Antimicrobial peptide hepcidin contributes to restoration of the intestinal flora after Aeromonas hydrophila infection in Acrossocheilus fasciatus.

Hepcidin is a cysteine-rich antimicrobial peptide that serves an important role in the immunity system of fishes. It exhibits antibacterial, antifungal, antiviral, and antitumor activities. However, the exact role of fish hepcidin in the regulation of the intestinal flora still remains a mystery. In our study, we sequenced and characterized hepcidin from the liver of Acrossocheilus fasciatus. Phylogenetic tree analysis showed that A. fasciatus hepcidin and Gobiocypris rarus hepcidin were the most closely related, and both belonged to the fish HAMP1 cluster. Studies conducted on in vivo tissue distribution showed that the expression of hepcidin was highest in healthy A. fasciatus liver. Aeromonas hydrophila infection was confirmed by the increased expression of pro-inflammatory cytokine genes and bacterial loads in A. fasciatus tissues. After A. hydrophila infection, hepcidin expression significantly increased in the liver, spleen, and head kidney. In vitro antibacterial assays showed that hepcidin exhibits strong broad spectrum antibacterial activity. Furthermore, we examined the regulatory effect of hepcidin on the intestinal flora and found that A. fasciatus hepcidin restored the reduced diversity and compositional changes in intestinal flora caused by A. hydrophila infection. Our results suggest that hepcidin could regulate the intestinal flora in fishes; however, the underlying mechanisms need to be explored in greater detail.

Molecular Analysis and Sex-specific Response of the Hepcidin Gene in Yellow Perch (Perca Flavescens) Following Lipopolysaccharide Challenge.

Hepcidin antimicrobial peptide (hamp) is active in teleosts against invading pathogens and plays important roles in the stress and immune responses of finfish. The response of hamp gene was studied in yellow perch (yp) (Perca flavescens) challenged with lipopolysaccharides to understand if this immunity response is sex-specifically different. The cloned hamp gene consists of an open-reading frame of 273 bp and encodes a deduced protein of 90 amino acids (a.a.), which includes a signal peptide of 24 a.a., a pro-domain of 40 a.a. and a mature peptide of 26 a.a. Yp hamp involves 8 cysteine residues with 4 disulfide bonds, and a protein with an internal alpha helix flanked with C- and N-terminal random coils was modeling predicted. RT-qPCR was used to analyze the relative abundances (RAs) of hamp mRNA in the livers of juvenile female and male yellow perch challenged with lipopolysaccharide. The expression levels of hamp were significantly elevated by 3 h (RA = 7.3) and then peaked by 6 h (RA = 29.4) post-treatment in females but the peak was delayed to 12 h (RA = 65.4) post-treatment in males. The peak mRNA level of challenged males was shown 7.6-fold higher than females. The post-treatment responses in both genders decreased to their lowest levels by 24 h and 48 h. Overall, female perch had an earlier but less-sensitive response to the lipopolysaccharide challenge than male.

The Antimicrobial Peptide LJ-hep2 from Lateolabrax japonicus Exerting Activities against Multiple Pathogenic Bacteria and Immune Protection In Vivo.

