Antimicrobial activity of histone-derived peptides H2A and H4 from silver pomfret (Pampus argenteus) against Photobacterium damselae subsp. damselae

Highlights

• •Two histone-derived peptides, designated as spH2A.2 and spH4, were identified from the silver pomfret.
• •The expression levels of both histone genes were significant upregulated at 1 day post-infection in the liver.
• •The spH4 and spH2A.2 peptides had potent antimicrobial activity against Gram-negative bacteria.
• •SEM analysis revealed that spH4 and spH2A.2 could destroy the PDD cell wall structure.

Abstract

The silver pomfret (Pampus argenteus) is a commercially valuable marine fish species in China. Photobacterium damselae subsp. damselae (PDD) is an opportunistic pathogen in aquaculture environments and has become a serious bacterial pathogen of the aquacultural industry in recent years. In this study, two functional fragments originated from histone-derived peptides (H2A and H4) of the silver pomfret, and were identified and designated as spH2A.2 and spH4. In addition, the full-length cDNA of H2A from silver pomfret (SPH2A) consists of 387 base pairs (bp) coding sequence (CDS) that encodes 128 amino acids (aa), while the full-length cDNA of SPH4 consists of 312 bp CDS and encodes 103 aa. Tissue distribution analysis showed that SPH2A had a higher expression pattern in the head kidney and intestine, while SPH4 was mainly expressed in the spleen. After PDD infection, both histone gene expression levels were significant upregulated at 1 day post-infection (dpi) (P < 0.01) in the liver. Furthermore, spH4 and spH2A.2 peptides had potent antimicrobial activity against Gram-negative bacteria, with the MIC value ranging from 24 to 95 µM and 66–133 µM, respectively. Cytotoxicity of spH4 and spH2A.2 was tested in silver pomfret primary cells (liver, spleen and head kidney), and the results showed that spH4 had no cytotoxicity, while spH2A.2 displayed cytotoxic activity on silver pomfret cells. Additionally, scanning electronic microscope (SEM) analysis revealed that spH4 and spH2A.2 could destroy the PDD cell wall structure. Overall, the spH4 and spH2A.2 peptides were likely prone to exert the antimicrobial activity and may be useful to defend against PDD in silver pomfret aquaculture.

Introduction

Silver pomfret (Pampus argenteus) is a commercially valuable marine fish species indigenous to the coastal regions of China, the Arabian Gulf, and the Indian Ocean [1]. Prior research has demonstrated successful advancements in silver pomfret aquaculture, encompassing aspects such as capacity, breeding, hatching, and larval rearing [2], [3], [4]. Along with the high-intensity fishing growth and the industrialized aquaculture development, diverse aquatic animal diseases such as Photobacterium damselae subsp. damselae (PDD), Streptococcus sp., Uronema sp., Amyloodinium ocellatum, Cryptocaryon irritans and iridovirus are becoming hazards to the farming of silver pomfret [4], [5], [6], [7], [8], [9]. Among these pathogens, PDD stands out as a subspecies of Gram-negative marine bacteria known as P. damselae, posing a threat to various organisms such as reptiles, cetaceans, crustaceans, mollusks, fish and humans [10]. Particularly, PDD is an opportunistic pathogen in aquaculture settings, demonstrating the capability to induce diseases across a wide spectrum of organisms [11]. In silver pomfret aquaculture, PDD has been identified as a newly emerging bacterial pathogen, leading to frequent outbreaks during the summer months when water temperatures range between 23 °C and 26 °C. This pathogen is known to cause severe external hemorrhages and exhibits high cytotoxic effects on the kidney cells of marine finfish [4].

To date, research concerning effective strategies to manage PDD in silver pomfret aquaculture has remained quite limited. Regarding disease control, antibiotics have gained widespread usage in aquaculture practices. For example, streptomycin has proven effective against all PDD isolates, but the indiscriminate application of antibiotics has led to the emergence of multidrug-resistant bacteria, as evidenced by various studies [11], [12], [13], [14]. Consequently, the research for viable alternatives to antibiotics that can address and mitigate aquatic illnesses has become an urgent priority. In this context, recent attention has turned towards novel antimicrobial peptides (AMPs) derived from histones, which hold substantial promise in combatting bacterial infections in fish. For instance, peptides such as LcH1–1 and LcH1–2 originating from Larimichthys crocea histone1-derived antimicrobial peptides (LcH1) exhibited notable antibacterial efficacy, with minimum inhibitory concentrations (MICs) ranging from 12 to 24 µM and 24–48 µM, respectively [15]. This emerging avenue opens up exciting prospects for addressing the bacterial challenge and aquatic disease control in a more environmentally friendly manner.

