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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 Δ cspB , Δ cspD , and the double Δ cspB Δ cspD mutants were constructed and characterized. A. salmonicida Δ cspB and Δ cspB Δ cspD mutants showed a faster growth at 28°C, and reduced virulence in lumpfish. A. salmonicida Δ cspD 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, Δ cspB Δ cspD 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 Δ cspB , Δ cspD , and Δ cspB Δ cspD, respectively. In Δ cspB and Δ cspB Δ cspD 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., Competing Interests: The authors declare no conflict of interest.

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., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Active flavins derived from riboflavin (vitamin B 2 ) 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.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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 V max of 17.06 μg/mL/min, and a K m 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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.

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.

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 GlcNAcβ1-3Gal 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.

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 × 10 8  CFU/mL), low-dose A. salmonicida infection (3.06 × 10 5  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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 .

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.

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% CO 2 /N 2 ) 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 % CO 2 /N 2 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., (© 2020 The Authors. Journal of Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

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 .

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&#95;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&#95;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., (© 2019 John Wiley & Sons Ltd.)

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., (© FEMS 2019.)

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.

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 (10 6 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.

The environmental strain Aeromonas salmonicida subsp. pectinolytica 34mel T produces abundant melanin through the homogentisate pathway in several culture media, but unexpectedly not when grown in a medium containing glycerol. Using this observation as a starting point, this study investigated the underlying causes of the inhibition of melanin synthesis by glycerol, to shed light on factors that affect melanin production in this microorganism. The effect of different carbon sources on melanin formation was related to the degree of oxidation of their C atoms, as the more reduced substrates delayed melanization more than the more oxidized ones, although only glycerol completely abolished melanin production. Glyphosate, an inhibitor of aromatic amino acid synthesis, did not affect melanization, while bicyclopyrone, an inhibitor of 4-hydroxyphenylpyruvate dioxygenase (Hpd), the enzyme responsible for the synthesis of homogentisate, prevented melanin synthesis. These results showed that melanin production in 34mel T depends on the degradation of aromatic amino acids from the growth medium and not on de novo aromatic amino acid synthesis. The presence of glycerol changed the secreted protein profile, but none of the proteins affected could be directly connected with melanin synthesis or transport. Transcription analysis of hpd, encoding the key enzyme for melanin synthesis, showed a clear inhibition caused by glycerol. The results obtained in this work indicate that a significant decrease in the transcription of hpd, together with a more reduced intracellular state, would lead to the abolishment of melanin synthesis observed. The effect of glycerol on melanization can thus be attributed to a combination of metabolic and regulatory effects.

Aeromonas salmonicida subsp. salmonicida is a major pathogen affecting fisheries worldwide and is a well-known pigmented member of the Aeromonas genus. This subspecies produces melanin at ≤22°C. However, melanogenesis decreases as the culture temperature increases and is completely suppressed at 30°C to 35°C, while bacterial growth is unaffected. The mechanism and biological significance of this temperature-dependent melanogenesis remain unclear. Heterologous expression of an A. salmonicida subsp. salmonicida 4-hydroxyphenylpyruvate dioxygenase (HppD), the most critical enzyme in the homogentisic acid (HGA)-melanin synthesis pathway, results in thermosensitive pigmentation in Escherichia coli , suggesting that HppD plays a key role in this process. In this study, we demonstrated that the thermolability of HppD is responsible for the temperature-dependent melanization of A. salmonicida subsp. salmonicida Substitutions of three residues, S18T, P103Q, and L119P, in A. salmonicida subsp. salmonicida HppD increased the thermostability of this enzyme and resulted in temperature-independent melanogenesis. Moreover, the replacement of the corresponding residues in HppD from Aeromonas media strain WS, which forms pigment independent of temperature, with those of A. salmonicida subsp. salmonicida HppD resulted in thermosensitive melanogenesis. A structural analysis suggested that mutations at these sites, especially at position P103, strengthen the secondary structure of HppD and greatly improve its thermal stability. Additionally, we found that the HppD sequences of all A. salmonicida subsp. salmonicida isolates were identical and that two of the three residues were clearly distinct from those of other Aeromonas strains. IMPORTANCE Aeromonas salmonicida subsp. s almonicida is the causative agent of furunculosis, a bacterial septicemia of cold-water fish of the Salmonidae family. Although other Aeromonas species can produce melanin, A. salmonicida subsp. salmonicida is the only member of this genus that has been reported to exhibit temperature-dependent melanization. Here, we demonstrated that thermosensitive melanogenesis in A. salmonicida subsp. salmonicida strains is due to the thermolability of 4-hydroxyphenylpyruvate dioxygenase (HppD). Additionally, we confirmed that this thermolabile HppD exhibited higher activity at low temperatures than its mesophilic homologues, suggesting this as an adaptive strategy of this enzyme to the psychrophilic lifestyle of A. salmonicida subsp. salmonicida The strictly conserved hppD sequences among A. salmonicida subsp. salmonicida isolates and the specific possession of P103 and L119 residues could be used as a reference for the identification of A. salmonicida subsp. salmonicida isolates., (Copyright © 2018 American Society for Microbiology.)

