Characterization of the rainbow trout (Oncorhynchus mykiss) mucosal glycosphingolipid repertoire and Aeromonas salmonicida binding to neutral glycosphingolipids.

Infections pose a challenge for the fast growing aquaculture sector. Glycosphingolipids are cell membrane components that pathogens utilize for attachment to the host to initiate infection. Here, we characterized rainbow trout glycosphingolipids from five mucosal tissues using mass spectrometry and nuclear magnetic resonance and investigated binding of radiolabeled Aeromonas salmonicida to the glycosphingolipids on thin-layer chromatograms. 12 neutral and 14 acidic glycosphingolipids were identified. The glycosphingolipids isolated from the stomach and intestine were mainly neutral, whereas glycosphingolipids isolated from the skin, gills and pyloric caeca were largely acidic. Many of the acidic structures were poly-sialylated with shorter glycan structures in the skin compared to the other tissues. The sialic acids found were Neu5Ac and Neu5Gc. Most of the glycosphingolipids had isoglobo and ganglio core chains, or a combination of these. The epitopes on the rainbow trout glycosphingolipid glycans differed between epithelial sites leading to differences in pathogen binding. A major terminal epitope was fucose, that occurred attached to GalNAc in a α1-3 linkage but also in the form of HexNAc-(Fuc-)HexNAc-R. A. salmonicida were shown to bind to neutral glycosphingolipids from the gill and intestine. This study is the first to do a comprehensive investigation of the rainbow trout glycosphingolipids and analyze binding of A. salmonicida to glycosphingolipids. The structural information paves the way for identification of ways of interfering in pathogen colonization processes to protect against infections in aquaculture and contributes towards understanding A. salmonicida infection mechanisms.

Role of cold shock proteins B and D in Aeromonas salmonicida subsp. salmonicida physiology and virulence in lumpfish (Cyclopterus lumpus).

Cold shock proteins (Csp) are pivotal nucleic acid binding proteins known for their crucial roles in the physiology and virulence of various bacterial pathogens affecting plant, insect, and mammalian hosts. However, their significance in bacterial pathogens of teleost fish remains unexplored. Aeromonas salmonicida subsp. salmonicida (hereafter A. salmonicida) is a psychrotrophic pathogen and the causative agent of furunculosis in marine and freshwater fish. Four csp genes (cspB, cspD, cspA, and cspC) have been identified in the genome of A. salmonicida J223 (wild type). Here, we evaluated the role of DNA binding proteins, CspB and CspD, in A. salmonicida physiology and virulence in lumpfish (Cyclopterus lumpus). A. salmonicida ΔcspB, ΔcspD, and the double ΔcspBΔcspD mutants were constructed and characterized. A. salmonicida ΔcspB and ΔcspBΔcspD mutants showed a faster growth at 28°C, and reduced virulence in lumpfish. A. salmonicida ΔcspD showed a slower growth at 28°C, biofilm formation, lower survival in low temperatures and freezing conditions (-20°C, 0°C, and 4°C), deficient in lipopolysaccharide synthesis, and low virulence in lumpfish. Additionally, ΔcspBΔcspD mutants showed less survival in the presence of bile compared to the wild type. Transcriptome analysis revealed that 200, 37, and 921 genes were differentially expressed in ΔcspB, ΔcspD, and ΔcspBΔcspD, respectively. In ΔcspB and ΔcspBΔcspD virulence genes in the chromosome and virulence plasmid were downregulated. Our analysis indicates that CspB and CspD mostly act as a transcriptional activator, influencing cell division (e.g., treB), virulence factors (e.g., aexT), and ultimately virulence.

Identification of the Csr global regulatory system mediated by small RNA decay in Aeromonas salmonicida.

