Bass hepcidin synthesis, solution structure, antimicrobial activities and synergism, and in vivo hepatic response to bacterial infections

Bass hepcidin was purified from the gill of hybrid striped bass (Morone chrysops x Morone saxatilis) based on antimicrobial activity against Escherichia coli. This 21-amino acid peptide has 8 cysteines engaged in 4 disulfide bonds and is very similar to human hepcidin, an antimicrobial peptide with iron regulatory properties. To gain insight into potential role(s) of bass hepcidin in innate immunity in fish, we synthesized the peptide, characterized its antimicrobial activities in vitro, determined its solution structure by NMR, and quantified hepatic gene expression in vivo following infection of bass with the fish pathogens, Streptococcus iniae or Aeromonas salmonicida. Its structure is very similar to that of human hepcidin, including the presence of an antiparallel beta-sheet, a conserved disulfide-bonding pattern, and a rare vicinal disulfide bond. Synthetic bass hepcidin was active in vitro against Gram-negative pathogens and fungi but showed no activity against key Gram-positive pathogens and a single yeast strain tested. Hepcidin was non-hemolytic at microbicidal concentrations and had lower specific activity than moronecidin, a broad spectrum, amphipathic, alpha-helical, antimicrobial peptide constitutively expressed in bass gill tissue. Good synergism between the bacterial killing activities of hepcidin and moronecidin was observed in vitro. Hepcidin gene expression in bass liver increased significantly within hours of infection with Gram-positive (S. iniae) or Gram-negative (A. salmonicida) pathogens and was 4-5 orders of magnitude above base-line 24-48 h post-infection. Our results suggest that hepcidin plays a key role in the antimicrobial defenses of bass and that its functions are potentially conserved between fish and human.     

Piscidin 4, a novel member of the piscidin family of antimicrobial peptides

Piscidins are linear, amphipathic, antimicrobial peptides (AMPs) with broad, potent, activity spectrum. Piscidins and other members of the piscidin family appear to comprise the most common group of AMPs in teleost fish. All piscidins and related members of the piscidin family described to date are 18–26 amino acids long. We report here the isolation of a novel 5329.25 Da, 44-residue (FFRHLFRGAKAIFRGARQGXRAHKVVSRYRNRDVPETDNNQEEP) antimicrobial peptide from hybrid striped bass (Morone chrysops female x M. saxatilis male). We have named this peptide “piscidin 4” since it has considerable (to > 65%) N-terminal sequence homology to piscidins 1–3 and this distinctive, 10 to 11-residue, N-terminus is characteristic of piscidins. The native peptide has a modified amino acid at position 20 that, based upon mass spectrometry data, is probably a hydroxylated tryptophan. Synthetic piscidin 4 (with an unmodified tryptophan at position 20) has similar antibacterial activity to that of the native peptide. Piscidin 4 demonstrates potent, broad-spectrum, antibacterial activity against a number of fish and human pathogens, including multi-drug resistant bacteria. Its potent antimicrobial activity suggests that piscidin 4 plays a significant role in the innate defense system of hybrid striped bass.     