Microorganisms play an important role in the spoilage of chilled chicken. In this study, a total of 53 isolates, belonging to 7 species of 3 genera, were isolated using a selective medium based on the capacity to spoil chicken juice. Four isolates, namely Aeromonas salmonicida 35, Pseudomonas fluorescens H5, Pseudomonas fragi H8 and Serratia liquefaciens 17, were further characterized to assess their proteolytic activities in vitro using meat protein extracts and to evaluate their spoilage potential in situ. The in vitro studies showed that A. salmonicida 35 displayed the strongest proteolytic activity against both sarcoplasmic and myofibrillar proteins. However, the major spoilage isolate in situ was P. fragi H8, which exhibited a fast growth rate, slime formation and increased pH and total volatile basic nitrogen (TVBN) on chicken breast fillets. The relative amounts of volatile organic compounds (VOCs) originating from the microorganisms, including alcohols, aldehydes, ketones and several sulfur compounds, increased during storage. In sum, this study demonstrated the characteristics of 4 potential spoilage bacteria on chilled yellow-feather chicken and provides a simple and convenient method to assess spoilage bacteria during quality management., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis in several fish species, produces acinetobactin and amonabactin as siderophores. In a previous study, we chemically characterized these siderophores and proposed a biosynthetic pathway based on genetic analysis. However, the internalization mechanisms of ferric-acinetobactin and ferric-amonabactin remain largely unknown. In the present study, we demonstrate that the outer membrane protein FstB is the ferric-acinetobactin receptor in A. salmonicida since an fstB defective mutant is unable to grow under iron limitation and does not use acinetobactin as an iron source. In order to study the effect that structural changes in acinetobactin have on its siderophore activity, a collection of acinetobactin-based analogues was synthesized, including its enantiomer and four demethylated derivatives. The biological activity of these analogues on an fstB(+) strain compared to an fstB(-) strain allowed structure-activity relationships to be elucidated. We found a lack of enantiomer preference on the siderophore activity of acinetobactin over A. salmonicida or on the molecular recognition by FstB protein receptor. In addition, it was observed that A. salmonicida could not use acinetobactin analogues when imidazole or a similar heterocyclic ring was absent from the structure. Surprisingly, removal of the methyl group at the isoxazolidinone ring induced a higher biological activity, thus suggesting alternative route(s) of entry into the cell that must be further investigated. It is proposed that some of the synthetic acinetobactin analogues described here could be used as starting points in the development of novel drugs against A. salmonicida and probably against other acinetobactin producers like the human pathogen Acinetobacter baumannii.