Hepcidin is widely present in many kinds of fish and is an important innate immune factor. A variety of HAMP2-type hepcidins have strong antimicrobial activity and immunomodulatory functions and are expected to be developed as substitutes for antibiotics. In this study, the antimicrobial activity of Hepc2 from Japanese seabass (Lateolabrax japonicus) (designated as LJ-hep2) was investigated using its recombinant precursor protein (rLJ-hep2) expressed in Pichia pastoris and a chemically synthesized mature peptide (LJ-hep2(66-86)). The results showed that both rLJ-hep2 and synthetic LJ-hep2(66-86) displayed broad antimicrobial spectrum with potent activity against gram-negative and gram-positive bacteria, and fungi. Especially, LJ-hep2(66-86) had stronger antimicrobial activity and exhibited potent activity against several clinically isolated multidrug-resistant bacteria, including Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Enterococcus faecium. Moreover, LJ-hep2(66-86) exerted rapid bactericidal kinetic (killed tested bacteria within 2 h), induced significant morphological changes and promoted agglutination of E. coli, P. aeruginosa and Aeromonas hydrophila. The activity of LJ-hep2(66-86) against E. coli, P. aeruginosa and A. hydrophila was stable and remained active when heated for 30 min. In addition, LJ-hep2(66-86) exhibited no cytotoxicity to the mammalian cell line HEK293T and fish cell lines (EPC and ZF4). In vivo study showed that LJ-hep2(66-86) could improve the survival rate of marine medaka (Oryzias melastigma) by about 40% under the challenge of A. hydrophila, indicating its immunoprotective function. Taken together, both rLJ-hep2 and LJ-hep2(66-86) have good prospects to be used as potential antimicrobial agents in aquaculture and medicine in the future.

Hepcidin discriminates sepsis from other critical illness at admission to intensive care.

Initial differential diagnosis and prognosis for patients admitted to intensive care with suspected sepsis remain arduous. Hepcidin has emerged as a potential biomarker for sepsis. Here we report data on the relevance of levels of hepcidin versus other biomarkers as a diagnostic and prognostic tool for sepsis. 164 adult patients admitted to the intensive care unit (ICU) within 24 h upon arrival to the hospital were included. Blood samples collected daily for seven consecutive days and hepcidin levels, heparin binding protein (HBP) levels and standard biomarkers were determined. Blood cultures were initiated at inclusion. Clinical scores were evaluated daily and mortality after 28- and 180-days was recorded. One hundred of the patients were found to fulfil the criteria for sepsis whereas 64 did not. Hepcidin levels at admission were significantly higher in the septic than in the non-septic patients. In septic patients hepcidin levels declined significantly already at 24 h followed by a steady decline. A significant negative correlation was observed between hepcidin levels and SAPS 3 in patients with sepsis. Hepcidin levels at inclusion were significantly higher among septic patients that survived 180-days and predicted mortality. Our data show that hepcidin levels are indicative of sepsis in patients admitted to the ICU and has a prognostic value for mortality.

Molecular characterization and antibacterial immunity functional analysis of the antimicrobial peptide hepcidin from Coregonus ussuriensis berg.

Antimicrobial peptides are immune system molecules existing in different organisms including mollusks, crustaceans and vertebrates. Hepcidins are a group of cysteine rich antimicrobial peptides, which plays an important role in fish response to a variety of pathogens. In this study, we cloned and identified Hepcidin from the Coregonus ussuriensis Berg, and its functions in vivo and in vitro was investigated. Our results showed that, CuHepc contains a 267 bp coding sequence (CDS) region that encodes 88 putative amino acids with a molecular weight of 9.77 kD. Hepcidin transcripts were most abundant in the liver of healthy C. ussuriensis Berg. The synthesized Hepcidin peptide exhibited a wide range of antibacterial activity against Gram-positive and Gram-negative bacteria in vitro, and the results of in vivo bacterial attack assays showed that the CuHepc gene was differentially up-regulated in the six tissues investigated after infection with Aeromonas hydrophila. To analyze the changes in protein levels in C. ussuriensis, we generated Hepc polyclonal antibodies in rabbits and verified that the protein expression was increased after bacterial infection with Western blot assay. MIC assay results showed a geometric mean value of 5.513 µM for CuHepc peptide. In the in vivo experiment, immune-related genes IL-10, NF-κB, TLR3 were up-regulated post-infection CuHepc peptide in liver and intestine. Finally, CuHepc peptide reduced the tissues microbial load compared to infection with Aeromonas hydrophila. The above results indicate that Hepc plays a role in the immune response of C. ussuriensis to exogenous disturbances, indicate that CuHepc might act a candidate for modulation of the innate immune system in C. ussuriensis.

BioMIPs: molecularly imprinted silk fibroin nanoparticles to recognize the iron regulating hormone hepcidin.