We investigated the presence and functionality of the carbon storage regulator (Csr) system in Aeromonas salmonicida SWSY-1.411. CsrA, an RNA-binding protein, shared 89% amino acid sequence identity with Escherichia coli CsrA. CsrB/C sRNAs exhibited a typical stem-loop structure, with more GGA motifs, which bind CsrA, than E. coli. CsrD had limited sequence identity with E. coli CsrD; however, it contained the conserved GGDEF and EAL domains. Functional analysis in E. coli demonstrated that the Csr system of A. salmonicida influences glycogen biosynthesis, biofilm formation, motility, and stability of both CsrB and CsrC sRNAs. These findings suggest that in A. salmonicida, the Csr system affects phenotypes like its E. coli counterpart. In A. salmonicida, defects in csr homologs affected biofilm formation, motility, and chitinase production. However, glycogen accumulation and protease production were unaffected. The expression of flagellar-related genes and chitinase genes was suppressed in the csrA-deficient A. salmonicida. Northern blot analysis indicated the stabilization of CsrB and CsrC in the csrD-deficient A. salmonicida. Similar to that in E. coli, the Csr system in A. salmonicida comprises the RNA-binding protein CsrA, the sRNAs CsrB and CsrC, and the sRNA decay factor CsrD. This study underscores the conservation and functionality of the Csr system and raises questions about its regulatory targets and mechanisms in A. salmonicida.

Characterization of caspase gene family in Sebastes schlegelii and their expression profiles under Aeromonas salmonicida and Vibrio anguillarum infection.

The caspase, functioning as a proteinase, plays a crucial role in eukaryotic cell apoptosis, regulation of apoptosis, cellular growth, differentiation, and immunity. The identification of caspase gene family in Sebastes schlegelii is of great help to understand its antimicrobial research. In S. schlegelii, we totally identified nine caspase genes, including four apoptosis initiator caspases (caspase 2, caspase 8, caspase 9 and caspase 10), four apoptosis executioners (caspase 3a, caspase 3b, caspase 6, and caspase 7) and one inflammatory executioner (caspase 1). The duplication of caspase 3 genes on chr3 and chr8 may have been facilitated by whole genome duplication (WGD) events or other complex evolutionary processes. In general, the number of caspase genes relatively conserved in high vertebrates, while exhibiting variation in teleosts. Furthermore, syntenic analysis and phylogenetic relationships analysis supported the correct classification of these caspase gene family in S. schlegelii, especially for genes with duplicated copies. Additionally, the expression patterns of these caspase genes in different tissues of S. schlegelii under healthy conditions were assessed. The results revealed that the expression levels of most caspase genes were significantly elevated in the intestine, spleen, and liver. To further investigate the potential immune functions of these caspase genes in S. schlegelii, we challenged individuals with A. salmonicida and V. anguillarum, respectively. After infection with A. salmonicida, the expression levels of caspase 1 in the liver and spleen of S. schlegelii remained consistently elevated throughout the infection time points. The expression levels of most caspase family members in the intestine exhibited significant divergence following V. anguillarum infection. This study provides a comprehensive understanding of the caspase gene families in S. schlegelii, thereby establishing a solid foundation for further investigations into the functional roles of these caspase genes.

The interaction of an effector protein Hap secreted by Aeromonas salmonicida and myofibrillar protein of meat: Possible mechanisms from structural changes to sites of molecular docking.

Microbial spoilage of meat products is a significant problem in the food industry. Aeromonas salmonicida is a significant microorganism responsible for spoilage in chilled meat. Its effector protein, hemagglutinin protease (Hap), has been identified as an effective substance for degrading meat proteins. The ability of Hap to hydrolyze myofibrillar proteins (MPs) in vitro demonstrated that Hap has obvious proteolytic activity, which could alter MPs' tertiary structure, secondary structure, and sulfhydryl groups. Moreover, Hap could significantly degrade MPs, focusing primarily on myosin heavy chain (MHC) and actin. Active site analysis and molecular docking revealed that the active center of Hap was bound to MPs via hydrophobic interaction and hydrogen bonding. It may preferentially cleave peptide bonds between Gly44-Val45 in actin, and Ala825-Phe826 in MHC. These findings suggest that Hap may be involved in the spoilage mechanism of microorganisms and provide crucial insights into the mechanisms of meat spoilage induced by bacteria.

Role of riboflavin biosynthesis gene duplication and transporter in Aeromonas salmonicida virulence in marine teleost fish.

