Revealing extracellular protein profile and excavating spoilage-related proteases of Aeromonas salmonicida based on multi-omics investigation.

Aeromonas is a ubiquitous aquatic bacteria, and it is a significant factor contributing to meat spoilage during processing and consumption. The abilities of Aeromonas salmonicida 29 and 57, which exhibit spoilage heterogeneity, to secrete protease, lipase, hemolysin, gelatinase, amylase, and lecithinase were confirmed by plate method. A total of 3948 proteins were identified by ITRAQ in extracellular secretions of A. salmonicida, and 16 proteases were found to be potentially related to spoilage ability. The complete genome sequence of A. salmonicida 57 consists of one circular chromosome and three plasmids, while A. salmonicida 29 consists of one circular chromosome, without a plasmid. Transcriptomic analysis revealed a significant number of DEGs were up-regulated in A. salmonicida 29, which were mainly enriched in metabolic pathways (e.g., amino acid metabolism, carbohydrate metabolism), indicating that A. salmonicida 29 had better potential to decompose and utilize nutrients in meat. Six protease genes (2 pepB, hap, pepA, ftsI, and pepD) were excavated by combined ITRAQ with transcriptome analysis, which potentially contribute to bacterial spoilage ability and exhibit universality among other dominant spoilage bacteria. This investigation provides new insights and evidence for elucidating metabolic and spoilage phenotypic differences and provides candidate genes and strategies for future prevention and control technology development.

Transcriptomic analysis of turbot (Scophthalmus maximus) treated with zymosan a reveals that lncRNAs and inflammation-related genes mediate the protection conferred against Aeromonas salmonicida.

Aeromonas salmonicida is one of the most harmful pathogens in finfish aquaculture worldwide. Immunostimulants such as ß-glucans are used to enhance the immunity of cultured fish. However, their effects on fish physiology are not completely understood. In the present work, we evaluated the effect of a single intraperitoneal (ip) injection of zymosan A on fish survival against A. salmonicida infection. A single administration of this compound protected fish against A. salmonicida challenge and reduce the bacterial load in the head kidney one week after its administration. Transcriptome analyses of head kidney samples revealed several molecular mechanisms involved in the protection conferred by zymosan A and their regulation by long noncoding RNAs. The transcriptome profile of turbot exposed only to zymosan A was practically unaltered one week after ip injection. However, the administration of this immunostimulant induced significant transcriptomic changes once the fish were in contact with the bacteria and increased the survival of the infected turbot. Our results suggest that the restraint of the infection-induced inflammatory response, the management of apoptotic cell death, cell plasticity and cellular processes involving cytoskeleton dynamics support the protective effects of zymosan A. All this information provides insights on the cellular and molecular mechanisms involved in the protective effects of this widely used immunostimulant.

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.

Aeromonas salmonicida, causative agent of salmonid furunculosis, isolated from the freshwater parasitic copepod, Salmincola californiensis.

Here, we provide evidence that the freshwater parasitic copepod, Salmincola californiensis, acts as a vector for Aeromonas salmonicida. While investigating the effects of S. californiensis on Chinoook salmon (Oncorhynchus tshawytscha), we tangentially observed that fish infected with the copepod developed furunculosis, caused by A. salmonicida. This occurred despite being reared in pathogen-free well water in a research facility with no prior history of spontaneous infection. We further investigated the possibility of S. californiensis to serve as a vector for the bacterium via detection of fluorescently labelled A. salmonicida inside the egg sacs from copepods in which the fish hosts were experimentally infected with GFP-A449 A. salmonicida. We then evaluated copepod egg sacs that were collected from adult Chinook salmon from a freshwater hatchery with A. salmonicida infections confirmed by either culture or PCR. The bacterium was cultured on tryptic soy agar plates from 75% of the egg sacs, and 61% were positive by PCR. These three separate experiments indicate an alternative tactic of transmission in addition to direct transmission of A. salmonicida in captivity. The copepod may play an important role in transmission of the bacterium when fish are more dispersed, such as in the wild.