Histone-derived peptides originate from the proteolytic cleavage of histones, essential protein constituents of chromatin, thereby occupying a distinct niche in cell biology [16]. A variety of histone-derived AMPs have been reported in aquaculture, such as buforin I, piscidins, and hipposin, boasting wide-ranging efficacy [17], [18], [19]. In teleosts, histone-derived peptides manifest as peptide fragments exceeding 10 kDa, exhibiting antimicrobial potency against a diverse array of pathogens spanning Gram-positive and Gram-negative bacteria [20]. Eukaryotic histones comprise four core histones (H2A, H2B, H3, and H4) and two linker histones (H1 and H5). Among these histone-derived peptides, AMPs stemming from H2A and H4 have garnered significant attention across both invertebrates and vertebrates [21]. Recently, the defensive capabilities of H2A variants against infections such as Aeromonas hydrophila in Carassius auratus [22], Streptococcus agalactiae in Danio rerio [23], and Flavobacterium columnare in grass carp [24]. Furthermore, PvH4a, derived from histone H4 of Penaeus vannamei, has demonstrated the capacity to dismantle cell walls in Gram-negative bacteria [25].

In recent years, the emergence of PDD outbreaks has emerged as a significant challenge in the silver pomfret aquaculture industry. Scare literature on the utilization of H2A and H4-derived AMPs for mitigating PDD has been reported. Therefore, this study investigates two histone-derived peptides (H2A and H4) sourced from silver pomfret to explore their antibacterial efficacy. The research aims to uncover the distinct expression patterns of these histone-derived genes across various tissues of the silver pomfret. Furthermore, the study involves the identification of functional fragments spH2A.2 and spH4 originating from silver pomfret H2A and H4-derived peptides (SPH2A and SPH4), while their impact on PDD is also examined. Simultaneously, this study could provide valuable insights into the development of novel therapeutic approaches against PDD infection through the application of molecular biological techniques and antimicrobial assessments. These findings will also serve as a valuable resource for furthering the understanding and exploration of strategies to control PDD in the aquaculture industry.

Materials and methods

Fish

Healthy silver pomfrets with a body weight of 90 ± 5 g were collected from Xiangshan Bay Sci-Tech Co., Ltd (Zhejiang, China) and subsequently nurtured at the aquatic base of Ningbo University. The fish were kept in tanks equipped with a continuous seawater circulation system, maintaining a temperature of 25 ± 1 °C and a salinity level of 24–26 ppt to mimic natural conditions in terms of photoperiod. The fish, ranging from 12 to 18 cm in length, were given a two-week period for acclimatization before the experiments and were fed with commercial fish pellets (larve love 6#, Hayashikane Sangyo, Yamaguchi-ken, Japan) mixed with freshwater (1:1 by weight) twice a day [26].

Bacteria

The PDD strain (silver pomfret isolate 69TBY1) and Escherichia coli (DH5α) were used in this study. All strains were generated as previously described with slight modifications [4,27]. Briefly, PDD was streaked onto tryptone soy agar plates supplemented with 1 % NaCl (TSA-1) prior to testing, followed by incubation at 25 °C for 48 h. The E. coli cells were streaked onto Luria-Bertani (LB) agar plates and incubated at 37 °C for 24 h. Dense growth was observed on both media plates, and a single colony was picked to culture in TSB-1 or LB medium overnight.

Experimental infection

A total of 120 healthy silver pomfrets were cultivated and divided into six tanks. Briefly, twenty fish from each of the three PDD-infected (PI) groups were injected intramuscularly with 2.6 × 10⁵ CFU/mL of PDD re-suspended in 100 µL sterile 1 × PBS, according to the previously reported LD₅₀ dose [4]. Twenty fish from each of the three PBS-injected (sham-injected; SI) groups were designated as control groups. A tricaine methane sulfonate (TMS) solution of 40–50 mg/L in seawater was used to anesthetize fish prior to handling in the experiments. Tissues (brain, head kidney, spleen, trunk kidney, gill, muscles, liver, intestine) were obtained after 1, 3, 7, and 14 days post-infection (dpi). All procedures involving animals and bacteria were authorized by the Institutional Animal Care and Use Committee of Ningbo University and carried out in accordance with its regulations for using animals in research under the protocol number NBU20210049.

