Jorge M.O. Fernandes

Crustins: enigmatic WAP domain-containing antibacterial proteins from crustaceans

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.

Isolation and characterisation of oncorhyncin II, a histone H1-derived antimicrobial peptide from skin secretions of rainbow trout, Oncorhynchus mykiss.

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.

Anti-microbial properties of histone H2A from skin secretions of rainbow trout, Oncorhynchus mykiss

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.

Selection of suitable reference genes for real-time PCR studies of Atlantic halibut development.

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.

Proteome reference map of the skin mucus of Atlantic cod (Gadus morhua) revealing immune competent molecules

The skin mucosal proteome of Atlantic cod (Gadus morhua) was mapped using a 2D PAGE, LC-MS/MS coupled approach. Mucosal proteins from naive fish were identified primarily by similarity searches across various cod EST databases. The identified proteins were clustered into 8 groups based on gene ontology classification for biological process. Most of the proteins identified from the gel are hitherto unreported for cod. Galectin-1, mannan binding lectin (MBL), serpins, cystatin B, cyclophilin A, FK-506 binding protein, proteasome subunits (alpha-3 and -7), ubiquitin, and g-type lysozyme are considered immune competent molecules. Five of the aforementioned proteins were cloned and their tissue distribution was analysed by RT-PCR.

Oncorhyncin III: a potent antimicrobial peptide derived from the non-histone chromosomal protein H6 of rainbow trout, Oncorhynchus mykiss

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.

Dietary lipid levels have a remarkable impact on the expression of growth-related genes in Senegalese sole (Solea senegalensis Kaup)

SUMMARY In Senegalese sole (Solea senegalensis Kaup), growth is negatively correlated to dietary lipid levels. To understand the molecular basis of this effect a molecular toolbox of 12 genes, including fgf6, fst, mstn1, myf5, mrf4, myod1, myod2, myog, myHC, mylc2, igf1r and insr, was developed. The expression profiles of these genes were investigated in white muscle and liver of fish fed with three dietary lipid levels (4%, 12% and 20%). The expression of igf-I and igf-II was also examined. MRFs and myosins were only expressed in the muscle and, except for myf5, the general trend was a decrease in expression with an increase in dietary lipids. Fgf6 was identified for the first time in liver and its expression augmented in hepatic tissues with increasing dietary lipid levels. A similar tendency was observed for mstn1 and igf-I. The opposite was observed for igf1r expression in muscle and liver. Myog, mrf4, mylc2 and igf1r were highly correlated with growth and nutrient utilisation indices. In addition to its practical implications, this work provides a valuable contribution towards our understanding of the genetic networks controlling growth in teleosts.

Application of antimicrobial polypeptide host defenses to aquaculture: Exploitation of downregulation and upregulation responses.

Antimicrobial polypeptides (AMPPs), consisting of peptides and small proteins with antimicrobial activity, are an integral component of innate immunity. Their often potent properties and widespread prevalence in fish suggests that designing means of manipulating their levels has considerable potential for maintaining or improving fish health. There is evidence that a number of chronic stresses lead to significant downregulation of AMPPs and thus their monitoring could be a highly sensitive measure of health status and risk of an infectious disease outbreak. Conversely, upregulation of AMPP expression could be used to enhance disease resistance in stressful environments, as well as improve the efficacy of traditional antimicrobial drugs. However, further work is required in linking levels of a number of AMPPs to physiological function since, while a number of studies have documented the down- or upregulation of AMPPs via gene expression, relatively few studies have quantitatively examined changes in protein expression. In addition, not all AMPPs appear to be expressed at microbicidal levels in vivo, suggesting that at least some may have functions other than being directly protective. Nonetheless, in fish, there is evidence that some constitutively expressed AMPPs, such as piscidins and histone-like proteins, are expressed at microbicidal levels and that they decline with stress. Furthermore, certain AMPPs derived from hemoglobin-β are upregulated to microbicidal levels after experimental challenge. The likely widespread distribution of these three AMPP groups in fish provides the opportunity to design strategies to greatly improve the health of cultured fish populations.

Atlantic Cod Piscidin and Its Diversification through Positive Selection

Piscidins constitute a family of cationic antimicrobial peptides that are thought to play an important role in the innate immune response of teleosts. On the one hand they show a remarkable diversity, which indicates that they are shaped by positive selection, but on the other hand they are ancient and have specific targets, suggesting that they are constrained by purifying selection. Until now piscidins had only been found in fish species from the superorder Acanthopterygii but we have recently identified a piscidin gene in Atlantic cod (Gadus morhua), thus showing that these antimicrobial peptides are not restricted to evolutionarily modern teleosts. Nucleotide diversity was much higher in the regions of the piscidin gene that code for the mature peptide and its pro domain than in the signal peptide. Maximum likelihood analyses with different evolution models revealed that the piscidin gene is under positive selection. Charge or hydrophobicity-changing amino acid substitutions observed in positively selected sites within the mature peptide influence its amphipathic structure and can have a marked effect on its function. This diversification might be associated with adaptation to new habitats or rapidly evolving pathogens.

Molecular evolution of zebrafish dnmt3 genes and thermal plasticity of their expression during embryonic development

DNA reprogramming by DNA (cytosine-5)-methyltransferases (dnmts) after fertilisation is a dynamic mechanism that is essential for early development. Amongst the three types of dnmt genes in vertebrates, dnmt3 is the one involved in de novo methylation and comprises three related genes, termed dnmt3a, dnmt3b and dnmt3L in mammals. Zebrafish (Danio rerio) has six dnmt3 paralogues, which have hitherto been termed dnmt3 to dnmt8. Bayesian inference of phylogeny and synteny analysis revealed that dnmt6 and dnmt8 are in fact duplicated dnmt3a genes, whereas the other paralogues are closely related to dnmt3b. Hence, we propose a revised nomenclature that more accurately reflects the relationship amongst zebrafish dnmt3 genes. Both dnmt3a genes were ubiquitously expressed in adult tissues, whilst the various dnmt3b paralogues were differentially expressed, with notably high expression levels in the gonads. The influence of embryonic temperature on dnmt3 expression was investigated, since it is known to have a significant impact in early development and a long-term effect on growth in some teleost species. Embryos were incubated at 23, 27 or 31°C and samples collected at six developmental stages from blastula until protruding mouth. Dnmt3 expression during early development was remarkably dynamic. In particular, mRNA levels of the two dnmt3a genes showed a marked increase throughout development and several significant differences in dnmt3a and dnmt3b transcript levels were found between temperatures at the same developmental point. Taken together, our data indicate that dnmt3 paralogues are diverging and that dnmt3a and dnmt3b may play different roles in thermal epigenetic regulation of gene expression during early development.