The possibility to prepare molecularly imprinted nanoparticles from silk fibroin was recently demonstrated starting from methacrylated silk fibroin and choosing a protein as template. Here, we attempted the imprinting of fibroin-based molecularly imprinted polymers (MIPs), called bioMIPs, using as a template hepcidin that is a iron-metabolism regulator-peptide, possessing a hairpin structure. A homogeneous population (PDI < 0.2) of bioMIPs with size ~50 nm was produced. The bioMIPs were selective for the template; the estimated dissociation constant for hepcidin was KD = 3.6 ± 0.5 10-7 M and the average number of binding sites per bioMIP was equal to 2. The bioMIPs used in a competitive assay for hepcidin in serum showed a detection range of 1.01 10-7- 6.82 10-7 M and a limit of detection of 3.29 10-8 M.

Hepcidin-Ferroportin Interaction Controls Systemic Iron Homeostasis.

Despite its abundance in the environment, iron is poorly bioavailable and subject to strict conservation and internal recycling by most organisms. In vertebrates, the stability of iron concentration in plasma and extracellular fluid, and the total body iron content are maintained by the interaction of the iron-regulatory peptide hormone hepcidin with its receptor and cellular iron exporter ferroportin (SLC40a1). Ferroportin exports iron from duodenal enterocytes that absorb dietary iron, from iron-recycling macrophages in the spleen and the liver, and from iron-storing hepatocytes. Hepcidin blocks iron export through ferroportin, causing hypoferremia. During iron deficiency or after hemorrhage, hepcidin decreases to allow iron delivery to plasma through ferroportin, thus promoting compensatory erythropoiesis. As a host defense mediator, hepcidin increases in response to infection and inflammation, blocking iron delivery through ferroportin to blood plasma, thus limiting iron availability to invading microbes. Genetic diseases that decrease hepcidin synthesis or disrupt hepcidin binding to ferroportin cause the iron overload disorder hereditary hemochromatosis. The opposite phenotype, iron restriction or iron deficiency, can result from genetic or inflammatory overproduction of hepcidin.

Hepcidin is a relevant iron status indicator in infancy: results from a randomized trial of early vs. delayed cord clamping.

BACKGROUND: We aimed to evaluate whether serum hepcidin is a useful indicator of iron status in infants. METHODS: Term infants (n = 400) were randomized to delayed (≥180 s) or early (≤10 s) cord clamping (CC). Iron status was assessed at 4 and 12 months. In all cases with iron depletion or iron deficiency (ID) (as defined in "Methods") (n = 30) and 97 randomly selected iron-replete infants, we analyzed hepcidin and explored its correlation to the intervention, iron status, and perinatal factors. RESULTS: Serum hepcidin concentrations were significantly lower in the early CC group at both time points and in ID infants at 4 months. Median (2.5th-97.5th percentile) hepcidin in non-ID infants in the delayed CC group (suggested reference) was 64.5 (10.9-142.1), 39.5 (3.5-157.7), and 32.9 (11.2-124.2) ng/mL in the cord blood and at 4 and 12 months, respectively. The value of 16 ng/mL was a threshold detecting all cases of iron depletion/ID at 4 months. No similar threshold for ID was observed at 12 months. The strongest predictor of hepcidin at both ages was ferritin. CONCLUSIONS: Hepcidin is relevant as iron status indicator in early infancy and may be useful to detect ID. Levels <16 ng/mL at 4 months of age indicates ID. IMPACT: Serum hepcidin is a relevant indicator of iron status in early infancy. Normal reference in healthy infants is suggested in this study. Serum hepcidin may be useful in clinical practice to detect iron deficiency.

COVID-19 and iron dysregulation: distant sequence similarity between hepcidin and the novel coronavirus spike glycoprotein.

