Mudskipper - Springer Link

Probiotics and Antimicrobial Proteins https://doi.org/10.1007/s12602-017-9352-0

Mudskipper (Boleophthalmus pectinirostris) Hepcidin-1 and Hepcidin-2 Present Different Gene Expression Profile and Antibacterial Activity and Possess Distinct Protective Effect against Edwardsiella tarda Infection Jie Chen 1,2 & Li Nie 1 & Jiong Chen 1,3

# Springer Science+Business Media, LLC, part of Springer Nature 2017

Abstract Hepcidins are small cysteine-rich antimicrobial peptides that play an important role in fish immunity against pathogens. Most fish species have two or more hepcidin homologs that have distinct functions. This study investigated the immune functions of mudskipper (Boleophthalmus pectinirostris) hepcidin-1 (BpHep-1) and hepcidin-2 (BpHep-2) in vitro and in vivo. Upon infection with Edwardsiella tarda, the expression of BpHep-1 and BpHep-2 mRNA in immune tissues was significantly upregulated, but the expression profiles were different. Chemically synthesized BpHep-1 and BpHep-2 mature peptides exhibited selective antibacterial activity against various bacterial species, and BpHep-2 exhibited a stronger antibacterial activity and broader spectrum than BpHep-1. BpHep-1 and BpHep-2 both inhibited the growth of E. tarda in vitro, with the latter being more effective than the former. In addition, both peptides induced hydrolysis of purified bacterial genomic DNA (gDNA) or gDNA in live bacteria. In vivo, an intraperitoneal injection of 1.0 μg/g BpHep-2 significantly improved the survival rate of mudskippers against E. tarda infection compared with 0.1 μg/g BpHep-2 or 0.1 and 1.0 μg/g BpHep-1. Similarly, only BpHep-2 treatment effectively reduced the tissue bacterial load in E. tarda-infected mudskippers. Furthermore, treatment with 1.0 or 10.0 μg/ml BpHep-2 promoted the phagocytic and bactericidal activities of mudskipper monocytes/macrophages (MO/MФ). However, only the highest dose (10.0 μg/ml) of BpHep-1 enhanced phagocytosis, and BpHep-1 exerted no obvious effects on bactericidal activity. In conclusion, BpHep-2 is a stronger bactericide than BpHep-1 in mudskippers, and acts not only by directly killing bacteria but also through an immunomodulatory function on MO/MФ. Keywords Antibacterial activity . Edwardsiella tarda . Hepcidin . Monocyte/macrophage . Mudskipper . Gene expression

Introduction Host defense peptides (HDPs), also known as antimicrobial peptides (AMPs), are short (18–46 amino acids), usually cationic, amphipathic peptides that defend the host against various pathogens [1]. The antimicrobial effects of HDPs are primarily mediated by their ability to interact with bacterial membrane* Jiong Chen [email protected] 1

Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, China

2

Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture, Ningbo University, Ningbo 315211, China

3

Key Laboratory of Applied Marine Biotechnology of Ministry of Education, Ningbo University, Ningbo 315211, China

associated protein targets or to penetrate the bacterial cytoplasm and interact with intracellular targets such as genomic DNA (gDNA) that directly induce cell death [2]. Furthermore, HDPs serve a broad range of immunomodulatory functions, including chemotaxis, apoptosis, gene transcription, and cytokine production, to promote in vivo bacterial clearance [1]. HDPs are widely distributed in nature, and fish are a great source of these peptides; fish express almost all major classes of HDPs, such as β-defensins, cathelicidins, hepcidins, histonederived peptides, and fish-specific piscidins [3]. Hepcidin, also known as liver-expressed antimicrobial peptide (LEAP) 1, is a cysteine-rich and highly disulfide-bonded β-sheet HDP that is highly conserved in different species [4]. Six to eight cysteine residues are found at conserved positions within hepcidin, which are essential for its antimicrobial activity [5]. Hepcidin exerts bactericidal activities against many pathogens, such as Escherichia coli, Staphylococcus aureus,

Probiotics & Antimicro. Prot.

