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Antibacterial and antiparasitic activities analysis of a hepcidin-like antimicrobial peptide from Larimichthys crocea
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Libing Zheng1, 3, Yuan Li2, Jun Wang1, Ying Pan1, Jia Chen1, Weiqiang Zheng1, Longshan Lin2, *
Acta Oceanologica Sinica | 2020, 39(10) : 129 - 139
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Acta Oceanologica Sinica | 2020, 39(10): 129-139
• Marine Biology •
Antibacterial and antiparasitic activities analysis of a hepcidin-like antimicrobial peptide from Larimichthys crocea
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Libing Zheng1, 3, Yuan Li2, Jun Wang1, Ying Pan1, Jia Chen1, Weiqiang Zheng1, Longshan Lin2, *
Affiliations
  • 1 State Key Laboratory of Large Yellow Croaker Breeding, Ningde 352103, China
  • 2 Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China
  • 3 National and Provincial Joint Laboratory of Exploration and Utilization of Marine Aquatic Genetic Resources, School of Marine Science and Technology, Zhejiang Ocean University, Zhoushan 316022, China
Published: 2020-10-25 doi: 10.1007/s13131-020-1580-6
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As an economically important marine fish, the large yellow croaker Larimichthys crocea suffered from marine white spot disease caused by the ectoparasite Cryptocaryon irritans in recent years. This disease not only could result in physiological damage, but also lead to secondary bacterial invasion. Reports indicated some AMPs (antimicrobial peptides) were of antiparasitic activity to C. irritans. Hepcidin-like (Lc-HepL) was one of the significant differential expression genes excavated from the transcriptome following a challenge with C. irritans. In this study, we characterized this AMP’s bioactivity based on the levels of mRNA and protein. After challenged by C. irritans, qRT-PCR showed Lc-HepL was significantly upregulated in six tissues, including gill, muscle, liver, head kidney and spleen during theront infection, trophont falling off, and secondary bacterial invasion stages, which implicated a role Lc-HepL played in the immune defense against C. irritans and secondary bacterial infection. Recombinant Lc-HepL (rLc-HepL) was induced and purified successfully. rLc-HepL exhibited antibacterial activity to certain bacteria in a dose- and time-dependent manners. Anti-C. irritans activity was explored for the first time and found it could cause the theronts membrane rupture and contents leakage. These results provided the first evidence that Lc-HepL had strong antiparasitic activity against marine fish ectoparasites C. irritans theronts. Together, data indicated that Lc-HepL might be an important component in the innate immune system against C. irritans and has the potential to be employed in future drug development.

Larimichthys crocea  /  Cryptocaryon irritans  /  hepcidin-like  /  antibacterial activity  /  antiparasitic activity
Libing Zheng, Yuan Li, Jun Wang, Ying Pan, Jia Chen, Weiqiang Zheng, Longshan Lin. Antibacterial and antiparasitic activities analysis of a hepcidin-like antimicrobial peptide from Larimichthys crocea[J]. Acta Oceanologica Sinica, 2020 , 39 (10) : 129 -139 . DOI: 10.1007/s13131-020-1580-6
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1 Introduction
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AMPs (antimicrobial peptides) are the central components in the humoral immunity of the innate immune system involving in the first defense line, and are either induced after being infected by pathogens or stored in the secretory cells (Valero et al., 2020). AMPs present diverse sequences and structures (Mookherjee and Hancock, 2007) and exhibit strong activity to damage membranes, inhibit the proliferation of bacteria, kill bacteria directly (Patel and Akhtar, 2017), or interact with intracellular bio-macromolecules to disturb cell metabolism (Yeaman and Yount, 2003). In addition, some certain AMPs also have immunomodulatory properties (Valero et al., 2020; Oppenheim et al., 2003; Bowdish et al., 2006; Nicholls et al., 2010).