Detection of Aeromonas salmonicida subsp. salmonicida infection in zebrafish by labelling bacteria with GFP and a fluorescent probe based on the siderophore amonabactin.

Zebrafish (Danio rerio) is an excellent model to study bacterial infections in fish and their treatment. We used zebrafish as a model of infection for Aeromonas salmonicida subsp. salmonicida (hereinafter A. salmonicida), the causative agent of fish furunculosis. The infection process of A. salmonicida was studied by immersion of zebrafish larvae in 2 different doses of the bacteria and the fish mortality was monitored for three days. The bacterium caused a high mortality (65 %) in zebrafish larvae only when they were exposed to a high bacterial concentration (107 bacterial cells/mL). To evaluate the use of fluorescence microscopy to follow A. salmonicida infection in vivo, two different fluorescent strains generated by labeling an A. salmonicida strain with either, the green fluorescent protein (GFP), or with a previously reported siderophore amonabactin-sulforhodamine B conjugate (AMB-SRB), were used. The distribution of both labeled bacterial strains in the larvae tissues was evaluated by conventional and confocal fluorescence microscopy. The fluorescent signal showed a greater intensity with the GFP-labeled bacteria, so it could be observed using conventional fluorescence microscopy. Since the AMB-SRB labeled bacteria showed a weaker signal, the larvae were imaged using a laser scanning confocal microscope after 48 h of exposure to the bacteria. Both fluorescent signals were mainly observed in the larvae digestive tract, suggesting that this is the main colonization route of zebrafish for waterborne A. salmonicida. This is the first report of the use of a siderophore-fluorophore conjugate to study a bacterial infection in fish. The use of a siderophore-fluorophore conjugate has the advantage that it is a specific marker and that does not require genetic manipulation of the bacteria.

Transcriptome analysis of turbot (Scophthalmus maximus) kidney responses to inactivated bivalent vaccine against Aeromonas salmonicida and Edwardsiella tarda.

Turbot (Scophthalmus maximus) is a commercially important marine flatfish for global aquaculture. With intensive farming, turbot production is limited by several diseases, in which Aeromonas salmonicida and Edwardsiella tarda are two main causative agents. Vaccination is an effective and safe alternative to disease prevention compared to antibiotic treatment. In the previous study, we developed an inactivated bivalent vaccine against A. salmonicida and E. tarda with relative percent survival (RPS) of 77.1 %. To understand the protection mechanism in molecular basis of the inactivated bivalent vaccine against A. salmonicida and E. tarda, we use RNA-seq to analyze the transcriptomic profile of the kidney tissue after immunization. A total of 391,721,176 clean reads were generated in nine libraries by RNA-seq, and 96.35 % of the clean reads were mapped to the reference genome of S. maximus. 1458 (866 upregulated and 592 downregulated) and 2220 (1131 upregulated and 1089 downregulated) differentially expressed genes (DEGs) were obtained at 2 and 4 weeks post-vaccination, respectively. The DEGs were enriched in several important immune-related GO terms, including cytokine activity, immune response, and defense response. In addition, the analysis of several immune-related genes showed upregulation and downregulation, including pattern recognition receptors, complement system, cytokines, chemokines and immune cell surface markers. Eight DEGs (ccr10, calr, casr, mybpha, cd28, thr18, cd20a.3 and c5) were randomly selected for qRT-PCR analysis, which confirmed the validity of the RNA-seq. Our results provide valuable insight into the immune mechanism of inactivated bivalent vaccine against A. salmonicida and E. tarda in Scophthalmus maximus.

Efficacy of bivalent vaccine against Aeromonas salmonicida and Edwardsiella tarda infections in turbot.

