Abstract: Crustins are antibacterial proteins of ca. 7-14 kDa with a characteristic four-disulphide core-containing whey acidic protein (WAP) domain, expressed by the circulating haemocytes of crustaceans. Over 50 crustin sequences have been now reported from a variety of decapods, including crabs, lobsters, shrimp and crayfish. Three main types seem to occur but all possess a signal sequence at the amino terminus and a WAP domain at the carboxyl end. Differences between types lie in the structure of the central region. Those crustins purified as the native protein or expressed recombinantly all kill Gram-positive bacteria, and gene studies have shown that they are constitutively expressed, often at high levels, but show no consistent patterns of change in expression following injection of bacteria. This variable response to infection is enigmatic but indicates that these proteins could perform additional functions, perhaps as immune regulators in recovery from wounding, trauma or physiological stress.
Abstract: The cee (conserved edge expressed protein) gene was recently identified in a genome-wide screen to discover genes associated with myotube formation in fast muscle of pufferfish. Comparative genomic analyses indicate that cee arose some 1.6-1.8 billion years ago and is found as a single-copy gene in most eukaryotic genomes examined. The complexity of its structure varies from an intronless gene in yeast and tunicates to nine exons and eight introns in vertebrates. cee is particularly conserved among vertebrates and is located in a syntenic region within tetrapods and between teleosts and invertebrates. Low dN/dS ratios in the cee coding region (0.02-0.09) indicate that the Cee protein is under strong purifying selection. In Atlantic salmon, cee is expressed in the superficial layers of developing organs and tissues. These data, together with functional screens in yeast and Caenorhabditis elegans, indicate that cee has a hitherto uncharacterized role in normal growth and development.
Abstract: Gene expression studies are fundamental to understand the molecular basis of severe malformations in fish development, particularly under aquaculture conditions. Real-time PCR (qPCR) is the most accurate method of quantifying gene expression, provided that suitable endogenous controls are used to normalize the data. To date, no reference genes have been validated for developmental gene expression studies in Atlantic halibut (Hippoglossus hippoglossus). We have determined the expression profiles of 6 candidate reference genes (Actb, Eef2, Fau, Gapdh, Tubb2 and 18S rRNA) in 6 embryonic and 5 larval stages of Atlantic halibut development. There were significant changes in expression levels throughout development, which stress the importance and complexity of finding appropriate reference genes. The three software applications (BestKeeper, geNorm and NormFinder) used to evaluate the stability of potential reference genes produced comparable results. Tubb2 and Actb were the most stable genes across the different developmental stages, whereas 18S rRNA and Gapdh were the most variable genes and thus inappropriate to use as reference genes. According to geNorm and NormFinder, the best two-gene normalization factors corresponded to the geometric average of Tubb2/Actb and Tbb2/Fau, respectively. We believe that either of these normalization factors can be used for future developmental gene expression studies in Atlantic halibut.
Abstract: FoxK1 is a member of the highly conserved forkhead/winged helix (Fox) family of transcription factors and it is known to play a key role in mammalian muscle development and myogenic stem cell function. The tiger pufferfish (Takifugu rubripes) orthologue of mammalian FoxK1 (TFoxK1) has seven exons and is located in a region of conserved synteny between pufferfish and mouse. TFoxK1 is expressed as three alternative transcripts: TFoxK1-alpha, TFoxK1-gamma and TFoxK1-delta. TFoxK1-alpha is the orthologue of mouse FoxK1-alpha, coding for a putative protein of 558 residues that contains the forkhead and forkhead-associated domains typical of Fox proteins and shares 53% global identity with its mammalian homologue. TFoxK1-gamma and TFoxK1-delta arise from intron retention events and these transcripts translate into the same 344-amino acid protein with a truncated forkhead domain. Neither are orthologues of mouse FoxK1-beta. In adult fish, the TFoxK1 splice variants were differentially expressed between fast and slow myotomal muscle, as well as other tissues, and the FoxK1-alpha protein was expressed in myogenic progenitor cells of fast myotomal muscle. During embryonic development, TFoxK1 was transiently expressed in the developing somites, heart, brain and eye. The relative expression of TFoxK1-alpha and the other two alternative transcripts varied with the incubation temperature regime for equivalent embryonic stages and the differences were particularly marked at later developmental stages. The developmental expression pattern of TFoxK1 and its localisation to mononuclear myogenic progenitor cells in adult fast muscle indicate that it may play an essential role in myogenesis in T. rubripes.