and Staphylococcus epidermidis [4–6]. Hepcidin does not disrupt the physical integrity of the bacterial membrane, but hydrolyzes the bacterial gDNA to kill specific bacteria [5, 7]. It also regulates iron metabolism by inhibiting the iron exporter ferroportin [8]. A recent study showed that porcine hepcidin could reduce intracellular bacterial growth, through the degradation of ferroportin [9]. Other than their antibacterial activity, mammalian hepcidins also have antifungal and antimalarial activities [4, 10, 11]. For example, human hepcidin exerts direct antimalarial activity against Plasmodium berghei in vivo and increases the survival rate of infected mice [11]. Fish hepcidins share high sequence conservation with mammal hepcidins [12, 13], and nuclear magnetic resonance (NMR) spectroscopic analyses indicated that hybrid-striped bass hepcidin and human hepcidin adopt a similar 3D structure and disulfide-bonding pattern [14]. In recent years, numerous fish hepcidins have been studied because of their important role in innate immunity and their potential use in the prevention and treatment of infection. To date, fish hepcidins have been identified and functionally studied in many species, such as Japanese flounder (Paralichthys olivaceus) [12], Mozambique tilapia (Oreochromis mossambicus) [15], turbot (Scophthalmus maximus) [16–18], orange-spotted grouper (Epinephelus coioides) [19, 20], European sea bass (Dicentrarchus labrax) [21, 22], convict cichlid (Amatitlania nigrofasciata) [23], Chinese rare minnow (Gobiocypris rarus) [24], spotted scat (Scatophagus argus) [25], Siberian taimen (Hucho taimen) [26], mudskipper (Boleophthalmus pectinirostris) [27], common ponyfish (Leiognathus equulus) [28], and roughskin sculpin (Trachidermus fasciatus) [29]. Most mammals contain only one hepcidin gene, whereas most fish species have two or more homologous hepcidin genes [30, 31]. Fish hepcidin genes are usually classified into two groups, namely, hamp1- and hamp2-type isoforms [31, 32]. The hamp1type isoform usually presents a single copy sharing a considerable degree of homology with its mammalian counterparts serving a dual function, including innate immunity and iron regulation; whereas the hamp2-type may have many copies with diversified sequences, and this type presents a singular role in the immune response against various pathogens [31]. The transcripts of fish hepcidins are highly abundant in the liver but are also present in other tissues [25–27]. Lipopolysaccharide (LPS) and polyinosinicpolycytidylic acid (poly I:C) stimulation or pathogen infection significantly upregulates the mRNA expression of fish hepcidins in different tissues [19, 26, 27, 33]. Fish hepcidins exhibit a broad spectrum of bactericidal activities and agglutination activities against Gram-positive and Gramnegative bacteria [12, 15, 18–26, 29]. For example, Siberian taimen hepcidin displays antimicrobial activities against E. coli, Micrococcus lysodeikticus, and S. aureus [26]. Roughskin sculpin hepcidin was capable of

agglutinating Vibrio anguillarum, E. coli, Bacillus t h u r i n g i e n s i s , B a c i l l u s s u b t i l i s , S . a u re u s , a n d Pseudomonas aeruginosa [29]. In addition, fish hepcidins also have antifungal [14], antiviral [18, 19, 25], and antitumor activities [34]. However, little is known about the roles of fish hepcidin in regulating innate immunity and protecting the host against bacterial infection. The mudskipper, an amphibious fish that has adapted to a wide range of temperatures and salinity, is widely distributed throughout China, Japan, Malaysia, Korea, and Vietnam. Although mudskippers inhabit intertidal mudflats containing abundant and diverse microbial populations, they rarely suffer from severe bacterial diseases. Edwardsiella tarda, a wellknown intracellular bacterium, is pathogenic to mudskippers [35]. Hepcidin is more highly expressed compared with other HDPs that act as the first line of defense, such as cathelicidins and piscidins [30]. A previous study identified two mudskipper hepcidin genes, namely, BpHepcidin-1 (BpHep-1) and BpHepcidin-2 (BpHep-2) [27]. LPS stimulation upregulates the expression of BpHep-1 and BpHep-2 mRNA in fish tissues [27], suggesting that these genes are involved in mudskipper immune responses to bacterial infection. However, the antimicrobial activities of BpHep-1 and BpHep-2 remain unclear. The present study identified changes in BpHep-1 and BpHep-2 transcript levels in mudskipper immune tissues upon E. tarda infection. The antibacterial activities of synthetic BpHep-1 and BpHep-2 were also determined in vitro and in vivo. Finally, the effects of BpHep-1 and BpHep-2 on the phagocytic and intracellular bactericidal activities of E. tarda by monocytes/macrophages (MO/MФ) were investigated.