Cloning of H2A and H4 cDNA in silver pomfret

Liver tissues of silver pomfret were taken and immediately flash-frozen in liquid nitrogen, and then stored at −80 °C. Total RNA was extracted using TRIzol Reagent (Gibco, USA) according to the manufacturer's instructions. The cDNA was synthesized using a first-strand cDNA synthesis kit (Transgen, China). The primers were designed according to the sequences obtained from the previously generated transcriptome database in our lab [28]. All primers employed in cloning and expression of histone H2A and H4 genes were listed in Supplementary Table 1 (Table S1) and synthesized by Youkang Biotechnology Co., Ltd (Zhejiang, China). PCR reactions were performed in a total volume of 50 µL using 2 × Taq MasterMix kits (CWBIO, China). According to the manufacturer's instructions, the PCR conditions were set as follows: 94 °C for 5 min, followed by 32 cycles at 94 °C for 30 s, 50–54 °C for 30 s, and 72 °C for 1 min; followed by an extension stage at 72 °C for 10 min. The PCR products were separated on a 1.0 % agarose gel stained with ethidium bromide, purified with PCR purification kit (Omega Bio-Tek, USA), ligated with pMD-19T vector (Takara, Japan), and then transformed into competent E. coli DH5α cells (Invitrogen, USA). The positive colonies were identified with a colony PCR reaction using M13 forward and reverse primers (Table S1). The positive clones were identified and sequenced by Youkang Biotechnology Co., Ltd (Zhejiang, China).

Sequence and phylogenetic analysis

Sequences generated were analyzed for similarity with other known sequences using the BLAST programs at the National Center for Biotechnology Information (http://www.ncbi.nlm.gov/blast) [29]. Homologous sequences and the alignment of multiple sequences were performed using MEGA X64. Evolutionary trees of the two histone sequences were constructed using the neighbor-joining method, and the reliability of the branching was tested using bootstrap resampling with 1000 replicates. Protein sequences used for analysis in this study were all obtained from GenBank (Table S2 and S3). Moreover, the molecular weight was assessed using SMS system (http://www.bio-soft.net/sms/). The 3D structures of AlphaFold DB model were predicted using Swissmodel (https://swissmodel.expasy.org/) and performed by PyMOL 2.6 software. Furthermore, the DNAstar Protean software and helical wheel modeling were used to determine the secondary structure. The arrangement of amino acids is projected by a helical wheel, and residue counts begin at the amino (N) terminus of teleosts.

Quantitative mRNA analysis

Tissue distribution of SPH2A and SPH4 genes was analyzed in healthy fish using PerfectStart Green qPCR SuperMix kits (Transgen, China). Moreover, the mRNA expression levels of SPH2A and SPH4 in control (PBS) and PDD infected tissues (head kidney and liver) were analyzed. The specific primers were designed using premier primer 6.0 software. The β-actin gene was used as an internal reference gene (Table S1). According to the manufacturer's instructions, the thermal profile for quantitative real-time PCR (qRT-PCR) was 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The analysis of each sample was conducted in three replicates. The relative expression levels of genes were calculated according to the comparative threshold cycle method (2^−ΔΔCT) and shown as mean ± standard deviation (SD) [30].

Active peptide characterization

Fi-His_1–21 (SRSSRAGLQFPVGRIHRLLRK), an antimicrobial peptide derived from histone H2A in Fenneropenaeus indicus, and PvH4a (TVTAMDVVYALK), derived from histone H4 in P. vannamei, served as models for the design of spH2A.2 and spH4 in P. argenteus [25,31]. According to multiple alignment of SPH2A and SPH4 with other species, the synthesis of synthetic peptides with a final purifty exceeding 95 % was conducted through solid-phase peptide synthesis at Sangon Biotechnology Co., Ltd (Shanghai, China). Subsequently, the lyophilized peptide powder was reconstituted in sterile PBS and stored at −20 °C.

In vitro antibacterial test

The antimicrobial activity of peptides was conducted in triplicate on separate occasions in 96-well flat-bottom plates using MIC and minimum bactericidal concentration (MBC) as previously described methods with slight modifications [15,25]. Briefly, Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (E. coli, Vibrio harveyi, PDD, Pseudomonas aeruginosa, V. alginayticus, V. vulnificus, V. parahaemolyticus) were diluted with sterile PBS (pH 7.2). Synthesized AMPs were diluted to a series of concentrations with sterile PBS. Next, peptides were mixed separately with bacteria in equal volume, followed by incubation for 24 h at 26 °C or 37 °C in the dark. After the incubation period, samples were measured in terms of absorbance at 600 nm using a microplate reader (FlexA-200, ALLSHENG). Subsequently, 100 µL cultures were added on plates (TSA-1 or LB) in triplicate and incubated at the appropriate temperature overnight. Finally, colonies were counted and recorded. The MIC of AMPs was defined as the lowest concentration that completely inhibits bacterial growth, while the MBC was defined as the minimum AMP concentration without bacterial growth.

