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Invasins and intimins, members of virulence-related adhesin family which is involved in attachment and adherence to epithelial cells during infection, are found in various pathogens. These pathogens can attach to enterocytes and lead to the formation of a pedestal-like structure. Invasins and intimins belong to type Ve secretion systems, and the N-terminal β-barrel domain acts as a translocation pore to secrete the C-terminal passenger domain. However, the relationship between invasins/intimins and type III secretion system (T3SS) has been poorly studied. Based on the transposon insertion mutant library of Edwardsiella piscicida, we got a transposon insertion mutant with significant T3SS defect and identified the mutated gene ETAE_0323 (named inV later). This gene encoded a protein with 2359 amino acid residues and was predicted to be an invasin. To study the relationship between InV and T3SS, strains with N-terminus or C-terminus deleted InV fragments were made. However, none of them was able to copy the phenotype of the transposon insertion mutant previously identified. The localization of InV in ΔT3SS strain was not significantly different from WT, suggesting that the T3SS defect in the transposon insertion mutant was likely to be caused by polar effect. Nevertheless, depletion of inV still showed dramatic internalization and virulence defect in HeLa cell and zebrafish model, respectively, suggesting InV as a virulence related protein.

Summary Bacterial pathogens are thought to activate expression of virulence genes upon detection of host-associated cues, but identification of the nature of such signals has proved difficult. We generated a genome-scale defined transposon mutant library in Edwardsiella piscicida, an important fish pathogen, to quantify the fitness of insertion mutants for intracellular growth in macrophages and in turbot (Scophthalmus maximus). These screens identified EvrA, a transcription activator that induces expression of esrB, a key virulence regulator. EvrA is directly bound and activated by mannose-6-phosphate (man-6P) derived from actively imported mannose. Mutants lacking EvrA or expressing an EvrA unable to bind man-6P were similarly attenuated in turbot. Exogenously added mannose promoted E. piscicida virulence, and high levels of mannose were detected in fish tissue. Together, these observations reveal that binding of a host-derived sugar to a transcription factor can facilitate pathogen sensing of the host environment and trigger virulence programs., Graphical Abstract, Highlights • An E. piscicida defined mutant library is generated and analyzed in vitro and in vivo • EvrA is a key transcriptional activator of the known virulence regulator esrB • EvrA is directly bound and activated by mannose-6-phosphate from imported mannose • Extracellular mannose augments E. piscicida virulence in an evrA-dependent manner, Pathogenic Organism; Microbiology; Fish Culture

This paper fully reviews Chinese Proficiency Grading Standards for International Chinese Language Education which is the latest comprehensive standards formulated by China to describe and evaluate the Chinese proficiency of foreign learners. After introducing and analyzing its motivations for formulation, core content, key features, innovations and significance, this paper outlines some important subsequent work worth doing related to its promotion and applications, like developing corresponding curriculum standards and textbooks, compiling a new dictionary based on “three levels and nine bands” and “four-dimension benchmarks”, developing a new version of Chinese Proficiency Test (HSK) and a new type of HSK Speaking Test (HSKK).

Vibrio harveyi is an opportunistic pathogen in seawater and can cause severe vibriosis. It is prevalent in hatcheries worldwide and can lead to severe economic losses. Therefore, there is an urgent need to develop a rapid detection method for monitoring this pathogen. In this study, to increase the detection sensitivity of our assay with monoclonal antibodies (Mabs) against V. harveyi, the conditions of the dot blot assay were optimized, and enhanced chemiluminescent (ECL) substrate replaced the traditional tetramethylbenzidine (TMB) substrate. Based on the optimization results, an ECL-based novel dot blot assay was developed for the rapid and sensitive detection of V. harveyi. Compared with the traditional dot blot assay, the incubation time was shortened from 8 h to 2 h. The limit of detection (LOD) for V. harveyi was 2×105 CFU ml−1 (103 CFU/spot) in pure bacterial suspension, which was 50-fold more sensitive than the traditional dot blot assay (1×107 CFU ml−1). Furthermore, when compared with indirect ELISA, the dot blot assay showed approximately 1000-fold higher sensitivity (CFU/CFU). After the test sample was pre-enriched in turbot homogenates for 6 h before the dot blot analysis, the LOD for V. harveyi was 10 CFU ml−1. This article is protected by copyright. All rights reserved.

Background Conjugation is a major type of horizontal transmission of genes that involves transfer of a plasmid into a recipient using specific conjugation machinery, which results in an extended spectrum of bacterial antibiotics resistance. However, there is inadequate knowledge about the regulator and mechanisms that control the conjugation processes, especially in an aquaculture environment where a cocktail of antibiotics may be present. Here, we investigated these with pEIB202, a typical multi-drug resistant IncP plasmid encoding tetracycline, streptomycin, sulfonamide and chloramphenicol resistance in fish pathogen Edwardsiella piscicida strain EIB202. Results We used transposon insertion sequencing (TIS) to identify genes that are responsible for conjugation transfer of pEIB202. All ten of the plasmid-borne type IV secretion system (T4SS) genes and a putative lipoprotein p007 were identified to play an important role in pEIB202 horizontal transfer. Antibiotics appear to modulate conjugation frequencies by repressing T4SS gene expression. In addition, we identified topA gene, which encodes topoisomerase I, as an inhibitor of pEIB202 transfer. Furthermore, the RNA-seq analysis of the response regulator EsrB encoded on the chromosome also revealed its essential role in facilitating the conjugation by upregulating the T4SS genes. Conclusions Collectively, our screens unraveled the genetic basis of the conjugation transfer of pEIB202 and the influence of horizontally acquired EsrB on this process. Our results will improve the understanding of the mechanism of plasmid conjugation processes that facilitate dissemination of antibiotic resistance especially in aquaculture industries.

