Functional analysis of OmpA and its contribution to pathogenesis of Edwardsiella tarda.

Outer membrane protein A (OmpA), a major component of outer membrane proteins in gram-negative bacteria, is considered to be an important virulence factor in various pathogenic bacteria, but its underlying mechanisms involved in pathogenic process of Edwardsiella tarda has not yet been fully elucidated. E. tarda is an important facultative intracellular pathogen with a broad host range. This bacterium could survive and replicate in macrophages as an escape mechanism from the host defense. To address the functions of OmpA and its potential roles in the pathogenesis of E. tarda, ΔompA mutant strain and ΔompA-C complementary strain were constructed by the allelic exchange method in this study. Here, we demonstrate that the abilities of motility, biofilm formation and adherence to RAW264.7 cells of ΔompA were significantly impaired, although there was no difference in growth between wild-type (WT) strain and ΔompA. Moreover, inactivation of ompA rendered E. tarda more sensitive to oxidative, heat shock and osmotic stress, which simulate the in vivo conditions that E. tarda encounters within the intramacrophage environment. Consist with this observation, ΔompA was also found to be markedly attenuated for growth within macrophages. In addition, compared with the WT strain, ΔompA activated macrophages to release more inflammatory mediators, including tumor necrosis factor alpha (TNF-α), reactive oxygen species (ROS) and nitric oxide (NO). However, flow cytometry analysis revealed that ΔompA induced less apoptosis of RAW264.7 cells as compared with WT strain, characterized by decreased Annexin V binding and the activation of caspase-3. Overall, our findings suggest an importance of OmpA to E. tarda and provide the first comprehensive insight into its functions and potential roles in the pathogenesis of E. tarda, including its effect on interaction with macrophages.

Edwardsiella tarda Attenuates Virulence upon Oxytetracycline Resistance.

The relationship between antibiotic resistance and bacterial virulence has not yet been fully explored. Here, we use Edwardsiella tarda as the research model to investigate the proteomic change upon oxytetracycline resistance (LTB4-ROTC). Compared to oxytetracycline-sensitive E. tarda (LTB4-S), LTB4-ROTC has 234 differentially expressed proteins, of which the abundance of 84 proteins is downregulated and 15 proteins are enriched to the Type III secretion system, Type VI secretion system, and flagellum pathways. Functional analysis confirms virulent phenotypes, including autoaggregation, biofilm formation, hemolysis, swimming, and swarming, are impaired in LTB4-ROTC. Furthermore, the in vivo bacterial challenge in both tilapia and zebrafish infection models suggests that the virulence of LTB4-ROTC is attenuated. Analysis of immune gene expression shows that LTB4-ROTC induces a stronger immune response in the spleen but a weaker response in the head kidney than that induced by LTB4-S, suggesting it's a potential vaccine candidate. Zebrafish and tilapia were challenged with a sublethal dose of LTB4-ROTC as a live vaccine followed by LTB4-S challenge. The relative percentage of survival of zebrafish is 60% and that of tilapia is 75% after vaccination. Thus, our study suggests that bacteria that acquire antibiotic resistance may attenuate virulence, which can be explored as a potential live vaccine to tackle bacterial infection in aquaculture.

Virulent and Multi-drug-Resistant Edwardsiella tarda Infection in Oscar Fish: Unveiling the Threat of Mass Mortality and AMR Dissemination.

