Metabolic proteins with crucial roles in Edwardsiella tarda antioxidative adaptation and intracellular proliferation.

IMPORTANCE: Edwardsiella tarda is a significant fish pathogen that can live in challenging environments of reactive oxygen species (ROS), such as inside the phagocytes. Metabolic reconfiguration has been increasingly associated with bacterial oxidative tolerance and virulence. However, the metabolic proteins of E. tarda involved in such processes remain elusive. By proteomic analysis and functional characterization of protein null mutants, the present study identified eight crucial proteins for bacterial oxidative resistance and intracellular infection. Seven of them are metabolic proteins dictating the metabolic flux toward the generation of pyruvate, a key metabolite capable of scavenging ROS molecules. Furthermore, L-aspartate uptake, which can fuel the pyruvate generation, was found essential for the full antioxidative capacity of E. tarda. These findings identified seven metabolic proteins involved in bacterial oxidative adaptation and indicate that metabolic reprogramming toward pyruvate was likely a pivotal strategy of bacteria for antioxidative adaptation and intracellular survival.

Characterization and expression profiling of fadd gene in response to exogenous Aeromonas hydrophila or Edwardsiella tarda challenge in the hybrid yellow catfish (Pelteobagrus fulvidraco ♀ × P. vachelli ♂).

In mammals, fas-associated protein with death domain (FADD) is involved in the process of cell apoptosis and plays a key role in innate immune signaling. Nevertheless, its detailed molecular mechanisms underlying apoptosis and immune responses to exogenous bacterial infections in teleosts remain largely unknown. In this study, a group of 60 hybrid yellow catfish (with the body weight of 25 ± 0.5 g) were used in subsequent experiments, we examined the expression profiling of fadd gene through comparative genomics and comparative immunological methods. Our results showed that fadd in the hybrid yellow catfish (hycfadd) exhibited similar gene and spatial structures to those in other vertebrates, and formed an independent clade in phylogeny. An expression pattern analysis revealed that hycfadd widely transcribed in various tissues, with the highest transcription level in the liver. Furthermore, expression profiling of hycfadd when intraperitoneally infected with 50 µL of exogenous Aeromonas hydrophila (2.0 × 107 CFU/mL) or Edwardsiella tarda (2.0 × 107 CFU/mL) within 48 h were significantly up-regulated in the kidney, spleen, liver and intestine. Important genes in the toll like receptor (tlr) 1-tlr2- myeloid differentiation primary response 88 (MyD88)-fadd-caspase (casp) 8 cascades of TLR signaling pathway in liver were significantly up-regulated after the A. hydrophila stimulation, suggesting that apoptosis through the TLR signaling pathway may have been triggered and activated, which were further verified in the liver, kidney, spleen, intestine and gill by a TUNEL assay. Overall, this study provides solid evidence for the bacterial induction of fadd-related apoptosis in teleosts.

Japanese Flounder pol-miR-155 Is Involved in Edwardsiella tarda Infection via ATG3.

MicroRNAs (miRNAs) are small RNA molecules that function in the post-transcriptionally regulation of the expression of diverse genes, including those involved in immune defense. Edwardsiella tarda can infect a broad range of hosts and cause severe disease in aquatic species, including Japanese flounder (Paralichthys olivaceus). In this study, we examined the regulation mechanism of a flounder miRNA, pol-miR-155, during the infection of E. tarda. Pol-miR-155 was identified to target flounder ATG3. Overexpression of pol-miR-155 or knockdown of ATG3 expression suppressed autophagy and promoted the intracellular replication of E. tarda in flounder cells. Overexpression of pol-miR-155 activated the NF-κB signaling pathway and further promoted the expression of downstream immune related genes of interleukin (IL)-6 and IL-8. These results unraveled the regulatory effect of pol-miR-155 in autophagy and in E. tarda infection.

Resistance Patterns of Frequently Applied Antimicrobials and Occurrence of Antibiotic Resistance Genes in Edwardsiella tarda Detected in Edwardsiellosis-Infected Tilapia Species of Fish Farms of Punjab in Pakistan.