Hepcidin is a type of AMP with four disulfide linkages to take shape of a β-sheet structure. Hepcidin was firstly isolated from human blood in 2000 (Krause et al., 2000) and urine in 2001 (Park et al., 2001). Next, a mature peptide fragment was obtained from the gill of hybrid striped bass (Morone chrysops × M. saxatils), and the full-length cDNA was cloned (Shike et al., 2002). To date, increasing amounts of hepcidin were isolated and cloned from many fish species, such as Danio rerio (Shike et al., 2004), Paralichthys olivaceus (Hirono et al., 2005) and Larimichthys crocea (Wang et al., 2009), and the mature peptides contain 19–27 amino acid residues. Hepcidin from various sources has different resistance activity against pathogens (Krause et al., 2000; Shike et al., 2002; Wang et al., 2009), and a report showed the chemically synthetic hepcidin of Tilapia mossambica could also inhibit the growth of human fibrosarcoma cells (Chen et al., 2009). Hepcidin was likely to be the first defense line against pathogens invasion (Hirono et al., 2005; Lee et al., 2005), and it could coordinate with other AMPs to generate a synergistic antibacterial effect (Shi and Camus, 2006). Hepcidin of L. crocea (Lc-Hepc) exhibited broad-spectrum antibacterial activity and might interact with the signal molecules of pathogens through the electric charge effect to cause the formation of holes through which the contents leak (Cai, 2010).
In general, certain fish have multiple copies of hepcidin. Yang (2006) isolated two hepcidin sequences from the liver and gill of Pagrosomus major after challenged by a bacterial mixture. Seven hepcidin sequences were identified from Sparus macrocephlu, and three hepcidin sequences found in Oreochromis mossambicus (Babitt et al., 2006). One of two hepcidin sequences of P. olivaceus was upregulated in the liver upon iron overload, while the other sequence was downregulated (Hirono et al., 2005).
Prior to our study, hepcidin and hepcidin-like genes were two differential expression AMPs excavated from the comparative liver transcriptome of L. crocea (Zheng et al., 2018). In this study, hepcidin-like was selected as the target gene, and we aimed to (1) detect the expression profiles after challenged by C. irritans; (2) construct a prokaryotic expression system with the pET-32a plasmid and host Escherichia coli BL21(DE3)pLysS, and induce expression of recombinant hepcidin-like (rLc-HepL); (3) purify the rLc-HepL; (4) analyze the antibacterial activity; and (5) analyze anti-C. irritans activity. The results might provide valuable data on the immune functions of Lc-HepL as a key component in the innate immune response against multiple pathogens.
2 Materials and methods
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2.1 Cryptocaryon irritans challenge experiment and tissue collection
The challenge experiment was performed according to the pre-constructed procedure from our team (Niu, 2013). In brief, the tomonts of C. irritans were collected from the spontaneously infected L. crocea cultured at Ningde Marineland during an outbreak of marine white spot disease, and these tomonts were cultured at room temperature in sterile seawater (salinity of 32) for hatching theronts. The healthy L. crocea (temporarily reared for two weeks) were challenged with a dose of 26 665 theronts per fish (weight (85.5±15.1) g, body length (19.2±1.3) cm) in 500 L seawater for 4 h and then transferred to fresh seawater. Next, five individuals were randomly selected to dissect for tissue collection at 6 h, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, and 7d post-infection (pi). The healthy individuals were used as the control group. Tissues including gill, muscle, head kidney, liver, spleen and intestine were dissected, homogenized in RNAfixer (BioTek Corporation) immediately at 4°C overnight and then transferred to –20°C for total RNA extraction.
2.2 Total RNA isolation and cDNA synthesis
The detailed procedure of total RNA isolation was carried out according to the reference (Zheng et al., 2018). In short, the tissues were crushed in the RNAiso Plus (TaKaRa, Japan) and extracted with chloroform, nucleotide acid was precipitated for approximately 1 h with isopropyl alcohol at –20°C, and washed with 75% ethanol. The quantity and purity of the RNAs were tested by spectrophotometer (A260/A280). cDNAs were reverse transcribed using total RNA as templates with PrimerScriptTM RT reagent and gDNA Eraser kit (TaKaRa, Japan) according to the protocol.