Active flavins derived from riboflavin (vitamin B2) are essential for life. Bacteria biosynthesize riboflavin or scavenge it through uptake systems, and both mechanisms may be present. Because of riboflavin's critical importance, the redundancy of riboflavin biosynthetic pathway (RBP) genes might be present. Aeromonas salmonicida, the aetiological agent of furunculosis, is a pathogen of freshwater and marine fish, and its riboflavin pathways have not been studied. This study characterized the A. salmonicida riboflavin provision pathways. Homology search and transcriptional orchestration analysis showed that A. salmonicida has a main riboflavin biosynthetic operon that includes ribD, ribE1, ribBA, and ribH genes. Outside the main operon, putative duplicated genes ribA, ribB and ribE, and a ribN riboflavin importer encoding gene, were found. Monocistronic mRNA ribA, ribB and ribE2 encode for their corresponding functional riboflavin biosynthetic enzyme. While the product of ribBA conserved the RibB function, it lacked the RibA function. Likewise, ribN encodes a functional riboflavin importer. Transcriptomics analysis indicated that external riboflavin affected the expression of a relatively small number of genes, including a few involved in iron metabolism. ribB was downregulated in response to external riboflavin, suggesting negative feedback. Deletion of ribA, ribB and ribE1 showed that these genes are required for A. salmonicida riboflavin biosynthesis and virulence in Atlantic lumpfish (Cyclopterus lumpus). A. salmonicida riboflavin auxotrophic attenuated mutants conferred low protection to lumpfish against virulent A. salmonicida. Overall, A. salmonicida has multiple riboflavin endowment forms, and duplicated riboflavin provision genes are critical for A. salmonicida infection.

Characterization of effector protein Hap determining meat spoilage process from meat-borne Aeromonas salmonicida.

The spoilage roles of effector proteins secreted by dominant spoilage bacteria during food spoilage remained unknown. In this investigation, a hemagglutinin protease (Hap) belonging to the M4 family metallopeptidase was identified from Aeromonas salmonicida 29 isolate. It, has a molecular weight of 33.5 kDa, a Vmax of 17.06 µg/mL/min, and a Km of 2.46 mg/mL, and is conserved in various dominant spoilage bacteria. The stability testing demonstrated that Hap could maintain specific activity in the common environments (pH, temperature, and metal ions) of chilled meat. It exhibited high spoilage ability on meat in situ, increasing TVB-N, pH values, and the production of volatile organic compounds (VOCs), which was consistent with proteolytic activity analysis, completely confirming the determinant role of Hap for meat spoilage. These observations will enrich the spoilage theory and provide new insights into the control of food quality and safety.

Aeromonas salmonicida binds α2-6 linked sialic acid, which is absent among the glycosphingolipid repertoires from skin, gill, stomach, pyloric caecum, and intestine.

Carbohydrates can both protect against infection and act as targets promoting infection. Mucins are major components of the slimy mucus layer covering the fish epithelia. Mucins can act as decoys for intimate pathogen interaction with the host afforded by binding to glycosphingolipids in the host cell membrane. We isolated and characterized glycosphingolipids from Atlantic salmon skin, gill, stomach, pyloric caeca, and intestine. We characterized the glycosphingolipids using liquid chromatography - mass spectrometry and tandem mass spectrometry and the glycan repertoire was compared with the glycan repertoire of mucins from the same epithelia. We also investigated Aeromonas salmonicida binding using chromatogram and microtiter well based binding assays. We identified 29 glycosphingolipids. All detected acid glycans were of the ganglio-series (unless shorter) and showed a high degree of polysialylation. The non-acid glycans were mostly composed of the neolacto, globo, and ganglio core structures. The glycosphingolipid repertoire differed between epithelia and the proportion of the terminal moieties of the glycosphingolipids did not reflect the terminal moieties on the mucins from the same epithelia. A. salmonicida did not bind the Atlantic salmon glycosphingolipids. Instead, we identified that A. salmonicida binding to sialic acid occurred to α2-6 Neu5Ac but not to α2-3 Neu5Ac. α2-6 Neu5Ac was present on mucins whereas mainly α2-3 Neu5Ac was found on the glycosphingolipids, explaining the difference in A. salmonicida binding ability between these host glycoconjugates. A. salmonicida´s ability to bind to Atlantic salmon mucins, but not the glycosphingolipids, is likely part of the host defence against this pathogen.

Genome-Wide Identification, Evolutionary Analysis, and Expression Patterns of Cathepsin Superfamily in Black Rockfish (Sebastes schlegelii) following Aeromonas salmonicida Infection.