Abstract: Muscleblind-like (Mbnl) proteins are required for terminal muscle differentiation in mammals. In this study we have identified two mbnl paralogues from the tiger pufferfish, tmbnl2a and tmbnl3, which are the first examples of non-mammalian mbnl genes. Tmbnl2a and tmbnl3 were found in regions of conserved synteny and had a high degree of global conservation with their mammalian homologues. Phylogenetic analysis showed that the T. rubripes genome contains one mbnl3 gene and two copies of mbnl1 and mbnl2. Moreover, the mbnl1 and mbnl3 paralogues are derived from duplication of a common ancestral gene. The average rates of synonymous substitutions between T. rubripes, mouse and human mbnl2 and mbnl3 genes were much higher than the corresponding rates of non-synonymous mutations, suggesting that Mbnl2 and Mbnl3 are subjected to strong purifying selection. Quantitation of tmbnl2a and tmbnl3 transcripts by real-time PCR revealed that these two paralogues are differentially expressed in fast and slow myotomal muscle, heart, liver, skin, brain and testes. Tmbnl2a was expressed at similar levels in all tissues examined, as was the mouse orthologue. Tmbnl3 was expressed at higher levels than tmbnl2a, with a ubiquitous tissue distribution. Expression of tmbnl3 remained high in adult pufferfish muscle whereas the mouse orthologue was down-regulated in adults, perhaps reflecting the indeterminate and determinate growth patterns of these taxa, respectively.
Abstract: cDNA libraries were constructed from the following developmental stages (tissues) of the Atlantic halibut (Hippoglossus hippoglossus): 2-cell stage (embryos), 1 day-old yolk sac larvae (trunk) and juvenile (fast skeletal muscle). A total of 4249 high quality expressed sequence tags from the three libraries were clustered into a partial transcriptome of 2124 putative genes. A large proportion of the gene clusters (48.3%) had no significant matches against known proteins. The most abundant ESTs of nuclear transcripts in the 2-cell library included sequences with high identity to zebrafish H1M, a linker histone-like protein involved in primordial germ cell specification, zinc finger protein, rRNA external transcribed spacer, thymosin beta-4, cyclin B1 and several predicted peptides from the Tetraodon nigroviridis genome assembly with unknown functions. 170 and 123 ESTs represented ribosomal proteins in the larval and juvenile libraries respectively, compared with only two sequences in the 2-cell library, which may reflect an abundance of maternally inherited pre-formed ribosomes in the yolk. Even though some clusters were common to all three libraries, most putative genes showed a developmental-stage specific distribution with 72% (2-cell embryo), 59% (larval) and 57% (juvenile) sequences having no significant matches against the 8400 adult halibut sequences in the EMBL nucleotide database. Comparison between the predicted halibut peptide data set and the human, zebrafish, and pufferfishes (T. nigroviridis and Takifugu rubripes) proteomes revealed that, as expected, the halibut sequences were more similar to the other two fish species than to human proteins. However, no clear bias towards the pufferfishes was observed, suggesting significant sequence variation between orthologues within the clade Acanthomorpha. The sequence information generated in the present study will represent a significant new resource for future studies on normal and abnormal development in Atlantic halibut.