Edwardsiella tarda is an important fish pathogen that causes extensive losses in farmed fish around the world. Various types of vaccines have been developed against E. tarda infection, such as inactivated vaccine, live attenuated vaccine and subunit vaccine. However, inactivated vaccine exhibits poor immune protection against edwardsiellosis in some reports. In this study, we aimed to evaluate the protective effect of formalin-killed cells (FKC) against E. tarda with flagellin, FlgD, as an adjuvant in turbot. As a result, the relative percent survival (RPS) of turbot vaccinated with FKC reached 70% in the presence of FlgD adjuvant. Moreover, specific serum antibodies reached the highest level at 3weeks post vaccination. Furthermore, the transcription of some immune response-related genes, such as MHC-I, IgM, IL-1β, TCR, and TNFα were up-regulated in turbot after vaccination, indicating that both humoral and cellular immune responses were induced by this vaccine. In summary, flagellin can enhance immunoprotection of the formalin-inactivated E. tarda vaccine, which can be used as a potential adjuvant in fish vaccine.

Inflammasome activation is an important innate immune defense mechanism against bacterial infection, and in return, bacteria express virulence determinants that counteract inflammasome activation. Many such effectors are secreted into host cells via specialized bacterial secretion systems. Here, the intracellular pathogenic bacterium Edwardsiella tarda was demonstrated to activate NLRC4 and NLRP3 inflammasomes via a type III secretion system (T3SS), and to inhibit NLRP3 inflammasome via a type VI secretion system (T6SS), indicating the antagonistic roles of these systems in inflammasome signaling. Furthermore, a non-VgrG T6SS effector, EvpP, was identified that significantly inhibited NLRP3 inflammasome activation. Subsequent studies revealed that EvpP significantly suppressed Jnk activation, thus impairing oligomerization of the inflammasome adaptor ASC. Moreover, EvpP counteracted cytoplasmic Ca2+ increase, which works upstream of Jnk activation to regulate the NLRP3 inflammasome. Finally, EvpP-mediated inflammasome inhibition promoted bacterial colonization in vivo. This work expands our understanding of bacterial T6SS in counteracting host immune responses.

Edwardsiella tarda is associated with edwardsiellosis in cultured fish, resulting in heavy losses in aquaculture. So far, different types of vaccine have been attempted against E. tarda. In this study, an optimized eukaryotic expression plasmid was developed and an optimized DNA vaccine co-encoding antigenic and adjuvant peptide using a bicistronic expression system was designed. As a result, a modified plasmid harbored cytomegalovirus (CMV) promoter attached with R region of long terminal repeat from human T-cell leukemia virus type 1 (CMV/R) and woodchuck hepatitis virus post-transcriptional response element (WPRE) component showed an increased antigenic gene expression compared with unmodified plasmid. Moreover, the designed system based on bicistronic system exhibited a stronger ability to express antigenic gene and the RPS achieved 87.3% compared with plasmid encoding antigentic gene. Finally, immunological analysis showed that the DNA vaccine induced both innate and adaptive immune responses. These results suggest that co-encoding antigenic and adjuvant proteins might be an efficient strategy to develop DNA vaccines in aquaculture in the future.

Edwardsiella piscicida is an important pathogen that infects a wide range of hosts from fish to human. Recent studies demonstrated that E. piscicida can invade and survive within multiple nonphagocytic cells, but the internalization mechanism remains poorly understood. Here, we used HeLa cells as a nonphagocytic cell model to investigate the endocytic strategy used by the pathogenic E. piscicida isolate EIB202. Using a combination of optical and electron microscopy, we observed obvious membrane ruffles and F-actin rearrangements in HeLa cells after EIB202 infection. We also revealed that EIB202 internalization significantly depended on the activity of Na(+)/H(+) exchangers and multiple intracellular signaling events related to macropinocytosis, suggesting that E. piscicida utilizes the host macropinocytosis pathway to enter HeLa cells. Further, using inhibitory drugs and shRNAs to block specific endocytic pathways, we found that a caveolin-dependent but not clathrin-dependent pathway is involved in E. piscicida entry and that its entry requires dynamin and membrane cholesterol. Together, these data suggest that E. piscicida enters nonphagocytic cells via macropinocytosis and caveolin-dependent endocytosis involving cholesterol and dynamin, improving the understanding of how E. piscicida interacts with nonphagocytic cells. IMPORTANCE Bacterial internalization is the first step in breaking through the host cell defense. Therefore, studying the mechanism of bacterial internalization improves the understanding of the pathogenic mechanism of bacteria. In this study, the internalization process on nonphagocytic cells by Edwardsiella piscicida was evaluated. Our results showed that E. piscicida can be internalized into nonphagocytic cells via macropinocytosis and caveolin-mediated endocytosis, and that cholesterol and dynamin are involved in this process. These results reveal a new method for inhibiting E. piscicida infection, providing a foundation for further studies of bacterial pathogenicity.

Vibrio anguillarum, a devastating pathogen causing vibriosis among marine fish, is prevailing in worldwide fishery industries and accounts for grievous economic losses. Therefore, a rapid on-site detection and diagnostic technique for this pathogen is in urgent need. In this study, two mouse monoclonal antibodies (MAbs) against V. anguillarum, 6B3-C5 and 8G3-B5, were generated by using hybridoma technology and their isotypes were characterized. MAb 6B3-C5 was chosen as the detector antibody and conjugated with quantum dots. Based on MAb 6B3- C5 labeled with quantum dots, a modified dot blot assay was developed for the on-site determination of V. anguillarum. It was found that the method had no cross-reactivity with other than V. anguillarum bacteria. The detection limit (LOD) for V. anguillarum was 1 × 10(3) CFU/ml in cultured bacterial suspension samples, which was a 100-fold higher sensitivity than the reported colloidal gold immunochromatographic test strip. When V. anguillarum was mixed with turbot tissue homogenates, the LOD was 1 × 10(3) CFU/ml, suggesting that tissue homogenates did not influence the detection capabilities. Preenrichment with the tissue homogenates for 12 h could raise the LOD up to 1 × 10(2) CFU/ml, confirming the reliability of the method.