Structure-activity relationships of piscidin 4, a piscine antimicrobial peptide

Piscidin 4, an antimicrobial peptide recently isolated from mast cells of hybrid striped bass (Morone chrysops female × Morone saxatilis male), is unusual in that it is twice as long (44 amino acids) as the typical members of the piscidin family. We previously showed that native piscidin 4 had a modified amino acid at position 20, but synthetic piscidin 4 (having an unmodified Trp at position 20) had similar potent activity against a number of both human and fish bacterial pathogens. In this study, the structure and membrane topology of synthetic piscidin 4 were examined using liposomes as model bilayers. Circular dichroism analyses revealed that it had a disordered structure in aqueous solution and folded to form a relatively weak α-helical structure in both membrane-mimetic trifluoroethanol solutions and liposome suspensions. Fluorescence data (piscidin 4 embedded in liposomes) and leakage experiments indicated that piscidin 4 interacted strongly with the hydrophobic part of the liposome. Binding of piscidin 4 to liposomes induced significant blue shifts of the emission spectra of the single Trp residue (Trp20). Quenching of Trp20 by water-soluble quencher (either acrylamide or I-) indicated that the fluorescence of Trp20 decreased more in the presence of liposomes than in buffer solution, thus revealing that Trp20 is less accessible to the quenchers in the presence of liposomes. The relative leakage abilities of piscidin 4 (1 μM) with liposomes were in the following order: DPPC (100%)≥EYPC (94%)>DPPC/DPPG (65%)>EYPC/EYPG (0%). This high activity against DPPC and EYPC liposomes was contrary to our data suggesting that piscidin 4 has a much weaker tendency to form an α-helix than other piscidins, such as piscidin 1. However, the structural similarity of protozoan membranes to EYPC liposomes might explain our discovery of the potent activity of piscidin 4 against the important skin/gill parasite ich (Ichthyophthirius multifiliis), but its negligible hemolytic activity against vertebrate membranes (hybrid striped bass or human erythrocytes). It also suggests that other conformation(s) in addition to the α-helix of this peptide may be responsible for its selective activity. This differential toxicity also suggests that piscidin 4 plays a significant role in the innate defense system of hybrid striped bass and may be capable of functioning extracellularly.     

Isolation and characterization of 149 novel microsatellite DNA markers for striped bass,Morone saxatilis,and cross‐species amplification in white bass,Morone chrysops,and their hybrid

To support detailed genetic analysis of striped bass (Morone saxatilis) and white bass (Morone chrysops), we isolated 153 microsatellite loci from repeat‐enriched striped bass DNA libraries. Of these, 147 markers amplified in striped bass (average 4.7 alleles per locus) and 133 in white bass (average 2.2 alleles per locus). One hundred twenty‐two markers amplified in their hybrid. Development of new microsatellite markers will facilitate evaluations of genetic structure in wild populations and will support pedigree analysis and linkage mapping for selective breeding.     

cDNA sequence and tissue expression of an antimicrobial peptide, dicentracin; a new component of the moronecidin family isolated from head kidney leukocytes of sea bass, Dicentrarchus labrax

A 483-bp cDNA was isolated from sea bass (Dicentrarchus labrax) head kidney leukocytes, dicentracin, using PCR primers designed from conserved moronecidin domains. Gene bank analysis revealed that dicentracin cDNA belongs to the moronecidin family. As deduced from alignment with Morone chrysops moronecidin, the precursor of 79 aa appeared to be composed of a signal peptide of 22 aa, followed by the mature AMP (antimicrobial peptide) of 22 aa named dicentracin, and a C-terminal extension of 35 aa. Dicentracin precursor displayed 3 aa substitutions with other moronecidin sequence but none in the mature peptide sequence. Using in situ hybridization assay, dicentracin gene expression was observed in 68–71% of peripheral blood leukocytes, kidney leukocytes or peritoneal cavity leukocytes without significant statistical differences. Dicentracin mRNA was observed in most of the granulocytes, as well as in monocytes from both peripheral blood and head kidney, and in macrophages from peritoneal cavity. No expression was observed in thrombocytes or in lymphocytes.     

Evidence for widespread distribution of piscidin antimicrobial peptides in teleost fish

Antimicrobial peptides (AMPs) are increasingly recognized as a critical component of the host's defense against infection. Several types of AMPs have been recently identified from mucosal tissues or immune cells of a number of teleosts. Among these are the piscidins, which are 22 residue, alpha-helical AMPs that were originally isolated from mast cells of hybrid striped bass Morone saxatilis male x Morone chrysops female. Using an antibody specific for the conserved N-terminal amino acid sequence of piscidin 1, we used immunohistochemistry to probe skin, gill, and gastrointestinal tract of 39 teleosts representing 7 different orders. Nine fish species were piscidin-positive, with all of these species being in the Perciformes, the largest and most evolutionarily advanced order of teleosts. Piscidin-positive cells were identified in species belonging to the families Moronidae, Serranidae, Sciaenidae, Siganidae and Belontidae. Immunopositive cells were usually most consistent with mast cells, although in some species, the granule appearance and tinctorial properties diverged somewhat from those of a typical piscine mast cell. In addition, rodlet cells were piscidin-positive in one member of the family Cichlidae; to our knowledge, it is the first time that a host-associated chemical biomarker has been identified in rodlet cells. Our data suggest that piscidins are present in many evolutionarily advanced teleosts. Piscidin-immunoreactive cells were most common at sites of pathogen entry, including the skin, gill and gastrointestinal tract. These results strongly suggest that piscidins are a widespread and important component of many fishes' defense against disease.     