2.3 Expression changes of Lc-HepL mRNA after challenged by C. irritans
According to the cDNA sequence of Lc-HepL, a pair of primers, qHep-F/R (Table 1), were designed for quantitative real-time PCR (qRT-PCR) detection. β-actin was used as the internal control gene. The reactions were run on an ABI Quantitative 6 Flex system with SYBR Premix Dimer Eraser Kit (TaKaRa, Japan) following the established protocol. The detailed programs were used as the reference (Niu, 2013).
The relative expression level was quantified by the 2–△△Ct method (Livak and Schmittgen, 2001). Each reaction was performed in triplicate, and all data were shown as the mean±SD (standard deviation of the mean). One asterisk indicated significant at p<0.05, and two asterisks indicated highly significant at p<0.01.
2.4 Construction of expression vector and induction of rLc-HepL
2.4.1 Construction of expression vector
To amplify the sequence encoding mature peptide, specific primers BHepL-F/R (Table 1) were designed according to the sequence that introduced EcoR I/Xho I sites into the 5′ end respectively. PCR was performed in a 25 μL volume with Reaction Mix (TransGen, Beijing), and the products were refined by gel purification. The purified products and pET-32a were digested with enzymes EcoR I and Xho I (Thermo Scientific, USA), and the digested PCR products were sub-cloned into the pET-32a. Next, recombinant plasmids were transferred into the host competent cells of E. coli BL21(DE3)pLysS, selected with a LB agar plate containing 100 μg/mL ampicillin. The positive clones were selected to perform bacterial PCR and sequenced with T7 primers (Table 1). The positive clone containing the correct target sequence was stored in 25% (v/v) glycerinum at –80°C.
2.4.2 Optimization of induction expression conditions
The recombinant bacteria were cultured in fresh LB medium containing 0.5% glucose at 37°C, 180 r/min overnight. This solution was later inoculated into the same fresh medium at a 1:50 ratio and cultured at 37°C until the OD600 reached 0.6–0.7; isopropyl β-D-thiogalacto-pyranoside (IPTG) was added to final concentrations of 0.1, 0.3, 0.5, and 1.0 mmol/L to initiate induction at 37°C, 28°C and 18°C, respectively. Auto-induction in a simplified medium (Zhang, 2013) was explored in the study with induction time of 6 h at 37°C, 28°C and overnight at 18°C.
At the same time, initial OD600 values 0.1, 0.3, 0.5, 1.0, and induction expression for 1, 3, 5, 7, 9, 11 h were also explored to determine the optimal induction condition, respectively. Two hundred microliters (μL) of induction solution was taken out to be centrifuged at 5000 g for 5 min, and the precipitates were prepared for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis.
2.5 Capture and purification of rLc-HepL
According to the above-mentioned optimum induction conditions, a large-scale (2 L) induction expression was performed. The induction solution was centrifuged at 12 000 r/min for 10 min at 4°C to harvest the cells, and the precipitates were resuspended in 500 mL pre-chilled Tris-HCl buffer (50 mmol/L Tris-HCl, 500 mmol/L NaCl, 20 mmol/L imidazole, pH 7.8). The mixture was sonicated with an ATS high pressure homogenizing machine (ATS Engineering Inc., Canada), centrifuged at 12 000 r/min for 30 min at 4°C to isolate the supernatant and precipitates, and they were analyzed by SDS-PAGE, respectively.
Throughout the detection process, the supernatant was filtered with a 0.22 μm filter membrane for purification by immobilized metal-chelating affinity chromatography (IMAC) mean. In brief, 15–25 mL sterile MilliQ water and Tris-HCl buffer were used to wash and equilibrate HisCap 6FF column (Smart-Lifesciences, Changzhou), respectively. The prepared supernatant was allowed to flow through the column by gravity at 4°C. The column was later washed with graded imidazole to remove nonobjective proteins and capture rLc-HepL through a UV detector in an AKTA Purifier 100 (GE Healthcare Life Sciences). All of the fluid collected from each elution peak was analyzed by SDS-PAGE.