Edwardsiella tarda is one of the most significant fish pathogens, causes edwardsiellosis in a variety of freshwater fish species, and its antibiotic resistance against multiple drugs has made it a health risk worldwide. In this study, we aimed to investigate the antibiotic resistance (ABR) genes of E. tarda and establish its antibiotic susceptibility. Thus, 540 fish (299 Oreochromis niloticus, 138 O.mossambicus, and 103 O. aureus) were collected randomly from twelve fish farms in three districts of Punjab in Pakistan. E. tarda was recovered from 147 fish showing symptoms of exophthalmia, hemorrhages, skin depigmentation, ascites, and bacteria-filled nodules in enlarged liver and kidney. Antimicrobial susceptibility testing proved chloramphenicol, ciprofloxacin, and streptomycin effective, but amoxicillin, erythromycin, and flumequine ineffective in controlling edwardsiellosis. Maximum occurrence of qnrA, blaTEM, and sul3 genes of E. tarda was detected in 45% in the liver, 58%, and 42% respectively in the intestine; 46.5%, 67.2%, and 55.9% respectively in O. niloticus; 24%, 36%, and 23% respectively in summer with respect to fish organs, species, and season, respectively. Motility, H2S, indole, methyl red, and glucose tests gave positive results. Overall, E. tarda infected 27.2% of fish, which ultimately caused 7.69% mortality. The Chi-squared test of independence showed a significant difference in the occurrence of ABR genes of E. tarda with respect to sampling sites. In conclusion, the misuse of antibacterial agents has led to the emergence of ABR genes in E. tarda, which in association with high temperatures cause multiple abnormalities in infected fish and ultimately resulting in massive mortality.

Cloning, DNA sequence, and expression of flagellins from high and low virulence strains of Edwardsiella tarda and their macrophage-stimulating activities.

Edwardsiella tarda is a causative pathogen of edwardsiellosis in fish. Our previous studies on high (NUF251) and low (NUF194) virulent strains of E. tarda demonstrated that NUF251 strain induced significantly higher levels of NO and TNF-α from fish and mouse macrophages than NUF194 strain. Subsequent studies suggested that a flagellin-like protein secreted from E. tarda might be a responsible factor for the macrophage-stimulating activities. To evaluate the activities of flagellins of E. tarda, in this study, the flagellin genes of NUF251 and NUF194 strains were isolated by PCR and cloned into pQE-30 and pCold I expression vectors, and then the recombinant flagellins of two strains were overexpressed in E. coli JM109 and pG-Tf/BL21, respectively. The molecular weight of the purified recombinant flagellins of NUF251 and NUF194 strains were estimated to be 45 kDa and 37 kDa, respectively on SDS-PAGE analysis. Referring the three-dimensional structure of Salmonella flagellin, which has been reported to have 4 domains (D0, D1, D2, and D3), high sequence homology between two flagellins of E. tarda was observed at conservative domain (D0 and D1) regions, whereas the sequences equivalent to D2 and D3 domains were different, and even equivalent to 57 amino acids were deleted in NUF194. Both recombinant flagellins induced NO production, mRNA expression level of inducible NO synthase (iNOS), and intercellular ROS production in mouse macrophage cell line RAW264.7 cells. Also, the secretion of TNF-α and its mRNA expression level were increased by treatment of both recombinant flagellins. These results indicate that the recombinant flagellins from different virulent E. tarda strains can stimulate macrophages with nearly equal levels as judged by the parameters tested, even though they are differences in the structure and molecular weight, suggesting that conservative D0 and D1 domains are sufficient structural elements for the recombinant flagellins to induce a certain level of macrophage-stimulation in vitro. Further studies are necessary focusing on the role of D2 and D3 domain regions of the recombinant flagellins as macrophage-stimulating agent as well as their influence on host immune system in vivo.

Edwardsiella tarda is an Important Causative Agent of Maternal-Fetal Infections in Pregnant Women: a Case Report and Japanese Literature Review.