Materials and Methods Fish Rearing Mudskipper individuals weighing 30–35 g were obtained from a commercial farm in Ningbo City, China. The fish were kept in brackish water (salinity 10) tanks at 24–26 °C in a recirculating system with filtered brackish water and were acclimatized to laboratory conditions for 2 weeks prior to experiments. All experiments were performed in accordance with the Experimental Animal Management Law of China and were approved by the Animal Ethics Committee of Ningbo University.

Edwardsiella tarda Infection of Fish Mudskippers were infected with E. tarda as previously described [35]. Briefly, E. tarda was cultured in Tryptic Soy Broth (TSB) medium (Sigma, Shanghai, China) at 28 °C, and were harvested in the logarithmic growth phase. Bacteria were washed and resuspended in sterile saline water


to obtain a concentration of 1.0 × 105 colony-forming units (CFU)/ml. Fish were injected intraperitoneally with 1.0 × 104 CFU live E. tarda per fish. Saline was used as a control.

Real-Time Quantitative PCR (qPCR) At 4, 8, 12, and 24 h post-infection (hpi), the kidney, liver, and spleen were collected. Total RNA was extracted from tissues using RNAiso reagent (TaKaRa, Dalian, China) and used as a template for first strand cDNA synthesis using AMV reverse transcriptase (TaKaRa). Specific primer pair, BpHep-1F (5′ACTCGTGCTGGCCTTTGTTTGC-3′) and BpHep-1R (5′GGACAGGTGGCTCTGGCGTTT-3′), was used to amplify a 169 base pair (bp) fragment from BpHep-1 cDNA ( K U 6 6 5 2 9 6 ) . P r i m e r p a i r, B p H e p - 2 F ( 5 ′ - A C T G GAATGTCCACGTCCTC-3′) and BpHep-2R (5′-CCAA CCAAATCCACAAGTCC-3′), was used to amplify a 176 bp fragment from BpHep-2 cDNA (KU665297). Primer pair, Bp18SF (5′-GGCCGTTCTTAGTTGGTGGA-3′) and Bp18SR (5′-CCCGGACATCTAAGGGCATC-3′), was used to amplify a 112 bp fragment from Bp18S rRNA (KX492897) [35]. qPCR amplification was carried out using SYBR premix Ex Taq (Perfect Real Time) (TaKaRa) to measure expression of BpHep-1, BpHep-2, and Bp18S mRNA. Amplification conditions were as follows: 94 °C for 5 min, 40 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, using a StepOne Real-Time PCR System (Applied Biosystems, Foster City, USA). Relative gene expression was analyzed using the comparative 2-△△Ct method, against that of Bp18S rRNA.

In Vitro Determination of the Antibacterial Activity of Synthetic BpHep-1 and BpHep-2 Peptides M a t u r e p e p t i d e s o f B p H e p - 1 (QSHLSMCRWCCNCCRGNKGCGPCCKF) and BpHep-2 (GIKCKFCCGCCTPGVCGLCCRF) containing four disulfide bonds were chemically synthesized with over 90% purity (SynPeptide, Shanghai, China). During mature peptide synthesis, oxidation of disulfide bonds was performed as previously described [7, 14]. Antibacterial activity of peptides was assayed against a panel of bacteria, including Aeromonas hydrophila, E. coli, E. tarda, Listeria monocytogenes, S. aureus, Streptococcus iniae, Vibrio vulnificus, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio anguillarum, and Vibrio harveyi. A modified twofold microdilution method was used to determine the minimal inhibitory concentration (MIC) of the various agents [36]. Twofold serial dilutions of peptide were made from 100 to 1.563 μg/ml final concentration in a 96-well plate. Kanamycin (Sigma) was used as a positive control. An 80-μl aliquot of each dilution was added to the corresponding well of a 96-well plate. Bacteria were cultivated to mid-logarithmic phase then diluted to a final

concentration of 1.0 × 105 CFU/ml in suitable medium. A 20-μl aliquot of the bacterial suspension was added to each well of the 96-well plate then incubated at an appropriate temperature for 24 h. MIC was confirmed by visual verification of microbial sedimentation and absorbance reading (600 nm).