In purpose of valid Streptococcus iniae detection, we established a colorimetric biosensor using gold nanoparticles (AuNPs) labeled with dual functional probes and along with loop-mediated isothermal amplification (LAMP) assay (LAMP-AuNPs). Based on the characteristics of self-aggregation and bio-conjugation with ligands, AuNPs were chosen for observable color change in tandem with LAMP amplification method to reach high sensitivity and easy operation. Meanwhile, the improvement of dual probes that could fully utilize the LAMP product gave the biosensor a stable result exhibition. LAMP-AuNPs targeting gene ftsB, one of the ATP transporter-related genes, turned out favorable specificity in cross reaction among other fish pathogens. The detect limit of 102 CFU revealed a better sensitivity compared with polymerase chain reaction (PCR) method and AuNPs lateral flow test strip (LFTS). It was also proved to be effective by zebrafish infection model trials with less than 2-h time consumption and nearly no devices which make it a convenient biosensor for point-to-care S. iniae detection.

Bacterial strains used as backbone for the generation of vaccine prototypes should exhibit an adequate and stable safety profile. Given the fact that live attenuated vaccines often contain some potential risks in virulence recovery and spread infections, new approaches are greatly needed to improve their biological safety. Here, a critically iron-regulated promoter PviuA was screened from Vibrio anguillarum, which was demonstrated to respond to iron-limitation signal both in vitro and in vivo. By using PviuA as a regulatory switch to control the expression of phage P22 lysis cassette 13-19-15, a novel in vivo inducible bacterial lysis system was established in V. anguillarum. This system was proved to be activated by iron-limitation signals and then effectively lyse V. anguillarum both in vitro and in vivo. Further, this controllable bacterial lysis system, after being transformed into a live attenuated V. anguillarum vaccine strain MVAV6203, was confirmed to significantly improve biological safety of the live attenuated vaccine without impairing its immune protection efficacy.

Vibrio anguillarum, which is part of normal microflora on fish, is the causative agent of vibriosis in aquaculture. It is speculated that V. anguillarum does not affect the host in most situations, but can cause a severe disease once the host is compromised. In the study reported herein, skin-injured and intestine-injured zebrafish, Danio rerio, were established as a model to mimic the natural infection caused by V. anguillarum when fish suffered an injury to a mucosal surface. Our results showed the lethal dose to 50% of the population (LD50) of skin-injured zebrafish was 6.8 × 103 colony-forming unit (CFU)/mL, which was much lower than intestine-injured zebrafish (1.9 × 106 CFU/mL) or non-injured zebrafish (5.5 × 106 CFU/mL). With the quantitative polymerase chain reaction and immunohistochemical analysis, we found that V. anguillarum proliferated rapidly in the skin and muscle after the bacteria entered into the host via the skin injury. The bacteria were subsequently transported to the immune organs and then caused a systemic infection in the fish. However, mortality of skin-injured zebrafish significantly decreased if the fish were allowed to heal. These results indicate that minimizing injury to the mucosal surfaces of fish, especially the skin, will reduce infections caused by V. anguillarum.

As important zoonotic organisms causing infections in humans, Edwardsiella bacteria are also notorious leading fish pathogens haunting worldwide aquaculture industries. However, the taxa are now widely recognized to be misclassified, which hurdled the understanding of the epidemiology and development of effective diagnostics and vaccines. Currently the genus Edwardsiella consists of three species Edwardsiella tarda, E. ictaluri, and E. hoshinae. Previous phylogenomic analysis revealed that E. tarda strains display two major highly divergent genomic types (genotypes), EdwGI and EdwGII, and the former represents a genotype of fish-pathogenic isolates and being recently proposed as a novel species E. piscicida, sp. nov. Here multiple phylogenetic analyses and the genome-level comparisons of EdwGI strains disclose that the phylogroup strains from diseased eel formed an obviously distinct cluster that could be equated with a new species status. The phylogenetic evidence for the new species assignment was also supported by corresponding DNA-DNA hybridization estimation values and by phenotypic characteristics. Interestingly, further comparative genomics reveals that these strains have acquired the locus of enterocyte effacement (LEE) genes and as a result these bacteria contain at least 2 sets of distinct T3SS and 3 sets of T6SS gene clusters, respectively. It is therefore proposed that the phylogroup strains from diseased eel should be classified as Edwardisella anguillarum sp. nov., and the type strain is ET080813(T) (=DSM27202(T)=CCUG 64215(T)=CCTCC AB2013118(T)=MCCC 1K00238(T)). These findings will contribute to development of species-specific control measures against Edwardsiella bacterium in aquatic animals, while also shedding light on the pathogenesis evolution in Edwardsiella bacterium.

