of H2B-derived antimicrobial peptide HLP1 and HLP2 were increased in the gill and skin but not in other tissues [22], indicating that environmental factors also resulted in the expression of antimicrobial peptide. Currently, most publications have been focused on that the expressions of many kinds of antimicrobial peptides were upregulated in response to bacterial challenge [50,51,52], and the similar finding was achieved in this study. It is well-known that the up-regulated expressions of antimicrobial peptides to bacterial challenge imply the initiation of antimicrobial peptide producing. Recently, it was found that liver-expressed antimicrobial peptide 2 could inhibit mRNAs expressions of TNF-a and IL-1b, in a teleost fish, Plecoglossus altivelis with V. anguillarum challenge [53]. Shan et al. (2016) found that the antimicrobial peptide SpHyastatin in Scylla paramamosain was up-regulated expressed with the challenge of Vibrio parahaemolyticus [54]. Silencing SpHyastatin mRNA transcripts could result in the decrease of the survival rate of crabs challenged with V. parahaemolyticus. It is demonstrated that the ability of immune protection decreases after mRNA expression of SpHyastatin is inhibited. In order to clarify the relationship between the expression and function of antimicrobial peptides, the more experiments need to be carried out in further study. In this study, the expression profile of Ca-L-hipposin at mRNA level was similar with that of H2A in the same tissue of C. aurutus with A. hydrophila challenge. Only based on these results, it could not support the hypothesis that the expression of Ca-L-hipposin was regulated independently. Therefore, with regard to the derivation of Ca-L-hipposin, it need more evidences at protein level.
5. Conclusion The H2A complete cDNA in C. aurutus is 908 bp, which includes an ORF of 387 bp, a 50 -UTR of 55 bp, and a 30 -UTR of 466 bp, encoding a polypeptide of 128 amino acids with a molecular weight of 13.7 kDa and an isoelectric point of 10.7. Ca-L-hipposin is a cationic peptide with 51 amino acids, and similar to the other orthologous H2A-derived antimicrobial peptides, and its secondary structure contains an a-helix of 17 amino acids and a b-corner of 8 amino acids. Ca-L-hipposin variation and molecular evolution analysis shows high conservation in two motif regions, probably related to antimicrobial function. H2A mRNA expression was high in immune organs, such as the kidney and spleen. After fish were artificially infected with A. hydrophila, the mRNA expression levels of H2A were upregulated in the kidney, spleen, and liver of C. aurutus. It is demonstrated that H2A serves an important function in preventing the invasion of A. hydrophila. As regards the application of Ca-L-hipposin as a prospective antimicrobial peptide in aquaculture, further research has to be conducted on recombinant expression, purification, and activity of Ca-L-hipposin, as well as the mechanism of killing bacteria.
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Acknowledgments This study was funded by Program for Innovative Research Team in Science and Technology in the University of Henan Province (Project No. 15IRTSTHN018) and The joint Fund of Natural Science Foundation of China and Henan Province (Project No. U1604104). The authors would like to thank their colleagues for the valuable suggestions on the overall manuscript preparation.
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Short communication
H2A and Ca-L-hipposin gene: Characteristic analysis and expression responses to Aeromonas hydrophila infection in Carassius aurutus Xianghui Kong*, Xiangmin Wu, Chao Pei, Jie Zhang, Xianliang Zhao, Li Li, Guoxing Nie, Xuejun Li College of Fisheries, Henan Normal University, Xinxiang 453007, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 November 2016 Received in revised form 13 February 2017 Accepted 17 February 2017 Available online 20 February 2017
Antimicrobial peptide is an important component of the host innate immune system and thus serves a crucial function in host defense against microbial invasion. In this study, H2A and derived antimicrobial peptide Ca-L-hipposin were cloned and characterized in Carassius aurutus. The gene H2A full-length cDNA is 908 bp and includes a 50 -terminal untranslated region (UTR) of 55 bp and a 30 -terminal UTR of 466 bp with a canonical polyadenylation signal sequence AATAA, as well as an open reading frame (ORF) of 387 bp encoding a polypeptide of 128 amino acids, with a molecular weight of 13.7 kDa, an isoelectric point of 10.7, and 94% homology with Danio rerio H2A. The secondary structure of H2A includes the a-spiral with 51 amino acids with a composition ratio of 39.8%, as well as a b-corner with 15 amino acids in a composition ratio of 11.7%. The online software ExPaSy predicted