Dialysis was used to remove high concentration imidazole which might influence the activity detection from the collected eluate containing pure rLc-HepL. The solution of interest was bathed in 50 mmol/L Tris-HCl buffer (50 mmol/L Tris-HCl, 50 mmol/L NaCl, pH 7.8) for 12 h at 4°C, and continually dialyzed in MilliQ water for 12 h at 4°C two times. The concentration was subsequently determined by the BCA method, and the eluate was finally freeze-dried.
The detailed procedure of Western blot analysis was based on previous research (Zheng et al., 2016). Following the Western blot transfer, the PVDF (polyvinylidene fluoride) membrane was blocked with 5% non-fat milk at room temperature for 2–3 h, incubated with anti-His mouse monoclonal antibody (abm, Canada) (diluted 1:1 000) overnight at 4°C, and continually bathed in goat anti-mouse IgG (H+L) horseradish peroxidase conjugated antibody (TransGen Biotech, China) (diluted 1:5 000) for 2 h at room temperature. After washed with PBST, the transformed stripe was dyed with a DAB Kit (Solarbio, China) according to the instructions and a picture was taken.
2.6 Antibacterial activity of rLc-HepL
2.6.1 Antibacterial assays
MIC (minimal inhibitory concentration) and MBC (minimal bactericidal concentration) were determined against a panel of microorganisms. In total, 13 types of bacteria were employed in the antibacterial tests, including Gram-positive Bacillus subtilis, Staphylococcus aureus, Corynebacterium glutamicum, Micrococcus lysodeikticus, and Gram-negative E. coli, Pseudomonas fluorescens, Pseudomonas aeruginosa, Photobacterium damselae, Shigella flexneri, Aeromonas hydrophila, Vibrio harveyi, Vibrio alginolyticus and Vibrio Parahaemolyticus. All non-marine bacteria were cultured in Muller-Hinton Broth (MHB), marine bacteria were cultured in saline peptone water (3% Tryptone, 3% NaCl, w/v) (Destoumieux et al., 1999).
A liquid growth inhibition assay on a 96-well flat-bottom tissue-culture plate was performed to determine the MIC as previously described (Niu, 2013; López-García et al., 2007; Lauth et al., 2005). rLc-HepL was gradient diluted to a concentration of 100–3.125 μmol/L with sterile MilliQ water. Logarithmic phase bacterial culture was washed with 10 mmol/L NaPB (68.4 mmol/L Na2HPO4, 31.5 mmol/L NaH2PO4), and suspended in working medium (MHB:NaPB=2:3) to a final OD600 of 0.003. Marine bacteria were suspended in saline peptone water to a final OD600 of 0.006. Next, 50 μL diluted rLc-HepL was mixed with 50 μL bacterial suspension. Samples without rLc-HepL or rTRX replacing rLc-HepL were used as blanks, respectively. After incubation for 24 h at 28°C, the MIC was calculated as a range between visible or no visible bacterial growth.
To determine the MBC, aliquots from the wells with no detectable growth were coated onto MHA and incubated overnight at 28°C or 37°C to assess viability. Each assay was performed in triplicate.
2.6.2 Dose-dependent antibacterial activity
According to the antibacterial spectrum results, C. glutamicum was chosen for the dose-dependent antibacterial tests. The bacterial suspension was adjusted to a final OD600 of 0.003 in working medium. Similarly, the final concentration range of rLc-HepL was 1.563–50 μmol/L. The mixtures were cultured for 3 h at 28°C, and 0.7 μL from each well was coated onto MHA and cultured at 30°C for 12–18 h, while rLc-HepL replaced by sterile MilliQ water or rTRX were used as the control, respectively. The colonies were counted, with the percentage of CFU (colony forming units) in the control group being treated as 100%, and the other groups were defined relative to the control group (Lauth et al., 2005).
2.6.3 Kill-curve analysis
Corynebacterium glutamicum and P. damselae were selected for studying the kill-curve as described previously (Niu, 2013; Supungul et al., 2008). Two-fold MBC concentration of rLc-HepL was incubated with the adjusted bacterial solution, rLc-HepL replaced by sterile MilliQ water or rTRX were used as controls, respectively. At various times, 0.7 μL of the mixture was taken from the well, coated on MHA, and incubated at 30°C for 12–18 h. The calculation method was the same as that mentioned above.