Abstract: Splice variants of the basic helix-loop-helix myoblast determination factor (myoD) have not been previously found in vertebrates. Here we report the identification and characterization of three alternative transcripts of a myoD paralogue from the tiger pufferfish (Takifugu rubripes). The T. rubripes myoD1 gene (TmyoD1) has 3 exons and 2 introns and it is present on scaffold 104, in a region of conserved synteny with zebrafish. The isoform TMyoD1-alpha is a putative protein of 281 residues that contains the basic, helix-loop-helix and helix III domains and shares 61%, 56%, 51%, 49% and 56% overall identity with zebrafish, Xenopus, mouse, human and chicken MyoD1, respectively. TMyoD1-beta arises from an alternative 3' splice site and differs from TMyoD1-alpha by a 26-residue insertion adjacent to helix III, which is one of the functional domains required for chromatin remodelling. The third alternative transcript, TmyoD1-gamma, retains intron I and has two premature termination codons far from the 3'-most exon-exon junction. TmyoD1-gamma is therefore likely to be degraded by nonsense-mediated decay, an important widespread post-transcriptional mechanism that regulates transcript levels. Analysis of gene expression by qPCR revealed that TmyoD1-alpha was the most abundant transcript in fast and slow myotomal muscle. TmyoD1-alpha expression was 2-fold higher in fast muscle of juvenile fish that were actively producing new myotubes compared to adult stages that had stopped recruiting fast muscle fibres. A similar expression pattern was observed for TmyoD1-alpha in slow muscle but the differences were not significant. Transcript levels of TmyoD1-gamma only varied significantly in fast muscle and were 5-fold higher in adult compared to juvenile stages. Significant differences in expression of TmyoD1 splice variants were also observed during embryonic development. The differential expression of three alternative transcripts of myoD1 in developing and adult myotomal muscle of T. rubripes supports the hypothesis that diversity generated by alternative splicing may be of functional significance in muscle development in this species.
Abstract: Little is known about the transcriptional networks that regulate myotube production in vertebrates. In the present study, we have used a genomic approach to discover novel genes associated with myotube formation in fast muscle of the tiger puffer fish, Takifugu rubripes. The number of fast muscle fibers per myotome increased until 1.2 kg body mass, and subsequent growth was by fiber hypertrophy alone. Forward and reverse subtracted cDNA libraries were prepared from a 180-g (myotube +) and a 3.4-kg (myotube -) fish, and 1,452 expressed sequence tags (ESTs) were obtained. After these ESTs were grouped into nonredundant clusters and housekeeping and structural genes were eliminated, 57 genes were selected and quantitative PCR was used to investigate their expression levels in different tissues from independent groups of myotube(-) and myotube(+) fish acclimated to the same environmental conditions and diet. Eleven novel genes were found to be consistently differentially expressed, but only four showed appropriate tissue-specific expression. These four genes were upregulated 5-25 times in fast muscle of myotube(-) relative to myotube(+) growth stages, while their expression remained unchanged in the other tissues studied. The novel genes identified, which are also present in other vertebrate genomes, may play a role in inhibiting myotube formation in vertebrate muscle.
Abstract: A potent antimicrobial peptide, tentatively named oncorhyncin II, was isolated from an acid extract of rainbow trout skin secretions. Amino acid sequencing showed that the first 17 residues of oncorhyncin II are identical to residues 138-154 of histone H1 from rainbow trout. Matrix-assisted laser desorption ionization mass spectrometry revealed that the purified peptide has a molecular mass of 7195.3Da. Taken together, these data indicate that oncorhyncin II is a 69-residue C-terminal fragment of histone H1, probably phosphorylated at two residues. Oncorhyncin II has minimal inhibitory concentrations in the submicromolar range against Gram-(+) as well as Gram-(-) bacteria and it does not display significant haemolytic activity towards trout erythrocytes. The purified peptide was found to induce a marked destabilisation of planar lipid bilayers without the formation of stable ion channels. Oncorhyncin II is possibly a cleavage product of histone H1 with a potentially important role in mucosal defence of rainbow trout.