In recent years, more than one pathogenic organism has usually been isolated from diseased turbot Scophthalmus maximus, creating a pressing need for the development of combination vaccines to prevent fish diseases brought on simultaneously by various infections. In this study, the inactivated bivalent vaccine of Aeromonas salmonicida and Edwardsiella tarda was prepared by the formalin inactivation method. After challenge with A. salmonicida and E. tarda at 4 weeks post-vaccination in turbot, the relative percentage survival (RPS) of the inactivated bivalent vaccine was 77.1%. In addition, we assessed the effects of the inactivated bivalent vaccine and evaluated the immunological processes after immunization in a turbot model. Serum antibody titer and lysozyme activity of the vaccinated group were both upregulated and higher than that in control group after vaccination. The expression levels of genes (TLR2, IL-1ß, CD4, MHCI, MHCⅡ) that related to antigen recognition, processing and presentation were also studied in the liver, spleen and kidney tissues of vaccinated turbot. All the detected genes in the vaccinated group had a significant upward trend, and most of them reached the maximum value at 3-4 weeks, which had significant differences from the control group, suggesting that antigen recognition, processing and presentation pathway was activated by the inactivated bivalent vaccine. Our study provides a basis for further application of the killed bivalent vaccine against A. salmonicida and E. tarda in turbot, making it good potential that can be applied in aquaculture.

High immunogenicity of virus-like particles (VLPs) decorated with Aeromonas salmonicida VapA antigen in rainbow trout.

The Gram-negative bacterium A. salmonicida is the causal agent of furunculosis and used to be one of the most loss-causing bacterial infections in the salmonid aquaculture industry with a mortality rate of about 90% until the 1990s, when an inactivated vaccine with mineral oil as adjuvant was successfully implemented to control the disease. However, the use of this vaccine is associated with inflammatory side effects in the peritoneal cavity as well as autoimmune reactions in Atlantic salmon, and incomplete protection has been reported in rainbow trout. We here aimed at developing and testing a recombinant alternative vaccine based on virus-like particles (VLPs) decorated with VapA, the key structural surface protein in the outer A-layer of A. salmonicida. The VLP carrier was based on either the capsid protein of a fish nodavirus, namely red grouper nervous necrotic virus (RGNNV) or the capsid protein of Acinetobacter phage AP205. The VapA and capsid proteins were expressed individually in E. coli and VapA was fused to auto-assembled VLPs using the SpyTag/SpyCatcher technology. Rainbow trout were vaccinated/immunized with the VapA-VLP vaccines by intraperitoneal injection and were challenged with A. salmonicida 7 weeks later. The VLP vaccines provided protection comparable to that of a bacterin-based vaccine and antibody response analysis demonstrated that vaccinated fish mounted a strong VapA-specific antibody response. To our knowledge, this is the first demonstration of the potential use of antigen-decorated VLPs for vaccination against a bacterial disease in salmonids.

Recoding UAG to selenocysteine in Saccharomyces cerevisiae.

Unique chemical and physical properties are introduced by inserting selenocysteine (Sec) at specific sites within proteins. Recombinant and facile production of eukaryotic selenoproteins would benefit from a yeast expression system; however, the selenoprotein biosynthetic pathway was lost in the evolution of the kingdom Fungi as it diverged from its eukaryotic relatives. Based on our previous development of efficient selenoprotein production in bacteria, we designed a novel Sec biosynthesis pathway in Saccharomyces cerevisiae using Aeromonas salmonicida translation components. S. cerevisiae tRNASer was mutated to resemble A. salmonicida tRNASec to allow recognition by S. cerevisiae seryl-tRNA synthetase as well as A. salmonicida selenocysteine synthase (SelA) and selenophosphate synthetase (SelD). Expression of these Sec pathway components was then combined with metabolic engineering of yeast to enable the production of active methionine sulfate reductase enzyme containing genetically encoded Sec. Our report is the first demonstration that yeast is capable of selenoprotein production by site-specific incorporation of Sec.