2.6.4 SEM observation of antibacterial effect
To observe the morphological changes on bacteria caused by rLc-HepL treatment, a SEM (scanning electron microscope) was employed in this study. Corynebacterium glutamicum and P. damselae were selected to represent the Gram-positive and Gram-negative bacteria, respectively. The bacteria were adjusted to 1×108 CUF/mL (OD600≈1.0) with working medium, and rLc-HepL was added to a final concentration of 50 μmol/L. rLc-HepL replaced by sterile MilliQ water or rTRX were used as controls, respectively. The mixture was incubated at 28°C, and samples were fixed with 2.5% glutaraldehyde at various times overnight at 4°C. All fixative solutions were washed with 0.01 mol/L PBS (pH 7.4), dehydrated in a graded ethanol series, freeze-dried, and gold sputtered. Prepared samples were observed in JSM-6390 LV SEM.
2.7 Antiparasitic activity observation of rLc-HepL with LM (light microscope)
To preliminarily observe the antiparasitic activity of rLc-HepL, an antiparasitic assay was designed to determine the morphologic changes in C. irritans theronts treated with 40 μmol/L rLc-HepL. The theronts released from tomonts in 3 h were used to perform the test. Approximately 300 theronts were added to a 48-well plate containing 40 μmol/L rLc-HepL solution to a total volume of 1 mL at room temperature, and 200 μL of the mixture was fixed with 5.0% glutaraldehyde at various times at 4°C overnight, while rLc-HepL replaced by sterile MilliQ water or rTRX were used as controls, respectively. Prepared samples were observed on LM (LEICA ICC50 W, Germany).
3 Results
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3.1 The sequence analysis
Lc-HepL consists of a 258 bp ORF encoding a peptide of 85 amino acids with eight cysteines in the mature peptide to form four disulfide linkages which was employed for antibacterial function. The putative peptide contains a conserve signal peptide, motif RENR. There were only two amino acids variation sites between Lc-HepL and Lc-hepc.
3.2 Expression changes of Lc-HepL mRNA after C. irritans infection (Fig. 1)
According to the sequence analysis, only two amino acids was different between hepcidin and hepcidin-like, but no available qRT-PCR primers were screened to identify the two variants, so we could only detect the overall expression changes. After challenged by C. irritans, the Lc-HepL mRNA was upregulated instantaneously to peak level in the gill at 6 h pi, which was one of the primary infected tissues, and recovered to the basal expression level from 46-fold (p<0.01) instantaneously at 1 d pi. In the intestine, upregulation occurred at 3 d pi, maximum expression was reached at 4 d pi when the level was 8.66-fold (p<0.01), and gradually returned to normal at 6 d pi. Secondly, highly significant expression peaks appearing at 7 d pi were found in such tissues as head kidney, spleen, liver and muscle, where the levels were 4.12-fold, 9.27-fold, 24.015-fold and 7.85-fold, respectively. In addition, the presence of expression peaks in the head kidney and muscle was instantaneous, while it was upregulated at 4 d pi and gradually reached the maximum expression in the liver. Therefore, as seen from the expression peaks, gill was the only tissue to reach a peak early in the infection, where the extent of upregulation was the greatest; intestine was the only tissue where the greatest upregulation occurred at 4 d pi when the trophonts of C. irritans were falling off; at 6–7 d pi, in the secondary bacterial infection phase, Lc-HepL mRNA was upregulated in the other four tissues. Therefore, the induction expression of Lc-HepL mRNA by C. irritans appeared to be a time-dependent and with instantaneous expression trend.
3.3 Capture of rLc-HepL
3.3.1 Optimization of induction expression conditions
The induction results by various IPTG concentrations or auto-induction means were shown in Fig. 2. Compared to the blank group, the induced groups presented a distinct band at the position of 25 kDa, which was consistent with the expected size. As seen from the pictures, there were no visible differences among the various IPTG concentrations at the same induction temperature. Induction expression levels by IPTG or auto-induction appeared to have a positive correlation with the temperature. Therefore, 0.1 mmol/L IPTG or auto-induction at 37°C were sufficient.