Edwardsiella tarda and Vibrio anguillarum are the two main pathogenic bacteria that cause edwardsiellosis and vibriosis in various species of fish raised in aquaculture. In our previous study, the live attenuated vaccines E. tarda WED and V. anguillarum MVAV6203 showed robust relative protection when vaccinated zebrafish or turbot were challenged with virulent E. tarda or V. anguillarum, respectively. Additionally, vaccinated fish processed the two vaccines through different pathways of antigen processing and presentation. Here, the immune protection of a combination vaccination consisting of E. tarda WED and V. anguillarum MVAV6203 was initially evaluated in zebrafish. After challenge with E. tarda and V. anguillarum at 1 month post-vaccination, the vaccinated zebrafish exhibited the relative protective survival of 70% and 90%, respectively. The expression of genes related to antigen recognition, processing and presentation were measured in the liver and spleen of vaccinated zebrafish. Gene expression profiling showed that more than one Toll-like receptor signaling pathway was activated and that both MHC I and II pathways of antigen processing and presentation were evoked. Later, the immune protection of the combination vaccine was evaluated in turbot and it showed similarly effective immune-mediated protection. By ELISA analysis, we found that the specific antibody levels in vaccinated turbot increased compared to those of fish vaccinated by a single vaccine during 2 months post-vaccination. Meanwhile, the expression levels of MHC I and II in the liver, spleen and kidney of vaccinated turbot were both up-regulated, suggesting that the MHC I and II pathways of antigen processing and presentation are activated in vaccinated turbot, similar to vaccinated zebrafish. In summary, a combination vaccine of live attenuated E. tarda WED and V. anguillarum MVAV6203 is effective and could be used widely in the future.

Edwardsiella tarda is a rod-shaped Gram-negative pathogenic bacterium that causes hemorrhagic septicemia in fish. Nucleoid-associated protein HU is a basic DNA-binding protein with structural specificity in regulating genes expression. In wild-type E. tarda EIB202, HU is composed of two subunits HUα (hupA) and HUβ (hupB), and exists in homodimer or heterodimer forms. Different from the wild-type and ΔhupB mutant, ΔhupA mutant was found to be defective in cell growth, H2S production, acid adaptation, and exhibited abnormal cell division resulting in a filamentous phenotype in log phase bacteria. The qRT-PCR result showed that deletion of hupA significantly up-regulated the transcription levels of recA and sulA, which in turn stimulated RecA-dependent pathway to prevent cell division, resulting in filamentous morphology in E. tarda. Furthermore, the elongated ΔhupA cells showed a striking defect in EPC cell invasion, and the adhesion and internalization rates were reduced to 25% and 27% of the wild-type in log phase cultures. Confocal laser scanning microscopy revealed that filamentous bacteria failed to adhere to and could not be internalized into EPC. When some of the bacteria regained the rod-shape morphology in stationary cultures, the ΔhupA mutants showed increased adhesion and internalization rates into EPC. Moreover, ΔhupA mutant exhibited delayed mortalities (for two days) in zebrafish but the LD50 increased 17 folds. Immunohistochemical analysis showed that ΔhupA mutant reduced proliferation abilities in the muscle, liver and intestine of zebrafish. This study indicates that HU protein and strains morphology play essential roles in the virulence network of E. tarda.

This study aimed to investigate the protective mucosal immunity elicited by live attenuated Vibrio anguillarum in fish. Zebrafish were immunized by bath or injection way, and undertook bath challenge at 28 days post vaccination. The results implied that bath vaccination was the better delivery route for inducing the protective immunity against bath challenge in zebrafish. The expressions of genes related to Th1, Th2 and Th17 cells were measured in the mucosal tissues of vaccinated and challenged zebrafish. Gene expression profiles showed that Th17-like responses were induced in mucosal immune system by vaccination via bath and injection routes while Th1 and Th2-like responses were not remarkable. Compared to injection vaccination, bath vaccination elicited the intense Th17-like immune responses in the gut tissue of zebrafish. Additionally, in gills and skin, Th17-like mucosal immunity elicited by injection vaccination occurred later than that by bath vaccination. Our results proved the immunological importance of gut in bath vaccination and the presence of two-compartmental model for immune response in zebrafish. In conclusion, bath vaccination more efficiently elicited protective Th17-like immunity than injection vaccination in mucosal tissues of vaccinated zebrafish. In turbot, effective immune protection against wild-type V. anguillarum was obtained by bath-vaccinated and the Th17-like responses were found in mucosal and systemic tissues.

Here, we report a simple and ultrasensitive upconversion fluorescent strip sensor based on NaYF4:Yb,Er nanoparticles (NPs) and the lateral flow immunochromatographic assay (LFIA). Carboxyl-modified β-NaYF4:Yb,Er NPs were successfully synthesized by a facile one-pot solvothermal approach, upon further coupling with monoclonal antibody, the resultant UCNPs-antibody conjugates probes were used in LFIA and served as signal vehicles for the fluorescent reporters. V. anguillarum was used as a model analyte to demonstrate the use of this strip sensor. The limit of the detection for the fluorescent strip was determined as 10(2) CFU mL(-1), which is 100 times lower than those displayed by enzyme-linked immunosorbent assays, while the time needed for the detection was only 15 min. Furthermore, no cross-reaction with other eight pathogens was found, indicating the good specificity of the strip. This developed LFIA would offer the potential as a useful tool for the quantification of pathogens analysis in the future.

Summary A live attenuated Vibrio anguillarum vaccine was recently established in the laboratory that induced immunoprotection against vibriosis in zebrafish, Danio rerio. To improve immunogenicity, the effects of different booster vaccination regimens were investigated using bath-vaccination in a zebrafish model. Zebrafish receiving booster doses at 2 weeks or at both 2 and 4 weeks after primary vaccination were better protected in comparison to fish that received a single vaccination. In addition, the booster vaccination induced a prolonged specific antibody response. No correlation between a weak specific antibody response and a strong protection was observed, indicating that the booster vaccination could enhance the affinity of Immunoglobulin M (IgM) rather than the amount. Moreover, changes in the immune-related gene expression of the booster-vaccinated group suggested that the booster enhanced the adaptive immune responses.