The Oscar fish (Astronotus ocellatus) is among the most commonly domesticated and exported ornamental fish species from Kerala. The ornamental fish industry faces a significant challenge with the emergence of diseases caused by multi-drug-resistant bacteria. In the present study, six isolates were resolved from the diseased Oscar fish showing haemorrhages, necrosis, and loss of pigmentation. After phenotypic and genotypic characterization, the bacteria were identified as Edwardsiella tarda, Klebsiella pneumoniae, Enterococcus faecalis, Escherichia coli, Brevibacillus borstelensis, and Staphylococcus hominis. Experimental challenge studies in healthy Oscar fish showed that E. tarda caused 100% mortality within 240 h with 6.99 × 106 CFU/fish as LD50 and histopathology revealed the typical signs of infection. The pathogen was re-recovered from the moribund fish thereby confirming Koch's postulates. E. tarda was confirmed through the positive amplification of tarda-specific gene and virulence genes viz., etfD and escB were also detected using PCR. Antibiotic susceptibility tests using disc diffusion displayed that the pathogen is multi-drug-resistant towards antibiotics belonging to aminoglycosides, tetracyclines, and quinolones categories with a MAR index of 0.32, which implicated the antibiotic pressure in the farm. Plasmid curing studies showed a paradigm shift in the resistance pattern with MAR index of 0.04, highlighting the resistance genes are plasmid-borne except for the chromosome-borne tetracycline resistance gene (tetA). This study is the first of its kind in detecting mass mortality caused by E. tarda in Oscar fish. Vigilant surveillance and strategic actions are crucial for the precise detection of pathogens and AMR in aquaculture.

Changes in immune genes expression, immune response, digestive enzymes -antioxidant status, and growth of catla (Catla catla) fed with Astragalus polysaccharides against edwardsiellosis disease.

The effect of four level of Astragalus polysaccharides (APs) supplementation diets, (CD: control diet and three experiment diet (E), EA: 100 mg kg-1 APs; EB: 200 mg kg-1 APs; EC: 300 mg kg-1 APs) on growth, changes in haemato-biochemical parameters and metabolic-digestive enzymes, enhancement of antioxidant activity, innate-adaptive immune response, and cytokine gene expression were studied in catla (Catla catla) against Edwardsiella tarda. The healthy and challenged groups fed the CD displayed no mortality, while fish fed EA or EC revealed 10% mortality, but the mortality was only 5% in diet EB. Fish fed diet EB and EC revealed significantly better growth rates and high RBC count during the experimental period. Albumin and globulin levels were significant improved when fish were fed the diet EB and EC from weeks 6-8. The superoxide dismutase (SOD) was significant ameliorated by EB feeding from weeks 4-8. In contrast, serum myeloperoxidase (MPO), catalase (CAT), malondialdehyde (MDA)/lipid peroxidation (LPO), glutathione peroxidase (GPx), respiratory burst activity (RBA), bactericidal action (BCA), serum lysozyme activity (SLA), nitric oxide synthase (NOS), head kidney leukocytes response proliferation (HKLP), hemolytic action (HLA), hydrogen peroxides (H2O2), and immunoglobulin (Ig) were significantly improved from week 6-8. Groups fed the APs enriched diets had significant ameliorated interleukin (IL)-1ß and interferon (IFN)-γ mRNA expression after 6 and 8 weeks of feeding. However, IL-10 and major histocompatibility complex (MHC)-1 mRNA expressions were significant enhanced in catla fed all APs diets on week 8. APs enriched diets revealed significant improved tumor necrosis factor (TNF)-α and TNF receptor-associated factor-6 (TRAF6) mRNA expression on week 4, but toll-like receptor-2 (TLR2) and TLR4 mRNA expression were significant enhanced by diet EB and EC after weeks 6 and 8. Similarly, the lysozyme (Lyz)-C and Lyz-G mRNA levels in the head kidney (HK) increased by APs feeding on weeks 6 and 8, whereas the EB diet, the expression of nucleotide binding oligomerization domain-1 (NOD1) was significantly improved on weeks 6 and 8, but NOD2 mRNA expression was only significant enhanced after 8 weeks of diet EB. By feeding healthy catla and E. tarda challenged fish fed diet EB, resulted in significantly increased growth, haemato-biochemical indices, metabolic-digestive enzymes, antioxidant activities, innate-adaptive immune responses, and cytokine gene expression mainly between 6 and 8 weeks.

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.

A teleost interleukin-16 is implicated in peripheral blood leukocytes recruitment and anti-bacterial immunity.