Scanning electron microscopy observation

The preparation procedure of SEM analysis was conducted as previously described by Yang et al. with modifications [25]. In brief, bacterial suspensions of E. coli and PDD at 10^5–6 CFU/mL were treated with 1MIC of spH2A.2 or spH4 peptide. An equal volume of sterile PBS was used as a control. All samples were incubated at 26 °C or 37 °C for 2 h. Each treatment was centrifuged at 2700 g for 10 min and washed twice with sterile PBS. Then, the supernatant of each treatment was removed, and 2.5 % formaldehyde was added for overnight at 4 °C. Subsequently, the bacteria were dehydrated in a sequential graded ethanol series (30 %, 50 %, 75 %, 90 %, 95 % and 100 %), and the ethanol was then replaced by 100 % tertiary butyl alcohol. Finally, the supernatant was placed on a poly-l-lysine glass slide and subjected to drying by Freeze Dryer (Christ Alpha 1–4 LD plus, Germany). The bacteria cells were then gold-coated and photographed using a stereoscopic Model S-3400 N Type I microscope (Hitachi, Japan).

Cytotoxicity of spH4 and spH2A.2

The cytotoxicity of peptide spH4 and spH2A.2 was determined by the lactate dehydrogenase (LDH) method [4]. First, the procedures for the isolation and cultivation of fish cells and the medium (NMGFL-15) used for their cultivation had been described previously [32,33]. More specifically, silver pomfret cells from either the liver, spleen, or head kidney were isolated by passing the tissue through a stainless steel mesh with a homogenization solution containing antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) and heparin (50 U/mL). The obtained cell suspension was centrifuged at 400 g for 10 min at room temperature. The cells were washed twice by centrifugation at 400 g for 10 min each time and resuspended in complete NMGFL-15 medium. Silver pomfret cells from four tissues were counted and diluted to 2 × 10⁵/mL by using a hemocytometer. Furthermore, 100 µL silver pomfret cells were plated onto 96-well plates and incubated at 26 °C, followed by adding 10 µL of gradient concentration of peptide spH2A.2 or spH4 to each well. Samples treated with nisin (BioDuly, China) were used as the positive control, while sterile PBS was used as the negative control [25]. Each concentration was measured in triplicate. Following this, samples were incubated for 24 h at 26 °C and centrifuged at 400 g for 5 min to obtain the supernatant. According to the protocol of the LDH cytotoxicity assay kit (Beyotime, China), LDH activity of each well was determined, and the absorbance at 490 nm was measured using a FLEXa-200 microplate reader (ALLSHENG, China).

Statistical analysis

One-way analysis of variance (one-way ANOVA) and Duncan's multiple comparison test were used to examine all data using the Statistical Package for the Social Sciences 17.0 (SPSS Inc., USA). The mean and SD of the data were displayed. A probability threshold of 0.05 (P < 0.05) was employed in the student t-test to assess the statistical difference between the control and experimental groups. At the level of P < 0.001, highly significant differences were identified. The software GraphPad Prism version 8.0 (GraphPad, USA) was used to perform the graphs.

Results

Sequence analysis of SPH2A and SPH4 gene

A 387 base pairs (bp) segment of SPH2A was firstly cloned in this study. The cDNA sequence and deduced amino acid sequences of SPH2A were shown in Fig. S 1A (GenBank Accession No. OQ459357). Additionally, phylogenetic relationship analysis indicated that the amino acid sequence of SPH2A was grouped with the branch of H2A.Z (Table S4). The identity of the amino acid sequence deduced from the silver pomfret histone H2A was 100 %, 96 %, 82 % similar to Echeneis naucrates (XP_029365670), Mus musculus (NP_001096135), and Terrapene carolina triunguis (XP_024063074), respectively (Fig. 1A).

Fig. 1.