Edwardsiella tarda is the etiologic agent of Edwardsiellosis, a devastating fish disease prevailing in worldwide aquaculture industries. Here we developed the Spe I-based pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) assays to investigate the genetic diversity of Edwardsiella isolates obtained from aquaculture farms and to get insights into the epidemiological relationships of E . tarda isolates in China. The results showed a strong congruence of clustering profiles obtained by PFGE and MLST methods. This investigation also indicated distinct phylogenetic profiles of E . tarda isolates from different hosts and different geographical regions in China with the following conclusions: i) E . tarda isolates collected from flatfish (turbot or flounder) in Northern China were phylogenetically distinct from the eel isolates from Southern China, and ii) E . tarda isolates from humans and fish (except strain DT from Oscar fish) were clustered into two distinct clades. dnaK -based phylogenetic analysis of the 67 Edwardsiella strains from all over the world further confirmed the above inference as well as the previous findings that there is misclassification in Edwardsiella species and Edwardsiella ictaluri is close to fish isolates of E . tarda while showing distant phylogenetic relationship to human isolates of E . tarda . Our data highlighted the intraspecies relationships of E . tarda and indicated potential application of PFGE and MLST methods in tracing source of infection and enhancing understanding of epidemiological relationships among isolates of various origins.

Live attenuated vaccines are a promising application to control bacterial fish diseases. A live attenuated Vibrio anguillarum vaccine candidate was established in our laboratory to protect fish against vibriosis. To elucidate the mechanism of immunoprotection, it is necessary to compare the different immune responses to infection between vaccinated and non-vaccinated fish. In this study, the expression levels of pathogen-specific antibodies and immune-related genes upon challenge at 28 days post-vaccination were compared between vaccinated and non-vaccinated zebrafish. In the results, the specific antibody levels against virulent V. anguillarum in the vaccinated group did not rise significantly following infection, which suggested that high-affinity antibodies were induced by the vaccine. In the non-vaccinated group, the specific IgM response was triggered at 3 days post-infection and showed a delayed antibody response. Meanwhile, the transcription levels of the genes encoding the pro-inflammatory cytokine IL-1β and the chemokine IL-8 were more highly up-regulated in non-vaccinated fish than in vaccinated fish. This suggests that the overwhelming inflammatory response trigged by infection in non-vaccinated zebrafish was controlled in vaccinated zebrafish. Interestingly, the expression levels of adaptive immune-related genes were increased in vaccinated fish after challenge, compared to the non-vaccinated fish. These results suggest that inoculation with the live attenuated vaccine triggered protection by curbing inflammation and strengthening the adaptive immune response.

Edwardsiella tarda is a Gram-negative pathogen which causes systemic infection in turbot. The increasing frequency of edwardsiellosis in turbot farming has stressed the need to understand the immune responses of fish, for the further development of prevention and control strategies. As a broad spectrum protective antigen, a recombinant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of E. tarda EIB202 has been proven to present remarkable protection against E. tarda, Aeromonas hydrophila, Vibrio anguillarum, Vibrio alginolyticus and Vibrio harvei in zebrafish. Here, the protection and immune responses of turbot vaccinated with this antigen were studied. Fish vaccinated with recombinant GAPDH via intraperitoneal injection exhibited a low cumulative mortality when challenged with E. tarda EIB202, while high levels of specific antibodies and enhanced bactericidal activities of the immunized sera were observed. In addition, significantly increased transcription levels of four immune-related genes including IL-1β, MHC Iα and IIα, and IgM in the liver, spleen and kidney tissues of vaccinated fish showed that both humoral and cellular immune responses were soundly aroused in the vaccinated fish. Moreover, the IgM antibodies induced by recombinant GAPDH exhibited obvious cross-reactions with the other four pathogens. These results suggested that recombinant GAPDH could present effective protective immunity not only against E. tarda but also other extracellular pathogens, and would be a potential vaccine candidate against polymicrobial infections in the aquaculture industry.

Inflammasome activation is an important innate immune defense mechanism against bacterial infection, and in return, bacteria express virulence determinants that counteract inflammasome activation. Many such effectors are secreted into host cells via specialized bacterial secretion systems. Here, the intracellular pathogenic bacterium Edwardsiella tarda was demonstrated to activate NLRC4 and NLRP3 inflammasomes via a type III secretion system (T3SS), and to inhibit NLRP3 inflammasome via a type VI secretion system (T6SS), indicating the antagonistic roles of these systems in inflammasome signaling. Furthermore, a non-VgrG T6SS effector, EvpP, was identified that significantly inhibited NLRP3 inflammasome activation. Subsequent studies revealed that EvpP significantly suppressed Jnk activation, thus impairing oligomerization of the inflammasome adaptor ASC. Moreover, EvpP counteracted cytoplasmic Ca

With the development of gene technology, expressing heterologous antigens in attenuated bacteria has become an important strategy to design multivalent vaccines. In our previous work, an attenuated Vibrio anguillarum named MVAV6203 was developed and proven to be an efficient live vaccine candidate. In this research, we aimed to express protective antigen glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Edwardsiella tarda in attenuated Vibrio anguillarum to establish a multivalent V. anguillarum vector vaccine. Several strategies were compared between low- vs. high-copy plasmid-mediated antigen expression, in vivo-inducible vs. constitutive antigen expression and intracellular vs. surface-displaying antigen expression. Zebrafish, Danio rerio (Hamilton), was applied as the fish model to evaluate the immune protection of the V. anguillarum vector vaccine candidates. Our results demonstrated that V. anguillarum MVAV6203 (pUTatLNG40), which harbours a low-copy plasmid-loaded antigen surface display system under the control of a constitutive promoter, presented the best protective efficacy against the infection of Vibrio anguillarum (relative per cent survival, RPS = 85%) and Edwardsiella tarda (RPS = 70%).