MQM1, a bacteriophage infecting strains of Aeromonas salmonicida subspecies salmonicida carrying Prophage 3.

Aeromonas salmonicida subsp. salmonicida is a Gam-negative bacterium responsible for furunculosis in fish. Because this aquatic bacterial pathogen has a rich reservoir of antibiotic-resistant genes, it is essential to investigate antibacterial alternatives, including the use of phages. Yet, we have previously demonstrated the inefficiency of a phage cocktail designed against A. salmonicida subsp. salmonicida strains due to a phage resistance phenotype associated to a prophage, namely Prophage 3. To bypass this resistance, one of the solutions is to isolate novel phages capable of infecting Prophage 3-bearing strains. Here we report on the isolation and characterization of the new virulent phage vB_AsaP_MQM1 (or MQM1), which is highly specific to A. salmonicida subsp. salmonicida strains. Phage MQM1 inhibited the growth of 01-B516, a strain carrying Prophage 3, including when combined to the previous phage cocktail. MQM1 infected 26 out of the 30 (87%) Prophage 3-bearing strains tested. Its linear dsDNA genome contains 63,343 bp, with a GC content of 50.2%. MQM1 genome can encode 88 proteins and 8 tRNAs, while no integrase or transposase-encoding genes were found. This podophage has an icosahedral capsid and a non-contractile short tail. We suggest that MQM1 may be a good addition to future phage cocktails against furunculosis to resolve the Prophage 3-resistance issue.

The Sigma Factor AsbI Is Required for the Expression of Acinetobactin Siderophore Transport Genes in Aeromonas salmonicida.

Aeromonas salmonicida subsp. salmonicida (A. salmonicida), a Gram-negative bacterium causing furunculosis in fish, produces the siderophores acinetobactin and amonabactins in order to extract iron from its hosts. While the synthesis and transport of both systems is well understood, the regulation pathways and conditions necessary for the production of each one of these siderophores are not clear. The acinetobactin gene cluster carries a gene (asbI) encoding a putative sigma factor belonging to group 4 σ factors, or, the ExtraCytoplasmic Function (ECF) group. By generating a null asbI mutant, we demonstrate that AsbI is a key regulator that controls acinetobactin acquisition in A. salmonicida, since it directly regulates the expression of the outer membrane transporter gene and other genes necessary for Fe-acinetobactin transport. Furthermore, AsbI regulatory functions are interconnected with other iron-dependent regulators, such as the Fur protein, as well as with other sigma factors in a complex regulatory network.

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.

Isolation of Aeromonas salmonicida subspecies salmonicida from aquaculture environment in India: Polyphasic identification, virulence characterization, and antibiotic susceptibility.

This study reports the polyphasic identification, characterization of virulence potential, and antibiotic susceptibility of Aeromonas salmonicida subspecies salmonicida COFCAU_AS, isolated from an aquaculture system in India. The physiological, biochemical, 16s rRNA gene sequencing and PAAS PCR test identified the strain as Aeromonas salmonicida. The MIY PCR tests established the subspecies as 'salmonicida'. The in vitro tests showed the isolated bacterium as haemolytic with casein, lipid, starch, and gelatin hydrolysis activity, indicating its pathogenic attributes. It also showed the ability to produce slime and biofilm, and additionally, it possessed an A-layer surface protein. In vivo pathogenicity test was performed to determine the LD50 dose of the bacterium in Labeo rohita fingerlings (14.42 ± 1.01 g), which was found to be 106.9 cells fish-1. The bacteria-challenged fingerlings showed skin lesions, erythema at the base of the fins, dropsy, and ulcer. Almost identical clinical signs and mortalities were observed when the same LD50 dose was injected into other Indian major carp species, L. catla and Cirrhinus mrigala. Out of the twelve virulent genes screened, the presence of nine genes viz., aerA, act, ast, alt, hlyA, vapA, exsA, fstA, and lip were detected, whereas ascV, ascC, and ela genes were absent. The A. salmonicida subsp. salmonicida COFCAU_AS was resistant to antibiotics such as penicillin G, rifampicin, ampicillin, and vancomycin while highly sensitive to amoxiclav, nalidixic acid, chloramphenicol, ciprofloxacin, and tetracycline. In summary, we have isolated a virulent A. salmonicida subsp. salmonicida from a tropical aquaculture pond which can cause significant mortality and morbidity in Indian major carp species.