that a peptide sequence with 51 amino acids from the 2nd to 52nd amino acids in histone H2A can be produced through hydrolization by protease chymotrypsin, which indicates a difference of only three amino acids, compared with the antimicrobial peptide hipposin in Hippoglossus hippoglossus with a homology of 94%. Ca-L-hipposin includes 51 amino acids with a molecular weight of 5.4 kDa and an isoelectric point of 12.0, the secondary structure of which contains an a-helix of 17 amino acids accounting for 33.3% and a b-corner of 8 amino acids accounting for 15.7%. H2A was extensively expressed in the mRNA levels of various tissues, with higher expression levels in kidney and spleen. After C. aurutus was challenged with Aeromonas hydrophila, the mRNA expression levels of H2A were upregulated in the kidney, spleen, and liver. H2A serves an important function in the defense against the invasion of A. hydrophila. In addition, sequence characteristics reveal that Ca-L-hipposin could be a potential antimicrobial peptide for use in killing pathogenic bacteria in aquaculture. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Histone H2A Hipposin-like antimicrobial peptide Aeromonas hydrophila Carassius aurutus
1. Introduction Histone, being highly conservative and thermally stable, serves an important function in the assembly of DNA molecules. Histone can prevent DNA digestion induced by nuclease and change in topological structure. Moreover, histones can regulate gene expression, transfer, posttranslation modification, cell apoptosis, and tumor lesions [1e3]. In aquatic animals, histone-derived antimicrobial peptides are a type of cationic polypeptide that can kill pathogenic bacteria; these peptides are mainly derived from histones H1, H2A, and H2B [4e7]. Histone-derived antimicrobial
* Corresponding author. No. 46, Jianshe Road, College of Fisheries, Henan Normal University, Xinxiang 453007, PR China. E-mail address: [email protected] (X. Kong). http://dx.doi.org/10.1016/j.fsi.2017.02.028 1050-4648/© 2017 Elsevier Ltd. All rights reserved.
peptides have been isolated from mollusks Chlamys farreri [8], Crassostrea gigas [9, 10], arthropod Macrobrachium rosenbergii [11], amphibians Rhacophorus schlegelii [12], and Bufo bufo gargarizans [13]. In fishes, H2A-derived antimicrobial peptides have been isolated in Hippoglossus hippoglossus [14], Parasilurus asotus [15], and Ictalurus punctatus [16]. These peptides are widely expressed in various tissues [17] and especially abundant in the surface mucus of fish [14,15,18]. H2A-derived antimicrobial peptides possess strong antimicrobial activity, which can protect against Gram-positive and Gram-negative bacteria [14,17], as well as kill fungi without hemolytic activity [15,19]. The antimicrobial activity of these peptides is 12e100 times higher in fish than in amphibians, which shows a minimum bacteriostatic concentration of 0.3 mM (1.6 mg/mL) [15], and achieves effective bacteriostatic activity in concentrations
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ranging from 0.25 mg/mL to 4 mg/mL. They can serve as the first barrier of natural immunity to the invasion of foreign pathogens [15,20,21] and are believed to be the components of innate defenses [22e28]. A novel 51-residue cationic antimicrobial peptide isolated from the skin mucus of Atlantic halibut, H. hippoglossus, was called hipposin, which is rich in Arg and Trp residues but lacks acidic amino acids [20]. A total of 50 of the 51 residues in hipposin were identical to those in the N-terminal region of H2A in rainbow trout. The fragment containing 1 to 19 amino acid residues in the N-terminal exhibits no remarkable antimicrobial activity, whereas the fragment consisting of 16e39 amino acids (similar to buforin II) indicates the antimicrobial activity of hipposin; such activity was found to be enhanced by the presence of a fragment of 40e51 amino acid residues [21]. Almost all previous reports have shown that H2A-derived antimicrobial peptides are similar to the hipposin sequence, indicating that antimicrobial activity is mainly attributable to the action of this fragment. With respect to the production of antimicrobial peptide derived from histone, two suggestions have been proposed as the following. One is that antimicrobial peptide is produced from histone, which is hydrolyzed into the smaller peptide by specific proteases to better serve its antimicrobial function [29]. For example, in Asian toad, B. bufo gargarizans, buforin I is produced by the action of pepsin isozymes called pepsin Ca and Cb, which can cleave the Tyr39eAla40 bond in histone H2A [30]. In catfish, the peptide bond between Ser19 and Arg20 in histone H2A is cleaved by cathepsin D to produce antimicrobial peptide parasin I [15,31,32]. Another viewpoint is that histone-derived antimicrobial peptide