The oxygen sensor histidine kinase AfGcHK from the bacterium Anaeromyxobacter sp. Fw 109-5 forms a two-component signal transduction system together with its cognate response regulator (RR). The binding of oxygen to the heme iron of its N-terminal sensor domain causes the C-terminal kinase domain of AfGcHK to autophosphorylate at His183 and then transfer this phosphate to Asp52 or Asp169 of the RR protein. Analytical ultracentrifugation revealed that AfGcHK and the RR protein form a complex with 2:1 stoichiometry. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) suggested that the most flexible part of the whole AfGcHK protein is a loop that connects the two domains and that the heme distal side of AfGcHK, which is responsible for oxygen binding, is the only flexible part of the sensor domain. HDX-MS studies on the AfGcHK:RR complex also showed that the N-side of the H9 helix in the dimerization domain of the AfGcHK kinase domain interacts with the helix H1 and the β-strand B2 area of the RR protein's Rec1 domain, and that the C-side of the H8 helix region in the dimerization domain of the AfGcHK protein interacts mostly with the helix H5 and β-strand B6 area of the Rec1 domain. The Rec1 domain containing the phosphorylable Asp52 of the RR protein probably has a significantly higher affinity for AfGcHK than the Rec2 domain. We speculate that phosphorylation at Asp52 changes the overall structure of RR such that the Rec2 area containing the second phosphorylation site (Asp169) can also interact with AfGcHK. Proteins 2016; 84:1375-1389. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Aeromonas salmonicida subsp. pectinolytica 34mel(T) can be considered an extremophile due to the characteristics of the heavily polluted river from which it was isolated. While four subspecies of A. salmonicida are known fish pathogens, 34mel(T) belongs to the only subspecies isolated solely from the environment. Genome analysis revealed a high metabolic versatility, the capability to cope with diverse stress agents, and the lack of several virulence factors found in pathogenic Aeromonas. The most relevant phenotypic characteristics of 34mel(T) are pectin degradation, a distinctive trait of A. salmonicida subsp. pectinolytica, and melanin production. Genes coding for three pectate lyases were detected in a cluster, unique to this microorganism, that contains all genes needed for pectin degradation. Melanin synthesis in 34mel(T) is hypothesized to occur through the homogentisate pathway, as no tyrosinases or laccases were detected and the homogentisate 1,2-dioxygenase gene is inactivated by a transposon insertion, leading to the accumulation of the melanin precursor homogentisate. Comparative genome analysis of other melanogenic Aeromonas strains revealed that this gene was inactivated by transposon insertions or point mutations, indicating that melanin biosynthesis in Aeromonas occurs through the homogentisate pathway. Horizontal gene transfer could have contributed to the adaptation of 34mel(T) to a highly polluted environment, as 13 genomic islands were identified in its genome, some of them containing genes coding for fitness-related traits. Heavy metal resistance genes were also found, along with others associated with oxidative and nitrosative stresses. These characteristics, together with melanin production and the ability to use different substrates, may explain the ability of this microorganism to live in an extremely polluted environment., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Aims: This study aimed to select and validate different methodological strategies to quantify the expression of the virulence genes ascC and ascV by qPCR in Aeromonas salmonicida subsp. salmonicida (Aer. salmonicida)., Methods and Results: Using the geNorm, Normfinder and BestKeeper algorithms, reference genes for the qPCR were selected based on their in vitro expression stabilities in three Aer. salmonicida strains. Gene amplification efficiency was calculated by Real-time PCR Miner and LinReg PCR programmes, which have not been used previously in the analysis of bacterial gene expression. The expression of the ascC and ascV virulence genes in a virulent Aer. salmonicida strain was evaluated by three quantification models, including single (least or most stable) or three most stable reference genes, combined with constant or specific gene amplification efficiency. The most stable reference genes were gyrB, proC and rpoC, while rpoD and fabD were the least stable. Quantification models showed different expression patterns., Conclusions: The optimal strategy to quantify mRNA expression was to use a combination of the three algorithms and the quantification model including the three most stable reference genes. Real-time PCR Miner or LinReg PCR were valuable tools to estimate amplification efficiency., Significance and Impact of the Study: The methods used in this study gave more reliable expression data using qPCR than previously published methods. The quantification and expression dynamics of virulence genes will contribute to a better understanding of how Aer. salmonicida interacts with its host and the environment, and therefore to the prevention of epizootics due to this pathogen., (© 2014 The Society for Applied Microbiology.)

Aeromonas salmonicida subsp. salmonicida is an important pathogen in salmonid aquaculture and is responsible for the typical furunculosis. The type-three secretion system (T3SS) is a major virulence system. In this work, we review structure and function of this highly sophisticated nanosyringe in A. salmonicida. Based on the literature as well as personal experimental observations, we document the genetic (re)organization, expression regulation, anatomy, putative functional origin and roles in the infectious process of this T3SS. We propose a model of pathogenesis where A. salmonicida induces a temporary immunosuppression state in fish in order to acquire free access to host tissues. Finally, we highlight putative important therapeutic and vaccine strategies to prevent furunculosis of salmonid fish., (© 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

The bacterium Aeromonas salmonicida, a fish pathogen, uses the type three secretion system (TTSS) to inject effector proteins into host cells to promote the infection. The study of the genome of A. salmonicida has revealed the existence of Ati2, a potential TTSS effector protein. In the present study, a structure-function analysis of Ati2 has been done to determine its role in the virulence of A. salmonicida. Biochemical assays revealed that Ati2 is secreted into the medium in a TTSS-dependent manner. Protein sequence analyses, molecular modelling and biochemical assays demonstrated that Ati2 is an inositol polyphosphate 5-phosphatase, which hydrolyses PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in a way similar to VPA0450, a protein from Vibrio parahaemolyticus having high sequence similarity with Ati2. Mutants of Ati2 with altered amino acids at two different locations in the catalytic site displayed no phosphatase activity. Wild-type and mutant forms of Ati2 were cloned into expression systems for Dictyostelium discoideum, a soil amoeba used as an alternative host to study A. salmonicida virulence. Expression tests allowed us to demonstrate that Ati2 is toxic for the host cell in a catalytic-dependent manner. Finally, this study demonstrated the existence of a new TTSS effector protein in A. salmonicida.