Phylogenetic analysis of SPH2A (A) and SPH4 (B) proteins with other homologous sequences. Numbers on the branches represent confidence values from bootstrap with 1000 replicates. A: The phylogenetic of SPH2A. Sequence accession numbers used in the study are indicated as follows: Oreochromis niloticus (XP_003451211), Sparus aurata (XP_030293901), Mastacembelus armatus (XP_026183860), Seriola dumerili (XP_022604923), Echeneis naucrates (XP_029365670), Larimichthys crocea (XP_010732740), Oryzias melastigma (XP_024145027), Oncorhynchus mykiss (XP_021449894), Salmo salar (XP_013992645), Danio rerio (XP_002666988), Pygocentrus nattereri (XP_017542589), Carassius auratus (XP_026059183), Takifugu rubripes (XP_003980051), Clupea harengus (XP_012683108), Pangasianodon hypophthalmus (XP_053098258), Acipenser ruthenus (XP_033851818), Gallus gallus (NP_001025924), Duttaphrynus melanostictus (ALS54715), Xenopus tropicalis (NP_001015968), Terrapene carolina triunguis (XP_024063074), Rattus norvegicus (NP_001019453), Mus musculus (NP_001096135), Bos taurus (DAA33791), Homo sapiens (NP_066544). B: The phylogenetic of SPH4. Sequence accession numbers used in the study are provided as follow: Acipenser ruthenus (XP_034766173), Bos taurus (NP_776305), Carassius auratus (XP_026124419), Cyprinus carpio (XP_042584802), Clupea harengus (XP_042559554), Echeneis naucrates (XP_029385479), Gallus gallus (XP_040516429), Larimichthys crocea (XP_027129607), Homo sapiens (NP_003486), Oryzias melastigma (XP_024145027), Danio rerio (XP_021333749), Takifugu rubripes (XP_029687832), Macaca mulatta (NP_001361474), Mastacembelus armatus (XP_033180989), Mus musculus (NP_291074), Pangasianodon hypophthalmus (XP_034169669), Oncorhynchus mykiss (XP_036805837), Pygocentrus nattereri (XP_037389488), Penaeus vannamei (XP_027223322), Rattus norvegicus (NP_073177), Sparus aurata (XP_030296530), Seriola dumerili (XP_022611590), Xenopus tropicalis (XP_031748985).

In addition, sequence analysis showed the ORF of SPH4 contains 312 bp and encodes 103 aa protein. The cDNA sequence and deduced amino acid sequences of the SPH4 were shown in Fig. S1B (GenBank Accession No. OQ623792). The result of BLASTp analysis showed that SPH4 was 98 % clustered with Xenopus tropicalis (XP_031748985), Takifugu rubripes (XP_029687832), Sparus aurata (XP_030296530), and Seriola dumerili (XP_022611590) (Fig. 1B).

SPH2A and SPH4 mRNA expression in different tissues

In this study, the mRNA expression level of SPH2A and SPH4 was detected through qRT-PCR in various tissues of healthy silver pomfret. As shown in Fig. 2, the mRNA expression level of SPH2A was mainly expressed in the head kidney and intestine, and a relatively lower level was observed in the muscle, trunk kidney, and liver (Fig. 2A), while SPH4 was mainly expressed in the spleen, liver, and intestine, with relatively lower expression level in the muscle and brain (Fig. 2B). After PDD infection, the mRNA expression level of SPH2A was significantly upregulated in the liver at 1 and 14 dpi (P < 0.01) (Fig. 3A). In contrast, the mRNA expression level of SPH2A was significantly upregulated in the head kidney at 7 and 14 dpi (P < 0.05), but downregulated at 3 dpi (P < 0.05) (Fig. 3B). Additionally, the mRNA expression level of SPH4 showed a significantly upregulation at 1 dpi in both liver and head kidney tissues (P < 0.01) (Fig. 3C and 3D).

Fig. 2.

Tissue distribution pattern and relative expression level of SPH2A and SPH4 in healthy silver pomfret. A: The tissue distribution pattern and relative expression level of the SPH2A gene in healthy tissues. B: The tissue distribution pattern and relative expression level of the SPH4 gene in healthy tissues. One-way ANOVA followed Duncan's multiple comparison test was used for statistical analysis. Significant differences between different tissues are indicated with different letters (P < 0.05).

Fig. 3.

SPH2A and SPH4 mRNA expression patterns from immune tissues after PDD challenge. A: The SPH2A mRNA expression pattern in the liver after PDD infection. B: The SPH2A mRNA expression pattern in the head kidney after PDD infection. C: The SPH4 mRNA expression pattern in the liver after PDD infection. D: The SPH4 mRNA expression pattern in the head kidney after PDD infection. Expression levels are represented as mean ± SD (n = 3), and are indicated with different letters. The numbers in each group represent the P value. The symbol * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001 using Duncan's multiple comparison.