is expressed in an independent pattern (communications), the expression of which is not limited to the expression of histone, i.e., the expressions of histone and its derivative antimicrobial peptide are independent. However, up to now, the process of histonederived antimicrobial peptide production remains unclear. Therefore, more evidence should be sought to elucidate the production of histone-derived antimicrobial peptide. In fish, not only the N-terminal segment of H2A, but also the complete H2A protein has antimicrobial activity [18]. Antimicrobial peptide can be induced in the mucus of Oncorhynchus kisutch after exposure to infectious agents [23]. In fish infected with different bacteria, the expression patterns of antimicrobial peptides differ [24,25]. Among a number of aquatic pathogens, the Gram-negative Aeromonas hydrophila has been discovered in a variety of freshwater fish and found to be capable of growing in both aerobic and anaerobic conditions. This bacteria can result in the outbreak of many fish diseases, such as ulcers, fin rot, tail rot, and hemorrhagic septicemia, indicating rapid outburst, high mortality, and difficulty to control, and such outbreak could finally result in serious economic losses in fisheries [26,27]. Research on Carassius aurutus infected with A. hydrophila has been previously conducted [28]. However, the responses of H2A and its derived antimicrobial peptide to bacteria remain unclear. The Qihe crucial carp C. aurutus is an important commercial fish that is widely cultivated in the north of Henan Province, China. In this study, the complete H2A cDNA sequence in C. aurutus was cloned through reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Meanwhile, H2A-derived antimicrobial peptide Ca-L-hipposin was also achieved. The expression responses of H2A and Ca-L-hipposin to A. hydrophila challenge were studied. The aims of this research are as follows: (1) to determine the gene characteristics of H2A and CaL-hipposin in C. aurutus, (2) to analyze the gene variation and molecular evolution of H2A and Ca-L-hipposin, (3) to understand the expression responses of H2A and Ca-L-hipposin to A. hydrophila challenge.
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2. Materials and methods 2.1. Fish The experimental fish C. aurutus with a body weight of 28 ± 2 g, obtained from aquaculture farms in Henan Normal University, were grouped and cultivated in different tanks (in triplicate), with 60 fish each tank. The fish were fed with commercial pellets twice a day (10:00 a.m. and 4:00 p.m.), and sufficient dissolved oxygen was supplied by an aerator. The water temperature was maintained at 23 ± 2 C. After acclimation for one week, the experiment was conducted. 2.2. RNA extraction and cDNA preparation After C. aurutus was dissected, the gill, liver, spleen, intestine, kidney, heart, brain, and muscle were immediately taken and placed in liquid nitrogen for RNA extraction. Total RNA was extracted using TRIzol Reagent (from TaKaRa Com) according to manufacturer's instructions. The quality of RNA was assessed by electrophoresis in a 1.0% agarose gel. The ratio of OD260/OD280 of total RNA was determined to be 1.8 to 2.0, and the concentration was determined to be 400 ng/mL to 500 ng/mL, using a microscale Nanodrop ND2000 spectrophotometer. The cDNA was synthesized using a first-strand cDNA synthesis kit (TaKaRa, No. DRR037A). 2.3. Cloning of H2A cDNA in C. aurutus The first strand of cDNA was used as a template to amplify the central fragments of histone H2A using the specific primers (PF and PR, as listed in Table 1) designed on the basis of conserved domains. PCR reactions were performed in a total volume of 20 mL, including 2 Taq Master Mix 10 mL (Taq DNA Polymerase, PCR Buffer, Mg2þ, dNTPs), 1.0 mL of each primer, 1.0 mL of cDNA, and the addition of double-distilled water (ddH2O) for a total of 20 mL. The PCR conditions were set as follows: 5 min at 95 C for predenaturation, 35 cycles consisting of 30 s at 95 C for denaturation, 30 s at 55 C for annealing, and 45 s at 72 C for extension, and 7 min at 72 C for final extension. 30 -RACE and 50 -RACE were used to obtain the 30 -terminal and 50 terminal sequences of H2A cDNA, respectively. The 30 -terminal, 50 terminal, and central sequences assembled the full-length cDNA sequences of H2A. Eight specific primers are listed in Table 1. These primers were designed on the basis of the amplified central sequences of H2A cDNA. 30 -RACE and 50 -RACE kits were purchased
Table 1 List of primers used in this study. Primer name
Primer sequences
PF PR 30 P-OUT-H2A 50 P-OUT-H2A 30 P-IN-H2A 50 P-IN-H2A 30 RACE Olig(T)-Adaptor 30 RACE Adaptor 50 RACE Olig(T)-Adaptor 50 RACE Adaptor FO-Ca-L-hipposin RE-Ca-L-hipposin FO-H2A RE-H2A FO-b-Actin RE-b-Actin