Biosurfactants are produced by hydrocarbon-degrading marine bacteria in response to the presence of water-insoluble hydrocarbons. This is believed to facilitate the uptake of hydrocarbons by bacteria. However, these diffusible amphiphilic surface-active molecules are involved in several other biological functions such as microbial competition and intra- or inter-species communication. We report the isolation and characterization of a marine bacterial strain identified as Cobetia sp. MM1IDA2H-1, which can grow using the sulfur-containing heterocyclic aromatic hydrocarbon dibenzothiophene (DBT). As with DBT, when the isolated strain is grown in the presence of a microbial competitor, it produces a biosurfactant. Because the obtained biosurfactant was formed by hydroxy fatty acids and extracellular lipidic structures were observed during bacterial growth, we investigated whether the biosurfactant at its critical micelle concentration can interfere with bacterial communication systems such as quorum sensing. We focused on Aeromonas salmonicida subsp. salmonicida, a fish pathogen whose virulence relies on quorum sensing signals. Using biosensors for quorum sensing based on Chromobacterium violaceum and Vibrio anguillarum, we showed that when the purified biosurfactant was mixed with N-acyl homoserine lactones produced by A. salmonicida, quorum sensing was inhibited, although bacterial growth was not affected. In addition, the transcriptional activities of A. salmonicida virulence genes that are controlled by quorum sensing were repressed by both the purified biosurfactant and the growth in the presence of Cobetia sp. MM1IDA2H-1. We propose that the biosurfactant, or the lipid structures interact with the N-acyl homoserine lactones, inhibiting their function. This could be used as a strategy to interfere with the quorum sensing systems of bacterial fish pathogens, which represents an attractive alternative to classical antimicrobial therapies in fish aquaculture., (© 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

The exploration of novel antibiotic resistance determinants in a particular environment may be limited because of the presence of uncultured microorganisms. In this work, a culture-independent approach based on functional metagenomics was applied to search for chloramphenicol resistance genes in agro-industrial wastewater in Lerma de Villada, Mexico. To this end, a metagenomic library was generated in Escherichia coli DH10B containing DNA isolated from environmental samples of the residual arsenic-enriched (10 mg/ml) effluent. One resistant clone was detected in this library and further analyzed. An open reading frame similar to a multidrug resistance protein from Aeromonas salmonicida and responsible for chloramphenicol resistance was identified, sequenced, and found to encode a member of the major facilitator superfamily (MFS). Our results also showed that the expression of this gene restored streptomycin sensitivity in E. coli DH10B cells. To gain further insight into the phenotype of this MFS family member, we developed a model of the membrane protein multiporter that, in addition, may serve as a template for developing new antibiotics.

A real-time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg (DNA), 2.2 × 10(4) ± 1 × 10(4) CFU g(-1) (without enrichment) and 40 ± 10 CFU g(-1) (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real-time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n = 750) and pink shubunkin, Carassius auratus (L.) (n = 157). The real-time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A. salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive and a reproducible method for the detection of A. salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes., (© 2012 Blackwell Publishing Ltd.)