Hydrolysis of Bacterial gDNA by BpHep-1 and BpHep-2 Hydrolysis of E. tarda gDNA by BpHep-1 and BpHep-2 was assayed as previously described [37, 38]. Bacterial gDNA was extracted using a MiniBEST Universal Genomic DNA Extraction Kit Ver. 5.0 (TaKaRa), and concentration was determined using a NanoDrop instrument (Thermo Fisher Scientific, Wilmington, USA). To determine the dosage effect of mudskipper hepcidins on hydrolysis of purified gDNA of E. tarda, 800 ng gDNA in 20 μl PBS was mixed with increasing amounts of BpHep1 or BpHep-2 peptide. After incubation at room temperature for 30 min, 2 μl 10 × loading buffer (TaKaRa) was added to the mixture, and 14 μl of each reaction was applied to a 1.0% agarose gel in 0.5 × Tris-acetate-EDTA buffer. To determine the effect of mudskipper hepcidins on gDNA hydrolysis of live bacteria, E. tarda was cultured and adjusted to a final concentration of 1.0 × 109 CFU/ml in PBS. The E. tarda suspension (100 μl) was mixed with 50 or 100 μg/ml BpHep-1, BpHep-2, or BSA (control) then incubated at 28 °C for 1, 2, 4, or 6 h. After incubation, E. tarda gDNA was extracted.

Fish Survival Assay Six groups, each containing 16 fish, were injected intraperitoneally with 1.0 × 104 CFU live E. tarda per fish. Thirty minutes post-infection, fish from the five experimental groups were injected intraperitoneally with 0.1 μg/g BpHep-1, 1.0 μg/g BpHep-1, 0.1 μg/g BpHep-2, 1.0 μg/ g BpHep-2, or 1.0 μg/g BpHep-1 plus 1.0 μg/g BpHep-2 of fish weight. The control group received saline. Fish were assessed every 24 h for the next 7 days for signs of death or moribund state. The Kaplan–Meier method was used to determine survival rate.

Bacterial Load Assay Five groups of six fish each were used in a bacterial load assay. Each fish was injected intraperitoneally with 1.0 × 104 CFU live E. tarda. Thirty minutes post-infection, fish from the four experimental groups were injected intraperitoneally with 0.1 μg/g BpHep-1, 1.0 μg/g BpHep-1, 0.1 μg/g BpHep-2, or 1.0 μg/g BpHep-2 of fish weight. The control group was injected with saline. Liver, kidney,


spleen, and blood samples were collected at 24 hpi. Tissue (0.1 g fresh tissue weight) and blood (0.1 ml) samples were homogenized, serially diluted in TSB, and then plated onto Tryptic Soy Agar (TSA) plates. After incubation at 28 °C for 18 h, colonies were counted and normalized to 0.1 g tissue weight or 0.1 ml blood volume.

plates to measure bacterial uptake values. Samples from the kill group were incubated for a further 1.5 h to allow for bacterial killing, then cells were lysed. After incubation at 28 °C for 18 h, colonies were counted. Bacterial survival rate was determined by dividing the CFU of the kill group by that of the uptake group.

Cell Preparation

Statistical Analysis

Mudskipper kidney-derived MO/MФ were isolated and cultured as previously described [35]. Briefly, fish kidney leukocyte-enriched fractions were obtained using Ficoll-Hypaque PREMIUM (1.077 g/ml) (GE healthcare, NJ, USA). Non-adherent cells were removed by washing, and adherent cells were incubated in complete medium (RPMI 1640, 5% mudskipper serum, 5% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin) at 24 °C with 5% CO2.

All data are presented as mean ± SEM. Statistical analysis was conducted using one-way ANOVA using SPSS version 13.0 (SPSS Inc., Chicago, USA). Statistical significance was considered at P < 0.05.