Comparative genomic analysis provides insights into taxonomy and temperature adaption of Aeromonas salmonicida.

Aeromonas salmonicida has long been known as psychrophiles since it is mainly isolated from cold water fish, and recent reports have revealed the existence of mesophilic strains isolated from warm sources. However, the genetic differences between mesophilic and psychrophilic strains remain unclear due to few complete genomes of mesophilic strain are available. In this study, six A. salmonicida (2 mesophilic and 4 psychrophilic) were genome-sequenced, and comparative analyses of 25 A. salmonicida complete genomes were conducted. The ANI values and phylogenetic analysis revealed that 25 strains formed three independent clades, which were referred as typical psychrophilic, atypical psychrophilic and mesophilic groups. Comparative genomic analysis showed that two chromosomal gene clusters, related to lateral flagella and outer membrane proteins (A-layer and T2SS proteins), and insertion sequences (ISAs4, ISAs7 and ISAs29) were unique to the psychrophilic groups, while the complete MSH type IV pili were unique to the mesophilic group, all of which may be considered as lifestyle-related factors. The results of this study not only provide new insights into the classification, lifestyle adaption and pathogenic mechanism of different strains of A. salmonicida, but also contributes to the prevention and control of disease caused by psychrophilic and mesophilic A. salmonicida.

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.

Time effect of experimental infection on Rainbow trout (Oncorhynchus mykiss) by immersion with Aeromonas salmonicida subsp. salmonicida.

Furunculosis caused by Aeromonas salmonicida subsp salmonicida (Ass) is a medically and economically important bacterial disease in salmonid farms that requires therapeutic measures to prevent and control the disease. Evaluation of the effectiveness of traditional measures such as antibiotics or vaccines usually requires infecting fish experimentally. The objective of this study is to develop a method of infectious challenge of large (250-g) Rainbow trout by immersion close to natural infection conditions. We compare mortality, morbidity and anti-Ass antibody production of Rainbow trout following different bathing times (2, 4, 8 and 24 h) at a final bacterial concentration of 106 CFU/mL. One hundred sixty fish divided in five groups corresponding to the 4 bathing times and the non-challenged group were studied. The 24 h contact duration resulted in the infection of all fish, with a mortality rate of 53.25%. The challenged fish developed acute infection with symptoms and lesions (inappetance, altering of swimming behaviour, presence of boils) similar to those observed in furunculosis, and produced antibodies against the bacterium at 4 weeks after challenging, in contrast with the non-challenged group.

Aeromonas salmonicida isolates from Canada demonstrate wide distribution and clustering among mesophilic strains.

All the 36 known species to date of the genus Aeromonas are mesophilic except the species Aeromonas salmonicida, which includes both psychrophilic and mesophilic subspecies. For 20 years, more and more mesophilic A. salmonicida strains have been discovered. Only A. salmonicida subsp. pectinolytica has officially been classified as a mesophilic subspecies. Most mesophiles have been isolated in hot countries. We present, for the first time, the characterization of two new mesophilic isolates from Quebec (Canada). Phenotypic and genomic characterizations were carried out on these strains, isolated from dead fish from a fish farm. Isolates 19-K304 and 19-K308 are clearly mesophiles, virulent to the amoeba Dictyostelium discoideum, a surrogate host, and close to strain Y577, isolated in India. To our knowledge, this is the first time that mesophilic strains isolated from different countries are so similar. The major difference between the isolates is the presence of plasmid pY47-3, a cryptic plasmid that sometimes presents in mesophilic strains. More importantly, our extensive phylogenetic analysis reveals two well-defined clades of mesophilic strains with psychrophiles associated with one of these clades. This helps to have a better understanding of the evolution of this species and the apparition of psychrophilic subspecies.