Responses of hybrid striped bass to waterborne and dietary copper in freshwater and saltwater

Mechanisms of copper toxicity and consequences of exposure vary due to uptake route and ionoregulatory status. The goal of this research was to develop a model fish system to assess the influence of different Cu exposure routes (waterborne or dietary) on bioavailability, uptake, and effects in hybrid striped bass (Morone chrysops x Morone saxatilis) acclimated to fresh- or saltwater. Initially, hybrid striped bass were exposed to dietary Cu concentrations of 571, 785, and 1013 mug Cu/g, along with a control (approximately 5 microg Cu/g), for 14 days in saltwater. Intestinal and liver Cu accumulated in a dose-dependent manner in fish exposed to increasing levels of dietary Cu. Chronic (42 days) experiments were then conducted to determine sub-lethal effects of aqueous, dietary, and combined aqueous and dietary Cu exposures to both freshwater- and saltwater-acclimated hybrid striped bass. Growth and Cu accumulation in the gill, intestine, and liver were measured. Although no significant effects were observed in fish exposed to waterborne Cu, those exposed through the diet accumulated significant liver and intestinal Cu but showed no significant change in growth. Overall, these results suggest that at the levels tested, exposure to elevated waterborne Cu did not cause significant long-term tissue Cu accumulation, whereas dietary Cu exposure caused significant liver and intestinal Cu accumulation in hybrid striped bass which was comparable in both freshwater and saltwater (15 g/L).     

Evolution of an MHC class Ia gene fragment in four North American Morone species

A nucleotide sequence analysis of a fragment of a Morone MHC class Ia gene detected high levels of polymorphism in striped bass Morone saxatilis, white perch Morone americana and yellow bass Morone mississippiensis. Extremely low levels of MHC diversity, however, were detected in white bass Morone chrysops, suggesting the possibility of a severe population bottleneck for this species.     

Use of cellular oncogene probes to identify Morone hybrids.

We tested the ability of cellular oncogene (c-onc) probes to identify F1 hybrids and the lineage of known backcrosses within the fish genus Morone. Total DNA was isolated from five to 14 individuals per North American Morone species (striped bass, white bass, white perch, and yellow bass). The DNA was digested with two restriction enzymes, Eco RI and Hin dIII, Southern blotted, and hybridized to six different c-onc probes including v-abl, v-erb B, c-myc, c-H-ras, c-K-ras, and v-src. We found fixed genotypic differences among the four species for all six probes in single restriction enzyme digests. The heritability of these nuclear DNA genotypes was evaluated in hatchery-produced F1 Morone hybrids (striped bass x white bass and striped bass x white perch) tested with the six informative single probe/restriction enzyme combinations. All F1 individuals exhibited heterozygosity in all diagnostic nuclear DNA fragments, confirming the Mendelian inheritance of these genotypes in these fish. Furthermore, analysis of these nuclear DNA genotypes in hatchery-produced backcrosses of F1 hybrids striped bass x (white bass x striped bass) detected both recombinant and parental genotypes at all six polymorphic c-onc sequences. The lineage of suspected Morone hybrids of unknown descent collected from Lewis Smith Lake, Alabama, and from the Occoquan River, Virginia, was determined using the c-onc probes. Our results suggest that c-onc probes are suitable markers to unequivocally identify F1 hybrids and backcrosses and to quantify introgression in natural populations of fishes. The addition of RFLP analysis of mtDNA provided a complete ancestral history of individual fish.     