Results Changes in BpHep-1 and BpHep-2 mRNA Expression upon E. tarda Infection

Phagocytosis Assay Phagocytosis of mudskipper MO/MФ was measured as previously described [35]. Briefly, E. tarda in midlogarithmic phase were labeled with fluorescein isothiocyanate (FITC) (Sigma, Saint Louis, USA), and bacteria were hereafter referred to as FITC-E. tarda. MO/MΦ were pretreated with 1.0 or 10.0 μg/ml of BpHep-1 or BpHep2 for 8 h. Heat-killed FITC-E. tarda were added at a multiplicity of infection (MOI) of 10 then incubated for a further 30 min. MO/MΦ were washed extensively with sterile PBS to remove extracellular particles then resuspended in FACS buffer (PBS, 0.2% BSA, 0.1% sodium azide). Engulfed bacteria were examined using a Gallios Flow Cytometer (Beckman Coulter, Miami, USA). Relative mean fluorescence intensity (MFI) of bacteria engulfed by cells was determined using FlowJo software. The MFI of the PBS-, BpHep-1- or BpHep-2-treated groups was expressed as fold change relative to the MFI of the nobacteria group, while the MFI of the PBS-treated group was assigned a value of 100.

Bacterial-Killing Assay The bacterial-killing assay of mudskipper MO/MΦ was performed as previously described [35]. Briefly, MO/MФ were pretreated with different concentrations 1.0 or 10.0 μg/ml of BpHep-1 or BpHep-2 for 8 h. MO/MΦ were then washed with sterile PBS and treated with live E. tarda at a MOI of 10. Bacterial phagocytosis was allowed to proceed for 30 min at 24 °C in an atmosphere of 5% CO2. Noninternalized E. tarda were removed by washing with sterile PBS. Samples from the uptake group were lysed in 1% Triton X-100 solution and plated onto TSA

After infection by E. tarda, the expression of both BpHep1 and BpHep-2 mRNA was significantly upregulated in the liver, spleen, and kidney, but they showed distinct gene expression profiles (Fig. 1). The biggest increases in BpHep-1 transcript levels were 57.38-fold in the spleen at 4 hpi, 20.44-fold in the kidney at 8 hpi, and 2.47-fold in the liver at 4 hpi (Fig. 1a–c), while the biggest changes in BpHep-2 transcript levels were 4.75-fold in the spleen at 4 hpi, 7.06-fold in the kidney at 12 hpi, and 80.54-fold in the liver at 24 hpi (Fig. 1d–f).

In Vitro Antibacterial Activity of BpHep-1 and BpHep-2 The MIC of BpHep-1 and BpHep-2 was measured against a panel of bacteria. BpHep-2 showed a stronger antibacterial activity and broader spectrum than BpHep-1 (Table 1). BpHep-2 displayed antimicrobial activities against V. vulnificus, V. parahaemolyticus, L. monocytogenes, and S. iniae, with MIC values of 50, 100, 6.25, and 25 μg/ml, respectively. Meanwhile, BpHep-1 exerted no antimicrobial activities against these bacteria at 100 μg/ml. BpHep-2 also displayed antimicrobial activities against V. alginolyticus, V. anguillarum, E. tarda, and S. aureus at MIC values of 25, 3. 75, 25, and 25 μg/ml, respectively, whereas BpHep-1 had an MIC value of 100 μg/ml against the abovementioned bacteria (Table 1).

Hydrolysis of Bacterial gDNA by BpHep-1 and BpHep-2 To determine the dosage effect of mudskipper hepcidins on DNA hydrolysis, gDNA from E. tarda was incubated with


Fig. 1 BpHep-1 and BpHep-2 mRNA expression in mudskippers challenged with E. tarda. Tissues were collected at different time points after bacterial infection. BpHep-1 (a-c) and BpHep-2 (d-f) transcript levels

were normalized to Bp18S rRNA. Data are expressed as mean ± SEM of results from four fish. *P < 0.05

different concentrations of BpHep-1 or BpHep-2 then subjected to agarose gel electrophoresis. The intensity of the gDNA band gradually diminished with increasing amounts of BpHep-1 and BpHep-2 peptide. gDNA treated with 75 or 100 μg/ml BpHep-2 was hydrolyzed more quickly than that treated with BpHep-1 (Fig. 2a, b). To determine the effect of mudskipper hepcidins on gDNA hydrolysis in live bacteria, BpHep-1 and BpHep-2 were incubated with E. tarda for several hours. gDNA was extracted and subjected to electrophoresis. The results show that treatment with 50

or 100 μg/ml of both peptides caused degradation of intracellular gDNA, whereas BSA treatment had no apparent effect (Fig. 2c–f).