We present a case study of a 6-day-old male infant who was referred to our hospital for fever, jaundice, and poor suckling, and was diagnosed with meningitis due to Edwardsiella tarda. We were able to detect E. tarda DNA after performing broad-range polymerase chain reaction targeting the bacterial 16S rRNA gene followed by a Basic Local Alignment Search Tool analysis of the DNA extracted from the cerebrospinal fluid. Furthermore, Japanese literature review showed that E. tarda caused perinatal invasive infections in mothers and infants and that the risk of fetal meningitis might be related to the food preference of Japanese people for raw fish. It is crucial to disseminate the information that pregnant women should refrain from eating high-risk raw fish, including freshwater fish.

Phenotypic differences between Edwardsiella piscicida and Edwardsiella anguillarum isolates in Japan.

OBJECTIVE: Edwardsiella tarda has been regarded as the causative agent of edwardsiellosis in cultured marine and freshwater fish species in Japan. Our previous study genetically classified an E. tarda-like isolate from diseased Olive Flounder Paralichthys olivaceus as E. piscicida and that from diseased Red Seabream Pagrus major as E. anguillarum. This study aimed to understand the phenotypic differences between E. piscicida and E. anguillarum. METHODS: Fourteen E. piscicida and seven E. anguillarum isolates were used in this study. The colonies of each isolate were grown on brain-heart infusion agar plates and then subjected to DNA extraction. The extracted DNA was amplified using PCR. carbohydrate fermentation of the isolates was examined using API 50 CH test kits. Moreover, the growth of the two species was examined in defined media. Also, free amino acids in Olive Flounder and Red Seabream sera were detected and quantified via high-performance liquid chromatography-mass spectrometry. Statistical differences in the concentrations of free amino acids were analyzed using Welch's t-tests. RESULT: The API 50 CH test revealed that L-arabinose and D-mannitol were fermented by E. anguillarum isolates but not E. piscicida isolates. Furthermore, the growth of E. piscicida and E. anguillarum was reduced in the defined medium without methionine and iron sulfate. The growth of E. piscicida was reduced in the defined medium without phenylalanine, tyrosine, alanine, or nicotinic acid, whereas the growth of E. anguillarum was reduced in the defined medium without serine, cysteine, leucine, threonine, or isoleucine. Tyrosine and alanine were present in higher concentrations in the Olive Flounder serum, whereas threonine and isoleucine were present in higher concentrations in the Red Seabream serum, suggesting favorable growth conditions for E. piscicida and E. anguillarum. CONCLUSION: This study characterizes a minimal defined medium that can be used for developing vaccines against E. piscicida and E. anguillarum.

Phenotypic and genotypic analysis of Edwardsiella isolates from Taiwan indicates wide variation with a particular reference to Edwardsiella tarda and Edwardsiella anguillarum.

Edwardsiella spp. is a gram-negative, facultatively anaerobic, intracellular bacteria threatening the aquaculture industry worldwide. Noticeably, E. tarda is now genotypically classified into three distinct groups (E. tarda, E. piscicida and E. anguillarum), but morphologically, it is unclear due to varying degrees of virulence in different fish hosts. Hence, to reclassify E. tarda, we investigated differences in genotypes, phenotypes and pathogenicity. We collected Edwardsiella isolates from five different counties of Taiwan between 2017 and 2021. At first, gyrB gene was amplified for a phylogenetic tree from 40 isolates from different fish and one reference isolate, BCRC10670, from the human. Thirty-nine strains clustered into E. anguillarum, 1 strain into E. piscicida and 1 strain into E. tarda from human strain. Second, all isolates were characterized using various phenotypic (API 20E biochemical profiles) and genotypic (pulsed-field gel electrophoresis [PFGE], and virulence-related gene detection). SpeI digestion revealed 10 pulsotypes and I-CeuI into 7 pulsotypes. Virulent genes (citC, gadB, katB, mukF and fimA) confirmed in 35, 31, 28, 37 and 38 isolates, respectively. Finally, in vivo challenge test in milkfish (Chanos chanos) indicated the highest mortality from E. anguillarum. Overall, results revealed unique features with Edwardsiella spp. genotypes and pathogenicity, which are relevant to the host and provide useful insights for future vaccine development.