Aims: The aims of this study were to reveal the roles of the gene locus qseEGF in the pathogenesis of Edwardsiella tarda. Methods and Results: Genome sequencing of fish pathogen E. tarda EIB202 reveals that the gene locus qseEGF, which encodes a novel two-component system QseEF, were located in E. tarda. The transcription of qseE, qseF and qseG was firstly characterized to be cotranscribed by reverse-transcribed PCR (RT-PCR). The mutant strains ΔqseE, ΔqseF and ΔqseG were constructed with in-frame deletion strategy. Compared with the wild type, all of the mutants showed attenuated virulence and impaired intracellular survival capabilities. Deletion in qseE, qseF and qseG resulted in different effects on hemolysin production in E. tarda. qRT-PCR results indicated that QseEF played a role in regulation of secretion systems, which in turn affected the virulence of E. tarda. Conclusions: The results manifested that QseEF system affected the virulence in E. tarda EIB202 by controlling the secretion system and hemolysin production. QseE, QseG and QseF in E. tarda serve for the physiological fitness and pathogenesis related to the bacterial survival in macrophage and in vivo of fish. Significance and Impact: The present results suggested that the important role of two-component system QseEF in regulation of E. tarda pathogenesis and its potential for attenuated live vaccine construction.

Edwardsiella tarda is a Gram-negative broad-host-range pathogen that causes hemorrhagic septicemia in many commercially important fish species. Its ability to adapt to and thrive in diverse environments outside and inside of its hosts prompts us to investigate the roles of the previously identified 33 putative two-component signal transduction systems (TCSs) in E. tarda. In this work, we successfully constructed deletion mutations in each of the response regulator genes, suggesting that none of the TCSs are essential for cell viability in E. tarda. The mutants were further examined for roles in biofilm formation, antibiotic resistance, stress response, expression and secretion of proteins involved in either the type III secretion system (T3SS) or type VI secretion system (T6SS), as well as virulence. Through these assays, we identified four regulators of biofilm development, two regulators of antibiotic resistance, and four regulators involved in stress responses. We found that two regulators, EsrB and PhoP, are essential for the pathogenicity of E. tarda and further demonstrated that these two regulators have codependent and independent contributions to E. tarda virulence. Mutation of EsrB resulted in the complete loss of both the T3SS and T6SS proteins, while PhoP partially regulated the expression of T3SS and T6SS genes through EsrB, and was essential for resistance to antimicrobial peptides. This work suggested that these two response regulators are involved in the regulation of the complex virulence network of this bacterium and merit as candidate genes for live attenuated vaccine construction.

Aims: The aim of this study was to reveal functional redundancy and variation of the two catalases KatB and KatG in Edwardsiella tarda. Methods and Results: Genome sequencing of fish pathogen Edw. tarda EIB202 reveals that it contains two genes putatively encoding catalases, katB (ETAE_1368) and katG (ETAE_0889). Under free-living conditions, single disruption in katB or katG resulted in no growth impairment, whereas double mutation of the two genes led to moderate decrease in growth, indicating that these two catalases were together essential for the physiological fitness by dissipating the endogenous H2O2. katG mutant exhibited much more elevated sensitivity to exogenous H2O2 than katB mutant did, indicating that KatG was quasi-essential in detoxifying external reactive oxygen species (ROS) in Edw. tarda EIB202. Further comparative analysis indicated that katB or katG disruption showed different effects on the virulence-related processes of Edw. tarda such as haemolysin production, bile and serum resistance, as well as the internalization within fish epithelial cells. Moreover, both of the katB and katG mutants exhibited incapacity to replicate in murine macrophage J774 cell model, although the deficiency was seen much severe for ΔkatB/katG mutant. With regard to in vivo virulence, katB and katG mutants displayed delayed lethality and increased LD50 values for zebrafish. Conclusions: KatB and KatG in Edw. tarda serve for the physiological fitness, and pathogenesis related the bacterial survival in macrophage and in vivo of fish. Significance and Impact of the Study: Counteracting ROS for systemic infection, Edw. tarda catalase KatG and KatB merits as potential targets for attenuated live vaccine construction.

Aims: Subcellullar localizations and cross-immunities of GAPDHs from six common pathogenic bacteria in aquaculture were investigated. Methods and Results: Subcellullar localizations of GAPDHs of Edwardsiella tarda EIB202, Edwardsiella ictaluri ATCC33202, Aeromonas hydrophila LSA34, Vibrio anguillarum MVM425, Vibrio alginolyticus EPGS020401 and Vibrio harveyi VIB647 were analysed with Western blotting, indirect immunofluorescence and flow cytometry examinations. Immunoprotections of different recombinant GAPDHs against these pathogens were investigated with zebrafish model. Western blotting of subcellular extractions showed that all GAPDHs were secreted into extracellular medium and periplasmic space. In addition, GAPDHs were demonstrated to distribute in the outer membranes except MVM425 and VIB647. And, GAPDHs were confirmed to be present on the surface of these bacteria with indirect immunofluorescence and flow cytometry examinations. The remarkable cross-protective immunities of these recombinant GAPDHs were induced in zebrafish, and the relative protective survivals were almost over 60%. Conclusions: Localizations of GAPDHs from these pathogenic bacteria were similar to many other causative agents. And, GAPDHs could be important protective antigens and give remarkable cross-immunity against different pathogens. Significance and Impact of the Study: Recombinant GAPDH could be designed as a broad spectrum vaccine candidate against multiple microbial infections in aquaculture.