Peptides purified from SPH2A and SPH4

To investigate the antimicrobial effects of SPH2A and SPH4 peptides in silver pomfret, two synthetic peptides corresponding to SPH2A and SPH4 were identified and subsequently synthesized using capillary HPLC, as detailed in Table 1. The information of chromatogram and MS spectrum was included in Fig. S2 and S3. As shown in Fig. S4A, other H2A-derived antimicrobial peptides that came from different animals aligned with spH2A.2. In the multiple alignment analysis, the sequences of spH2A.2 showed a notable similarity to buforin IIb, showcasing two conservative amino acids (G, G) shared with other species. Concerning spH4, the multiple alignment revealed that all other species shared the same conservative amino acids (TVTAMDVVYALK) (Fig. S4B). The homology modeling of SPH2A and SPH4, depicted in white in Fig. 4A and 4C, underscores their α-helical structures. Notably, spH2A.2 and spH4 are recognized as fragmentary derivatives of SPH2A and SPH4, respectively. The Schiffer-Edmundson helical wheel diagrams for spH2A.2 (Fig. 4B) and spH4 (Fig. 4D) illustrate their amphipathic α-helical configurations. The amino acid residues are sequentially numbered starting from the amino terminal end. In the helical wheel diagram of spH2A.2, the distribution of hydrophilic residues (arginine [R], histidine [H], lysine [K], glutamine [Q]) and hydrophobic residues (alanine [A], glycine [G], proline [P], valine [V], leucine [L], isoleucine [I], phenylalanine [F]) is such that they are oppositely aligned. Similarly, in the helical wheel diagram of spH4, hydrophilic residues (threonine [T], tyrosine [Y], aspartic acid [D], lysine [K]) are aligned opposite to hydrophobic residues (alanine [A], valine [V], leucine [L], methionine [M]), illustrating their respective amphipathic arrangements.

Table 1.

Properties of silver pomfret H2A and H4 protein derived antimicrobial peptide.

Seq.ID	Sequence	Length(aa)	modification	MW by MS	Predict from reference
spH4	TVTAMDVVYALK	12	C-terminal amide	1310.1	(Yang et al., 2021)
spH2A.2	RAGLQFPVGRIHRHLK	16	N-terminal amide	1885.2	(Kong et al., 2017)

Fig. 4.

The homology modeling and Schiffer-Edmundson helical wheel diagram of spH2A.2 and spH4 peptides are shown. A: The white part represents SPH2A, and the green model represents spH2A.2. The hydrogen bonds within 2.8 to 3.4 Å are shown as yellow lines in spH2A.2. B: The Schiffer-Edmundson helical wheel diagram of spH2A.2. C: The white part represents SPH4, and the green model represents spH4. The hydrogen bonds within 2.9 to 3.4 Å are shown as yellow lines in spH4. D: The Schiffer-Edmundson helical wheel diagram of spH4.

Antimicrobial activity assays

To assess the antimicrobial activity of the synthesized peptides, spH2A.2 and spH4, various microorganisms were subjected to incubation with these peptides individually. The results, as revealed through MIC and MBC estimations, were documented in Table 2, and showed that the spH4 and spH2A.2 peptides exhibited notable antimicrobial activity against Gram-negative bacteria. However, their impact on Gram-positive bacteria, such as S. aureus, did not exhibit obvious antimicrobial activity. The peptide spH4 demonstrated the capability to impede the proliferation of V. parahaemolyticus and P. aeruginosa, attaining a MIC value of 24 µM and a MBC value of 48 µM. It is noteworthy that spH4 exhibited antimicrobial activity against PDD and E. coli with MIC and MBC values of 48 µM and 95 µM, respectively. Moreover, it was effective against V. vulnificus, V. harveyi, and V. alginayticus with MIC and MBC values of 95 µM and 191 µM. Similarly, peptide spH2A.2 demonstrated robust antimicrobial attributes against V. parahaemolyticus, PDD, and E. coli with corresponding MIC and MBC values of 66 µM and 133 µM.

Table 2.

Antimicrobial activity of histone derived peptide against bacteria (µM).

Bacteria	MIC (µM)		MBC (µM)
	spH4	spH2A.2	spH4	spH2A.2
V. parahaemolyticus	24	66	48	133
V. vulnificus	95	133	191	133
V. alginayticus	95	133	191	265
P. aeruginosa	24	133	48	265
PDD	48	66	95	133
V. harveyi	95	133	191	>265
E. coli	48	66	95	133
S. aureus	>191	>265	>191	>265

Note: MIC: Minimal inhibitory concentration; MBC: Minimal bactericidal concentration.

Scanning electron microscopy examination of the bacterial membrane

Morphological alterations of spH2A.2 and spH4-treated Gram-negative bacteria (E. coli and PDD) were observed by scanning electron microscopy (SEM) (Fig. 5). In contrast to the smooth surface structure observed in the control group for E. coli (Fig. 5A), treatment with 1MIC spH2A.2 induced the dissolution of cell wall (Fig. 5B), yielding an abnormality rate of approximately 10 %. In addition, exposure of E. coli to spH4 at 1MIC led to observable destruction of the membrane structure (Fig. 5C), with an estimated abnormality rate of 20 %. Conversely, SEM observations demonstrated that the characteristic presence of two flagellates and smooth surfaces in normal PDD (Fig. 5D). In addition, the application of 1MIC spH2A.2 caused irregular surface features and discernible membrane deformation in PDD (Fig. 5E), with an abnormality rate of 33 %, while treatment with 1MIC spH4 led to notable and profund morphological changes, characterized by membrane disruption (Fig. 5F), and an abnormality rate of 25 %.