Table 1

Effect of BpHep-1 or BpHep-2 Treatment on Survival of E. tarda-Infected Mudskippers To investigate the in vivo effects of synthesized hepcidins against E. tarda in mudskippers, a survival analysis was conducted. Fish treated with 1.0 μg/g BpHep-1 plus 1.0 μg/g

MIC values of BpHep-1and BpHep-2 against bacteria

Bacteria

Isolates/strains

BpHep-1 MIC (μg/ml)

BpHep-2 MIC (μg/ml)

Kanamycin MIC (μg/ml)

Vibrio vulnificus Vibrio alginolyticus

ATCC279562 ATCC17749

100

50 25

25 25

Vibrio parahaemolyticus Vibrio harveyi Vibrio anguillarum Edwardsiella tarda Escherichia coli Staphylococcus aureus Aeromonas hydrophila Listeria monocytogenes Streptococcus iniae

ATCC33847 ATCC33866 ATCC19264 Et-CD K12 ATCC6538 ATCC7966 ATCC19115 ATCC29178

12.5 100 100 100 -

100 12.5 3.75 25 25 6.25 25

50 1.563 25 25 50 12.5 6.25 1.563 6.25

B-^ no inhibition detected at 100 μg/ml


Fig. 2 Hydrolytic activity of BpHep-1 and BpHep-2 on bacterial gDNA. a, b Various concentrations of peptides were incubated with 800 ng gDNA of E. tarda at room temperature for 30 min, then gDNA was analyzed by electrophoresis on a 1.0% agarose gel. c–f Live E. tarda were

incubated with either 50 μg/ml or 100 μg/ml BpHep-1 or BpHep-2 for 0, 1, 2, 4, and 6 h or with the same volume of BSA for 6 h, then gDNA was extracted and subjected to electrophoresis on a 1.0% agarose gel

BpHep-2 or 1.0 μg/g BpHep-2 alone exhibited a better survival rate compared with other groups in a 7-day test. On day 7, fish treated with 1.0 μg/g BpHep-1 plus 1.0 μg/g BpHep-2 or 1.0 μg/g BpHep-2 alone displayed a 50% survival rate, whereas no significant differences were observed among the saline-, 0.1 μg/g BpHep-1-, 1.0 μg/g BpHep-1-, and 0.1 μg/g BpHep-2-treated groups (Fig. 3).

Effect of BpHep-1 or BpHep-2 Treatment on Bacterial Load in E. tarda-Infected Mudskippers Bacterial load was evaluated in the immune tissues and blood of E. tarda-infected mudskippers following treatment with BpHep-1 or BpHep-2. The BpHep-2-treated group showed a significant reduction in bacterial load compared with the


Fig. 3 Effect of BpHep-1 or BpHep-2 treatment on survival rate of E. tarda-infected mudskippers. Fish were injected intraperitoneally with equal volumes of saline, 0.1 μg/g BpHep-1, 1.0 μg/g BpHep-1, 0.1 μg/g BpHep-2, 1.0 μg/g BpHep-2, or 1.0 μg/g BpHep-1 plus 1.0 μg/g BpHep2, at 30 min after E. tarda infection. Fish mortality was monitored daily for 7 days. n = 16. *P < 0.05

saline- and BpHep-1-treated groups (Fig. 4). Meanwhile, no significant differences were observed between the saline- and BpHep-1-treated groups (Fig. 4).