Cathepsins are lysosomal cysteine proteases belonging to the papain family and play crucial roles in intracellular protein degradation/turnover, hormone maturation, antigen processing, and immune responses. In the present study, 18 cathepsins were systematically identified from the fish S. schlegelii genome. Phylogenetic analysis indicated that cathepsin superfamilies are categorized into eleven major clusters. Synteny and genome organization analysis revealed that whole-genome duplication led to the expansion of S. schlegelii cathepsins. Evolutionary rate analyses indicated that the lowest Ka/Ks ratios were observed in CTSBa (0.13) and CTSBb (0.14), and the highest Ka/Ks ratios were observed in CTSZa (1.97) and CTSZb (1.75). In addition, cathepsins were ubiquitously expressed in all examined tissues, with high expression levels observed in the gill, intestine, head kidney, and spleen. Additionally, most cathepsins were differentially expressed in the head kidney, gill, spleen, and liver following Aeromonas salmonicida infection, and their expression signatures showed tissue-specific and time-dependent patterns. Finally, protein-protein interaction network (PPI) analyses revealed that cathepsins are closely related to a few immune-related genes, such as interleukins, chemokines, and TLR genes. These results are expected to be valuable for comparative immunological studies and provide insights for further functional characterization of cathepsins in fish species.

Atlantic Salmon Mucins Inhibit LuxS-Dependent A. Salmonicida AI-2 Quorum Sensing in an N-Acetylneuraminic Acid-Dependent Manner.

One of the most important bacterial diseases in salmonid aquaculture is furunculosis, caused by Aeromonas salmonicida. Bacterial communication through secreted autoinducer signals, quorum sensing, takes part in the regulation of gene expression in bacteria, influencing growth and virulence. The skin and mucosal surfaces, covered by a mucus layer, are the first point of contact between fish and bacteria. Mucins are highly glycosylated and are the main components of mucus. Here, we validate the Vibrio harveyi BB170 bioreporter assay for quantifying A. salmonicida quorum sensing and study the effects of Atlantic salmon mucins as well as mono- and disaccharides on the AI-2 levels of A. salmonicida. Atlantic salmon mucins from skin, pyloric ceca, proximal and distal intestine reduced A. salmonicida AI-2 levels. Among the saccharides abundant on mucins, fucose, N-acetylneuraminic acid and GlcNAcß1-3Gal inhibited AI-2 A. salmonicida secretion. Removal of N-acetylneuraminic acid, which is the most abundant terminal residue on mucin glycans on Atlantic salmon mucins, attenuated the inhibitory effects on AI-2 levels of A. salmonicida. Deletion of A. salmonicida luxS abolished AI-2 production. In conclusion, Atlantic salmon mucins regulate A. salmonicida quorum sensing in a luxS and N-acetylneuraminic acid-dependent manner.

Genome-wide integrated analysis reveals functions of lncRNA-miRNA-mRNA interactions in Atlantic salmon challenged by Aeromonas salmonicida.

Aeromonas salmonicida (A. salmonicida) is a pathogenic bacterium that causes serious problems in the global Atlantic salmon aquaculture industry. In this study, we comprehensively analyzed the profiles of lncRNAs, miRNAs and mRNAs in gills of Atlantic salmon at high-dose A. salmonicida infection (3.06 × 108 CFU/mL), low-dose A. salmonicida infection (3.06 × 105 CFU/mL), and a PBS (100 µL) control. We identified 65 differentially expressed lncRNAs, 41 miRNAs, and 512 mRNAs between the control group and infection groups. Functional analysis showed that these genes were significantly enriched in the p53 signaling pathway, Wnt signaling pathway, mTOR signaling pathway, JAK-STAT signaling pathway, and Toll-like receptor signaling pathway. In addition, we predicted key genes in immune-related pathways and constructed a lncRNA-miRNA-mRNA network based on whole transcriptomic analysis. We further predicted three lncRNA-miRNA-mRNA axes as potential novel biomarkers in regulating the immune response of Atlantic salmon against A. salmonicida infection.

Aeromonas salmonicida activates rainbow trout IgM+ B cells signalling through Toll like receptors.

As B cells are singularly equipped with a B cell receptor (BCR) and a range of innate receptors, they are able to integrate both antigen-specific and innate signals, with the latter being essential to reach an adequate level of activation. Whether teleost B cells sense pathogens through innate mechanisms has not yet been explored, despite the fact that fish B cells display a wider array of innate receptors than many mammalian B cell subsets. Hence, in the current study, we have investigated the effects of inactivated Aeromonas salmonicida, a Gram negative rainbow trout pathogen, on trout splenic IgM+ B cells in vitro in the presence or absence of different inhibitors of Toll-like receptor (TLR) signalling, to establish to what degree innate signals are contributing to the activation of B cells in teleosts. Our results demonstrate that most of the effects that A. salmonicida exerts on trout IgM+ B cells are significantly blocked in the presence of inhibitors of MyD88 and TRIF, important nodes in TLR signal pathways. Thus, the data presented demonstrates that, also in teleost, TLR signalling is essential for the activation of IgM+ B cells. These results will be useful for the future optimization of novel vaccines and adjuvants.