Aeromonas salmonicida subsp. salmonicida is the etiological agent of furunculosis; a serious infectious disease in aquaculture raised salmonids. Iron acquisition has been shown to be critical for the survival of pathogenic bacteria during the course of infection. Previous work has demonstrated that A. salmonicida expresses iron-repressible IROMP proteins, suggesting the presence of iron acquisition systems that are under the control of a ferric uptake regulator (Fur). In this study, the A. salmonicida fur has been sequenced and a fur deletion strain generated. The A. salmonicida fur gene has an open reading frame of 428 bp, coding for a protein of 143 amino acids, and with high homology to previously described Fur proteins. The Fur protein product had a 94% sequence identity and 96% sequence similarity to the Aeromonas hydrophila Fur protein product. Transcription of the A. salmonicida fur gene was not regulated by the iron status of the bacterium and is not autoregulated, as in Escherichia coli. Proteomic analysis of the A. salmonicida fur mutant, fails to repress iron-regulated outer membrane proteins in the presence of iron. The A. salmonicida fur::KO mutant shows significantly reduced pathogenicity compared to the wild-type parental strain. In addition, the A. salmonicida fur mutant provides an important tool for further investigation of the iron acquisition mechanisms utilized by A. salmonicida., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The iron-sulfur cluster protein ISCU is a scaffold protein tasked with the building and mediation of iron-sulfur [Fe-S]-clusters. These are crucial for [Fe-S]-enzymes, which are involved in essential biological cell processes like metabolism or ion transport. Analysis of ISCU in rainbow trout (Oncorhynchus mykiss) and maraena whitefish (Coregonus maraena) revealed the existence of two gene variants in each of the two salmonids. This study presents the characterization of the duplicated ISCU cDNA sequences in both species as well as the comparative functional analysis of the genes in healthy and affected fish of two rainbow trout strains differing in trait robustness under regional aquaculture conditions. Coding sequences of trout ISCUA and ISCUB genes are spanning over five exons. Open reading frames (ORF) of trout (ISCUA: 495bp, ISCUB: 498bp) and whitefish (ISCUA and ISCUB: 495bp) genes encode for evolutionary highly conserved proteins and share 72% sequence similarity with human ISCU. Transcriptome analyses comparing healthy fish of the local rainbow trout strain BORN and the import strain TCO revealed strain-specific expression patterns for ISCU. Expression analyses by quantitative RT-PCR indicated remarkable differences between the transcript level of the gene variants ISCUA and ISCUB. Moderate temperature challenge (8°C and 23°C) suggests a generally higher transcript level of the two gene variants at 8°C in the liver, spleen, and gill of both strains. However, no remarkable differences between the strains occurred in the temperature-dependent ISCU gene expression profiles. The experimental infection with Aeromonas salmonicida resulted in a different ISCU gene expression in the gill and trunk kidney of both strains after two weeks, suggesting a specific role of the scaffold gene in rainbow trout strain BORN, regarding the recovery after infection. Although results partially reflect the expected strain- and tissue-specific ISCUA and ISCUB regulation in rainbow trout, the data do not support the assumed association of ISCU with the trait robustness., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Virulence factors for Aeromonas salmonicida subsp. salmonicida (ASS) strains isolated from cultured turbot Psetta maxima L. are unknown with regard to this host. The presence of virulence genes associated with different stages of ASS infection in salmonids (vapA, tapA, fla, ascV, ascC, aexT, satA and aspA) was analysed using a polymerase chain reaction (PCR) technique in ASS strains isolated from turbot. Other ASS strains isolated from salmonids and environmental A. salmonicida (AS) strains were included for comparison. The presence of the genes was evaluated with respect to ASS virulence in turbot based on intraperitoneal and bath challenges. The genetic profile, including all of the genes studied, that was linked to virulent behaviour after intraperitoneal challenge was significantly more frequent in strains isolated from turbot than in those from salmonids or the environment. The data prove that it is not possible to predict the virulence of ASS in turbot based only on the presence of all genes tested. Moreover, the combined PCR results of vapA, aexT, ascV and ascC were useful for separating most of the ASS from environmental A. salmonicida strains. An association between virulence or genetic profile and the geographical or facility origin of the strains was not found.

This work describes an efficient, simple, and green bioprocess for obtaining 5-halogenated pyrimidine nucleosides from thymidine by transglycosylation using whole cells. Biosynthesis of 5-fluoro-2'-deoxyuridine (floxuridine) was achieved by free and immobilized Aeromonas salmonicida ATCC 27013 with an 80% and 65% conversion occurring in 1 h, respectively. The immobilized biocatalyst was stable for more than 4 months in storage conditions (4 °C) and could be reused at least 30 times without loss of its activity. This microorganism was able to biosynthesize 2.0 mg L(-1) min(-1) (60%) of 5-chloro-2'-deoxyuridine in 3 h. These halogenated pyrimidine 2'-deoxynucleosides are used as antitumoral agents., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Mass spectrometry provides a rapid and reliable method for characterization of bacterial polysaccharides. Application of the in-source fragmentation technique to promote the formation of structurally relevant repeating units of heterogeneous capsular polysaccharides and O-chain polysaccharides has proven to be particularly useful for detection of non-carbohydrate functionalities and subtle differences arising across bacterial serotypes. Here we discuss application of these methods to the direct analysis of bacterial cells allowing for rapid analysis of cell surface polysaccharide antigens and providing a basis for serological typing and epidemiological surveillance studies of human and animal pathogens.

The core lipopolysaccharide (LPS) of Aeromonas hydrophila AH-3 and Aeromonas salmonicida A450 is characterized by the presence of the pentasaccharide alpha-d-GlcN-(1-->7)-l-alpha-d-Hep-(1-->2)-l-alpha-d-Hep-(1-->3)-l-alpha-d-Hep-(1-->5)-alpha-Kdo. Previously it has been suggested that the WahA protein is involved in the incorporation of GlcN residue to outer core LPS. The WahA protein contains two domains: a glycosyltransferase and a carbohydrate esterase. In this work we demonstrate that the independent expression of the WahA glycosyltransferase domain catalyzes the incorporation of GlcNAc from UDP-GlcNAc to the outer core LPS. Independent expression of the carbohydrate esterase domain leads to the deacetylation of the GlcNAc residue to GlcN. Thus, the WahA is the first described bifunctional glycosyltransferase enzyme involved in the biosynthesis of core LPS. By contrast in Enterobacteriaceae containing GlcN in their outer core LPS the two reactions are performed by two different enzymes.