Interleukin-16 (IL-16), as a lymphocyte chemoattractant cytokine, plays a crucial role in regulating cellular activities and anti-pathogen immunity. In teleost, the information about the antibacterial effect of IL-16 is scarce. In our study, we examined the immune functions of an IL-16 homologue (CsIL-16) from tongue sole Cynoglossus semilaevis. The CsIL-16 precursor (proCsIL-16) is comprised of 1181 amino acid residues, sharing 21.1%-67.3% identities with IL-16 precursor from invertebrate and vertebrate. The C-terminal proCsIL-16 containing two PDZ domains was designated as mature CsIL-16 which was released into the supernatant of peripheral blood leukocytes (PBLs). CsIL-16 was expressed in various tissues and regulated by bacterial invasion. Recombinant CsIL-16 (rCsIL-16), as a homodimer, was able to bind to the membrane of PBLs and played essential roles in regulating chemotaxis and activation of PBLs, which in vitro inhibited intracellular survival of E. tarda. Under in vivo condition, rCsIL-16 could dramatically regulate the induction of inflammatory genes, and suppress the bacterial dissemination in fish tissues. Collectively, our results reveal that CsIL-16 plays positive roles in antibacterial immunity, and provide insights into the immune function of CsIL-16.

Acute Cholecystitis, Sepsis, and Disseminated Intravascular Coagulation Caused by Edwardsiella tarda in an Elderly Woman.

Edwardsiella tarda is a gram-negative bacillus associated with gastrointestinal diseases. It is rarely responsible for sepsis; however, the fatality is very high. Only two cases of E. tarda infections in patients over 90 years of age have been reported; these are not cases of sepsis associated with acute cholecystitis. We report a case of acute cholecystitis, sepsis, and disseminated intravascular coagulation (DIC) caused by E. tarda in a super-elderly woman aged over 90 years. There could be a possibility for recovery from sepsis and DIC if antimicrobial treatment responsiveness is ensured in the super-elderly.

Identification and Characterization of Long Non-coding RNAs in the Intestine of Olive Flounder (Paralichthys olivaceus) During Edwardsiella tarda Infection.

Long non-coding RNAs (lncRNAs) play widespread roles in fundamental biological processes, including immune responses. The olive flounder (Paralichthys olivaceus), an important economical flatfish widely cultured in Japan, Korea, and China, is threatened by infectious pathogens, including bacteria, viruses, and parasites. However, the role of lncRNAs in the immune responses of this species against pathogen infections is not well-understood. Therefore, in this study, we aimed to identify lncRNAs in the intestine of olive flounder and evaluate their differential expression profiles during Edwardsiella tarda infection, which is an important zoonotic and intestinal pathogen. A total of 4,445 putative lncRNAs were identified, including 3,975 novel lncRNAs and 470 annotated lncRNAs. These lncRNAs had shorter lengths and fewer exons compared with mRNAs. In total, 115 differentially expressed lncRNAs (DE-lncRNAs) were identified during E. tarda infection. To validate the expression pattern of lncRNAs, six DE-lncRNAs were randomly selected for quantitative real-time PCR. The co-located and co-expressed mRNAs of DE-lncRNAs were predicted, which were used to conduct the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The target genes of DE-lncRNAs enriched numerous immune-related processes and exhibited a strong correlation with immune-related signaling pathways. To better understand the extensive regulatory functions of lncRNAs, the lncRNA-miRNA-mRNA regulatory networks were constructed, and two potential competing endogenous RNA (ceRNA) networks, LNC_001979-novel_171-Potusc2 and LNC_001979-novel_171-Podad1, were preliminarily identified from the intestine of olive flounders for the first time. In conclusion, this study provides an invaluable annotation and expression profile of lncRNAs in the intestine of olive flounder infected with E. tarda; this forms a basis for further studies on the regulatory function of lncRNAs in the intestinal mucosal immune responses of olive flounder.

