Novel mannose binding natterin-like protein in the skin mucus of Atlantic cod (Gadus morhua)

Author's accepted version (post-print).Available from 23/07/2018.acceptedVersio

Differentially expressed proteins in the skin mucus of Atlantic cod (Gadus morhua) upon natural infection with Vibrio anguillarum

BACKGROUND: Vibriosis caused by V. anguillarum is a commonly encountered disease in Atlantic cod farms and several studies indicate that the initiation of infection occurs after the attachment of the pathogen to the mucosal surfaces (gut, skin and gills) of fish. Therefore it is necessary to investigate the role of different mucosal components in fish upon V. anguillarum infection. The present study has two parts; in the first part we analyzed the differential expression of skin mucus proteins from Atlantic cod naturally infected with V. anguillarum using two dimensional gel electrophoresis coupled with mass spectrometry. In the second part, a separate bath challenge experiment with V. anguillarum was conducted to assess the mRNA levels of the genes in skin tissue, corresponding to the selected proteins identified in the first part. RESULTS: Comparative proteome analysis of skin mucus of cod upon natural infection with V. anguillarum revealed key immune relevant proteins like calpain small subunit 1, glutathione-S-transferase omega 1, proteasome 26S subunit, 14-kDa apolipoprotein, beta 2-tubulin, cold inducible RNA binding protein, malate dehydrogenase 2 (mitochondrial) and type II keratin that exhibited significant differential expression. Additionally a number of protein spots which showed large variability amongst individual fish were also identified. Some of the proteins identified were mapped to the immunologically relevant JNK (c-Jun N-terminal kinases) signalling pathway that is connected to cellular events associated with pathogenesis. A bath challenge experiment with V. anguillarum showed differential expression of beta 2-tubulin, calpain small subunit 1, cold inducible RNA binding protein, flotillin1, and glutathione S-transferase omega 1 transcripts in the skin tissue of cod during early stages of infection. CONCLUSIONS: Differentially expressed proteins identified in the cod skin mucus point towards their possible involvement in V. anguillarum pathogenesis. The role of some of these proteins in vibriosis in cod described in this paper can be considered unconventional with respect to their established functions in higher vertebrates. Based on the differential expression of these proteins they are possibly important components of fish defence against bacteria and innate immunity at large. The feasibility of utilizing these proteins/genes as markers of bacterial infection or stress in cod needs to be explored further

Dietary Yeast Cell Wall Extract Alters the Proteome of the Skin Mucous Barrier in Atlantic Salmon (Salmo salar) : Increased Abundance and Expression of a Calreticulin-Like Protein

Funding: This work was supported by a studentship from BioMar Ltd. to GM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Details of feeding period and tank replicates for the feeding trials.

<p>The base diet was manufactured by Biomar and described in three terms: the product range name (CPK), the pellet diameter and the oil to protein ratio. The control and experimental diets were designed to be iso-nitrogenous and iso-lipidic. YCW = yeast cell wall extracts. FOS = fructooligosaccharides.</p

Phylogenetic trees showing the evolutionary relationship of the calreticulin family of proteins in vertebrates, rooted by the mammalian calreticulin-3.

<p>The tree was used to name the calreticulin isoforms of <i>Salmo salar</i>. The tree was constructed using multiple alignments of the vertebrate calreticulin. The neighbour-joining method in MEGA 5 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169075#pone.0169075.ref037" target="_blank">37</a>] was selected. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (10,000 replicates) is shown next to the branches [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169075#pone.0169075.ref040" target="_blank">40</a>]. The salmon sequences are boxed. Each entry is described by the species common name and protein name, followed by the Genbank or Ensembl accession number.</p

Primer sequences, together with their optimum annealing temperature and product size, used for the gene expression studies in Atlantic salmon skin cDNA.

<p>Primer sequences, together with their optimum annealing temperature and product size, used for the gene expression studies in Atlantic salmon skin cDNA.</p

Validation of proteomic results by analysing the gene expression of the skin mRNA corresponding to the respective proteins.

<p>Fold changes at the proteomic level was calculated on SameSpots using normalised spot volume data of two-dimensional gels (2DGE). Gene expression fold changes were calculated from C_t values of qPCR assays by deriving arbitrary units using the standards graph equation and normalising using two housekeeping genes, EF-1α and β-actin. (n = 9).</p

Differentially expressed proteins in the skin of Atlantic salmon fed prebiotic dietary supplements, as identified by two-dimensional electrophoresis.

<p>The relevant spots were cut out from the gel and sequenced by LC-MS/MS, after which the peptides were identified by a MASCOT search. The spot numbers refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169075#pone.0169075.g001" target="_blank">Fig 1</a>, MW stands for molecular weight and fold change represents the expression level in the experimental fish group as compared to the control.</p

Effect of dietary YCW extracts on <i>calrl</i> expression in the gut of Atlantic salmon during Trial#1.

<p>Fish were fed either a control diet (Control) or the same base diet supplemented with 0.4% YCW. Expression is given as a ratio of the arbitrary unit for CALRL to the arbitrary units of two reference genes, elongation factor 1-alpha and beta-actin. Data are present as Means ± SEM (n = 6). The expression of CALRL is not significantly different between diets (p>0.05), as established by independent sample t-test after checking for normality and outliers.</p