50 -ATGWGYGGRMGWGGCAAGAC-30 50 -CVGTCTTCTTKGGSAGCAG-30 50 -GGGAACGCAGCCCGAGACAACA-30 50 -TCTTGTTGTCTCGGGCTGCGTTCC-30 50 -CAGTGCTGCTGCCCAAGAAGACGG-30 50 -CCCCGGTTTTACCACTCCCAGACAT-30 50 -CTGATCTAGAGGTACCGGATCCTTTTTTTTTTTTTT-30 50 -CTGATCTAGAGGTACCGGATCC-30 50 -GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-30 50 -GACTCGAGTCGACATCG-30 50 -TCTGGGAGTGGTAAAAC-30 50 -CGCCAGATACACAGGGGCT-30 50 -TGAGTCCAAGTAATGTTATGTTCCCTA-30 50 -TTAAGTGGTGGTGTAGCTCTGTT-30 50 -CATTGACTCAGGATGCGGAAACT-30 50 -CTGTGAGGGCAGAGTGGTAGACG-30
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from TaKaRa, Japan. The amplified PCR product was verified by electrophoresis on a 1.0% agarose gel, which was then purified using an E.Z.N.A.™ gel extraction kit (TaKaRa, Japan) and ligated into PMD19-T vector (TaKaRa, Japan). Then, 10 mL of PMD19-T vector-connected objective cDNA was transferred into 100 mL of competent cells and then cultured on a solid tablet for 10 h. Eight single bacterial colonies were selected and cultured in LB liquid culture. The target sequence was verified by PCR. Two selected colonies were sequenced by the Sangon Biotech Company (Shanghai, China). 2.4. Expression of H2A and Ca-L-hipposin in mRNA levels in C. aurutus The temporal and spatial expressions of H2A and Ca-L-hipposin mRNA were studied using real-time fluorescence quantitative PCR (RT-qRT). Total RNA was extracted from the liver, spleen, heart, kidney, gill, muscle, brain, and intestine using Trizol reagent as described above. To avoid genomic DNA interference on RT-qPCR, the total RNA was incubated with RNase-free DNaseI (TaKaRa, Japan). Based on the cloned H2A cDNA sequence, the specific primers (FO-H2A, RE-H2A, FO-Ca-L-hipposin, and RE-Ca-Lhipposin) were designed and are listed in Table 1. The primer FOCa-L-hipposin and RE-Ca-L-hipposin were designed within Ca-Lhipposin cDNA to detect the expression of Ca-L-hipposin, and the primer FO-H2A and RE-H2A were designed within H2A cDNA and out of Ca-L-hipposin cDNA to detect the expression of H2A. RTqPCR was performed on a 7500 Real-time Fluorescent Quantitative Instruments. The reaction mixture of the PCR consisted of 10 mL of SYBR Premix Ex Taq TMII (TaKaRa, Japan), 0.8 mL of specific primer (10 mM), and 2.0 mL of cDNA template for a total volume of 20 mL. Real-time PCR was performed using a three-step method, which was held at 95 C for 30 s, followed by 40 cycles (at 95 C for 5 s, 60 C for 42 s, and 72 C for 30 s), and the melting curve temperature was finally maintained at 95 C for 15 s, 60 C for 15 s, and 95 C for 15 s. At the end of each PCR reaction, melting curve analysis of amplification products was performed to verify whether the PCR product was single. The b-actin gene (GenBank Accession No. AY690421.1) was used as an internal control. The relative expression levels of the target genes were expressed as the copy number ratio of the target gene relative to b-actin gene. The relative expressions of H2A and Ca-L-hipposin were presented using the 2DDct analysis method [33], with triplication for each PCR. 2.5. Responses of H2A and Ca-L-hipposin to bacterial challenge The experimental fish were randomly divided into two groups. One group was intraperitoneally injected with 100 mL of A. hydrophila (2 108 CFU/mL), and the other group was injected with 0.65% physiological saline at 100 mL. After 3, 6, 9, 12, 15, and 18 h, respectively, the gill, kidney, spleen, and liver of fish were taken out. The same tissues from three fish were mixed as one sample. The experiments were carried out in triplicate. Total RNA was extracted, and cDNA was synthesized as previously described. RT-qPCR was performed using the same protocol as described above. 2.6. Analysis of sequence characteristics and phylogeny Nucleic acid and amino acid sequence alignments were implemented in DNAman software. A phylogenetic tree was constructed using MEGA 6.0 software with a neighbor joining method. The
peptidase cutting locus was analyzed online at http://web.expasy. org/peptide cutter/. The phosphorylation site was analyzed online at http://www.cbs.dtu.dk/services/NetPhos/. The spatial structure was predicted online at http://www.sbg.bio.ic.ac.uk/phyre2/. 2.7. Statistical analysis The significant difference in gene expression between the experimental and control groups was statistically analyzed using one-way analysis of variance, which was implemented in EXCEL 2010 software. The significance level was set at P < 0.05. 