Multiplex PCR assay for correct identification of the fish pathogenic species of Edwardsiella genus reveals the presence of E. anguillarum in South America in strains previously characterized as E. tarda.

AIMS: Develop a species-specific multiplex PCR to correctly identify Edwardsiella species in routine diagnostic for fish bacterial diseases. METHODS AND RESULTS: The genomes of 62 Edwardsiella spp. isolates available from the National Center for Biotechnology Information (NCBI) database were subjected to taxonomic and pan-genomic analyses to identify unique regions that could be exploited by species-specific PCR. The designed primers were tested against isolated Edwardsiella spp. strains, revealing errors in commercial biochemical tests for bacterial classification regarding Edwardsiella species. CONCLUSION: Some of the genomes of Edwardsiella spp. in the NCBI platform were incorrectly classified, which can lead to errors in some research. A functional mPCR was developed to differentiate between phenotypically and genetically ambiguous Edwardsiella, with which, we detected the presence of Edwardsiella anguillarum affecting fish in Brazil. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that the misclassification of Edwardsiella spp in Brazil concealed the presence of E. anguillarum in South America. Also, this review of the taxonomic classification of the Edwardsiella genus is a contribution to the field to help researchers with their sequencing and identification of genomes, showing some misclassifications in online databases that must be corrected, as well as developing an easy assay to characterize Edwardsiella species in an end-point mPCR.

AroC, a chorismate synthase, is required for the formation of Edwardsiella tarda biofilms.

Biofilms contribute to the resistance of Edwardsiella tarda to antibiotics and host immunity. AroC in the shikimate pathway produces chorismate to synthesize crucial intermediates such as indole. In this study, the differences between biofilms produced by aroC mutants (△aroC), wild-type (WT) strains, and △aroC complementary strains (C△aroC) were detected both in vitro with 96-well plates, tubes, or coverslips and in vivo using a mouse model of subcutaneous implants. When examining potential mechanisms, we found that the diameters of the movement rings in soft agar plates and the flagellar sizes and numbers determined by silver staining were all lower for △aroC than for WT and C△aroC. Moreover, qRT-PCR showed that the transcription levels of flagellar synthesis genes, fliA and fliC, were reduced in △aroC. AroC, FliC, or FliA may accompany the motility of △aroC strains. In addition, compared with the WT and C△aroC, the amounts of indole in △aroC were significantly decreased. Notably, the formation of biofilms by these strains could be promoted by exogenous indole. Therefore, the aroC gene could affect the biofilm formation of E. tarda concerning its impact on flagella and indole.

Selection of References for microRNA Quantification in Japanese Flounder (Paralichthys olivaceus) Normal Tissues and Edwardsiella tarda-Infected Livers.

MicroRNA (miRNA) plays essential roles in post-transcriptional regulation of protein coding genes, and the quantitative real-time polymerase chain reaction (qRT-PCR) is the powerful and broadly employed tool to conduct studies of miRNA expression. Identifying appropriate references to normalize quantitative data is a prerequisite to ensure the qRT-PCR accuracy. Until now, there has been no report about miRNA reference for qRT-PCR in Japanese flounder (Paralichthys olivaceus), one important marine cultured fish along the coast of Northern Asia. In this study, combined with miRNA-Seq analysis and literature search, 10 candidates (miR-34a-5p, miR-205-5p, miR-101a-3p, miR-22-3p, miR-23a-3p, miR-210-5p, miR-30c-5p, U6, 5S rRNA, and 18S rRNA) were chosen as potential references to test their expression stability among P. olivaceus tissues, and in livers of P. olivaceus infected with Edwardsiella tarda at different time points. The expression stability of these candidates was analyzed by qRT-PCR and evaluated with Delta CT, BestKeeper, geNorm, as well as NormFinder methods, and RefFinder was employed to estimate the comprehensive ranking according to the four methods. As the result, miR-22-3p and miR-23a-3p were proved to be the suitable combination as reference miRNAs for both P. olivaceus normal tissues and livers infected with E. tarda, and they were successfully applied to normalize miR-7a and miR-221-5p expression in P. olivaceus livers in response to E. tarda infection. All these results provide valuable information for P. olivaceus miRNA quantitative expression analysis in the future.