Plasmids pRAS3.1 and pRAS3.2 are two closely related, natural variants of the IncQ-2 plasmid family that have identical plasmid backbones except for two differences. Plasmid pRAS3.1 has five 6-bp repeat sequences in the promoter region of the mobB gene and four 22-bp iterons in its oriV region, whereas pRAS3.2 has only four 6-bp repeats and three 22-bp iterons. Plasmid pRAS3.1 was found to have a higher copy number than pRAS3.2, and we show that the extra 6-bp repeat results in an increase in mobB and downstream mobA/repB expression. Placement of repB (primase) behind an arabinose-inducible promoter in trans resulted in an increase in repB expression and an approximately twofold increase in the copy number of plasmids with identical numbers of 22-bp iterons. The pRAS3 plasmids were shown to have a previously unrecognized toxin-antitoxin plasmid stability module within their replicons. The ability of the pRAS3 plasmids to mobilize the oriT regions of two other plasmids of the IncQ-2 family, pTF-FC2 and pTC-F14, suggested that the mobilization proteins pRAS3 are relaxed and can mobilize oriT regions with substantially different sequences. Plasmids pRAS3.1 and pRAS3.2 were highly incompatible with plasmids pTF-FC2 and pTC-F14, and this incompatibility was removed on inactivation of an open reading frame situated downstream of the mobCDE mobilization genes rather than being due to the 22-bp oriV-associated iterons. We propose that the pRAS3 plasmids represent a third, gamma incompatibility group within the IncQ-2 family plasmids.

Comparison between the lipopolysaccharide (LPS) core structures of Aeromonas salmonicida subsp. salmonicida A450 and Aeromonas hydrophila AH-3 shows great similarity in the inner LPS core and part of the outer LPS core but some differences in the distal part of the outer LPS core (residues ld-Hep, d-Gal, and d-GalNAc). The three genomic regions encoding LPS core biosynthetic genes in A. salmonicida A450, of which regions 2 and 3 have genes identical to those of A. hydrophila AH-3, were fully sequenced. A. salmonicida A450 region 1 showed seven genes: three identical to those of A. hydrophila AH-3, three similar but not identical to those of A. hydrophila AH-3, and one without any homology to any well-characterized gene. A. salmonicida A450 mutants with alterations in the genes that were not identical to those of A. hydrophila AH-3 were constructed, and their LPS core structures were fully elucidated. At the same time, all the A. salmonicida A450 genes identical to those of A. hydrophila AH-3 were used to complement the previously obtained A. hydrophila AH-3 mutants for each of these genes. Combining the gene sequence and complementation test data with the structural data and phenotypic characterization of the mutant LPSs enabled a presumptive assignment of all LPS core biosynthesis gene functions in A. salmonicida A450. Furthermore, hybridization studies with internal probes for the A. salmonicida-specific genes using different A. salmonicida strains (strains of different subspecies or atypical strains) showed a unique or prevalent LPS core type, which is the one fully characterized for A. salmonicida A450.

In this study we applied the Fur titration assay (FURTA) to isolate iron regulated genes in Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis in fish. We have identified genes for siderophore biosynthesis and for ferri-siderophore transport, some of which have been previously described in this species. A gene for a hitherto uncharacterized functional hemin receptor HutE was identified, but its inactivation did not affect significantly the use of hemin as sole iron source, suggesting that redundant genes encoding hemin receptors exist in the A. salmonicida genome. Additional FURTA positive clones contained genes encoding a Flavoprotein, a transcriptional regulator protein, a DNA binding protein Fis, as well as genes encoding putative DapD and Flp/Fap-pilin proteins. A screening of gene distribution demonstrated that all the analyzed strains shared genes for siderophore biosynthesis and transport and for heme utilization, indicating that these two systems of iron acquisition are a conserved trait in this subspecies, whereas other genes are not common to all the isolates. Thus, the Fur regulon of A. salmonicida subsp. salmonicida includes both conserved and differentially occurring genes, suggesting that the genetic diversity within this fish pathogen might be higher than expected.

Strain H1, Aeromonas salmonicida ssp. masoucida isolated from an ulcer on the sea cucumber Apostichopus japonicus proved to be virulent toward this holothurian at 12 degrees C. Extracellular products (ECP) of strain H1 were prepared, and an extracellular peptidase of 42 kDa was purified from the ECP using Sephadex G-100 gel filtration chromatography and diethylaminoethyl Sepharose ion exchange chromatography. Stepwise virulence tests on sea cucumbers indicated that the 42 kDa peptidase was the virulence factor of strain H1, and its LD50 for these holothurians was 1.12 microg g(-1). A peptidase activity test using azocasein as a substrate showed that optimum pH and temperature for the peptidase are 8.0 and 40 degrees C, respectively; the peptidase was completely inactivated above 70 degrees C. The peptidase was inhibited strongly by 5 mM phenylmethanesulphonyl fluoride, indicating that the enzyme is a serine peptidase.