The spike glycoprotein of the SARS-CoV-2 virus, which causes COVID-19, has attracted attention for its vaccine potential and binding capacity to host cell surface receptors. Much of this research focus has centered on the ectodomain of the spike protein. The ectodomain is anchored to a transmembrane region, followed by a cytoplasmic tail. Here we report a distant sequence similarity between the cysteine-rich cytoplasmic tail of the coronavirus spike protein and the hepcidin protein that is found in humans and other vertebrates. Hepcidin is thought to be the key regulator of iron metabolism in humans through its inhibition of the iron-exporting protein ferroportin. An implication of this preliminary observation is to suggest a potential route of investigation in the coronavirus research field making use of an already-established literature on the interplay of local and systemic iron regulation, cytokine-mediated inflammatory processes, respiratory infections and the hepcidin protein. The question of possible homology and an evolutionary connection between the viral spike protein and hepcidin is not assessed in this report, but some scenarios for its study are discussed.

Structure of hepcidin-bound ferroportin reveals iron homeostatic mechanisms.

The serum level of iron in humans is tightly controlled by the action of the hormone hepcidin on the iron efflux transporter ferroportin. Hepcidin regulates iron absorption and recycling by inducing the internalization and degradation of ferroportin1. Aberrant ferroportin activity can lead to diseases of iron overload, such as haemochromatosis, or iron limitation anaemias2. Here we determine cryogenic electron microscopy structures of ferroportin in lipid nanodiscs, both in the apo state and in complex with hepcidin and the iron mimetic cobalt. These structures and accompanying molecular dynamics simulations identify two metal-binding sites within the N and C domains of ferroportin. Hepcidin binds ferroportin in an outward-open conformation and completely occludes the iron efflux pathway to inhibit transport. The carboxy terminus of hepcidin directly contacts the divalent metal in the ferroportin C domain. Hepcidin binding to ferroportin is coupled to iron binding, with an 80-fold increase in hepcidin affinity in the presence of iron. These results suggest a model for hepcidin regulation of ferroportin, in which only ferroportin molecules loaded with iron are targeted for degradation. More broadly, our structural and functional insights may enable more targeted manipulation of the hepcidin-ferroportin axis in disorders of iron homeostasis.

Hepcidin, an overview of biochemical and clinical properties.

Hepcidin is a peptide hormone which helps in regulating iron homeostasis in the human body. Iron obtained from daily diet is passed through the intestinal enterocyte apical membrane via divalent metal transporter 1 (DMT1), which is either stored as ferritin or moved into the plasma by hepcidin-ferroportin (Fpn) as an exporter. Hepcidin (hepatic bactericidal protein) is a cysteine rich peptide, was initially identified as a urinary antimicrobial peptide. It contains 25 amino acids and four disulfide bridges. It has significant role in regulation of iron in the body. Stimulation of iron in plasma and further its storage is linked with the production of hepcidin. This enhancement of iron hampers the absorption of iron from the diet. The cause of hereditary recessive anemia also known as Iron-refractory iron deficiency anemia (IRIDA) is characterized by increased hepcidin production due to a gene mutation in the suppressor matriptase-2/TMPRSS6. During infection, hepcidin plays a defensive role against various infections by depleting the extracellular iron from the body. Moreover, hepcidin lowers the concentrations of iron from the duodenal enterocytes, macrophages and also decrease its transport across the placenta.This review highlights the significant role of hepcidin in the iron homeostasis and as an antimicrobial agent.

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

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.

Cellular binding analysis of recombinant hybrid heteropolymer of camel hepcidin and human ferritin H chain. The unexpected human H-ferritin binding to J774 murine macrophage cells.