Construction of Genomic Library and Screening of Edwardsiella tarda Immunogenic Proteins for Their Protective Efficacy Against Edwardsiellosis.

Edwardsiella tarda is a severe aquaculture pathogen that can infect many hosts including humans, animals, and fish. Timely diagnosis and treatment are crucial for the control of edwardsiellosis in the aqua industry. By using rabbit polyclonal antibody, an expression gene library of virulent Edwardsiella tarda strain ED-BDU 1 isolated in south India was constructed and screened. The identified immune expressive proteins were characterized, and the corresponding coding sequences were cloned, expressed, and the purified recombinant proteins were used as antigens. The identified immunoreactive proteins namely HflC, HflK, and YhcI were studied for their immune protective potential in vivo by challenge experiments. The protective efficacy of HflC, HflK, and YhcI showed that the clearance of Edwardsiella from the host with ~ 60% survivability. Further, the immunoreactive proteins induce a strong immune response upon infection and elicit the significant production of IL-10, IFN-γ, Th1, and Th2 mediated mRNA expression and were therefore effective in vaccine production for edwardsiellosis.

Acid resistance system CadBA is implicated in acid tolerance and biofilm formation and is identified as a new virulence factor of Edwardsiella tarda.

Edwardsiella tarda is a facultative intracellular pathogen in humans and animals. The Gram-negative bacterium is widely considered a potentially important bacterial pathogen. Adaptation to acid stress is important for the transmission of intestinal microbes, so the acid-resistance (AR) system is essential. However, the AR systems of E. tarda are totally unknown. In this study, a lysine-dependent acid resistance (LDAR) system in E. tarda, CadBA, was characterized and identified. CadB is a membrane protein and shares high homology with the lysine/cadaverine antiporter. CadA contains a PLP-binding core domain and a pyridoxal phosphate-binding motif. It shares high homology with lysine decarboxylase. cadB and cadA are co-transcribed under one operon. To study the function of the cadBA operon, isogenic cadA, cadB and cadBA deletion mutant strains TX01ΔcadA, TX01ΔcadB and TX01ΔcadBA were constructed. When cultured under normal conditions, the wild type strain and three mutants exhibited the same growth performance. However, when cultured under acid conditions, the growth of three mutants, especially TX01ΔcadA, were obviously retarded, compared to the wild strain TX01, which indicates the important involvement of the cadBA operon in acid resistance. The deletion of cadB or cadA, especially cadBA, significantly attenuated bacterial activity of lysine decarboxylase, suggesting the vital participation of cadBA operon in lysine metabolism, which is closely related to acid resistance. The mutations of cadBA operon enhanced bacterial biofilm formation, especially under acid conditions. The deletions of the cadBA operon reduced bacterial adhesion and invasion to Hela cells. Consistently, the deficiency of cadBA operon abated bacterial survival and replication in macrophages, and decreased bacterial dissemination in fish tissues. Our results also show that the expression of cadBA operon and regulator cadC were up-regulated upon acid stress, and CadC rigorously regulated the expression of cadBA operon, especially under acid conditions. These findings demonstrate that the AR CadBA system was a requisite for the resistance of E. tarda against acid stress, and played a critical role in bacterial infection of host cells and in host tissues. This is the first study about the acid resistance system of E. tarda and provides new insights into the acid-resistance mechanism and pathogenesis of E. tarda.

Cytokines Induced by Edwardsiella tarda: Profile and Role in Antibacterial Immunity.

