Elsevier

Fish & Shellfish Immunology

Volume 98, March 2020, Pages 551-563
Fish & Shellfish Immunology

Full length article
Antibacterial and immunoregulatory activity of a novel hepcidin homologue in diploid hybrid fish (Carassius auratus cuvieri ♀ × Carassius auratus red var ♂)

https://doi.org/10.1016/j.fsi.2020.01.038Get rights and content

Highlights

  • We characterized a novel hepcidin gene in hybrid crucian carp.

  • The up-regulation of WR-hepcidin was observed following bacterial challenge.

  • WR-hepcidin can bind to gram positive and gram negative bacteria.

  • WR-hepcidin can restrict the bacterial growth and reduce bacteria-stimulated inflammatory response.

Abstract

Hepcidin, a multifunctional hormone oligopeptide, not only exhibits a regulatory role in iron metabolism, but also participates in the regulation of teleostean immunity. In this study, ORF sequence of WR-hepcidin was 258 bp and encoded 85 amino acid residues. Tissue-specific analysis revealed that the highest expression of WR-hepcidin was observed in liver. Aeromonas hydrophila challenge can sharply increased WR-hepcidin mRNA expression in liver, trunk kidney and spleen. The purified WR-hepcidin fusion peptide can directly bind to A. hydrophila and Streptococcus agalactiae, reduce the relative bacterial activity, limit bacterial growth and attenuate their dissemination to tissues in vivo. In addition, the treatment of WR-hepcidin fusion protein can diminish the production of pro-inflammatory cytokines. These results indicated that WR-hepcidin can play a negative regulatory role in bacteria-stimulated pro-inflammatory cytokines production and MyD88-IRAK4 activation.

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).

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