Ferritin is a molecule with enormous potentiality in biotechnology that have been already used to encapsulate molecules, as contrast in magnetic resonance imaging and to carry epitopes. We proposed to use it to carry another key protein of iron metabolism, hepcidin that is a small hormone peptide that control systemic iron homeostasis. In this work, we purified the previously produced camel hepcidin and human H-ferritin heteropolymer (HepcH-FTH) and to monitor its binding capability toward J744 cell line in presence or absence of ferric ammonium citrate. Fused camel hepcidin and human H-ferritin monomer (HepcH) as well as the assembled HepcH-FTH heteropolymer (ratio 1:5) was easily purified by a one-step purification using size exclusion chromatography. SDS-PAGE electrophoresis of HepcH, purified from soluble and insoluble fractions, showed a single band of 24 kDa with an estimated purity of at least 90%. The purification yields of HepcH from the soluble and insoluble fractions was, respectively, of about 6.80 and 2 mg/L of bacterial culture. Time curse cellular binding assays of HepcH-FTH revealed its great potential to bind the J774 cells after 15 min of incubation. Furthermore, HepcH-FTH was able to degrade ferroportin, the unique hepcidin receptor, even after 30 min of incubation with J774 cells treated with 100 µM ferric ammonium citrate. In conclusion, we proposed ferritin as a peptide carrier to promote the association of the hybrid HepcH-FTH nanoparticle with a particular type of cell for therapeutic or diagnostic.

DNA methylation suppresses liver Hamp expression in response to iron deficiency after bariatric surgery.

BACKGROUND: Iron deficiency is extremely common after bariatric surgery. HEPCIDIN, encoded by Hamp, is a hormone that negatively regulates iron homeostasis. OBJECTIVES: We aimed to investigate the alteration of Hamp expression and related regulatory factors to explore the probable role of DNA methylation in modulating Hamp expression in the context of iron deficiency after bariatric surgery. SETTING: Laboratories of Diabetes Institute. METHODS: RNA-seq was performed using rat liver tissue after either Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy surgery to identify differentially expressed genes between the bariatric surgery and sham group. Hamp expression were measured by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The DNA methylation level was determined using MassARRAY EpiTYPER. Iron status, erythrocyte parameters, and inflammation factors were assessed. RESULTS: RNA-seq data showed that liver Hamp expression changed most dramatically in RYGB-operated rats. Both the mRNA expression of Hamp and the abundance of its protein product HEPCIDIN-25 decreased markedly after bariatric surgery compared with sham, while sleeve gastrectomy-operated rats showed marginally higher Hamp expression than RYGB-operated rats. The DNA methylation level of the Hamp promoter region was significant higher in RYGB-operated rats than sham, while sleeve gastrectomy rats increased slightly in DNA methylation. Consistent with the change of HEPCIDIN-25, serum iron was significantly lower for both bariatric groups than sham and particularly low in RYGB. CONCLUSIONS: Our data demonstrate that elevated DNA methylation of the Hamp promoter region suppresses its expression, this epigenetic modification likely occurs in reaction to iron deficiency after bariatric surgery, helping to maintain system iron homeostasis.

Biochemical and molecular identification of a novel hepcidin type 2-like antimicrobial peptide in the skin mucus of the pufferfish Takifugu pardalis.

Fish skin mucus is considered to act as the first line of defense against waterborne pathogens and to be potential source of novel antimicrobial components. Here we report the purification and characterization of a novel hepcidin type 2-like antimicrobial peptide (TpHAMP2) from the skin mucus of the pufferfish Takifugu pardalis. The purified TpHAMP2 comprised of 23 amino acids (AAs) with eight Cys residues that form four intramolecular disulfide bonds. The TpHAMP2 gene shared overall structural characteristics with all known hepcidins, which have a tripartite exon-intron gene organization and three structural signatures in the precursor protein. Phylogenetically, TpHAMP2 was classified as HAMP2 class in acanthopterygian fish. Interestingly, the AA sequence of TpHAMP2 did not contain a proprotein cleavage site (RXXR motif) that conserved in most hepcidins and showed a highly positive charged (RKR-) short N-terminus and Val18 and Gly22 residues, which are distinctive structures compared to other known active hepcidins. Recombinant TpHAMP2 identical to the native form exhibited a broad spectrum and potent antimicrobial activity against tested gram-positive and -negative bacteria. Expression of TpHAMP2 mRNA was predominant in the liver and was upregulated in the liver, the spleen, the intestine, and the skin of T. pardalis post immune challenge. Thus, our findings suggests that TpHAMP2 might be of importance in the framework of discovering the fish hepcidins, especially type 2s, and provide noteworthy insight into its gene structure and expression and in the innate immunity as well as the mucosal immunity in regard to hepcidins' evolutionary history in fish species.