3.3.2 Exploration of initial OD600
The induction expression results at various initial OD600 were shown in Fig. 3. With increasing of OD600 from 0.1 to 0.9, the expression level apparently decreased, namely the expression level appeared to have a negative correlation with the initial OD600. Therefore, an initial OD600 of 0.1 was sufficient to induce rLc-HepL.
3.3.3 Exploration of induction time
SDS-PAGE analysis of rLc-HepL induction expression for different times was shown in Fig. 4. Within 7 h, the expression level increased as the time extended; from 7 h to 11 h, the expression level showed no visual difference. Therefore, 7 h induction was sufficient for rLc-HepL expressioin.
3.3.4 Purification of rLc-HepL
According to the explored induction conditions, a large-scale induction was carried out. Prior to purification, the expression form must be detected. Figure 5 showed that rLc-HepL existed both in the supernatants and inclusion bodies. During the purification process, the graded imidazole was used to remove the nonobjective protein contamination and capture pure rLc-HepL. The eluent containing pure rLc-HepL was dialyzed to remove the imidazole, and the concentration was 0.986 mg/mL.
3.3.5 Western blot analysis
The Western blot result was shown in Fig. 6, 6×His-tag was able to interact with the mouse monoclonal antibody. Thus, the purified rLc-HepL was adequate for the following tests.
3.4 Antibacterial activity of rLc-HepL
3.4.1 MIC and MBC detection
The antibacterial spectrum of rLc-HepL was shown in Table 2. The results showed rLc-HepL was highly active against certain bacteria, including C. glutamicum, M. lysodeikticus, and P. damselae. These three bacteria were all susceptible to rLc-HepL (<3.125 μmol/L), while the MIC for other tested bacteria was >50 μmol/L. For these three bacteria, the MBC was either equal to or two times of the MIC.
3.4.2 Dose-dependent antibacterial activity
The C. glutamicum was incubated with rLc-HepL at 28°C for 3 h, and the relative percentage of CFU was shown in Fig. 7. Evidently, as the concentration of rLc-HepL increased, the survival rate decreased. In other words, the antibacterial activity of rLc-HepL to C. glutamicum appeared to be strictly dose-dependent.
3.4.3 Kinetics of killing of C. glutamicum and P. damselae
Figure 8 showed the survival rate of C. glutamicum and P. damselae upon exposure to rLc-HepL with 2-fold MBC at various times. Within 2 h, approximately 70% of the bacteria were killed. During the sampled times, the two bacteria had not been completely killed. In fact, the kinetics study showed that rLc-HepL killed the bacteria gradually and slowly.
3.4.4 SEM observation on antibacterial effect to C. glutamicum
Corynebacterium glutamicum, representing Gram-positive bacteria, was treated to perform SEM, and the results were shown in Fig. 9. Corynebacterium glutamicum showed a smooth surface and dispersed over the microscopic field in the control groups. When treated by rLc-HepL, the bacteria were found aggregated and surrounded by floccule materials. A mass of C. glutamicum gathered gradually, and a small amount of floccule material appeared around part of the bacteria at 15 min (Fig. 9b); a larger amount of floccule material wrapped the bacteria after 30 min incubation, and the surface of the bacteria became rough with the appearance of processes indicating the development of content leakage; floccule materials grew gradually and formed into a honeycomb structure at 2 h (Figs 9c-e). All of these data indicated the antibacterial effect of rLc-HepL on C. glutamicum was already functioning at 15 min.