Abstract: Two novel antibacterial muramidases were purified to homogeneity from skin exudates of rainbow trout (Oncorhynchus mykiss). Unusually, one has an acidic isoelectric point and it is the first anionic muramidase to be reported for fish. Its molecular mass is 14,268 Da, as determined by mass spectrometry. The other muramidase is cationic with a mass of 14,252 Da. Partial N-terminal amino acid sequencing and peptide mapping strongly point to it being a c-type lysozyme, the first to be purified and characterised from skin of a salmonid. Its optimum pH ranges from 4.5 to 5.5 and its optimum temperature, at pH 5.0, is 33-49 degrees C, although it still exhibits activity at 5 degrees C. It is strongly bactericidal to the Gram-(+) bacterium Planococcus citreus, with a minimum bactericidal concentration of 100 U ml(-1), but is neither chitinolytic nor haemolytic. These two muramidases probably contribute to epithelial defence of the fish against microbes, either alone or in synergism with antibacterial peptides.
Abstract: Antimicrobial peptides are natural antibiotics known to be present in both myeloid cells and epithelial surfaces of vertebrates. Nevertheless, the reports of antimicrobial peptides isolated from blood cells of teleosts are scarce. In this paper we show that acid-soluble erythrocyte extracts from rainbow trout, Oncorhynchus mykiss, display antibacterial activity against Planococcus citreus on a radial diffusion assay. Following tC18 solid phase extraction, cationic exchange chromatography and C18 reversed phase HPLC, two groups of fractions with antibacterial properties were obtained. This antibacterial activity is thermostable and susceptible to digestion by proteinase K, thus showing that the antibacterial agents have a proteinaceous nature. The factors eluted from a C18 column with circa 33% acetonitrile are active against P. citreus and Escherichia coli, with minimal inhibitory concentrations in the range 7-14 microg ml(-1) and 14-28 microg ml(-1), respectively; the ones eluted with approximately 44% acetonitrile on the same column only displayed activity against P. citreus, with a minimal inhibitory concentration of 1-2 microg ml(-1). These results raise the possibility that trout erythrocytes may contain antimicrobial factors not previously considered to be part of the innate immune system.
Abstract: The partial N-terminal amino acid sequence of the antimicrobial peptide reported in the present paper has been submitted to the TrEMBL database under the accession number P83338. A 6.7 kDa antimicrobial peptide was isolated from trout skin secretions using acid extraction followed by cation-exchange chromatography, (t)C(18) solid-phase extraction, and C(18) reversed-phase HPLC. The molecular mass of this peptide, which is tentatively named oncorhyncin III, is 6671 Da, as determined by matrix-assisted laser-desorption ionization MS. N-terminal amino acid sequencing revealed that the first 13 residues of oncorhyncin III are identical with those of the non-histone chromosomal protein H6 from rainbow trout. Hence these data combined with the MS results indicate that oncorhyncin III is likely to be a cleavage product of the non-histone chromosomal protein H6 (residues 1-66) and that it probably contains two methylated residues or one double methylation. The purified peptide exhibits potent antibacterial activity against both Gram-positive and Gram-negative bacteria, with minimal inhibitory concentrations in the submicromolar range. The peptide is sensitive to NaCl, and displays no haemolytic activity towards trout erythrocytes at concentrations below 1 microM. Scanning electron microscopy revealed that oncorhyncin III does not cause direct disruption of bacterial cells. Reconstitution of the peptide in planar lipid bilayers strongly disturbs the membranes, but does not induce the formation of stable ion channels. Taken together, these results support the hypothesis that oncorhyncin III plays a role in mucosal innate host defence.
Abstract: An antimicrobial peptide was purified from skin secretions and epithelial cells of rainbow trout by cation exchange and reversed phase chromatography. Partial N-terminal amino acid sequence of the purified peptide revealed 100% identity with the first 11 residues of a 40S ribosomal peptide from medaka fish. Its molecular mass, determined by matrix-associated laser desorption/ionisation mass spectrometry, was found to be 6676.6Da. These results indicate that this antimicrobial peptide is likely to be the 40S ribosomal protein S30. It is active at submicromolar concentrations, with an effective 50% reduction concentration of 0.02-0.04 microM against Planococcus citreus. Thus, in addition to its conventional function in the cell as part of the small ribosomal subunit, this peptide may play a role in protection against intracellular or extracellular pathogens.