3. Results 3.1. Molecular characterization of H2A and Ca-L-hipposin in C. aurutus The sequences of the H2A cDNA conserved region, 30 -untranslated region (UTR), and 50 -UTR were respectively achieved through RT-PCR, 30 -RACE, and 50 -RACE in C. aurutus. The complete cDNA of H2A, registered in GenBank with Accession No. KJ704981, was 908 bp (Fig. 1) and contained an ORF of 387 bp encoding a peptide of 128 amino acids, a 50 -UTR of 55 bp, and a 30 -UTR of 466 bp. The polyadenylation signal (AATAA) was located at the upstream position of 71 bp ahead of the poly A tail. ExPaSy software was used to predict that the molecular weight of H2A is 13.7 kDa, isoelectric point is 10.7, and instability index is 48.6. The estimated half-life of H2A is 30 h in mammalian reticulocytes in vitro, >20 h in yeast in vivo, and >10 h in Escherichia coli in vivo. As regards the secondary structure, an a-helix composed of 51 amino acids accounts for 39.8% of the total length, whereas the b-corner involved in 15 amino acids accounts for 11.7%. H2A-derived antimicrobial peptide Ca-L-hipposin was predicted online by ExPaSy software. The results indicated that the molecular weight is 5.4 kDa, isoelectric point is 12.0, and instability index is 42.5. The secondary structure a-helix of Ca-L-hipposin, which is composed of 17 amino acids, accounts for 33.3%, and the b-corner containing eight amino acids accounts for 15.7%. The estimated half-life is 1.9 h in mammalian reticulocytes in vitro, >20 h in yeast in vivo, and >10 h in E. coli in vivo. Therefore, H2A and its derived Ca-L-hipposin belong to the instability proteins. H2A contains alkaline amino acid residues (13 Lys, 12 Arg, and 2 His), acidic amino acid residues (7 Glu and 2 Asp), and 6 Pro without Cys. Ca-L-hipposin contains alkaline amino acid residue (5 Lys, 8 Arg, and 1 His), acidic amino acid residues (1 Glu), and 2 Pro without Cys. NetPhos2.0 Server online analysis showed that histone H2A phosphorylation sites were located at positions Ser17/19/20/127, Thr7/121, and Tyr40. Ca-L-hipposin phosphorylation sites were located at positions Ser16/18/19, Thr6, and Tyr39. 3.2. Spatial structure of H2A and Ca-L-hipposin in C. aurutus The spatial structures of histone H2A and Ca-L-hipposin were predicted using online Phyre2 software. The spatial structure prediction of H2A was based on the model d1kx5c, and the prediction of Ca-L-hipposin was based on the model d1eqza. The model adopted for the prediction has a coverage of 100%, with a credibility of more than 99%. The predicted spatial structures of H2A and Ca-Lhipposin are shown in Fig. 2. The spatial structure of H2A contains a-helix and b-corner, without a b-sheet, and that of Ca-L-hipposin was similar but with a reduction in quantity. It was postulated that
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Fig. 1. Histone H2A gene and deduced amino acid sequence in C. aurutus. The H2A-derived antimicrobial peptides Ca-L-hipposin are shown in the gray background.
the a-helix structure serves an important function in antimicrobial activity. 3.3. Molecular variations and phylogenetic analysis The 51 Aa of H2A N-terminal (52 Aa minus 1 Met) in C. aurutus corresponds to the antimicrobial peptide hipposin in H. hippoglossus with a homology of 94% and is therefore called Ca-Lhipposin (like hipposin in C. aurutus). Other H2A-derived antimicrobial peptides, such as parasin I (19 Aa), buforin I (39 Aa), buforin II (21 Aa), artificially designed buforin IIA (17 Aa), and buforin IIB (21 Aa) based on buforin II, himantura (39 Aa), abhisin (40 Aa), Rainbow Tr (51 Aa), scallop A (40 Aa), litopenaeu (38 Aa), and molluskin (25 Aa) [10,14,15,21,29,34], which were derived from other animals, were aligned as shown in Fig. 3. Most of these peptides were derived from the N-terminal region of H2A. Ca-Lhipposin showed only a three amino acid difference with hipposin in H. hippoglossus, indicating that the Arg/3rd, Thr/16th, and His/
41st in hipposin respectively correspond to Ser, Ser, and Glu in Ca-Lhipposin. Ca-L-hipposin has high similarity with N-terminal 2e40 Aa of Buforin I . Ca-L-hipposin is the same as 17e37 Aa in Buforin II with the exception of the 17th Aa. Similarly, Ca-L-hipposin, was found to be highly homologous with H2A-derived antimicrobial peptide sequences in other animals. The orthologous sequences of H2A cDNA were searched in GenBank using the searching tool Blastn on the NCBI website. Based on the aligned H2A sequence matrix, a phylogenetic relationship was constructed using the neighbor-joining method implemented in MEGA 6.0 software (Fig. 4). The confidence of the phylogenetic tree was assessed by performing 1000 bootstrap repetitions. Phylogenetic analyses indicated that fish were clustered into two clades in teleosts: one clade includes the fish species in Cypriniformes and Salmoniformes, and another clade includes the fish species of two other orders. In Cypriniformes, C. aurutus shared the highest homology of H2A with Danio rerio, indicating the closest relationship in the phylogenetic tree. The H2A sequence was highly conserved, with C. aurutus being 94% homologous with D. rerio and moderately similar to Oryzias latipes (83%), Salmo salar and Oncorhynchus mykiss (82%), and Haplochromis burtoni (81%).