The sea lamprey (Petromyzon marinus) is a parasitic cartilaginous fish of the North American Great Lakes and a predator of many bony fish species of commercial importance to the fishing industry. Mannose-binding C-type lectin (MBL) was isolated by mannan-agarose affinity chromatography from sea lamprey plasma. Mannose-binding lectin has not before been identified and quantitated in the plasma of this sea lamprey species. The affinity-purified and 2-ME reduced lamprey MBL showed two bands of 35kDa and 65kDa by SDS-PAGE and Western blotting using guinea pig anti-MBL IgG as the primary antibody. Amino acid composition analysis (mol%) of the purified lamprey MBL found high amounts of histidine, threonine, tyrosine and phenylalanine present when compared with three other vertebrate MBLs. N-terminal amino acid sequencing by Edman degradation for the first 10 residues gave XXXTKGCPDA. Lamprey plasma contained 261mug of MBL/ml of plasma. Plasma protein concentration was 40.1mg/ml. Lamprey MBL was present then in plasma at 6.5mug MBL/mg total protein. The sea lamprey MBL also specifically binds to mannose on the surface of the pathogen Aeromonas salmonicida. The presence of MBL in high concentration in lamprey plasma could be important in their innate immunity and resistance to infection. This study describes the presence of MBL in sea lamprey plasma and evidence for a C-type lectin complement pathway of innate immunity.

Aeromonas salmonicida subsp. salmonicida, the causative agent of furunculosis in fish, can use heme as the sole iron source. We applied the Fur Titration Assay to isolate a cluster including six genes hutAZXBCD that showed similarity to heme uptake genes of other Gram-negative bacteria, and three genes orf123 of unknown function. The spatial organization of these nine genes, arranged in five transcriptional units, was similar to that of a homologous cluster in A. hydrophila. When a TonB system was provided, this cluster allowed Escherichia coli 101ESD (an ent mutant, unable to synthesize enterobactin) to utilize hemin and hemoglobin as iron sources. Mutation of hutB, a gene that encodes a predicted periplasmic hemin-binding protein, caused a drastic defect in the ability of A. salmonicida to grow with hemin as unique source of iron. Interestingly, a mutant for hutA gene (encoding the outer membrane hemin receptor) showed initially a reduced ability to grow with hemin as sole iron source, but after 24 h it achieved growth levels similar to parental strain. Thus mutation of hutA could not abolish the growth with hemin as iron source, suggesting that redundant outer membrane heme transport functions might be encoded in the A. salmonicida genome.

Aeromonas salmonicida subsp. salmonicida, the etiological agent of furunculosis in fish, produces a catechol-type siderophore under iron-limiting conditions. In this study, the Fur titration assay (FURTA) was used to identify a cluster of six genes, asbG, asbF, asbD, asbC, asbB, and asbI, encoding proteins similar to components of the siderophore biosynthetic machinery in other bacteria. Reverse transcriptase PCR analyses showed that this cluster consists of four iron-regulated transcriptional units. Mutants with deletions in either asbD (encoding a multidomain nonribosomal peptide synthetase), asbG (encoding a histidine decarboxylase), or asbC (encoding a predicted histamine monooxygenase) did not grow under iron-limiting conditions and did not produce siderophores. Growth of the DeltaasbG strain under iron starvation conditions was restored by addition of histamine, suggesting that the siderophore in this species could contain a histamine-derived moiety. None of the mutants could grow in the presence of transferrin, indicating that A. salmonicida uses the catechol-type siderophore for removal of iron from transferrin rather than relying on a receptor for this iron-binding protein. All 18 A. salmonicida strains analyzed by DNA probe hybridization were positive in tests for the presence of the asbD gene, and all of them promoted the growth of asbD, asbG, and asbC mutants, suggesting that this siderophore-mediated iron uptake system is conserved among A. salmonicida isolates. This study provides the first description of siderophore biosynthesis genes in this fish pathogen, and the results demonstrate that the asbD, asbG, and asbC genes are necessary for the production of a catecholate siderophore that is essential for the growth of A. salmonicida under iron limitation conditions.