Structure and mechanism of action of the antimicrobial peptide piscidin

Piscidin, an antibacterial peptide isolated from the mast cells of striped bass, has potent antimicrobial activity against a broad spectrum of pathogens in vitro. We investigated the mechanism of action of this 22-residue cationic peptide by carrying out structural studies and electrophysiological experiments in lipid bilayers. Circular dichroism experiments showed that piscidin was unstructured in water but had a high alpha-helix content in dodecylphosphocholine (DPC) micelles. 1H NMR data in water and TFE confirmed these results and demonstrated that the segment of residues 8-17 adopted an alpha-helical structure in a micellar environment. This molecule has a marked amphipathic character, due to well-defined hydrophobic and hydrophilic sectors. This structure is similar to those determined for other cationic peptides involved in permeabilization of the bacterial membrane. Multichannel experiments with piscidin incorporated into azolectin planar bilayers gave reproducible I-V curves at various peptide concentrations and unambiguously showed that this peptide permeabilized the membrane. This pore forming activity was confirmed by single-channel experiments, with well-defined ion channels obtained at different voltages. The characteristics of the ion channels (voltage dependence, only one or two states of conductance) clearly suggest that piscidin is more likely to permeabilize the membrane by toroidal pore formation rather than via the "barrel-stave" mechanism.     

Host site of activity and cytological effects of histone-like proteins on the parasitic dinoflagellate Amyloodinium ocellatum

Histone-like proteins (HLPs) are broad-spectrum, endogenously produced antibiotics which we have isolated from tissues of rainbow trout Oncorhynchus mykiss and hybrid striped bass (Morone saxatilis male x M. chrysops female). Here, we show that HLP-1, which has high sequence homology to histone H2B, equally inhibited both young and mature trophonts of the important ectoparasite Amyloodinium ocellatum. In addition to direct killing of Amyloodinium trophonts, there was evidence that HLP-1 from both rainbow trout and hybrid striped bass caused severe developmental abnormalities, including delayed development, in both the parasitic trophont stage as well as the reproductive tomont stage. The deleterious effects of HLP-1 also were manifested in what appeared to be 'delayed mortality', where parasites of normal appearance would die later in development. Similar serious damage was also seen with calf histone H2B and the unrelated peptide antibiotic magainin 2. A comparison of the antibiotic activity in mucus versus epidermis compartments of the skin of hybrid striped bass suggested that the majority of antibiotic (including HLP-1) activity resided in the epidermis, although some activity was present in the mucus. These data suggest that normal, nonimmune fish skin contains potent defenses against protozoan ectoparasites and that the effects of these defenses may extend beyond their transient interactions with the parasites, which has important implications for this host-parasite relationship.     

SNP panel development for genetic management of wild and domesticated white bass (Morone chrysops)

White bass (Morone chrysops), striped bass and their interspecific hybrid are important game fishes, whereas the hybrid striped bass is an important aquaculture species in the US. Numerous state, federal and private hatcheries, therefore, rear these species for stocking purposes as well as for food fish. Although striped bass populations (both wild and domesticated) have been extensively evaluated, relatively little effort has been directed toward the study and improvement of white bass. In this study, we developed SNP resources to examine the genetic relationships among a long‐term domesticated white bass line and five potential founder stocks for selective breeding collected from drainages in Arkansas, Texas and Alabama. Using genotyping‐by‐sequencing, we generated 13 872 genome‐wide SNP loci across the six populations. Stringent filtering of SNP‐calling parameters identified 426 informative SNP loci. Population genetic and structure analyses using these loci revealed only moderate genetic differentiation between populations (global F_st = 0.083) and indicated two major genetic clusters. A final 57‐SNP assay was successfully designed and validated using the MassARRAY system. The developed SNP panel assigned 96 additional genotyped individuals to their population of origin with 100% accuracy. The SNP resources developed in this study should facilitate ongoing efforts in selective breeding and conservation of white bass.     