Aims: The aim of this study was to classify Edwardsiella strains isolated from China aquaculture based on biochemical and molecular methods. Methods and Results: In this study, biochemical characterization of 19 Edwardsiella tarda isolates and two Edwardsiella ictaluri isolates was performed with API 20E system. Other pathogenicity-related phenotypes such as haemagglutination, haemolytic activities and lethality to fish were also examined in these strains. As it was difficult to categorize the subgroups of Edw. tarda according to their origins or phenotypic properties, three PCR-based methods, i.e. PCR amplification of virulence genes, Enterobacterial repetitive intergenic consensus-PCR and BOX-PCR, were carried out to further resolve the relatedness of the Edw. tarda isolates. As a result, all Edw. tarda isolates could be generally grouped into pathogenic and nonpathogenic branches before being classified into strain-specific or origin-specific clades. Conclusions: Biochemical characterization was sensitive for interspecific typing, while PCR-based approaches permitted a more accurate discrimination for intraspecific typing resulting in pathogenic and nonpathogenic clusters and further more delicate clades for Edwardsiella. Significance and Impact of the Study: PCR-based genomic fingerprinting to study the relatedness and trace the pathogenicity of the Edwardsiella strains will be helpful in investigating the virulence factors of Edwardsiella and in the development of vaccines and diagnostics for edwardsiellosis.

Aims: The aims of this study were to construct and evaluate the live attenuated vaccine against edwardsiellosis on zebra fish model. Methods and Results: In this study, the deletion mutant of aroC gene for the biosynthesis of chorismic acid in Edwardsiella tarda EIB202 was firstly constructed by allelic exchange strategy. According to the genome information, 19 double mutants and one multiple mutant were successively constructed by deleting virulence-associated genes based on the ΔaroC mutant. Zebra fish model was used to assay the virulence of the mutants by intramuscular (i.m.) injection. Fourteen mutants were significantly attenuated with accumulated mortality ranged from 0 to 63% (P

Edwardsiella tarda is the causative agent of edwardsiellosis in fish. The genome sequence of a virulent strain EIB202 has been determined. According to the genome sequence, the lipopolysaccharide (LPS) synthesis cluster containing a putative O-antigen ligase gene waaL was identified. Here, the in-frame deletion mutant ΔwaaL was constructed to analyze the function of WaaL in E. tarda EIB202. The ΔwaaL mutant displayed absence in O-antigen side chains in the LPS production. The ΔwaaL mutant exhibited an increased sensitivity to hydrogen peroxide indicating that the LPS was involved in the endurance to the oxidative stress in hosts during infection. In addition, the resistance of ΔwaaL to serum and polymyxin B decreased remarkably. The ΔwaaL mutant was also attenuated in virulence, showed an impaired ability in internalization of epithelioma papulosum cyprinid (EPC) cells and a comparatively poor ability of proliferation in vivo, which was in line with the increased LD(50) value. These results indicated that waaL gene was a functional member of the gene cluster involved in LPS synthesis and highlighted the importance of the O-antigen side chains to stress adaption and virulence in E. tarda, signifying the gene as a potential target for live attenuated vaccine against this bacterium.

Edwardsiella tarda is a gram-negative pathogen for hemorrhagic septicemia in a broad range of hosts. The type VI secretion system (T6SS) has recently been dissected in E. tarda to secrete EvpC, EvpI and a novel effector protein EvpP. In this study, sequencing and genetic alignments showed that evpP genes from different E. tarda isolates were highly similar and an evpP homolog was also found in Aeromonas hydrophila 0865 isolated from a diseased eel, suggesting the possible lateral gene transfer of evpP or the whole T6SS gene island. With reporter strains carrying gfp gene fused to the evpP promoter region, flow cytometric analysis revealed that transcription of evpP was positively regulated by either the two-component system EsrA-EsrB in E. tarda or the iron concentration in media. Compared with the parental strain, in-frame deletion of evpP in E. tarda EIB202 led to the significantly increased 50% lethal doses in zebrafish (Danio rerio) and Japanese flounder (Paralichthys olivaceus), decreased hemolytic activities, failure to adhere to mucus and reduced serum resistance, and complementation of an intact evpP gene restored these phenotypes in the evpP mutant. Investigation of infection kinetics indicated that the evpP deletion mutant was unable to proliferate in vivo, particularly in immune organs of fish. Moreover, the evpP deletion mutant exhibited incapacity to internalize in EPC cell model in vitro, demonstrating that EvpP in T6SS plays critical roles for invasion mechanism of E. tarda and merits as potential target for attenuated live vaccine construction.

In this study, rpoS gene was identified from Edwardsiella tarda EIB202 and its functional role was analyzed by using an in-frame deletion mutant rpoS and the complemental strain rpoS (+). Compared with the wild type and rpoS (+), rpoS was impaired in terms of the ability to survive under oxidative stress and nutrient starvation, as well as the resistance to 50% serum of Scophthalmus maximus in 3 h, demonstrating essential roles of RpoS in stress adaptation. The rpoS mutant also displayed markedly increased chondroitinase activity and biofilm formation. Real-time polymerase chain reaction revealed that the expression level of quorum sensing autoinducer synthetase genes luxS and edwI was increased by 3.7- and 2.5-fold in the rpoS mutant strain. Those results suggested that rpoS might be involved in the negative or positive regulation of chondroitinase and biofilm formation, or quorum sensing networks in E. tarda, respectively. Although there were no obvious differences between the wild-type and the rpoS mutant in adherence of epithelioma papulosum cyprini (EPC) cell and in the lethality on fish model, rpoS deletion leads to the drastically reduced capacity for E. tarda to internalize in EPC cells, indicating that RpoS was, while not the main, the factor required for the virulence network of E. tarda.