Edwardsiella tarda is a Gram-negative bacterial pathogen with a broad range of hosts, including fish and mammals. In the present study, we used an advanced antibody array technology to identify the expression pattern of cytokines induced by E. tarda in a mouse infection model. In total, 31 and 24 differentially expressed cytokines (DECs) were identified in the plasma at 6 h and 24 h post-infection (hpi), respectively. The DECs were markedly enriched in the Gene Ontology (GO) terms associated with cell migration and response to chemokine and in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with immunity, diseases, and infection. Ten key DECs, including IL6 and TNF-α, were found to form extensive protein-protein interaction networks. IL6 was demonstrated to inhibit E. tarda infection and be required for E. tarda-induced inflammatory response. TNF-α also exerted an inhibitory effect on E. tarda infection, and knockdown of fish (Japanese flounder) TNF-α promoted E. tarda invasion in host cells. Together, the results of this study revealed a comprehensive profile of cytokines induced by E. tarda, thus adding new insights into the role of cytokine-associated immunity against bacterial infection and also providing the potential plasma biomarkers of E. tarda infection for future studies.

Cloning and characterization of two chondroitin sulfate ABC lyases from Edwardsiella tarda LMG2793.

Chondroitinase ABC can be used to prepare chondroitin sulfate (CS) oligosaccharides efficiently and environmentally. It also promotes nerve recovery through enzymatic degradation of glycosaminoglycan chains in damaged nerve tissue. In this study, two new chondroitin sulfate ABC lyases were expressed and characterized from Edwardsiella tarda LMG2793, with molecular weight of 116.8 kDa and 115.9 kDa, respectively. Two lyases ChABC I and ChABC II belonged to the polysaccharide lyase (PL) family 8. ChABC I and ChABC II showed enzyme activity towards chondroitin sulfate A (CS-A), CS-B, CS-C and CS-D, but had no activity towards hyaluronan (HA). The optimal temperature for ChABC I to exhibit the highest activity against CS-A was 40 °C and the optimal pH was 7.0. ChABC II showed the highest activity to CS-A at optimal temperature of 40 °C and pH of 9.0. ChABC I and ChABC II were stable at 37 °C and remained about 90 % of activity after incubation at 37 °C for 3 h. Many metal ions had no effect on the activity of ChABC I and ChABC II. These properties were beneficial to their further basic research and application. ChABC I was an endo-type enzyme while ChABC II was an exo-type enzyme. A group of amino acids were selected for further study by evaluating the sequence homology with other CS degradation lyases. Mutagenesis studies speculated that the catalytic residues in ChABC I were His522, Tyr529 and Arg581. The catalytic residues of ChABC II were His498, Tyr505 and Arg558. This work will contribute to the structural and functional characterization of biomedically relevant CS and promote the application of CS lyase in further basic research and therapeutics.

Target genes directly regulated by Eha are required for Edwardsiella tarda survival within macrophages.

Eha is a virulence regulator in Edwardsiella tarda (E. tarda). The present study examined how Eha regulated its target genes to affect the bacterial survival within the cells. We constructed the reporter a pGEX-4T-ehaflag plasmid expressing Eha tagged at its C terminus with the flag epitope, and introduced the plasmid into an eha mutant ET13 strain, and obtained a Cehaflag strain. The expression and activity of an EhaFlag fusion protein restored the survival of the Cehaflag as the wild type in macrophages by Western blotting and intracellular survival experiments. We used a monoclonal anti-Flag antibody to precipitate EhaFlag-DNA complexes using chromatic immunoprecipitation (ChIP). We then designed primers based on the differentially-expressed genes identified from RNA-sequencing, and identified ten Eha-interacting genes by qPCR. We amplified the promoter regions of the ten genes and the eha gene from ET13 strain by PCR, constructed pBD-PtargetlacZ and pBD-PehalacZ plasmids. The eha gene directly and positively regulated these target genes, and be negatively auto-regulated by Eha in E. tarda, as determined by comparing their ß-Galactosidase activities. These target genes were distributed in the categories involved in the bacterial growth, movement and resistance to H2O2 or acid. We further constructed a ETATCC_RS15225 mutant (△dcuA1), a ETATCC_ RS14855 mutant (△flgK) anda ETATCC_RS07650 mutant (ΔtnaA), and a partial complementary strains of △eha-tnaA and △eha-flgK and the complementary strains of CΔflgK and CΔtnaA. The ETATCC_RS15225 gene probably encoded a transporter protein DcuA1 at outer membrane with SDS-PAGE and RT-PCR. The ETATCC _RS14855 gene probably encoded FlgK protein and affected the bacterial motility. The ETATCC_RS07650 gene encoded Tryptophanase, which affected the bacterial survival within macrophages. With the assistance of these above strains, our results showed that the eha gene was able to regulate the ETATCC_RS15225 gene to express its outer membrane protein DcuA1, the ETATCC _RS14855 gene to control the flagellar motility and the ETATCC_RS07650 to affect the bacterial survival within macrophages. With the combination of other functions of above three genes, our results suggested that Eha directly regulates the target genes to affect E. tarda to survive within the cells.