Regulatory Role of Fc Receptor in mIgM+ B Lymphocyte Phagocytosis in Flounder (Paralichthys olivaceus).

Fc receptor (FcR) is an important opsonin receptor on the surface of immune cells, playing an important role in antibody-dependent cell-mediated immunity. Our previous work found that the FcR of flounder showed a marked expression response in phagocytizing IgM+ B cell, which suggested that FcR might participate in regulating Ig-opsonized phagocytosis. In this paper, in order to elucidate the potential role of FcR in mediating phagocytosis of IgM+ B cell, flounder anti-E. tarda serum was prepared and complement-inactivated for the use of E. tarda opsonization, and the sera of healthy flounder were used as control. Flow cytometric analysis showed that the phagocytosis rates of antiserum-opsonized E. tarda in peripheral blood mIgM+ B lymphocytes were significantly higher than the control group, and higher phagocytosis rates of mIgM+ B lymphocyte could be detected with an increasing incubation time ranging from 1 to 5 h. The phagocytosis rates of antiserum-opsonized E. tarda by mIgM+ B lymphocyte for an incubation time of 1, 3 or 5 h were 51.1, 63.0, and 77.5% respectively, which were significantly higher than the phagocytosis rates in the control groups with 40.2, 50.9, and 63.8%, respectively. While the Fc fragment of IgM on the surface of opsonized E. tarda was blocked by rabbit anti-flounder IgM polyclonal antibodies, phagocytosis rates of mIgM+ B lymphocyte decreased significantly compared with the unblocked group. Moreover, the proportion of mIgM+ B lymphocytes with higher intracellular reactive oxygen species (ROS) levels rose to 32.1% from the control level of 23.0% after phagocytosis of antiserum-opsonized E. tarda. FcγRII and Syk were found to be significantly upregulated, while FcγRIII was significantly downregulated in the mIgM+ B lymphocytes post phagocytosis. Furthermore, when FcγRII of mIgM+ B lymphocytes was blocked by the prepared antibodies, their phagocytosis rate of antiserum-opsonized E. tarda was 39.0%, which was significantly lower than the unblocked group of 54.0%. These results demonstrate that FcR plays a critical role in mediating phagocytosis and bactericidal activity of mIgM+ B lymphocytes, which would facilitate an improved understanding of the regulatory roles of FcR in phagocytosis of teleost B lymphocytes.

Macrophage colony stimulating factor (MCSF) of Japanese flounder (Paralichthys olivaceus): Immunoregulatory property, anti-infectious function, and interaction with MCSF receptor.

Macrophage colony-stimulating factor (MCSF) is an essential growth factor to control the proliferation, differentiation and survival of the macrophage lineage in vertebrates. Sequences of MCSF have been identified in multiple teleost species, however, the functional investigations of MCSF were documented in only a few species. In this study, we examined the biological activity and the immunomodulatory property of a MCSF homologue, PoMCSF, from Japanese flounder (Paralichthys olivaceus). Structural analysis showed that PoMCSF possesses conserved structural characteristics of MCSF proteins, including a signal peptide, a CSF-1 domain, and a transmembrane region closed to the C-terminal. Under normal physiological condition, PoMCSF expression distributes in all the examined tissues, the highest three tissues are blood, muscle, and head kidney. When infected by extracellular and intracellular bacterial pathogens and viral pathogen, the PoMCSF expression patterns vary with different types of microbial pathogens infection and different immune tissues. In vitro experiment showed recombinant PoMCSF promoted the activity of macrophage. In vivo experiment indicated that PoMCSF overexpression boosted the defensive ability of flounder against Edwardsiella piscicida, a severe fish pathogen that infects multiple species of economically important fish, and regulated the expression of multiple immune-related genes. To explore the relationship between PoMCSF and its receptor PoMCSFR, anti-PoMCSFR antibody was prepared and PoMCSFR knockdown was conducted. The neutralization assay showed that when PoMCSFR was neutralized by its antibody, the role of PoMCSF on host defense against E. piscicida was weakened. Knockdown of PoMCSFR impaired the phagocytic capacity of macrophages. Collectively, these findings suggest that PoMCSF plays a crucial role in the immune defense system of Japanese flounder and the effect of PoMCSF is dependent on PoMCSFR. This study provides new insights into the biological activity of MCSF and the relationship between MCSF and MCSFR in teleost.