Effect of BpHep-1 or BpHep-2 Treatment on Phagocytic and Bactericidal Activities of Mudskipper MO/MФ We further investigated the effect of BpHep-1 and BpHep-2 on the biological functions of mudskipper kidney-derived MO/MФ in vitro. Our results show that treatment with 10.0 μg/ml BpHep-1 increased phagocytosis of E. tardaFITC by mudskipper MO/MФ up to 1.40-fold compared to controls, whereas treatment with 1.0 μg/ml BpHep-1 had no significant effect (Fig. 5a). By contrast, treatment with 1.0 or 10.0 μg/ml BpHep-2 increased phagocytosis by MO/MФ up to 1.31- and 1.64-fold, respectively, compared to controls (Fig. 5b). Treatment with BpHep-1 had no significant effect on E. tarda survival rate in MO/MФ (Fig. 5c), whereas treatment with 1.0 or 10.0 μg/ml BpHep-2 reduced bacterial survival in MO/MФ to 0.66- and 0.53-fold of control levels, respectively (Fig. 5d).

Discussion Hepcidins play an important role in host immunity against pathogenic organisms. In contrast to most mammals that have a single hepcidin gene, fish species commonly have

Fig. 4 Effect of BpHep-1 or BpHep-2 treatment on bacterial load of E. tarda-infected mudskippers. Each fish was first injected intraperitoneally with 1.0 × 104 CFU of E. tarda and an equal volume of saline, 0.1 μg/ g BpHep-1, 1.0 μg/g BpHep-1, 0.1 μg/g BpHep-2, or 1.0 μg/g BpHep-2 after 30 min. Fish were euthanized 24 h later. Liver (a), blood (b), kidney (c), and spleen (d) were collected and homogenized before culturing on TSA plates. CFUs were normalized to 0.1 ml of blood or 0.1 g of tissue weight (for the liver, spleen, and kidney). n = 6. *P < 0.05

two or more homologous hepcidin genes [30, 31]. A previous study revealed that the mudskipper has two isoforms of hepcidin (BpHep-1 and BpHep-2), both of which are highly expressed in the liver. Upon LPS stimulation, BpHep-1 (hamp1-type isoform) mRNA expression is upregulated during the early immune response and immediate recovery to control levels, whereas BpHep-2 (hamp2-type isoform) mRNA expression exhibits clear time-coursedependent upregulation [27]. In our study, the expression of BpHep-1 and BpHep-2 mRNA was significantly upregulated in the liver, spleen, and kidney of mudskippers following E. tarda infection, but they showed distinct gene expression profiles. The change in BpHep-1 mRNA expression in the spleen and kidney was much greater than that of BpHep-2, while the change in BpHep-2 mRNA expression in the liver was much greater than that of BpHep-1. These results reveal that BpHep-1 and BpHep2 gene expression responds quickly to pathogen infection, but their functions in immune responses may differ. In recent years, in vitro studies have attributed antibacterial activity to fish hepcidins [12, 15, 18–26, 29]. In Japanese flounder, synthesized hepcidin-2 (hamp1-type isoform) shows


Fig. 5 Effect of BpHep-1 or BpHep-2 treatment on phagocytic and bactericidal activities of mudskipper MO/MФ. a, b MO/MФ were pretreated with PBS, 1.0 μg/ml BpHep-1, 10.0 μg/ml BpHep-1, 1.0 μg/ml BpHep2, or 10.0 μg/ml BpHep-2 for 8 h before adding heat-killed FITC-E. tarda at an MOI of 10. After 30 min, bacterial uptake by MO/MФ was analyzed using flow cytometry. MFI is presented as fold change relative to the PBS-treated group, which was assigned a value of 100. c, d MO/MФ

were pretreated with 1.0 μg/ml BpHep-1, 10.0 μg/ml BpHep-1, 1.0 μg/ ml BpHep-2, or 10.0 μg/ml BpHep-2 for 8 h before adding live E. tarda at an MOI of 10. Phagocytosis was allowed to proceed for 30 min, and kill groups were incubated for a further 1.5 h to allow for killing of bacteria. Bacterial survival rate was calculated by dividing the CFU of the kill group by that of the uptake group. Data are expressed as mean ± SEM. n = 4. *P < 0.05

