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RNA (New York, N.Y.) Sep 2023Unique chemical and physical properties are introduced by inserting selenocysteine (Sec) at specific sites within proteins. Recombinant and facile production of...
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 using translation components. tRNA was mutated to resemble tRNA to allow recognition by seryl-tRNA synthetase as well as 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.
Topics: Codon, Terminator; Saccharomyces cerevisiae; Aeromonas salmonicida; Protein Engineering; RNA, Transfer, Cys; Humans; Nucleic Acid Conformation
PubMed: 37279998
DOI: 10.1261/rna.079658.123 -
Microorganisms Nov 2023The plasmid known as pAsa5 is present in subsp. , a fish pathogen. The pAsa5 plasmid carries genes that are essential for the bacterium's virulence. Recombination...
The plasmid known as pAsa5 is present in subsp. , a fish pathogen. The pAsa5 plasmid carries genes that are essential for the bacterium's virulence. Recombination events are known to occur in pAsa5, resulting in the loss of certain segments or the acquisition of additional genetic elements. For example, the transposon carried by the large pAsa8 plasmid was found to be inserted into the pAsa5 plasmid in the SHY16-3432 strain, enabling the addition of antibiotic resistance genes to this plasmid, which does not normally possess any. In this study, we present the isolation of additional strains carrying pAsa8. Further analyses of these strains revealed that a fusion between pAsa5 and the complete version of pAsa8 is possible. The pAsa8 transposon insertion in pAsa5 seen in the SHY16-3432 strain appears to be an aberrant event compared to the fusion of the two full-length plasmids. A 22-nucleotide sequence, present in both plasmids, serves as the site for the fusion of the two plasmids. Moreover, it is possible to introduce pAsa8 through conjugation into naive strains of subsp. and once the plasmid is within a new strain, the fusion with pAsa5 is detectable. This study reveals a previously unexplored aspect of pAsa5 plasmid biology, highlighting an additional risk for the spread of antibiotic resistance genes in subsp.
PubMed: 38004697
DOI: 10.3390/microorganisms11112685 -
Fish & Shellfish Immunology Aug 2023In recent years, more than one pathogenic organism has usually been isolated from diseased turbot Scophthalmus maximus, creating a pressing need for the development of...
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-1β, 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.
Topics: Animals; Enterobacteriaceae Infections; Edwardsiella tarda; Aeromonas salmonicida; Flatfishes; Antibodies, Bacterial; Vaccines, Combined; Fish Diseases; Bacterial Vaccines
PubMed: 37269913
DOI: 10.1016/j.fsi.2023.108837 -
International Microbiology : the... Dec 2023Aeromonas salmonicida is the typical pathogen causing furunculosis, reported widely in salmonids. Because of multiple serotypes, the control of A. salmonicida-caused...
Aeromonas salmonicida is the typical pathogen causing furunculosis, reported widely in salmonids. Because of multiple serotypes, the control of A. salmonicida-caused disease has increasingly received much attention. Recently, A. salmonicida infection was reported in non-salmonid fish species. Here, a pathogenic A. salmonicida, named as As-s, was isolated from cultured snakehead (Channa argus) in a local fish farm in Shandong, China. As-s displayed clear hemolysis, amylase, and positive catalase activities, and grew at a wide range of temperatures (10-37 °C) and pH values (5.5-8.5). As-s was highly sensitive to cefuroxime sodium, ceftriaxone, ceftazidime, piperacillin, and cefoperazone and also apparently sensitive to chloramphenicol, erythromycin, and 25% cinnamaldehyde. The Virulence array protein gene cloning' results suggested that As-s has this gene compared with the other two vapA-containing strains, despite a close relationship of these strains via phylogenetic analysis. Severe ulcers on skin, muscle, and abnormal liver, and hemorrhage in pectoral/ventral fins and anal region were observed, and exophthalmos were also noticed in infected juvenile snakehead, as well as necrosis and infiltration of blood cells emerged in the internal organs using pathological section. In addition, As-s caused high mortality in snakehead, consistently with its immune gene response. This study reports the first isolation of vapA-absent A. salmonicida in snakehead.
PubMed: 38062211
DOI: 10.1007/s10123-023-00455-w -
Microorganisms Dec 2023Furunculosis, caused by , poses a significant threat to both salmonid and non-salmonid fish in diverse aquatic environments. This study explores the genomic intricacies...
Furunculosis, caused by , poses a significant threat to both salmonid and non-salmonid fish in diverse aquatic environments. This study explores the genomic intricacies of re-emergent outbreaks in Atlantic salmon (). Previous clinical cases have exhibited pathological characteristics, such as periorbital hemorrhages and gastrointestinal abnormalities. Genomic sequencing of three Chilean isolates (ASA04, ASA05, and CIBA_5017) and 25 previously described genomes determined the pan-genome, phylogenomics, insertion sequences, and restriction-modification systems. Unique gene families have contributed to an improved understanding of the psychrophilic and mesophilic clades, while phylogenomic analysis has been used to identify mesophilic and psychrophilic strains, thereby further differentiating between typical and atypical psychrophilic isolates. Diverse insertion sequences and restriction-modification patterns have highlighted genomic structural differences, and virulence factor predictions can emphasize exotoxin disparities, especially between psychrophilic and mesophilic strains. Thus, a novel plasmid was characterized which emphasized the role of plasmids in virulence and antibiotic resistance. The analysis of antibiotic resistance factors revealed resistance against various drug classes in Chilean strains. Overall, this study elucidates the genomic dynamics of re-emergent and provides novel insights into their virulence, antibiotic resistance, and population structure.