Edwardsiella tarda induces enteritis in farmed seahorses (Hippocampus erectus): An experimental model and its evaluation.

Bacterial enteritis is an important deadly threat to farmed seahorses. However, its pathogenesis is obscure because of the paucity of reproducible experimental intestinal inflammation models. Herein, a strain of Edwardsiella tarda YT1 from farmed seahorse Hippocampus erectus was isolated and identified by morphological, phylogenetic, and biochemical analysis, and confirmed as a pathogen of enteritis for the first time by challenge experiment. Two E. tarda concentrations (1 × 105 and 1 × 107 colony forming units [cfu] ml-1) were confirmed suitable for an enteritis model by intraperitoneal injection. To develop and evaluate the experimental model, we challenged seahorses with E. tarda and found that (1) the infection inhibited body length increase, significantly decreased body weight (P < 0.05), and induced typical pathological features including anorexia, anal inflammation, and intestinal fluid retention; (2) 19 external (weight, height, anal inflammation, feeding status, and intestinal fluid retention), histological (goblet and inflammatory cell numbers and thickening of lamina propria and muscularis mucosae), and molecular (hepcidin, liver-expressed antimicrobial peptide, lysozyme, piscidin, interleukin [IL]-1ß, IL-1ß receptor, IL-2, IL-10, interferon1, tumor necrosis factor [TNF]-α, and toll-like receptor 5 [TLR5]) indicators were suitable for model evaluation, as they could sensitively respond and varied similarly throughout the experiment, indicating the high sensitivity of seahorses against pathogen invasion; (3) TLR5 may play an essential role in triggering host immune responses during E. tarda-induced chronic enteritis, and (4) the evaluating system could reflect the pattern and intensity of disease progression. Thus, we developed an experimental model and an evaluating system of bacterial enteritis in farmed seahorses, helping us to reveal the pathogenesis of bacterial enteritis, identify potential therapeutic drugs, and search suitable genetic markers for seahorse molecular breeding.

Fish borne Edwardsiella tarda eha involved in the bacterial biofilm formation, hemolytic activity, adhesion capability and pathogenicity.

Edwardsiella tarda (E. tarda) is distributed widely in a variety of hosts including humans, other mammals and fish, and it is worthwhile to notice that E. tarda -caused fish infections lead to the most important bacterial disease in fish. Considering Eha acting as a transcriptional regulator in E. tarda strain ET13 have been reported previously, to better understand its pathogenesis due to this, a type of cell of epithelial cell line (Caco-2) infection model for the pathogen was established in the laboratory. We focused on studying various parameters such as lactate dehydrogenase release (to measure cytotoxicity) and cell adhesions, both of which are related to the bacterial pathogenesis. Furthermore biofilm formation, hemolytic activity, and adhesion to Caco-2 cells were decreased in an E.tarda mutant strain with deletion in-frame isogenic gene eha (∆eha) compared to the wild-type and the complementary strain eha+ (an engineered construct of ∆eha expressing eha); Meanwhile, we found that hemolytic activity and biofilm formation were significantly enhanced in the strain eha+. Moreover, the ∆eha strain had attenuated pathogenicity in the zebrafish infection model. The data also demonstrated that the series of genes fimA, esrB, gadB, mukF, katB, and katG are regulated by eha based on a quantitative reverse transcription polymerase chain reaction tests and analysis. Thus our research data indicated that eha has an impact on hemolytic activity, biofilm formation, adhesion, and pathogenicity of pathogenic strain ET13 and plays an essential role in manifesting the virulence factors.