3.4. H2A mRNA expression in different tissues and organs of C. aurutus
Fig. 2. Predicted protein spatial structures of H2A and Ca-L-hipposin in C. aurutus. The predicted spatial structure is based on the model d1kx5c for H2A and the model for CaL-hipposin.
The expression of histone H2A was detected through RT-qPCR in various tissues and organs (kidney, spleen, gill, liver, brain, intestine, heart, and muscle) of healthy C. aurutus (Fig. 5). The results indicated that H2A was predominantly expressed in kidney, spleen, and gill (10- to 31-fold), secondarily expressed in liver and brain (<5-fold), and less expressed in intestine, heart, and muscle (<2fold).
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Fig. 3. Comparison of H2A-derived antimicrobial peptides. Gray region is on behalf of conservative amino acids.
3.5. mRNA expressions of H2A and Ca-L-hipposin in C. aurutus injected A. hydrophila After injection of A. hydrophila, H2A mRNA expression levels were upregulated in kidney, spleen, gills, and liver of C. aurutus at most time points, as shown in Fig. 6. In the liver, the expression level of H2A mRNA gradually increased and reached the highest value at 15 h after injection, indicating significant differences compared with the controls (P < 0.01). A similar tendency was observed in the spleen and kidney, indicating that the expression level of H2A mRNA reached the highest value at 15 h after injection (P < 0.01) and then significantly decreased and returned to normal levels. However, in the gills, a significant upregulation of H2A mRNA expression was observed at 3 and 12 h after bacterial challenge
(P < 0.01). After injection of A. hydrophila, the expression profile of Ca-Lhipposin at mRNA level was similar with that of H2A in the same tissue of C. aurutus (data not shown). 4. Discussions Recent studies have reported that almost all histone H2As and their derivative antimicrobial peptides are cationic antimicrobial peptides with an amphiphilic a-helix structure [35]. H2A-derived antimicrobial peptides, with remarkable specificity to prokaryotes and low toxicity to eukaryotic cells, have been favored for application as a new antibiotic with antimicrobial activity against a broad spectrum of pathogenic organisms, capability to resist high
Fig. 4. Phylogenetic tree constructed by NJ based on histone H2A implemented in software Mega 6.0. This phylogenetic tree is the unrooted tree. + indicates the position of C. aurutus; The used H2A gene sequences are from the species Xenopus tropicalis (XM_002944612.1), Danio rerio (BX_120005.6), Oryzias latipes (XM_004065529.1), Oncorhynchus mykiss (BT_073517.1), Salmo salar (BT_047220.1), Haplochromis burtoni (XM_005928954.1), Sus scrofa (XM_001927727.3), Rattus norvegicus (XM_003749860.2), Homo sapiens (NM_002105.2), Mus musculus (NM_175662.1), Tursiops truncates (XM_004330511.1), Orcinus orca (XM_004284025.1), Equus caballus (XM_005603620.1), Capra hircus (XM_005696813.1), Microtus ochrogaster (XM_005369911.1), Cavia porcellus (XM_003468798.2), Chrysochloris asiatica (XM_006872785.1).
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Based on the spatial structures of H2A and Ca-L-hipposin, the ahelix was the main structural component. Therefore, a-helical structures in Ca-L-hipposin serve an important function in antimicrobial function through interaction with the cell membrane. Previous studies have reported that a-helical structures help antimicrobial peptides facilitate to combine with and damage the cell membrane [19,36].