antimicrobial activity against E. coli, S. aureus, Latococcus garvieae, and S. iniae, whereas hepcidin-1 (hamp2-type isoform) does not [12]. Similarly, in European sea bass, synthetic hepcidin-2 exhibits clear antimicrobial activity against V. anguillarum, L. garviae, Streptococcus parauberis, and Photobacterium damselae in vitro, but hepcidin-1 does not [21]. In the present study, we confirmed that BpHep-2 has a stronger antibacterial activity and broader antibacterial spectrum than BpHep-1 in vitro (Table 1), suggesting that BpHep2 has a more specialized role in immunity. Both BpHep-1 and BpHep-2 can inhibit the growth of E. tarda, a known pathogen of mudskippers. As previously described, HDPs do not only exert their function by perturbing the bacterial cell membrane; several cationic HDPs can also enter the cytoplasm of target microbes and exert antimicrobial activity by interacting with microbial nucleic acids and other target factors, causing cell death [2, 39]. For example, human and rainbow trout (Oncorhynchus mykiss) hepcidins do not disrupt the physical integrity of the bacterial membrane but instead kill specific bacteria by hydrolyzing the bacterial gDNA [5, 7]. In the present study, both BpHep-1 and BpHep-2 were able to hydrolyze the gDNA of E. tarda, which is consistent with previous reports [5, 7]. These results suggest that mudskipper hepcidins can directly kill bacteria, possibly by hydrolyzing

the bacterial gDNA. We further evaluated the antimicrobial activities of BpHep-1 and BpHep-2 against E. tarda in vivo. Administration of 1.0 μg/g BpHep-2 significantly increased the survival rate of E. tarda-infected mudskippers in a 7-day survival test, which was accompanied by a lower bacterial burden in the tissues and blood of the fish. The administration of BpHep-1 could neither decrease fish mortality upon E. tarda infection nor lower bacterial burden. Similar results were reported with Mozambique tilapia hepcidins, which decrease the mortality and bacterial burden of zebrafish (Danio rerio) after V. vulnificus infection [40, 41]. Furthermore, we evaluated whether these two hepcidins had a synergistic effect on survival rate. The results showed that co-administration of 1.0 μg/g BpHep-1 and 1.0 μg/g BpHep-2 was not significantly different from treatment with 1.0 μg/g BpHep-2 alone, indicating no synergistic effect against E. tarda in vivo (Fig. 3). Recent studies showed that HDPs in combination generally have an additive or synergistic effect against bacteria in vitro [42, 43]. However, the synergistic effects of these HDPs have not been observed in vivo. The reason for the synergistic effects and their functional details are not clear and require further elucidation. Studies on the biological functions of HDPs suggest that they are not only involved in the direct killing of bacteria


in vivo, but also possibly exert immunomodulatory effects [1]. Human cathelicidin peptide LL-37 can activate macrophages, leading to enhanced phagocytosis of bacteria [44]. Ayu cathelicidin can promote the phagocytic and bactericidal activities of MO/MФ through mediation of P2X7R [45]. Mudskipper LEAP-2 cannot kill E. tarda directly but can effectively protect the host against E. tarda infection by modulating immune cell functions [35]. In the present study, treatment with 1.0 or 10.0 μg/ml BpHep-2 significantly enhanced the phagocytic and bactericidal activities of MO/MФ. BpHep1 was only able to promote phagocytosis at high concentrations, but exerted no significant effect on the bactericidal activity of MO/MФ. These results demonstrate that mudskipper hepcidins have immunomodulatory effects on MO/MФ, and BpHep-2 is much more effective than BpHep-1. In conclusion, we determined the antibacterial activities of BpHep-1 and BpHep-2 both in vitro and in vivo. BpHep-1 and BpHep-2 may improve the survival of E. tarda-infected mudskippers by directly killing bacteria and having an immunomodulatory effect on MO/MФ. Further investigation is needed to identify the underlying mechanism that accounts for the antimicrobial role of BpHep-2. Funding information This project was supported by the Program for the National Natural Science Foundation of China (31772826), the Natural Science Foundation of Zhejiang Province (LZ18C190001, LQ17C190001), the Natural Science Foundation of Ningbo City of China (2017A610284), the Scientific Innovation Team Project of Ningbo (2015C110018), the Scientific Research Foundation of Graduate School of Ningbo University (G16023), and the KC Wong Magna Fund in Ningbo University.

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Compliance with Ethical Standards All experiments were performed in accordance with the Experimental Animal Management Law of China and were approved by the Animal Ethics Committee of Ningbo University.

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Conflict of Interest The authors declare that they have no conflicts of interest.

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