3.4.5 SEM observation on antibacterial effect to P. damselae
After treated with rLc-HepL, SEM observed the changes in P. damselae, and the result was shown in Fig. 10. Photobacterium damselae presented a smooth, clean surface and dispersed over the microscopic field when the cells were incubated with sterile MilliQ water or rTRX in the control group (Fig. 10a). After treated with rLc-HepL, the two obvious changes were the appearance of the flocculent structures and the aggregation of the bacteria. After incubation for 15 min, the surface became rough with the appearance of different extent processes, and the bacteria were covered by a small amount of floccule materials (Fig. 10b). Many P. damselae were found aggregated wrapped by a larger amount of floccule materials within 30 min after incubation, and some bacteria were disformed (Fig. 10c). The flocculent structures began to form into the honeycomb structure and coated onto the bacteria at 1 h incubation (Fig. 10d). Continuously, the honeycomb structure became highly significant at 2 h (Fig. 10e), but the number of misshapen bacteria did not increase from 1 h to 2 h.
3.5 Antiparasitic activity of rLc-HepL
The LM results of rLc-HepL effect on C. irritans theronts were shown in Fig. 11. Compared to the theronts with rounded bodies and clear cilia in the control group, there were no integral or detailed changes after incubation for 15 min (Fig. 11b). The visible changes began at 30 min, with some cilia no longer erect, instead lying down on the surface, and obvious processes appeared (Fig. 11c). The processes increased in number and size at different positions, which caused the cells disformed at 1 h (Fig. 11d). Similar damages continued, with the cells being completely deformed at 1.5 h, membrane ruptured at different positions causing contents leakage (Fig. 11e). These data indicated that rLc-HepL had antiparasitic activity on C. irritans theronts.
4 Discussion
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The signal peptide of hepcidin was highly conserved in fish, except for the zebrafish (Shike et al., 2004) and the channel catfish (Hu et al., 2007). The putative motif RENR was conserved in the Lc-HepL and Lc-hepc (Wang et al., 2009), which was different from the typical RX(K/R)R found in most other fish (Shike et al., 2002; Douglas et al., 2003). There may be several hepcidin copies in some species (Mu et al., 2018), the reason why there were multiple hepcidin copies in some fish was not clear. It was proposed that in fish, different hepcidins may have different functions, and the additional copies may serve to support innate immunity (Hirono et al., 2005).
qRT-PCR detection illustrated that Lc-HepL was involved in the immune process of C. irritans infection, and the mRNA was significantly upregulated at different times post infection. The results were the first proof demonstrating Lc-HepL was involved in the anti-C. irritans immune response at the mRNA level. To date, reports showed many AMPs took part in the antiparasitic process, Lc-pis expression was significantly upregulated in all tested tissues at 6 h or one day after C. irritans infection, indicating that Lc-pis was not only constitutively expressed but also exhibited an increased response to parasite infection. Additionally, Lc-hepc was significantly enhanced in spleen, heart, and stomach but not in the liver and kidney after challenged by LPS (Lipopolysaccharides). Hepcidin 1 and 2 of the Japanese flounder P. olivaceus were induced to higher levels in liver (Hirono et al., 2005). Hepcidin in the Lates calcarifer was modulated in response to infection, and the most significantly up-regulated gene observed was hepcidin 1 precursor post 2 d infection when the expression level increased 91.72-fold (Khoo et al., 2012). After challenged by C. irritans, the increase in the liver was smaller but greater in the gill and head kidney. Therefore, the induction expression profiles of fish hepcidin caused by bacteria or C. irritans were significantly different.