Prediction of genomic breeding values based on pre-selected SNPs using ssGBLUP, WssGBLUP and BayesB for Edwardsiellosis resistance in Japanese flounder.

BACKGROUND: Edwardsiella tarda causes acute symptoms with ascites in Japanese flounder (Paralichthys olivaceus) and is a major problem for China's aquaculture sector. Genomic selection (GS) has been widely adopted in breeding industries because it shortens generation intervals and results in the selection of individuals that have great breeding potential with high accuracy. Based on an artificial challenge test and re-sequenced data of 1099 flounders, the aims of this study were to estimate the genetic parameters of resistance to E. tarda in Japanese flounder and to evaluate the accuracy of single-step GBLUP (ssGBLUP), weighted ssGBLUP (WssGBLUP), and BayesB for improving resistance to E. tarda by using three subsets of pre-selected single nucleotide polymorphisms (SNPs). In addition, SNPs that are associated with this trait were identified using a single-SNP genome-wide association study (GWAS) and WssGBLUP. RESULTS: We estimated a heritability of 0.13 ± 0.02 for resistance to E. tarda in Japanese flounder. One million SNPs at fixed intervals were selected from 4,978,724 SNPs that passed quality controls. GWAS identified significant SNPs on chromosomes 14 and 24. WssGBLUP revealed that the putative quantitative trait loci on chromosomes 1 and 14 contained SNPs that explained more than 1% of the genetic variance. Three 50 k-SNP subsets were pre-selected based on different criteria. Compared with pedigree-based prediction (ABLUP), the three genomic methods evaluated resulted in at least 7.7% greater accuracy of predictions. The accuracy of these genomic prediction methods was almost unchanged when pre-selected trait-related SNPs were used for prediction. CONCLUSIONS: Resistance to E. tarda in Japanese flounder has a low heritability. GWAS and WssGBLUP revealed that the genetic architecture of this trait is polygenic. Genomic prediction of breeding values performed better than ABLUP. It is feasible to implement genomic selection to increase resistance to E. tarda in Japanese flounder with 50 k SNPs. Based on the criteria used here, pre-selection of SNPs was not beneficial and other criteria for pre-selection should be considered.

Genome-wide characterization of Toll-like receptors in black rockfish Sebastes schlegelii: Evolution and response mechanisms following Edwardsiella tarda infection.

As one of the key components of pattern recognition receptors, Toll-like receptors (TLRs) play pivotal roles in the innate immune system. However, little information is available about the TLR genes in Sebastes schlegelii. In the present study, 17 TLR genes were identified and classified based on the whole genome database. Tandem duplication events in TLR1, TLR2, TLR5 and TLR13 played major role in the expansion of S. schlegelii TLR genes; both TLR2-3 and TLR2-4 had the same largest number of introns/exons, 11 exons and 10 introns. The syntenic analysis showed neighboring genes of TLR genes were most conserved in S. schlegelii and in L. crocea. Phylogenetic and evolutionary analysis showed that these TLR genes were divided into five subfamilies and exhibited different selection pressures. Meanwhile, the expression patterns of TLR genes in the intestine after E. tarda infection were investigated by qRT-PCR. Finally, protein and protein interaction (PPI) network analysis indicated that TLR genes interacted with IFN-inducible genes, inflammatory cytokines, and participated in MyD88-dependent pathway. In summary, this study provided valuable information for further functional characterization of TLR genes in S. schlegelii.

Transcriptome Analysis of Paralichthys olivaceus Erythrocytes Reveals Profound Immune Responses Induced by Edwardsiella tarda Infection.

