Full length articleAntibacterial and immunoregulatory activity of a novel hepcidin homologue in diploid hybrid fish (Carassius auratus cuvieri ♀ × Carassius auratus red var ♂)
Introduction
Mammalian cell killing mechanism is playing a key role in immune defense system, showing a cytotoxic effect on invading pathogens, infected tissues and malignant cells [1]. In fish, innate immunity is the first line of immune defense against pathogenic infection [2]. In general, acute phase response is a non-specific and complex reaction, playing a regulatory role in the processes of infection or tissue injury [3]. Among known acute phase proteins (APPs), mammalian hepcidin, a cysteine-rich oligopeptide secreted by liver, is previously discovered as iron regulatory hormone, which can modulate cellular iron metabolism by binding to ferroportin and its expression is highly associated with acute or chronic inflammation during pathological condition [4,5]. In addition, hepcidin is also an innate antimicrobial molecule that can attenuate the proliferative activity of invading pathogens by restrict their iron acquisition ability [6,7].
Evidences are emerging that invertebrate innate immunity possesses various pathogen-recognizing properties, but it is a fundamental defense in teleost. For instances, teleost contains a developed complement system and immune-related pathways, which shows a close similarity to those of mammals [[8], [9], [10], [11]]. Although most studies focus on function of mammalian genes synchronizing innate immunity with adaptive immune responses, only a few reports study on the architecture and expression of teleostean hepcidin, such as sea bass [12], turbot [13], seabream [14], common carp [15] and yellow catfish [16]. As is well known, chimeric genes may form through the fusion of pieces of various genomes [17]. Chimeric genes and nonsynonymous mutation may generate structural changes at transcriptional level that can alter enzyme activities or fidelities [18]. In addition, a high level of genome reconstruction may occur in hybrid offsprings, such as genetic recombination, offspring-specific mutation and change of gene expression [19]. Compared to distant hybridization, intraspecific hybridization triggers limited genetic variation, whereas it can generate hybrid offsprings harboring the excellent traits derived from their parents and establish a steady lineage [20]. Although several chimeric genes are observed and validated in hybrids by intraspecific hybridization [21], the data on the immune-related genetic variants of pro-inflammatory cytokines or MyD88-IRAK4 signaling and immunoregulatory function of chimeric hepcidin in hybrids are sparse.
Hybrid crucian carp (WR), novel diploid hybrid fish (2n = 100), are generated by crossing white crucian carp (Carassius auratus cuvieri, WCC, ♀) with red crucian carp (Carassius auratus red var, RCC, ♂), which belong to Cypriniformes, Cyprinidae (genus Carassius) [20,21]. Additionally, crucian carp is also one of the most important economic fish in China. However, the emergence of global climate anomaly may be one of the abnormal phenomena and elicits a lingering effect in the expansion of water-borne pathogenic diseases, therefore posing a great threat to the survival of aquatic organisms [22]. In general, increasing bacterial population in water may highly link to elevated level of fecal pollution. Previous studies indicate that accumulative mortality of allogynogenetic crucian carp increases significantly following Aeromonas hydrophila challenge [23]. Recently, Streptococcus agalactiae was reported to pose a great threat to the survival of ya-fish, Nile tilapia and crucian carp [24]. Thus, the study on the immune response to gram-positive and gram-negative bacterial infection in hybrid crucian carp may be propitious to the sustainable development of aquaculture.
In this study, the aims were to characterize the architecture of WR-hepcidin in hybrid crucian carp and measure tissue distribution of WR-hepcidin mRNA. We also assessed the effect of bacterial infection on the expression patterns of WR-hepcidin mRNA in various immune tissues. To further characterize its function, we also studied binding activity of WR-hepcidin to both gram-positive and gram-negative bacteria and its potential effect on bacterial growth in vitro and in vivo, which may provide a new insight to the immune regulation of hybrid fish.
Section snippets
Animals
According to previous studies, diploid hybrid fish (WR) was generated by crossing white crucian carp (Carassius auratus cuvieri, WCC, ♀) and red crucian carp (Carassius auratus red var, RCC, ♂) [20,21]. WRs with average length 13.2 ± 0.52 cm and average weight 28.50 ± 0.79 g were obtained from an aquaculture base in Wang Cheng district (Changsha, Hunan province, China). WRs were acclimatized in 70 × 65 × 65 cm plastic aquarium (25 fishes/aquarium) with the diluted freshwater for two weeks. WRs
Characterization of WR-hepcidin sequence and its chimeric validation in hybrid and parents
The obtained ORF sequence of WR-hepcidin was 258 bp and encoded 85 amino acid residues with a estimated molecular mass of 9.69 KDa and a predicted isoeletric point of 8.02. As shown in Supplementary Fig. 1A and Supplementary Fig. 2, comparative analysis of hepcidin gene revealed that WR-hepcidin gene possessed the chimeric fragments composed of continuous, alternating variations from paternal/maternal-specific genetic types with or without offspring-specific mutations, which may alter the amino
Discussion
Hepcidin is a cysteine-rich peptide hormone of hepcidin family containing a conserved cleavage site “RXXR” and a predicted iron binding motif “QSHLS”, which can participate in both immune regulation and iron metabolism [6,42]. In this study, chimeric hepcidin in hybrid offsprings contained the continuous and alternating variations derived from parent-specific genes with or without offspring-specific mutations, which may subsequently alter the amino acid level in hybrid fish-originating
Acknowledgements
This research was supported by the National Natural Science Foundation of China, China (grant no. 31902363) and the Doctoral Publishing Fund of Hunan Normal University, China (grant no. 368002).
References (66)
- et al.