Subfunctionalization and evolution of liver-expressed antimicrobial peptide 2 (LEAP2) isoform genes in Siberian sturgeon (Acipenser baerii), a primitive chondrostean fish species.

Two liver-expressed antimicrobial peptide 2 (LEAP2) isoforms were characterized in a primitive chondrostean sturgeon species, Acipenser baerii (Acipenseriformes). A. baerii LEAP2 isoforms represented essentially common structures shared by their vertebrate orthologs at both genomic (i.e., tripartite organization) and peptide (two conserved disulfide bonds) levels. A. baerii LEAP2 isoforms (designed LEAP2AB and LEAP2C, respectively) phylogenetically occupy the most basal position in the actinopterygian lineage and represent an intermediate character between teleostean and tetrapodian LEAP2s in the sequence alignment. Molecular phylogenetic analysis including LEAP2s from extant primitive fish species indicated that the evolutionary origin of ancestral LEAP2 in vertebrate groups should date back to earlier than the actinopterygian-sarcopterygian split. Gene expression assays under both basal and stimulated conditions suggested that A. baerii LEAP2 isoforms have undergone substantial subfunctionalization in tissue distribution pattern, developmental/ontogenetic expression, and immune responses. LEAP2AB showed a predominant liver expression, while LEAP2C exhibited the highest level of expression in the intestine. LEAP2C was a more dominantly expressed isoform during embryonic development and prelarval ontogeny. The LEAP2AB isoform is more closely associated with innate immune response to microbial invasion, compared with LEAP2C, as evidenced by results from LPS, poly(I:C) and Aeromonas hydrophila challenges. Synthetic mature peptides of LEAP2AB displayed a more potent antimicrobial activity than did LEAP2C. Data from this study could be useful not only to provide deeper insights into the evolutionary mechanism of LEAP2 in the actinopterygian lineage but also to better understand the innate immunity of this commercially important chondrostean species.

Cloning, characterization and tissue-specific expression of the antimicrobial peptide hepcidin from caspian trout (Salmo caspius) and the antibacterial activity of the synthetic peptide.

Antimicrobial peptides have a wide range of antimicrobial activity and widely occur in different organisms including mollusks, crustaceans and vertebrates. Hepcidins are a group of cysteine-rich antimicrobial peptides that are active against a variety of pathogens including gram-positive and gram-negative bacteria, as well as viruses. In this study, the hepcidin gene of Caspian trout (CtHep) was identified and characterized. Our results showed that CtHep cDNA has a 267-bp Open Reading Frame (ORF), which is translated to 88 amino acids. The CtHep was classified in the HAMP1 class of hepcidins. Comparison of DNA and cDNA sequences showed that CtHep has 3 exons and 2 introns. The signal, prodomain and mature part of CtHep have 24, 39 and 25 amino acids, respectively. The mature peptide has a molecular weight of 2881.43 Da and a theoretical isoelectric point of 8.53. The expression of CtHep mRNA was detected in different tissues of healthy and infected fish. CtHep expression in the liver, head kidney, spleen and skin was significantly enhanced after bacterial challenge. Expression of CtHep in different embryonic development stages was also substantial. Antibacterial activity of synthetic CtHep peptides was investigated against a number of Gram-positive and Gram-negative bacteria. CtHep inhibited some pathogenic bacteria such as Streptococcus iniae and Aeromonas hydrophila. In the in vivo experiment, CtHep upregulated the cytokines IL-6 and TNF-α in both kidney and spleen tissues after 24 h of the peptide injection. In conclusion, our study showed that CtHep plays an important role in the immune system of Caspian trout and also in the embryonic stages. Moreover, CtHep peptide has a potential to be used as an antimicrobial therapeutic agent as well as an immunostimulant in aquaculture.