Unlike mammalian red blood cells (RBCs), fish RBCs are nucleated and thus capable of gene expression. Japanese flounder (Paralichthys olivaceus) is a species of marine fish with important economic values. Flounder are susceptible to Edwardsiella tarda, a severe bacterial pathogen that is able to infect and survive in flounder phagocytes. However, the infectivity of and the immune response induced by E. tarda in flounder RBCs are unclear. In the present research, we found that E. tarda was able to invade and replicate inside flounder RBCs in both in vitro and in vivo infections. To investigate the immune response induced by E. tarda in RBCs, transcriptome analysis of the spleen RBCs of flounder challenged with E. tarda was performed. Six sequencing libraries were constructed, and an average of 43 million clean reads per library were obtained, with 85% of the reads being successfully mapped to the genome of flounder. A total of 1720 differentially expressed genes (DEGs) were identified in E. tarda-infected fish. The DEGs were significantly enriched in diverse Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, especially those associated with immunity, disease, and infection. Ninety-one key DEGs involved in 12 immune-related pathways were found to form extensive interaction networks. Twenty-one genes that constituted the hub of the networks were further identified, which were highly regulated by E. tarda and involved in a number of immune processes, notably pathogen recognition and signal transduction, antigen processing and presentation, inflammation, and splicing. These results provide new insights into the immune role of flounder RBCs during bacterial infection.

Isolation and characterization of phage (ETP-1) specific to multidrug resistant pathogenic Edwardsiella tarda and its in vivo biocontrol efficacy in zebrafish (Danio rerio).

Edwardsiella tarda phage (ETP-1) was isolated from marine fish farm water to characterize its effect against pathogenic multidrug-resistant E. tarda. According to transmission electron microscopy results, ETP-1 is classified as a member of the family Podoviridae. ETP-1 showed MOI dependent E. tarda growth inhibition, a latent period of 60 min, and burst size of 100 PFU per infected cells. In host range tests, five out of eight E. tarda strains were sensitive to ETP-1 which had efficiency of plating index in the range 1-1.28. ETP-1 was stable over a broad range of pH and temperature. The size of the ETP-1 genome was predicted to be approximately 40 kb. Zebrafish exposed to ETP-1 showed no adverse gene responses to the inflammatory mediator cytokines, il1-ß, tnf-α, il-6, and il-10, the chemokine, cxcl-8a, and reactive oxygen species, sod-1. When zebrafish were bath exposed to ETP-1 for 12 days and simultaneously challenged with E. tarda (1.08 × 105 CFU fish-1), the survival rate was higher in phage exposed fish (68%) compared to that of the control (18%) until 4 days post challenge. Our results suggest that ETP-1 can be used as a potential bio-therapeutic candidate to control multi-drug resistant E. tarda infection in aquaculture.

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.

A Comparative Analysis of Edwardsiella tarda-Induced Transcriptome Profiles in RAW264.7 Cells Reveals New Insights into the Strategy of Bacterial Immune Evasion.

Edwardsiella tarda is a Gram-negative bacterial pathogen with a broad host range, including fish, reptiles, and mammals. One prominent virulence feature of E. tarda is its ability to survive and replicate in host phagocytes, but the relevant molecular mechanism is largely unknown. In this study, we examined the transcriptome profiles of RAW264.7 cells, a murine macrophage cell line, infected with live E. tarda or stimulated with dead E. tarda for 4 h and 8 h. Eighteen libraries were constructed, and an average of 69 million clean reads per library were obtained, with ~81.63% of the reads being successfully mapped to the reference genome. In total, 208 and 232 differentially expressed genes (DEGs) were identified between live and dead E. tarda-treated cells at 4 h and 8 h post-infection, respectively. The DEGs were markedly enriched in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with immunity. Live E. tarda differed strikingly from dead E. tarda in the regulation of immune related genes. Compared with dead E. tarda-treated cells, live E. tarda-treated cells exhibited marked and significant suppression in the induction of a large amount of immune genes, including RIG-I-like receptors, cytokines, and interferon-related genes. Furthermore, some of the immune genes highly regulated by live E. tarda formed complicated interaction networks with each other. Together, the results of this study revealed a transcriptome profile specifically induced by the active virulence elements of live E. tarda during the infection process, thus adding new insights into the intracellular infection mechanism of E. tarda. This study also provided a valuable set of target genes for further study of the immune evasion strategy of E. tarda.