Recent Insights into Aeromonas salmonicida and Its Bacteriophages in Aquaculture: A Comprehensive Review.

The emergence and spread of antimicrobial resistance in pathogenic bacteria of fish and shellfish have caused serious concerns in the aquaculture industry, owing to the potential health risks to humans and animals. Among these bacteria, Aeromonas salmonicida, which is one of the most important primary pathogens in salmonids, is responsible for significant economic losses in the global aquaculture industry, especially in salmonid farming because of its severe infectivity and acquisition of antimicrobial resistance. Therefore, interest in the use of alternative approaches to prevent and control A. salmonicida infections has increased in recent years, and several applications of bacteriophages (phages) have provided promising results. For several decades, A. salmonicida and phages infecting this fish pathogen have been thoroughly investigated in various research areas including aquaculture. The general overview of phage usage to control bacterial diseases in aquaculture, including the general advantages of this strategy, has been clearly described in previous reviews. Therefore, this review specifically focuses on providing insights into the phages infecting A. salmonicida, from basic research to biotechnological application in aquaculture, as well as recent advances in the study of A. salmonicida.

Degradation of the mixture of ethyl formate, propionic aldehyde, and acetone by Aeromonas salmonicida: A novel microorganism screened from biomass generated in the citric acid fermentation industry.

Microorganisms play important roles in the degradation of volatile organic compounds. Aeromonas salmonicida strain (AEP-3) generated from biomass in the citric acid fermentation industry was screened and subjected to denaturing gradient gel electrophoresis (DGGE) fingerprinting and 16S rDNA gene sequencing. The growth conditions of AEP-3 in Luria-Bertani broth were optimized at 25 °C and approximately pH 7. AEP-3 was used to degrade ethyl formate, propionic aldehyde, or acetone alone and their mixture. The concentrations of ethyl formate, propionic aldehyde, and acetone were below 7500, 600, and 800 mg L-1, respectively, and their maximum degradation efficiencies were 100%, 92.41%, and 34.75%. AEP-3 first degraded acetone and propionic aldehyde in the mixture, followed by ethyl formate. The degradation pathways of these organic compounds in the mixture and their substrate interactions during degradation were explored. Propionic aldehyde was first converted into propionic acid in the metabolic process and was involved in the subsequent carboxylic acid cycle. By contrast, ethyl formate was first hydrolyzed into formic acid and ethanol. Then, formic acid participated in the cyclic metabolism of carboxylic acid, whereas, ethanol was hydrolyzed into acetaldehyde and acetic acid through alcohol and aldehyde dehydrogenase. Additionally, acetone directly interacted with nitrate in the medium under the action of hydrogen ions and produced carbon dioxide, water, and nitrogen. Overall, this study provides a new degrading bacterium biodegradability toward the exhaust gas of citric acid fermentation.

Growth and spoilage metabolites production of a mesophilic Aeromonas salmonicida strain in Atlantic salmon (Salmo salar L.) during cold storage in modified atmosphere.

AIMS: The aim of the study was to quantify the growth kinetic parameters and spoilage-associated metabolites of an inoculated strain of Aeromonas salmonicida in pre-rigor filleted Atlantic salmon (Salmo salar L.) stored in vacuum (VP) or modified atmosphere (MAP 60/40% CO2 /N2 ) at 4 and 8°C. METHODS AND RESULTS: The maximum growth rate of A. salmonicida in VP salmon stored at 4°C was 0·56 ± 0·04 day-1 with no detectable lag-phase and the concentration of Aeromonas reached 8·33 log CFU per g after 10 days. The growth rates and maximum population density of Aeromonas in MAP salmon were lower but the applied atmosphere did not inhibit the growth. A selection of metabolites associated with fish spoilage were quantified using 1 H nuclear magnetic resonance (NMR) spectroscopy. The concentration of trimethylamine (TMA) was significantly affected by storage time and temperature, packaging atmosphere and inoculation with A. salmonicida (General Linear Model (GLM), P < 0·001 for all factors). CONCLUSION: The study presents preliminary results on A. salmonicida as a potential spoilage organism in vacuum-packaged salmon during cold storage. The combination of refrigeration and a packaging atmosphere consisting of 60/40 % CO2 /N2 did not completely inhibit the growth but prevented the formation of TMA. SIGNIFICANCE AND IMPACT OF THE STUDY: Little information is available on the spoilage potential of Aeromonas spp. in minimally processed salmon products under different packaging conditions. The study clearly demonstrates the importance of hurdle technology and provides data to further elucidate the significance of Aeromonas spp. as a spoilage organism.