Piscidin-1, an antimicrobial peptide from fish (hybrid striped bass morone saxatilis x M. chrysops), induces apoptotic and necrotic activity in HT1080 cells

Piscidin-1, a 22-residue cationic peptide isolated from mast cells of a hybrid striped bass, has potent antimicrobial activities against both gram-positive and -negative bacteria. To date, there is no report of its antitumor activity on any tumor cell lines. In this study, we examined the antitumor activity of a synthetic piscidin-1 peptide against several human cancer cell lines using an MTS assay and soft-agar colony-formation assay. We found that a low dose of piscidin induces both apoptosis and necrosis in HT1080 cells, as shown by annexin-V/propidium iodide and acridine orange/ethidium bromide staining, and triggers a necrotic cell death pathway in a short period with high-dose treatment. The destruction of cell membranes by piscidin-1 was demonstrated by transmission electron microscopy. Furthermore, piscidin-1 also inhibits the migration of HT1080 cells in a dose-dependent manner. This study provides the first evidence of the anticancer activity of the antimicrobial peptide, piscidin-1, with potential implications for the treatment of cancer.     

Expression and purification of antimicrobial peptide adenoregulin with C-amidated terminus in Escherichia coli

Adenoregulin is a 33 amino acid antimicrobial peptide isolated from the skin of the arboreal frog Phyllomedusa bicolor. Natural adenoregulin is synthesized with an amidated valine residue at C-terminus and shows lethal effects against filamentous fungi, as well as a broad spectrum of pathogenic microorganisms. A synthetic gene for adenoregulin (ADR) with an additional amino acid glutamine at C-terminus was cloned into pET32a vector to allow expression of ADR as a Trx fusion protein in Escherichia coli BL21(DE3). The resulting expression level of the fusion protein could reach up to 20% of the total cell proteins. The fusion protein could be purified effectively by Ni2+-chelating chromatography. Released from the fusion protein by enterokinase cleavage and purified to homogeneity, the recombinant ADR displayed antimicrobial activity similar to that of the synthetic ADR reported earlier. Comparing the antimicrobial activities of the recombinant adenoregulin with C-amidated terminus to that without an amidated C-terminus, we found that the amide of glutamine at C-terminus of ADR improved its potency on certain microorganisms such as Tritirachium album and Saccharomyces cerevisiae.     

Apolipoprotein A-I from striped bass (Morone saxatilis) demonstrates antibacterial activity in vitro

HDL and apolipoprotein A-I from teleostean fishes demonstrate in vitro activity against gram-positive and gram-negative bacteria. In this study, we purified ApoA-1 from striped bass (Morone saxatilis) plasma and examined its in vitro antibacterial activity against Streptococcus sp., Escherichia coli, and Mycobacterium marinum. In addition, we obtained sequence for a putative striped bass ApoA-1 gene, which when translated contained the identical sequence generated from N-terminal sequencing of the purified ApoA-1. The predicted secondary and tertiary structures contained the characteristic proline residues and high alpha-helical content conserved between mammals and fishes. Purified ApoA-1 exhibited antibacterial activity against the bacteria assayed. Concentrations of 125 microg/mL for E. coli, 250 microg/mL for Streptococcus sp., and 250 microg/mL for M. marinum, inhibited bacterial growth by 50% compared to control. ApoA-1 plasma concentrations in experimental and wild fish ranged from undetectable levels to greater than 5 mg/mL, indicating that striped bass ApoA-1 is an effective antibacterial agent at concentrations below the range of physiological concentrations in striped bass plasma. We therefore conclude that ApoA-1 could play a role in innate defense against bacterial pathogens in striped bass.     

Viral encephalopathy and retinopathy outbreak in freshwater fish farmed in Italy

Viral encephalopathy and retinopathy (VER), otherwise known as viral nervous necrosis (VNN), is a neuropathological condition affecting > 40 species of fish. Although VER affects mainly marine fish, the disease has also been detected in certain species reared in freshwater environments. There are relatively few reports concerning the disease in freshwater species, and there is not much information on clinical signs. Nevertheless, the most common clinical findings reported from affected freshwater species are consistent with the typical signs observed in marine species. In this paper we describe the main clinical signs and the laboratory results associated with the detection of a betanodavirus in hybrid striped bass x white bass (Morone saxatilis x Morone chrysops) and largemouth bass Micropterus salmoides, reared in a freshwater environment. We also detected the virus by real-time PCR and isolated it in cell culture from a batch of pike-perch Sander lucioperca farmed in the same system.