Aeromonas salmonicida subsp. salmonicida, a bacterial pathogen of Atlantic salmon, has no visible pili, yet its genome contains genes for three type IV pilus systems. One system, Tap, is similar to the Pseudomonas aeruginosa Pil system, and a second, Flp, resembles the Actinobacillus actinomycetemcomitans Flp pilus, while the third has homology to the mannose-sensitive hemagglutinin pilus of Vibrio cholerae. The latter system is likely nonfunctional since eight genes, including the gene encoding the main pilin subunit, are deleted compared with the orthologous V. cholerae locus. The first two systems were characterized to investigate their expression and role in pathogenesis. The pili of A. salmonicida subsp. salmonicida were imaged using atomic force microscopy and Tap- and Flp-overexpressing strains. The Tap pili appeared to be polar, while the Flp pili appeared to be peritrichous. Strains deficient in tap and/or flp were used in live bacterial challenges of Atlantic salmon, which showed that the Tap pilus made a moderate contribution to virulence, while the Flp pilus made little or no contribution. Delivery of the tap mutant by immersion resulted in reduced cumulative morbidity compared with the cumulative morbidity observed with the wild-type strain; however, delivery by intraperitoneal injection resulted in cumulative morbidity similar to that of the wild type. Unlike the pili of other piliated bacterial pathogens, A. salmonicida subsp. salmonicida type IV pili are not absolutely required for virulence in Atlantic salmon. Significant differences in the behavior of the two mutant strains indicated that the two pilus systems are not redundant.

The cell envelope of Aeromonas salmonicida contains a lipopolysaccharide (LPS) essential for the physical integrity and functioning of bacterial cell membrane. Using a recently developed in-source fragmentation technique, we screened 39 typical and atypical isolates of A. salmonicida and established their O-chain polysaccharide structure by capillary electrophoresis-mass spectrometry (CE-MS), compositional and linkage analyses and comparison to the previously determined O-chain polysaccharide structure of A. salmonicida strain A449. These studies have demonstrated that A. salmonicida isolates fall into three distinct structural types, types A-C, based on chemical structures of their respective O-chain polysaccharide components. Subsequent immunoblotting and serological studies with salmon polyclonal antisera produced to formalin-fixed cells of A. salmonicida strains A449, N4705 and 33659 representing three structural types A-C revealed that variations in the O-chain polysaccharide structure have led to significant serological differences between strains belonging to type A and non-type A, where non-type A species include chemically separated structural types B and C. Due to the presence of common antigenic determinants shared by their respective O-chain polysaccharide components, serological cross-reactions were observed between A. salmonicida strains belonging to structural types B and C. These findings suggest the possibility of developing LPS-based classification system of A. salmonicida sub-species consisting of two serologically distinct types, type A and non-type A.

Aeromonas salmonicida subsp. salmonicida contains a functional type III secretion system that is responsible for the secretion of the ADP-ribosylating toxin AexT. In this study, the authors identified AopP as a second effector protein secreted by this system. The aopP gene was detected in both typical and atypical A. salmonicida isolates and was found to be encoded on a small plasmid of approximately 6.4 kb. Sequence analysis indicates that AopP is a member of the YopJ family of effector proteins, a group of proteins that interfere with mitogen-activated protein kinase (MAPK) and/or nuclear factor kappa B (NF-kappaB) signalling pathways. AopP inhibits the NF-kappaB pathway downstream of IkappaB kinase (IKK) activation, while a catalytically inactivated mutant, AopPC177A, does not possess this inhibitory effect. Unlike other effectors of the YopJ family, such as YopJ and VopA, AopP does not inhibit the MAPK signalling pathway.

The recently described type III secretion system (TTSS) of Aeromonas salmonicida subsp. salmonicida has been linked to virulence in salmonids. In this study, three TTSS effector genes, aexT, aopH or aopO, were inactivated by deletion, as was ascC, the gene encoding the outer-membrane pore of the secretion apparatus. Effects on virulence were assayed by live challenge of Atlantic salmon (Salmo salar). The DeltaascC mutant strain was avirulent by both intraperitoneal (i.p.) injection and immersion, did not appear to establish a clinically inapparent infection and did not confer protection from subsequent rechallenge with the parental strain. 1H NMR spectroscopy-based metabolite profiling of plasma from all fish showed significant differences in the metabolite profiles between the animals exposed to the parental strain or DeltaascC. The experimental infection by immersion with DeltaaopO was indistinguishable from that of the parental strain, that of DeltaaexT was delayed, whilst the virulence of DeltaaopH was reduced significantly but not abolished. By i.p. injection, DeltaaexT, DeltaaopH and DeltaaopO caused an experimental disease indistinguishable from that of the parental strain. These data demonstrate that while the TTSS is absolutely essential for virulence of A. salmonicida subsp. salmonicida in Atlantic salmon, removal of individual effectors has little influence on virulence but has a significant effect on colonization. The DeltaascC i.p. injection data also suggest that in addition to host invasion there is a second step in A. salmonicida pathogenesis that requires an active TTSS.