Abstract: Skin exudates of rainbow trout contain a potent 13.6 kDa anti-microbial protein which, from partial internal amino acid sequencing, peptide mass fingerprinting, matrix-associated laser desorption/ionization MS and amino acid analysis, seems to be histone H2A, acetylated at the N-terminus. The protein, purified to homogeneity by ion-exchange and reversed-phase chromatography, exhibits powerful anti-bacterial activity against Gram-positive bacteria, with minimal inhibitory concentrations in the submicromolar range. Kinetic analysis revealed that at a concentration of 0.3 microM all test bacteria lose viability after 30 min incubation. Weaker activity is also displayed against the yeast Saccharomyces cerevisiae. The protein is salt-sensitive and has no haemolytic activity towards trout erythrocytes at concentrations below 0.3 microM. Reconstitution of the protein in a planar lipid bilayer strongly disturbs the membrane but does not form stable ion channels, indicating that its anti-bacterial activity is probably not due to pore-forming properties. This is the first report to show that, in addition to its classical function in the cell, histone H2A has extremely strong anti-microbial properties and could therefore help contribute to protection against bacterial invasion.
Abstract: Antibacterial proteins are an important part of the innate immune system for all animals. They have been extensively studied in mammals, amphibians and invertebrates, but have received only scant attention in fish. Their expression and processing, however, provide a way of monitoring defence vigour during development or with seasonal changes in physiology. The aim of the present work was to identify and characterise antibacterial proteins in rainbow trout. In vitro analyses of extracts of the peripheral blood leucocytes, head kidney leucocytes and mucus from adult unstimulated (non-immune) fish showed marked antibacterial activity against Gram positive bacteria. Fractionation by ion exchange chromatography and RP-HPLC of head kidney extracts showed the presence of two forms of lysozyme but no constitutively expressed antimicrobial proteins of < 10 kDa. By contrast, chromatographic analyses of mucus revealed at least four antibacterial proteins. Two are conventional lysozymes, a third is an unusual lysozyme-like protein with a low isoelectric point, and the fourth is a highly hydrophobic, cationic peptide of c. 3 kDa.
Abstract: Antimicrobial peptides are potent natural antibiotics present in myeloid cells or mucosal tissues of most multicellular organisms. It is now well established that these peptides are crucial components of the innate immune system, operating as a first-line of host defence against potential pathogens, either individually or in synergism with other defence factors, such as lysozyme.
This thesis reports the purification and characterisation of 5 low molecular weight, cationic antimicrobial proteins present in skin secretions, liver or erythrocytes of rainbow trout, Oncorhynchus mykiss. One is a novel 3 kDa peptide with a predicted amphipathic α-helical structure and active against the Gram-(+) bacterium Planococcus citreus. Another is a 13.6 kDa protein, active against Gram-(+) bacteria, which was identified as being histone H2A. A third is a 6.7 kDa peptide that displays activity against P. citreus and is likely to be the 40S ribosomal protein S30. The fourth is a 7.2 kDa C-terminal fragment of histone H1, with broad-spectrum activity and the fifth is a 6.7 kDa N-terminal fragment of histone H6 that is also active against both Gram-(+) and Gram-(-) bacteria. Histone H2A and the histone H6-derived peptide are inhibited by high NaCl concentrations (maximum 3.2 % (w/v)), do not display significant haemolytic activity to trout erythrocytes and are unable to form stable pores on artificial membranes.
Additionally, skin mucus was found to contain two muramidases: a novel muramidase with an acidic isoelectric point and a c-type lysozyme that was purified and characterised. Antibacterial activity was likewise detected in liver ribosomes and in erythrocyte extracts; the antibacterial, cationic factors were partially purified.
Taken together, these data demonstrate that rainbow trout expresses a multitude of novel antimicrobial peptides and proteins that are likely to confer protection against microbial exploitation, particularly at the mucosal surfaces.