Fig. 5.

Scanning electron micrographs of E. coli and PDD are shown. E. coli and PDD were incubated with 1MIC of spH2A.2 and spH4 separately. Micrographs show E. coli treated with PBS (A) as control, or peptide spH2A.2 (B) and spH4 (C). PDD treated with control (D), or peptide spH2A.2 (E) and spH4 (F). The bacteria reveal clear morphological changes (red arrows).

Cytotoxicity of peptide spH4 and spH2A.2

The cytotoxicity of spH4 and spH2A.2 was tested using a gradient MIC concentration ranging from 1MIC to 8MIC in silver pomfret cells (liver, spleen, and head kidney). As illustrated in Fig. 6, it is evident that spH4 did not exhibit any cytotoxic effects on the silver pomfret cells, whereas spH2A.2 at a higher concentration did influence the cells. Notably, at the highest tested concentration (8MIC) of spH2A.2, a significant growth inhibition of 75 % was observed in liver cells (Fig. 6A), and a range of 80–90 % inhibition was observed in the remaining cell types (Fig. 6B, 6C and 6D).

Fig. 6.

Effect of spH4 and spH2A.2 on the viability of normal silver pomfret cells (A: Liver, B: Spleen, C: Head kidney). The 1MIC of spH4 used was 48 µM. The 1MIC of spH2A.2 used was 66 µM. An equal concentration of nisin (1MIC=58 µM) was used as positive control, while PBS was used as the negative control. The numbers in each group represent the P value. Symbol * represents a P < 0.05, ** represents a P < 0.01, and *** represents a P < 0.001 using a Duncan's multiple comparison.

Discussion

In aquaculture, the understanding of the host-pathogen interaction holds the potential to yield effective preventative measures for curbing residual antibiotic levels in the environment [34,35]. AMPs form a cornerstone of the innate immune system and are being increasingly advocated as ‘harmless alternative to antibiotics’ [36]. Previous research has illuminated the pivotal role of histone proteins within the innate immune system, contributing to the host defense by cleaving into truncated fragments upon infection [31,34]. Within the context of silver pomfret aquaculture, this study delves into H2A-derived and H4-derived antimicrobial peptides, identified from the silver pomfret, as a strategy to counteract PDD. Additionally, an initial exploration into the antibacterial mechanism underlying the actions of spH2A.2 and spH4 against pathogens is presented.

Histone H2A exhibits a multitude of fragments across various animal species, encompassing parasin I, buforin I, buforin II, buforin IIa, buforin IIb, himantura, abhisin, rainbow Tr, scallop A, litopenaeu, molluskin, Ca-l-hipposin, Hw-Hip, and Fi-Histin [19,21,22,31,[37], [38], [39], [40]]. These H2A-derived fragments, when subjected to phylogenetic analysis, unveil the presence of conserved residues. Intriguingly, the divergence between spH2A.2 and buforin IIb was least pronounced within this study. Moreover, buforin IIb, a synthetic analog derived from buforin II [41,42], exhibits a propensity for selectively targeting cancer cells by binding to negatively charged gangliosides [43]. Additionally, buforin II has demonstrated efficacy in combating microorganisms by penetrating cellular membranes and impeding their functional processes [38]. Remarkably, spH2A.2 exhibits a significant potential for a wide range of antimicrobial activities. Structurally, in comparison to spH2A.2, spH4 is distinguished by its composition of highly conserved amino acids, exhibiting substantial homology with analogous sequences in various species. However, the potential influence of minor impurities or contaminants within the spH2A.2 peptide cannot be disregarded, as evidenced by additional peaks observed in the MS spectrum. Notably, in vitro experimentation has revealed that histone H4 possesses antimicrobial properties against the Gram-negative bacterium E. coli, suggesting its beneficial utility in fish aquaculture [44,45]. It has been elucidated that a specific short peptide sequence (TVTAMDVVYALK) derived from H4 may exhibit a broad spectrum of antimicrobial activity within the host. This study serves to underscore the enhanced stability and heightened homology of H4-derived AMPs in comparison to H2A, particularly in terms of their efficacy across diverse species in exerting the antimicrobial activity.

Based on the 3D structure models and the Schiffer-Edmundson helical wheel diagram, both spH2A.2 and spH4 exhibit a shared characteristic of an amphipathic structure and linear α-helical peptides devoid of cysteine residues. Meanwhile, it was demonstrated that spH2A.2 and spH4 respectively stem from truncated fragments of SPH2A and SPH4. In alignment with numerous cationic AMPs, α-helical peptides possess notable flexibility in an aqueous environment, adopting amphipathic conformations exclusively when engaging with membranes [35,46]. Previous studies reveal that α-helical structure inherently equips histone-derived AMPs with the capacity to neutralize pathogens [47,48]. Our present findings on the study of spH2A.2 and spH4 corroborate these prior observations.