In this study, we observed that rLc-HepL exhibited antibacterial activity to certain bacteria, including C. glutamicum, M. lysodeikticus and P. damselae. Compared to the antibacterial activity of Lc-hepc, rLc-HepL showed significant limitations, because Lc-hepc exhibited potent antibacterial activity to most of the tested Gram-positive and Gram-negative bacteria, particularly to V. harveyi which is a serious pathogen infecting marine fish and invertebrates (Di Pinto et al., 2008); V. parahaemolyticus which is an enteric pathogen typically responsible for acute human gastroenteritis associated with consuming contaminated seafood (Vizioli and Salzet, 2002); and S. aureus which includes resistant strains (Ruangsri et al., 2012). In Atlantic cod (Gadus morhua), piscidin 2 also exhibited poorer antibacterial properties than piscidin 1 due to the disrupted amphipathic structure, which is caused by the substitution of two basic residues in piscidin 1 for leucine and glycine at positions 14 and 18 (Lauth et al., 2002). Similarly, rLc-HepL showed weaker antibacterial activity than rLc-hepc, which might be the variation of phenylalanine to leucine in the mature region of Lc-HepL and lead to the instability of the disulfide linkages or even the damage of the β-sheets structure. In view of the hepcidin from different fish species, the antibacterial activity was significantly different (Balebona et al., 1998). The morphological changes of the bacteria observed with SEM in this study were similar to those described in previous Lc-hepc researches (Cai, 2010). However, we observed a large amount of floccule materials emerging at later time in our study. Because the content leakage of certain bacteria might not be sufficient to cause this phenomenon, we proposed that most of the floccule materials were likely to be secreta which were caused by rLc-HepL. In addition, Lc-hepc caused damage to the nucleus of S. aureus. Therefore, damaging the membrane by gathering on the membrane to a certain concentration causing holes formation, and disturbing biological processes through entering into cells were the main bactericidal mechanisms of hepcidin (Cai, 2010).
Being an antimicrobial and systemic regulator of iron homeostasis, hepcidin combats the invading pathogen by sequestering the free iron, and ultimately limits the proliferative capability of the pathogen (Nemeth et al., 2004). Regarding the antiparasitic activity, this study was the first attempt to learn the activity of hepcidin against C. irritans. Increasing number of studies found some AMPs were of resistance to C. irritans infection; therefore, they were also called antiparasitic peptides (APPs). Lc-pis was effective against C. irritans trophonts, killing all trophonts in a short time at 6 μmol/L. The synthetic Lc-pis-His was active against trophonts and theronts in a dose- and time-dependent manner, causing the rupture of the membrane and content leakage in both trophonts and theronts (Niu, 2013). The serum of Siganus oramin was found to have strong killing effect on C. irritans, and reversed phase high-performance liquid chromatography (RP-HPLC) was used to isolate L-amino acid oxidase-like protein (SR-LAAO) from serum that was poisonous to C. irritans theronts. SR-LAAO located primarily on the cell membrane and nucleus of the theronts (Wang et al., 2010, 2011), and this protein had a significantly cytotoxic effect on C. irritans theronts resulting in the cilia detaching from theronts, the cells becoming rounded, the membranes eventually lysing in different cell positions and the cytoplasmic contents leaking out of the cell (Li et al., 2013). The results suggested that these proteins may contribute considerably to the host non-specific immune defense mechanism to combat microbes, and have the potentials in future drug development.
Funding
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  • The National Key Research and Development Program of China under contract No. 2018YFC1406302; the Local Science and Technology Development Project Guide by the Central Government under contract No. 2017L3019; the Development Project Guide by the Central Government under contract No. 2017L3019; the Technical Innovation Platform for Large Yellow Croaker under contract No. XDHT2018143A; the Major Special Projects of Fujian Province under contract No. 2016NZ0001.
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Year 2020 volume 39 Issue 10
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Article Info
doi: 10.1007/s13131-020-1580-6
  • Receive Date：2019-12-24
  • Online Date：2026-03-31
  • Published：2020-10-25
Article Data
Affiliations
History
  • Received：2019-12-24
  • Accepted：2020-02-14
Funding
The National Key Research and Development Program of China under contract No. 2018YFC1406302; the Local Science and Technology Development Project Guide by the Central Government under contract No. 2017L3019; the Development Project Guide by the Central Government under contract No. 2017L3019; the Technical Innovation Platform for Large Yellow Croaker under contract No. XDHT2018143A; the Major Special Projects of Fujian Province under contract No. 2016NZ0001.
Affiliations
    1 State Key Laboratory of Large Yellow Croaker Breeding, Ningde 352103, China
    2 Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China
    3 National and Provincial Joint Laboratory of Exploration and Utilization of Marine Aquatic Genetic Resources, School of Marine Science and Technology, Zhejiang Ocean University, Zhoushan 316022, China

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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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