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.

A case of necrotizing fasciitis following Edwardsiella tarda septicemia with gastroenteritis.

Edwardsiella tarda is an uncommon pathogen that causes gastroenteritis in humans and is found in the aquatic environment. In rare cases, it also causes fatal infections, including sepsis and necrotizing fasciitis. However, it remains unknown whether E. tarda gastroenteritis could lead to these lethal diseases via hematogenous spread. Here we have reported a previously healthy 64-year-old woman with necrotizing fasciitis consecutively caused by E. tarda septicemia with gastroenteritis. The patient was transferred to the emergency department due to disturbance of consciousness and hypotension after suffering from diarrhea for a month. As whole-body computed tomography (CT) revealed an edematous change in the small intestine, septic shock following gastroenteritis was suspected, and the patient was immediately started on empiric antibiotic therapy and provided critical care. Her general physical conditions gradually began improving, but, on day 7, rapidly appearing blisters on both the lower limbs were noted, and she was accordingly examined again by conducting a CT scan. Based on the results, she was diagnosed with necrotizing fasciitis in both lower extremities, and surgical debridement was rapidly performed. Microbiological analysis of the specimens revealed E. tarda bacteremia, which suggested that E. tarda caused a series of infections in this patient. Finally, she fully recovered and was discharged within 3 months. Cumulatively, we proposed that gastroenteritis by E. tarda could directly result in fatal infections through the blood stream.

EsR240, a non-coding sRNA, is required for the resistance of Edwardsiella tarda to stresses in macrophages and for virulence.

Bacterial small non-coding RNAs (sRNAs) are gene expression modulators that respond to environmental changes and pathogenic conditions. In this study, 13 novel sRNAs were identified in the intracellular pathogen, Edwardsiella tarda (E. tarda) ET13 strain, based on RNA sequencing and bioinformatic analyses. Eight of the 13 putative sRNAs from the ET13 strain were transcribed (as indicated by RT-PCR) following exposure to different stresses. The transcription levels of three sRNAs (EsR128, EsR139 and EsR240) were all highly induced under these stress conditions. Northern blot hybridization was employed to verify that EsR240 was expressed in the ET13 strain under both logarithmic and stationary growth phases, and that it formed a single copy transcript in the chromosomes of the ET13 strain. The precise start and end points of EsR240 were determined using 5'and 3' RACE. The conservation of EsR240 was in agreement with the characteristics of sRNA, as indicated by a BLAST analysis. Furthermore, the survival rates of EsR240 mutant were lower than the rates of the wild type ET13 under stress conditions. When the infection time was extended 4 or 6 h, the CFUs of the wild type bacteria increased more significantly within macrophages compared to the mutant. When the intra-peritoneal (i.p.) route of infection was used in mice, the bacterial loads of the tissues in the mice infected with the wild type bacteria were significantly higher than in the mice infected with the mutants. The virulence of the EsR240 mutant was 6.79-fold lower than the wild type bacterium based on the LD50. In addition, the IntaRNA program was used to predict the target genes of EsR240. Out of the top 10 predicted target genes, 9 genes were regulated by EsR240. These target genes may encode FtsH protease modulator YccA, Na+ and H+ antiporters, FtsX-like permease family protein, glycoside hydrolases or various other proteins. Therefore, EsR240 may positively regulate its target genes in E. tarda to maintain intracellular survival within host macrophages and to increase its virulence.