In the current investigation, we evaluated SPH2A and SPH4 mRNA expression levels in various organs, and found that the SPH2A gene has a high expression in the head kidney, while SPH4 is mainly expressed in the spleen. Furthermore, insights from the report on C. aurutus highlighted histone H2A gene expression in the kidney [22]. This observation implies that the expression of histone H2A may mainly generate from the silver pomfret head kidney after PDD infection. Interestingly, the extensive size and extra functional regions may render the antimicrobial activity of intact histone proteins relatively lower than that of their fragments [49]. Nevertheless, histone H2A extracted from the skin secretions of rainbow trout has been found to possess antibacterial activity [50]. Consequently, it remains unclear whether complete SPH2A and SPH4 have antibacterial properties or are specific to particular organs. Additionally, the liver and head kidney are two crucial immune organs in silver pomfret following PDD infection [4]. SPH4 and SPH2A exhibited elevated mRNA expression levels in the liver at 1 dpi. These findings were consistent with earlier research indicating that PDD infiltration can lead to petechial hemorrhages in the liver [4,51]. As such, it can be inferred that SPH2A and SPH4 in the liver might be induced to protect silver pomfret during the PDD infection.

Furthermore, the SEM observation revealed that synthetic spH2A.2 and spH4 could induce considerable morphological distortions and disrupt the membranes of both PDD and E. coli. The morphology is similar to that observed in the Pityrosporum ovale cells treated with Abhisin, which is a histone H2A-derived AMP known to compromise structural integrity [37]. Moreover, other histone H2A-derived AMPs, such as Fi-His_1–21 and Sphistin, have demonstrated the ability to rupture membranes, causing extensive in bacterial lysis [27,31]. These findings strongly suggest that spH2A.2 exerts a disruptive impact on bacterial cell membranes. Conversely, in the case of spH4, the morphological changes of PDD and E. coli following treatment indicate membrane perforation and mirror those observed with spH2A.2. On the contrary, PvH4a derived from P. vannamei induces the destruction of V. parahaemolyticus by provoking slender vesicles formation with the efflux of intracellular content [25]. Hence, it is plausible that the bacterial responses to AMPs treatment may diverge.

According to the cytotoxicity of spH4 and spH2A.2 in silver pomfret cells, spH2A.2 exhibited obvious cytotoxicity when the concentration exceeded 2MIC. In accordance with a pervious study, nisin, as a human food additive, served as a positive control for human cells [25]. Additionally, the combination of buforin I and nisin has shown potential as a promising strategy for bacterial prevention, displaying robust antimicrobial activity [52]. However, our observations revealed that nisin only had a marginal impact on silver pomfret liver cells at 4MIC. It is believed that the lower concentrations of spH2A.2 and nisin would pose no harm to fish cells. In contrast, the results of the cytotoxicity assessment demonstrated that spH4 did not exert any detrimental effects on silver pomfret cells. In previous research, PvH4a did not induce cytotoxicity in normal human cells [25]. Notably, in comparison to other research, spH4 and spH2A.2 have undergone initial assessemnts regarding their safety in fish cellular environments. Consequently, this supports the hypothesis that spH4 possesses considerable potential as a therapeutic agent and an effective bioactive peptide in the realm of silver pomfret aquaculture, presenting minimal cytotoxic risks.

In summary, two histone-derived antimicrobial peptides (H2A and H4) were identified from silver pomfret. The synthetic peptides, spH2A.2 and spH4, demonstrated potent antimicrobial activity against Gram-negative bacteria, effectively compromising the integrity of the PDD membrane. Furthermore, our investigation into fish cell cytotoxicity indicated that spH4 emerges as a promising candidate for preclinical exploration, with potential applications in the realm of fishery medicine. Moreover, our study sheds light on the antimicrobial properties of histone-derived peptides, introducing an innovative approach for controlling PDD in the context of silver pomfret aquaculture.

CRediT authorship contribution statement

Kejing Huang: Writing – original draft, Methodology, Investigation, Data curation. Lu Yuan: Methodology, Investigation. Xiongling Li: Methodology, Investigation. Rongrong Ma: Resources, Project administration. Suming Zhou: Resources, Project administration. Jianhu Jiang: Resources, Project administration. Yajun Wang: Supervision, Resources, Project administration. Jiasong Xie: Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

• Data will be made available on request.