Temperature-mediated processes in teleost immunity: in vitro immunosuppression induced by in vivo low temperature in channel catfish
Vet. Immunol. Immunopathol.
(1991) - et al.
The acute phase response and innate immunity of fish
Dev. Comp. Immunol.
(2001) Hepcidin, a key regulator of iron metabolism and mediator of anemia of inflammation
Blood
(2003)- et al.
Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein
Blood
(2003) - et al.
Hepcidin antimicrobial peptide transgenic mice exhibit features of the anemia of inflammation
Blood
(2007) - et al.
Complement system of bony and cartilaginous fish
Fish Shellfish Immunol.
(2000) - et al.
The complement system in teleosts
Fish Shellfish Immunol.
(2002) - et al.
Molecular cloning, characterization and expression analysis of complement component C8 beta in the orange-spotted grouper (Epinephelus coioides) after the Vibrio alginolyticus challenge
Gene
(2015) - et al.
Dual function of fish hepcidin: response to experimental iron overload and bacterial infection in sea bass (Dicentrarchus labrax)
Dev. Comp. Immunol.
(2006) - et al.
Molecular cloning and expression analysis of a hepcidin antimicrobial peptide gene from turbot (Scophthalmus maximus)
Fish Shellfish Immunol.
(2007)
The antimicrobial peptide hepcidin exerts an important role in the innate immunity against bacteria in the bony fish gilthead seabream
Mol. Immunol.
Molecular characterization of hepcidin gene in common carp (Cyprinus carpio L.) and its expression pattern responding to bacterial challenge
Fish Shellfish Immunol.
cDNA cloning and expression analysis of a hepcidin gene from yellow catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae)
Fish Shellfish Immunol.
Effects of mannan oligosaccharide on the physiological responses, HSP70 gene expression and disease resistance of Allogynogenetic crucian carp (Carassius auratus gibelio) under Aeromonas hydrophila infection
Fish Shellfish Immunol.
Molecular cloning, characterization and expression analysis of PPAR gamma in the orange-spotted grouper (Epinephelus coioides) after the Vibrio alginolyticus challenge
Fish Shellfish Immunol.
Molecular cloning and characterization of PTEN in the orange-spotted grouper (Epinephelus coioides)
Fish Shellfish Immunol.
Effects of a recombinant complement component C3b functional fragment α 2 MR (α 2-macroglobulin receptor) additive on the immune response of juvenile orange-spotted grouper (Epinephelus coioides) after the exposure to cold shock challenge
Fish Shellfish Immunol.
Transcriptome profiling of grass carp (Ctenopharyngodon idellus) infected with Aeromonas hydrophila
Fish Shellfish Immunol.
Studies on expression pattern of toll-like receptor 5 (TLR5) in Edwardsiella tarda infected Pangasianodon hypophthalmus
Fish Shellfish Immunol.
Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method
Methods
Molecular cloning, characterization and expression analysis of (B-cell lymphoma-2 associated X protein) Bax in the orange-spotted grouper (Epinephelus coioides) after the Vibrio alginolyticus challenge
Dev. Comp. Immunol.
Molecular cloning, characterization and expression analysis of (B-cell lymphoma-2) Bcl-2 in the orange-spotted grouper (Epinephelus coioides)
Dev. Comp. Immunol.
N-terminal domain of EcC1INH in Epinephelus coioides can antagonize the LPS-stimulated inflammatory response
Fish Shellfish Immunol.
Optimal conditions for expressing a complement component 3b functional fragment (α2-macroglobulin receptor) gene from Epinephelus coioides in Pichia pastoris
Protein Expr. Purif.
Characterization of a CD59 in orange-spotted grouper (Epinephelus coioides)
Fish Shellfish Immunol.
Antimicrobial activities of a proline-rich proprotein from Spodoptera litura
Dev. Comp. Immunol.
Molecular characterization of the liver-expressed antimicrobial peptide 2 (LEAP-2) in a teleost fish, Plecoglossus altivelis: antimicrobial activity and molecular mechanism
Mol. Immunol.
Characterization and functional analysis of a novel C1q-domain-containing protein in Japanese flounder (Paralichthys olivaceus)
Dev. Comp. Immunol.
Hepcidin: a direct link between iron metabolism and immunity
Int. J. Biochem. Cell Biol.
Identification and expression analysis of hepcidin-like antimicrobial peptides in bony fish
Dev. Comp. Immunol.
The morphology of the immune system in teleost fishes
Fish Shellfish Immunol.
Trunk kidney of grass carp (Ctenopharyngodon idella) mediates immune responses against GCRV and viral/bacterial PAMPs in vivo and in vitro
Fish Shellfish Immunol.
Importance of the disulfide bridges in the antibacterial activity of human hepcidin
Peptides
Cited by (17)
Antibacterial and immunoregulatory activity of an antimicrobial peptide hepcidin in loach (Misgurnus anguillicaudatus)
2023, International Journal of Biological MacromoleculesAntimicrobial peptide hepcidin contributes to restoration of the intestinal flora after Aeromonas hydrophila infection in Acrossocheilus fasciatus
2023, Comparative Biochemistry and Physiology Part - C: Toxicology and PharmacologyFerritin H can counteract inflammatory response in hybrid fish and its parental species after Aeromonas hydrophila infection
2021, Comparative Biochemistry and Physiology Part - C: Toxicology and PharmacologyCitation Excerpt :To investigate the effect of RCC-Ferritin H, WCC-Ferritin H and WR-Ferritin H on inflammatory response following A. hydrophila infection, above fusion protein treated samples were used for RNA isolation and cDNA synthesis. Then, qRT-PCR assay was performed as described above (Luo et al., 2020c). pET32a tag treated group was used as the control.