4.2. Function of H2A-derived antimicrobial peptides
Fig. 5. Relative H2A mRNA expression levels in various tissues and organs of C. aurutus. The different letters above the columns denote the significance between the different tissues and organs (P < 0.05), and the same letter signs no significant difference (P > 0.05).
temperatures, and widely distributed in organisms, especially in the lower animals (fish and amphibians) [7,21].
4.1. H2A and derived antimicrobial peptide Ca-L-hipposin Based on the composition and structure of H2A and Ca-Lhipposin in C. aurutus, the 51 Aa of H2A N-terminal coding region (minus 1 Met) in C. aurutus corresponds to the antimicrobial peptide hipposin in H. hippoglossus, with a homology of 94%. Therefore, it was suggested that antimicrobial peptide Ca-L-hipposin is derived from H2A. Compared with the H2A-derived antimicrobial peptides in other animals [10,14,15,21,30,34], the variations of sequences might result in differences in antimicrobial activities. Therefore, such peptides could supply the reference data for the redesign to achieve a high-activity antimicrobial peptide.
Most studies have focused on the antimicrobial activities of cationic antimicrobial peptides [37,38]. Antimicrobial peptide activity mainly depends on amino acid composition, amphipathicity, cationic charges, and structure size. These properties can cause these peptides to adhere to the surface of bacteria, insert into membrane bilayers to form pores, and finally, to break down the bacteria [37,39,40]. Some antimicrobial peptides (e.g., magainins and cecropins) preferentially act against bacteria, whereas others (e.g., melittin and gramicidins) interact favorably with both bacteria and eukaryotic cells [41]. Although the majority of antimicrobial peptides can kill bacteria through membrane disruption, some antimicrobial peptides can enter bacterial cells and interfere with physiological function. Examples are pyrrhocoricin, which inhibits ATPase actions and prevents chaperone-assisted protein folding, and indolicidin, which inhibits DNA synthesis in E. coli [42]. H2A-derived buforin II can enter cells to combine with DNA/ RNA, the binding force of which with nucleic acids is 20 times that of magainin 2 [13]. Antimicrobial peptides could inhibit the synthesis of nucleic acids, proteins, and cell walls as well as reduce enzymatic activity [43]. Buforin II can also damage the cell membrane of bacteria, causing a leakage of contents, the intensity of which is 100 times that of magainin 2 [34]. In this study, buforin II corresponds to the partial fragment of Ca-L-hipposin, which was
Fig. 6. mRNA expression levels of H2A in gill, kidney, spleen, and liver of C. aurutus after A. hydrophila infection. All values present with mean ± standard deviation (n ¼ 3). The statistically significant differences between the experimental group (gray bar) and the control (white bar) are indicated with asterisk above the gray bar (*, P < 0.05; **, P < 0.01).
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highly homologous with H2A-derived antimicrobial peptide sequences in other animals, indicating Ca-L-hipposin had functions in antimicrobial activity. 4.3. Phosphorylation sites and prolines in Ca-L-hipposin The phosphorylated form of protein can strengthen its function and stability. Histone H1, which has three phosphorylation sites, can not only improve the capability to bind DNA but can also resist the hydrolysis of protein, and without phosphorylation, can be digested by protease [44]. Phosphorylation in both end residues of H1-derived antimicrobial peptide oncorhyncin II can prevent damage from bacterial proteases. In this study, Ca-L-hipposin has phosphorylation sites at Ser/16th/18th/19th, Thr/6th, and Tyr/39th. These phosphorylation sites are postulated to serve important contributions to physiological function and protection in C. aurutus. The H2A-derived antimicrobial peptide buforin II can cause cell death by translocation across membranes and interaction with nucleic acids [45]. To understand the function of proline in buforin II, the effects of changes in proline position on antimicrobial activity were further studied using four mutants obtained through proline substitution, indicating that the variation of Pro could have an important influence on antimicrobial activity [34]. In this study, the antimicrobial peptide Ca-L-hipposin in C. aurutus contains Pro, the antimicrobial activity of which could be improved by altering its position and number in the future. 4.4. Variations of acidic and alkaline amino acids in H2A-derived antimicrobial peptides The cationic antimicrobial peptides generally have charges of þ2 to þ9, whereas the amino acids are at 10 to 50 [46]. The basic amino acid is essential for antimicrobial peptides, which serve an important function in maintaining antimicrobial activity [47]. Actually, the change in a single amino acid indicates a negligible effect on antimicrobial activity. For example, defenin in rabbits, with the lack of Arg residue, also showed similar antimicrobial activity [48]. However, an increase in cationic peptides could decrease antimicrobial activity. The amino acid fragments from positions 16 to 36 in Ca-Lhipposin, corresponding to buforin II, and positions 40 to 51 are the main antimicrobial motifs. Outside of the two fragments, the mutation implies a slight effect on antimicrobial activity, for example, a mutation in the third amino acid [26]. In this study, the variations of amino acids, such as that at the 16th and 41st positions are studied. In particular, we examine that of the 41st position, which is the basic amino acid in H. hippoglossus, and is the acidic amino acid in C. aurutus. Given that the acidic amino acid reduces the number of net positive charges, we speculated that acidic amino acids could have an important influence on antimicrobial activity. 