Immunity of turbot Induced by inactivated vaccine of Aeromonas salmonicida from the perspective of DNA methylation.

Introduction: DNA methylation was one of the most important modification in epigenetics and played an important role in immune response. Since the introduction of Scophthalmus maximus, the scale of breeding has continued to expand, during which diseases caused by various bacteria, viruses and parasites have become increasingly serious. Therefore, the inactivated vaccines have been widely researched and used in the field of aquatic products with its unique advantages. However, the immune mechanism that occurred in turbot after immunization with inactivated vaccine of Aeromonas salmonicida was not clear. Methods: In this study, differentially methylated regions (DMRs) were screened by Whole Genome Bisulfite Sequencing (WGBS) and significantly differentially expressed genes (DEGs) were screened by Transcriptome sequencing. Double luciferase report assay and DNA pull-down assay were further verified the DNA methylation state of the gene promoter region affected genes transcriptional activity after immunization with inactivated vaccine of Aeromonas salmonicida. Results: A total of 8149 differentially methylated regions (DMRs) were screened, in which there were many immune-related genes with altered DNA methylation status. Meanwhile, 386 significantly differentially expressed genes (DEGs) were identified, many of which were significantly enriched in Toll-like receptor signaling pathway, NOD-like receptor signaling pathway and C-type lectin receptor signaling pathway. Combined analysis of WGBS results and RNA-seq results, a total of 9 DMRs of negatively regulated genes are located in the promoter region, including 2 hypermethylated genes with lower expression and 7 hypomethylated genes with higher expression. Then, two immune-related genes C5a anaphylatoxin chemotactic receptor 1-like (C5ar1-Like) and Eosinophil peroxidase-like (EPX-Like), were screened to explore the regulation mechanism of DNA methylation modification on their expression level. Moreover, the DNA methylation state of the gene promoter region affected genes transcriptional activity by inhibiting the binding of transcription factors, which lead to changes in the expression level of the gene. Discussion: We jointly analyzed WGBS and RNA-seq results and revealed the immune mechanism that occurred in turbot after immunized with inactivated vaccine of A. salmonicida from the perspective of DNA methylation.

Isolation of vB_AsaM_LPM4 reveals the dynamics of Prophage 3 in Aeromonas salmonicida subsp. salmonicida.

Aeromonas salmonicida subsp. salmonicida causes furunculosis, a major infection that affects fish farms worldwide. We isolated phage vB_AsaM_LPM4 (LPM4) from a diseased fish. Based on its DNA sequence, LPM4 is identical to the uncharacterized Prophage 3, a prophage present mostly in North American A. salmonicida subsp. salmonicida isolates that bear the genomic island AsaGEI2a. Prophage 3 and AsaGEI2a are inserted side by side in the bacterial chromosome. The LPM4/Prophage 3 sequence is similar to that of other prophages found in various members of the genus Aeromonas. LPM4 specifically infects A. salmonicida subsp. salmonicida strains that do not already bear Prophage 3. The presence of an A-layer on the surface of the bacteria is not necessary for the adsorption of phage LPM4 but seems to facilitate its infection process. We also successfully produced lysogenic strains that bear Prophage 3 using sensitive strains with different genetic backgrounds, suggesting that there is no interdependency between LPM4 and AsaGEIs. PCR analysis of the excision dynamics of Prophage 3 and AsaGEIs revealed that these genetic elements can spontaneously excise themselves from the bacterial chromosome independently of one another. Through the isolation and characterization of LPM4, this study reveals new facets of Prophage 3 and AsaGEIs.

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.