Desialylation by Edwardsiella tarda is the initial step in the regulation of its invasiveness.

Edwardsiella tarda is a gram-negative bacterium causing significant economic losses to aquaculture. E. tarda possesses NanA sialidase which removes sialic acids from α2-3 sialo-glycoprotein of host cells. However, the relationship between NanA sialidase activity and E. tarda invasiveness remains poorly understood. Furthermore, the pathway of sialic acid metabolism in E. tarda remains to be elucidated. We studied sialidase activity in several E. tarda strains and found that the pathogenic strains exhibited higher sialidase activity and greater up-regulation of the NanA mRNA level than non-pathogenic strain. Pathogenic strains also showed higher rates of infection in GAKS cells, and the infection was drastically suppressed by sialidase inhibitor. Additionally, NanA gene overexpression significantly increased infection and treatment of E. tarda with free sialic acid enhanced the rate of infection in GAKS cells. Sialic acid treatment enhanced mRNA levels of two N-acetylneuraminate lyases and one N-acetylneuraminate cytidylyltransferase. E. tarda uses sialic acid as a carbon source for growth via N-acetylneuraminate lyases. The strains with high N-acetylneuraminate cytidylyltransferase level showed greater sialylation of the lipopolysaccharides and glycoproteins. Our study establishes the significance of desialylation by E. tarda sialidase in the regulation of its invasiveness.

Characterization of balofloxacin-stressed proteomics and identification of balofloxacin-binding proteins pre-peptidase and integration host factor in Edwardsiella tarda.

The overuse of antibiotics to control bacterial pathogens leads to the generation of their antibiotic-resistant strains including Edwardsiella tarda. Understanding of mechanisms of the antibiotic resistance is crucial to develop novel methods to manage the infection. Here, two-dimensional electrophoresis-based proteomics was used to characterize balofloxacin-responsive proteins. The altered proteome consisted of 19 proteins with differential abundance, where six metabolic pathways were enriched. The metabolic modulation activated the central carbon metabolism with elevation of NADH, PMF, and ATP. Among the 19 proteins, ETAE_1987 (pre-peptidase) and ETAE_2174 (integration host factor beta subunit) were bound with balofloxacin directly. This was further confirmed by the binding of balofloxacin with recombinant ETAE_1987 and ETAE_2174 using Oxford cup method. Compared with bovine serum albumin, a known balofloxacin-binding protein, ETAE_1987 and ETAE_2174 increased the binding capability by 3.3- and 22-fold, respectively. The combination was validated by microscale thermophoresis. These data characterize the balofloxacin-stressed proteome as a result of the increased central carbon metabolism and energy metabolism and determine ETAE_1987 and ETAE_2174 as balofloxacin-binding proteins. These findings have significant implications in understanding bacterial antibiotic-resistant and drug action mechanisms based on balofloxacin-binding proteins. SIGNIFICANCE: Antibiotic-resistant Edwardsiella tarda strains are frequently isolated and cause a great loss in aquaculture since these bacterial strains are insensitivity to antibiotics. The present study showed that the increased central carbon metabolism forms a characteristic feature of the balofloxacin-stressed proteomics. Furthermore, two proteins, ETAE_1987 (pre-peptidase) and ETAE_2174, of the balofloxacin-stressed proteome were identified as balofloxacin-binding proteins. The binding capability is 0.39 ±â€¯0.017 and 2.67 ±â€¯0.066 ng/µg proteins for ETAE_1987 and ETAE_2174, respectively. These results reveal the elevated central carbon metabolism as a key feature of the balofloxacin-stressed proteomics and pre-peptidase and integration host factor as balofloxacin-binding proteins in E. tarda. These findings are useful in the understanding of bacterial balofloxacin-stressed mechanisms and providing new targets for controlling antibiotic-resistant bacteria.