The Outer Membrane Protein FstC of Aeromonas salmonicida subsp. salmonicida Acts as Receptor for Amonabactin Siderophores and Displays a Wide Ligand Plasticity. Structure-Activity Relationships of Synthetic Amonabactin Analogues.

Amonabactins are a group of four related catecholate siderophores produced by several species of the genus Aeromonas, including A. hydrophila and the fish pathogen A. salmonicida subsp. salmonicida. Although the gene cluster encoding amonabactin biosynthesis also contains a gene that could encode the ferri-siderophore receptor (fstC), to date there is no experimental evidence to explain its role. In this work, we report the identification of the amonabactins' outer membrane receptor and the determination of the minimal structural parts of these siderophores involved in the molecular recognition by their cognate receptor. The four natural amonabactin forms (P750, T789, P693, and T732) and some mono and biscatecholate amonabactin analogues were chemically synthesized, and their siderophore activity on A. salmonicida FstC(+) and FstC(-) strains was evaluated. The results showed that each amonabactin form has quite different growth promotion activity, with P750 and T789 the most active. The outer membrane receptor FstC recognizes more efficiently biscatecholate siderophores in which the length of the linker between the two iron-binding catecholamide units is 15 atoms (P750 and T789) instead of 12 atoms (P693 and T732). Analysis of the siderophore activity of synthetic analogues indicated that the presence of Phe or Trp residues is not required for siderophore recognition. The results together point toward evidence that the amonabactin receptor FstC admits a high degree of ligand plasticity. We also showed that FstC is present in most Aeromonas species, including relevant human and animal pathogens as A. hydrophila. From the results obtained, we concluded that the ferri-amonabactin uptake pathway involving the outer membrane transporter FstC possesses a considerable functional plasticity that could be exploited for delivery of antimicrobial compounds into the cell. This would allow the use of the siderophore-based iron uptake mechanisms to combat infections caused by species of the genus Aeromonas.

Beyond the A-layer: adsorption of lipopolysaccharides and characterization of bacteriophage-insensitive mutants of Aeromonas salmonicida subsp. salmonicida.

Aeromonas salmonicida subsp. salmonicida is a fish pathogen that causes furunculosis. Antibiotherapy used to treat furunculosis in fish has led to resistance. Virulent phages are increasingly seen as alternatives or complementary treatments against furunculosis in aquaculture environments. For phage therapy to be successful, it is essential to study the natural mechanisms of phage resistance in A. salmonicida subsp. salmonicida. Here, we generated bacteriophage-insensitive mutants (BIMs) of A. salmonicida subsp. salmonicida, using a myophage with broad host range and characterized them. Phage plaques were different depending on whether the A-layer surface array protein was expressed or not. The genome analysis of the BIMs helped to identify mutations in genes involved in the biogenesis of lipopolysaccharides (LPS) and on an uncharacterized gene (ASA_1998). The characterization of the LPS profile and gene complementation assays identified LPS as a phage receptor and confirmed the involvement of the uncharacterized protein ASA_1998 in phage infection. In addition, we confirmed that the presence of an A-layer at the bacterial surface could act as protection against phages. This study brings new elements into our understanding of the phage adsorption to A. salmonicida subsp. salmonicida cells.

Biogeography of the fish pathogen Aeromonas salmonicida inferred by vapA genotyping.