4.5. mRNA expression responses of H2A and Ca-L-hipposin to the injected A. hydrophila As regards the tissue expression of H2A mRNA, the expression levels of H2A mRNA were higher in kidney, spleen, gill, liver, and brain, which indicated that the higher expression levels of H2A mRNA were maintained in immune organs (the highest in the kidney and the second highest in the spleen). This result was in agreement with the suggestions of previous studies [49]. After aquatic animals are infected by pathogenic bacterium, antimicrobial peptide expression presents a series of responses to
prevent and kill bacteria [49]. In this study, after A. hydrophila was injected into C. aurutus, the expression levels of H2A increased in the kidney, spleen, and liver, which indicated that the expression first increased and then decreased with the extension of time. The expression responses of H2A to A. hydrophila are sensitive to C. aurutus in the early stages. These results are similar to the responses of hepcidin to the challenge of Vibrio anguillarum in common carp (Cyprinus carpio L.) in that the expression level of hepcidin was quickly upregulated in liver, spleen, head kidney, and hindgut, indicating that H2A serves an important function in innate immunity [49]. After sea bass (Dicentrarchus labrax) was acutely stressed by crowding, the expressions of H2B-derived antimicrobial peptide HLP1 and HLP2 were increased in the gill and skin but not in other tissues [22], indicating that environmental factors also resulted in the expression of antimicrobial peptide. Currently, most publications have been focused on that the expressions of many kinds of antimicrobial peptides were upregulated in response to bacterial challenge [50,51,52], and the similar finding was achieved in this study. It is well-known that the up-regulated expressions of antimicrobial peptides to bacterial challenge imply the initiation of antimicrobial peptide producing. Recently, it was found that liver-expressed antimicrobial peptide 2 could inhibit mRNAs expressions of TNF-a and IL-1b, in a teleost fish, Plecoglossus altivelis with V. anguillarum challenge [53]. Shan et al. (2016) found that the antimicrobial peptide SpHyastatin in Scylla paramamosain was up-regulated expressed with the challenge of Vibrio parahaemolyticus [54]. Silencing SpHyastatin mRNA transcripts could result in the decrease of the survival rate of crabs challenged with V. parahaemolyticus. It is demonstrated that the ability of immune protection decreases after mRNA expression of SpHyastatin is inhibited. In order to clarify the relationship between the expression and function of antimicrobial peptides, the more experiments need to be carried out in further study. In this study, the expression profile of Ca-L-hipposin at mRNA level was similar with that of H2A in the same tissue of C. aurutus with A. hydrophila challenge. Only based on these results, it could not support the hypothesis that the expression of Ca-L-hipposin was regulated independently. Therefore, with regard to the derivation of Ca-L-hipposin, it need more evidences at protein level.
5. Conclusion The H2A complete cDNA in C. aurutus is 908 bp, which includes an ORF of 387 bp, a 50 -UTR of 55 bp, and a 30 -UTR of 466 bp, encoding a polypeptide of 128 amino acids with a molecular weight of 13.7 kDa and an isoelectric point of 10.7. Ca-L-hipposin is a cationic peptide with 51 amino acids, and similar to the other orthologous H2A-derived antimicrobial peptides, and its secondary structure contains an a-helix of 17 amino acids and a b-corner of 8 amino acids. Ca-L-hipposin variation and molecular evolution analysis shows high conservation in two motif regions, probably related to antimicrobial function. H2A mRNA expression was high in immune organs, such as the kidney and spleen. After fish were artificially infected with A. hydrophila, the mRNA expression levels of H2A were upregulated in the kidney, spleen, and liver of C. aurutus. It is demonstrated that H2A serves an important function in preventing the invasion of A. hydrophila. As regards the application of Ca-L-hipposin as a prospective antimicrobial peptide in aquaculture, further research has to be conducted on recombinant expression, purification, and activity of Ca-L-hipposin, as well as the mechanism of killing bacteria.
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Acknowledgments This study was funded by Program for Innovative Research Team in Science and Technology in the University of Henan Province (Project No. 15IRTSTHN018) and The joint Fund of Natural Science Foundation of China and Henan Province (Project No. U1604104). The authors would like to thank their colleagues for the valuable suggestions on the overall manuscript preparation.
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