PubMed: 38257891
DOI: 10.3390/microorganisms12010064 -
Microbial Pathogenesis Dec 2023Zebrafish (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...
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 (10 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.
Topics: Animals; Siderophores; Zebrafish; Fluorescent Dyes; Green Fluorescent Proteins; Aeromonas salmonicida; Fish Diseases
PubMed: 37858632
DOI: 10.1016/j.micpath.2023.106394 -
Virulence Dec 2023Active flavins derived from riboflavin (vitamin B) are essential for life. Bacteria biosynthesize riboflavin or scavenge it through uptake systems, and both mechanisms...
Active flavins derived from riboflavin (vitamin B) 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. , 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 riboflavin provision pathways. Homology search and transcriptional orchestration analysis showed that has a main riboflavin biosynthetic operon that includes , , , and genes. Outside the main operon, putative duplicated genes , and , and a riboflavin importer encoding gene, were found. Monocistronic mRNA , and encode for their corresponding functional riboflavin biosynthetic enzyme. While the product of conserved the RibB function, it lacked the RibA function. Likewise, 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. was downregulated in response to external riboflavin, suggesting negative feedback. Deletion of , and showed that these genes are required for riboflavin biosynthesis and virulence in Atlantic lumpfish (). riboflavin auxotrophic attenuated mutants conferred low protection to lumpfish against virulent . Overall, has multiple riboflavin endowment forms, and duplicated riboflavin provision genes are critical for infection.
Topics: Animals; Aeromonas salmonicida; Gene Duplication; Virulence; Riboflavin; Fishes; Fish Diseases
PubMed: 36895132
DOI: 10.1080/21505594.2023.2187025 -
Virus Research Sep 2023Aeromonas salmonicida subsp. salmonicida is a Gam-negative bacterium responsible for furunculosis in fish. Because this aquatic bacterial pathogen has a rich reservoir...
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.
Topics: Animals; Bacteriophages; Furunculosis; Prophages; Aeromonas salmonicida; Fishes
PubMed: 37385348
DOI: 10.1016/j.virusres.2023.199165 -
Microorganisms Oct 2023is the causative agent of septicemia in fish, and it is associated with significant economic losses in the aquaculture industry. While piscine infections are mainly...
is the causative agent of septicemia in fish, and it is associated with significant economic losses in the aquaculture industry. While piscine infections are mainly treated with antibiotics, the emergence of resistance in bacterial populations requires the development of alternative methods of treatment. The use of phages can be one of them. A novel jumbo phage, AerS_266, was isolated and characterized. This phage infects only mesophilic strains and demonstrates a slow lytic life cycle. Its genome contains 243,674 bp and 253 putative genes: 84 encode proteins with predicted functions, and 3 correspond to tRNAs. Genes encoding two multisubunit RNA polymerases, chimallin and PhuZ, were identified, and AerS_266 was thus defined as a phiKZ-like phage. While similar phages with genomes >200 kb specific to and have been previously described, AerS_266 is the first phiKZ-like phage found to infect .
PubMed: 38004661
DOI: 10.3390/microorganisms11112649 -
Cellular and Molecular Biology... Oct 2023The present study was designed to assess the influence of dietary supplementation with chestnut bee pollen at various levels in rainbow trout, Oncorhynchus mykiss. For...
Dietary chestnut bee pollen as an immunostimulant for rainbow trout (Oncorhynchus mykiss): Effects on the growth, haematological values, immune response, oxidant/antioxidant status, and survival against Aeromonas salmonicida subsp. achromogenes.
The present study was designed to assess the influence of dietary supplementation with chestnut bee pollen at various levels in rainbow trout, Oncorhynchus mykiss. For two weeks feeding period, a total of 300 fish were allocated into 12 fiberglass tanks and divided into four equal groups, three replicates each, with chestnut bee pollen (BP) dietary inclusion as follows; the fish group was given a basal diet (C); fish group fed a diet supplemented with BP 1% (BP-1); fish group fed a diet supplemented with BP 2% (BP-2); and fish group fed a diet supplemented with BP 4% (BP-3). At the end of the experiment, growth, haematological values, immune status, antioxidant status, and survival rate against Aeromonas salmonicida subsp. achromogenes were evaluated. Dietary supplementation with chestnut bee pollen significantly improves growth performance. Fish fed the diets containing chestnut bee pollen had higher the haematological values than those fed the control diet. The results showed that all the immunological parameters in the groups fed with chestnut bee pollen were significantly higher when compared to the control group. Moreover, dietary chestnut bee pollen increased disease resistance against Aeromonas salmonicida subsp. achromogenes compared to the control group. The tissue SOD, CAT and GSH-Px activities of groups fed with chestnut bee pollen significantly enhanced when compared with the control groups. In contrast, the tissue MDA levels in all groups fed with chestnut bee pollen were significantly decreased. The best values for the antioxidant parameters were determined in the groups fed with 2 and 4% of chestnut bee pollen. Overall, these findings suggest that dietary chestnut bee pollen enhances the growth, the haematological values, the immune and antioxidant response and increases disease resistance against rainbow trout.
Topics: Animals; Bees; Antioxidants; Oncorhynchus mykiss; Disease Resistance; Oxidants; Adjuvants, Immunologic; Dietary Supplements; Diet; Pollen; Animal Feed; Fish Diseases
PubMed: 37953565
DOI: 10.14715/cmb/2023.69.10.26