A recently described typing system based on sequence variation in the virulence array protein (vapA) gene, encoding the A-layer surface protein array, allows unambiguous subtyping of Aeromonas salmonicida. In the present study, we compile A-layer typing results from a total of 675 A. salmonicida isolates, recovered over a 59-year period from 50 different fish species in 26 countries. Nine novel A-layer types (15-23) are identified, several of which display a strong predilection towards certain fish hosts, including e.g. Cyprinidae and Pleuronectidae species. Moreover, we find indications that anthropogenic transport of live fish may have aided the near global dissemination of two cyprinid-associated A-layer types. Comparison of whole genome phylogeny and A-layer typing for a subset of strains further resulted in compatible tree topologies, indicating the utility of vapA as a phylogenetic as well as an epizootiological marker in A. salmonicida. A Microreact project (microreact.org/project/r1pcOAx9m) has been created, allowing public access to the vapA analyses and relevant metadata. In sum, the results generated provide valuable insights into the global population structure of A. salmonicida, particularly in relation to its piscine host spectrum and the geographic distribution of these hosts.

Phosphoproteomic analyses of kidneys of Atlantic salmon infected with Aeromonas salmonicida.

Aeromonas salmonicida (A. salmonicida) is a pathogenic bacterium that causes furunculosis and poses a significant global risk, particularly in economic activities such as Atlantic salmon (Salmo salar) farming. In a previous study, we identified proteins that are significantly upregulated in kidneys of Atlantic salmon challenged with A. salmonicida. Phosphoproteomic analyses were conducted to further clarify the dynamic changes in protein phosphorylation patterns triggered by bacterial infection. To our knowledge, this is the first study to characterize phosphorylation events in proteins from A. salmonicida-infected Atlantic salmon. Overall, we identified over 5635 phosphorylation sites in 3112 proteins, and 1502 up-regulated and 77 down-regulated proteins quantified as a 1.5-fold or greater change relative to control levels. Based on the combined data from proteomic and motif analyses, we hypothesize that five prospective novel kinases (VRK3, GAK, HCK, PKCδ and RSK6) with common functions in inflammatory processes and cellular pathways to regulate apoptosis and the cytoskeleton could serve as potential biomarkers against bacterial propagation in fish. Data from STRING-based functional network analyses indicate that fga is the most central protein. Our collective findings provide new insights into protein phosphorylation patterns, which may serve as effective indicators of A. salmonicida infection in Atlantic salmon.

Vaccine-Induced Protection Against Furunculosis Involves Pre-emptive Priming of Humoral Immunity in Arctic Charr.

With respect to salmonid aquaculture, one of the most important bacterial pathogens due to high mortality and antibiotic usage is the causative agent of typical furunculosis, Aeromonas salmonicida spp. salmonicida (Asal). In Atlantic salmon, Salmo salar, the host response during infections with Asal is well-documented, with furunculosis outbreaks resulting in significant mortality in commercial settings. However, less is known about the host-pathogen interactions in the emerging aquaculture species, Arctic charr Salvelinus alpinus. Furthermore, there is no data on the efficacy or response of this species after vaccination with commonly administered vaccines against furunculosis. To this end, we examined the immunological response of S. alpinus during infection with Asal, with or without administration of vaccines (Forte Micro®, Forte Micro® + Renogen®, Elanco Animal Health). Artic charr (vaccinated or unvaccinated) were i.p.-injected with a virulent strain of Asal (106 CFUs/mL) and tissues were collected pre-infection/post-vaccination, 8, and 29 days post-infection. Unvaccinated Arctic charr were susceptible to Asal with 72% mortalities observed after 31 days. However, there was 72-82% protection in fish vaccinated with either the single or dual-vaccine, respectively. Protection in vaccinated fish was concordant with significantly higher serum IgM concentrations, and following RNA sequencing and transcriptome assembly, differential expression analysis revealed several patterns and pathways associated with the improved survival of vaccinated fish. Most striking was the dramatically higher basal expression of complement/coagulation factors, acute phase-proteins, and iron hemostasis proteins in pre-challenged, vaccinated fish. Remarkably, following Asal infection, this response was abrogated and instead the transcriptome was characterized by a lack of immune-stimulation compared to that of unvaccinated fish. Furthermore, where pathways of actin assembly and FcγR-mediated phagocytosis were significantly differentially regulated in unvaccinated fish, vaccinated fish showed either the opposite regulation (ForteMicro®), or no impact at all (ForteMicro®Renogen®). The present data indicates that vaccine-induced protection against Asal relies on the pre-activation and immediate control of humoral immune parameters that is coincident with reduced activation of apoptotic (e.g., NF-κB) and actin-associated pathways.