Hepcidin cDNA evolution in vertebrates.

Hepcidin, belonging to the ß-defensin family, was isolated for the first time from plasma and human urine. It is a cationic peptide, rich in cysteine bound with four disulfide bridges, which plays a major role in innate immunity and iron homeostasis. Some vertebrate species have multiple hepcidin homolog genes and each contains only one copy that functions as an iron regulator except hepcidin sequences in the pigeon (Columba livia). The aim of this chapter is to investigate the molecular evolution of several hepcidin gene from searches of the literature and public genomic databases from 17 different species, all among the vertebrates.

Identification and expression of the hepcidin gene from brown trout (Salmo trutta) and functional analysis of its synthetic peptide.

Hepcidin, a hepatic antimicrobial peptide, is a key player of the nonspecific immune system. The structure of hepcidin gene from brown trout (Bthepc) has been characterized at the molecular level. The 1158-bp mRNA generates a coding sequence (CDS) of 267 bp, which encodes an 88-amino acid protein. Molecular evolution analysis classified Bthepc to the family Salmonidae. Amino acid sequence homologies between Bthepc and hepcidin in other species such as Oncorhynchus mykiss, Salmo salar, and Hucho taimen were found to be 93.18%, 96.59%, and 92.05% respectively. The mature peptide and the signal peptide of Bthepc are made of 25 and 24 amino acids, respectively. Similar to the other species, eight conserved cysteines in the mature peptide of Bthepc are held together by four disulphide bonds. Expression profiling of Bthepc indicated its highest expression in the liver. Further, iron levels or inflammation did not induce the age-dependent expression of Bthepc. Bthepc mRNA expression analysis in six immune tissues (liver, gill, spleen, skin, head kidney and intestine) indicated different levels of increase when challenged with Aeromonas salmonicida and Aeromonas hydrophila. The antimicrobial activity of synthetic Bthepc to typical pathogens was verified in vitro. In addition, Bthepc showed moderate haemolytic activity to mammalian erythrocytes. The antimicrobial activity of Bthepc was attributed to the disruption of the bacterial outer membrane integrity, which was evident from our scanning electron microscopy results. In summary, hepcidin gene of brown trout was characterized, and its antimicrobial activity was verified on different levels.

Host defense peptide LEAP-2 contributes to monocyte/macrophage polarization in barbel steed (Hemibarbus labeo).

The liver-expressed antimicrobial peptide 2 (LEAP-2) plays a vital role in host immunity against pathogenic organisms. In the present study, cDNA of the LEAP-2 gene was cloned and sequenced from the barbel steed (Hemibarbus labeo). The predicted amino acid sequence of the barbel steed LEAP-2 comprises a signal peptide and a prodomain, which is followed by the mature peptide. Sequence analysis revealed that barbel steed LEAP-2 belongs to the fish LEAP-2A cluster and that it is closely related to zebrafish LEAP-2A. We found that barbel steed LEAP-2 transcripts were expressed in a wide range of tissues, with the highest mRNA levels detected in the liver. In response to lipopolysaccharide (LPS) treatment, LEAP-2 was significantly upregulated in the liver, head kidney, spleen, gill, and mid intestine. A chemically synthesized LEAP-2 mature peptide exhibited selective antimicrobial activity against several bacteria in vitro. Moreover, LEAP-2, alone or in combination with LPS or phorbol 12-myristate 13-acetate, strongly induced a pro-inflammatory reaction in barbel steed monocytes/macrophages (MO/MФ), involving the induction of iNOS activity, respiratory burst, and the pro-inflammatory cytokines IFN-γ, TNF-α, and IL-1ß. Collectively, the results of this study indicate the importance of fish LEAP-2 in the M1-type polarization of MO/MΦ.