The causative agent was isolated from diseased turbots (Scophthalmus maximus) stricken by a high-mortality outbreak of bacterial septicaemia occurring in a mariculture farm in Yantai, a northern coastal city of China. Seven pure isolates, namely EH-15, EH-103, EH-107, EH-202, EH-203, EH-305 and EH-306, belonged to Edwardsiella tarda. The phenotypic features of the cultures were analysed extensively. Three of the isolates showed high 16S rDNA sequence similarities with E. tarda sequence (GenBank accession no. EF467289). However, unlike the E. tarda ATCC 15947, all the isolates, except EH-15, contained a novel large plasmid sized about 23.7 kb. Furthermore, pathogenicity of the isolates was addressed by experimental challenges with fish models. The isolates exhibited strong virulence to swordtail fish with LD50 ranging between 3.8 × 103 and 3.8 × 105 CFU g−1, and EH-202 displaying the lowest LD50 value among them. Antibiotic susceptibilities of E. tarda isolates were assayed. Compared with E. tarda ATCC 15947, the isolates displayed strong resistance to chloramphenicol, and the probable dominant chloramphenicol resistance determinant was cat III. Depicting the main biological properties of turbot-borne E. tarda strains in China, the study provided useful information for further unveiling their pathogenic mechanisms.

Edwardsiella piscicida is an important causative agent of hemorrhagic septicemia in fish and infects both cultured and wild fish species. Type VI secretion system (T6SS) was proved to play important roles in pathogenesis of E. piscicida. In this study, it was demonstrated that the expression of T6SS genes evpB and evpC was under control of the global regulator H-NS in E. piscicida and the transcriptional level of evpB and evpC was significantly down-regulated by H-NS. Compared to the wild type, the transcriptional levels of evpB and evpC were up-regulated in hns null mutant, while down-regulated in hns overexpression strain. The results of EMSA and DNase I footprinting revealed that H-NS protein directly bound to upstream region of evpC at multiple sites. A high-affinity motif with a 9-nucleotide sequence 5'-ATATAAAAT-3' was defined for H-NS preferential recognition based on the feature of the binding sites. These results indicated that H-NS acted cooperatively to form extended nucleoprotein filaments on target DNA. Site-directed mutagenesis of H-NS further showed that R86 played an essential role in T6SS gene binding. These findings highlighted the mechanisms underlying the complex regulation network of T6SS by H-NS in E. piscicida.

The quorum sensing (QS) system, as a well-functioning population-dependent gene switch, has been widely applied in many gene circuits in synthetic biology. In our work, an efficient cell density-controlled expression system (QS) was established via engineering of the Vibrio fischeri luxI-luxR quorum sensing system. In order to achieve in vivo programmed gene expression, a synthetic binary regulation circuit (araQS) was constructed by assembling multiple genetic components, including the quorum quenching protein AiiA and the arabinose promoter P araBAD , into the QS system. In vitro expression assays verified that the araQS system was initiated only in the absence of arabinose in the medium at a high cell density. In vivo expression assays confirmed that the araQS system presented an in vivo -triggered and cell density-dependent expression pattern. Furthermore, the araQS system was demonstrated to function well in different bacteria, indicating a wide range of bacterial hosts for use. To explore its potential applications in vivo , the araQS system was used to control the production of a heterologous protective antigen in an attenuated Edwardsiella tarda strain, which successfully evoked efficient immune protection in a fish model. This work suggested that the araQS system could program bacterial expression in vivo and might have potential uses, including, but not limited to, bacterial vector vaccines.

Background Delivery of antigens by live bacterial carriers can elicit effective humoral and cellular responses and may be an attractive strategy for live bacterial vaccine production through introduction of a vector that expresses an exogenous protective antigen. To overcome the instability and metabolic burden associated with plasmid introduction, alternative strategies, such as the use of in vivo-inducible promoters, have been proposed. However, screening an ideal in vivo-activated promoter with high efficiency and low leak expression in a particular strain poses great challenges to many researchers. Results In this work, we constructed an in vivo antigen-expressing vector suitable for Edwardsiella tarda, an enteric Gram-negative invasive intracellular pathogen of both animals and humans. By combining quorum sensing genes from Vibrio fischeri with iron uptake regulons, a synthetic binary regulation system (ironQS) for E. tarda was designed. In vitro expression assay demonstrated that the ironQS system is only initiated in the absence of Fe2+ in the medium when the cell density reaches its threshold. The ironQS system was further confirmed in vivo to present an in vivo-triggered and cell density-dependent expression pattern in larvae and adult zebrafish. A recombinant E. tarda vector vaccine candidate WED(ironQS-G) was established by introducing gapA34, which encodes the protective antigen glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the fish pathogen Aeromonas hydrophila LSA34 into ironQS system, and the immune protection afforded by this vaccine was assessed in turbot (Scophtalmus maximus). Most of the vaccinated fish survived under the challenge with A. hydrophila LSA34 (RPS = 67.0%) or E. tarda EIB202 (RPS = 72.3%). Conclusions Quorum sensing system has been extensively used in various gene structures in synthetic biology as a well-functioning and population-dependent gene circuit. In this work, the in vivo expression system, ironQS, maintained the high expression efficiency of the quorum sensing circuit and achieved excellent expression regulation of the Fur box. The ironQS system has great potential in applications requiring in vivo protein expression, such as vector vaccines. Considering its high compatibility, ironQS system could function as a universal expression platform for a variety of bacterial hosts.

As a leading pathogen, Edwardsiella piscicida can cause hemorrhagic septicemia in fish and gastro-intestinal infections in humans. The two-component regulatory system EsrA-EsrB plays essential roles in pathogenesis through the type III and type VI secretion systems, and hemolysin production in E. piscicida It is unclear whether other virulence- or stress response-associated genes are regulated by EsrA-EsrB. In this study, the proteomes of wild-type E. piscicida EIB202 and esrB mutant strains were compared to reveal EsrB regulon components after growth in Luria-Bertani broth (LB). Overall, the expression levels of nine genes exhibited significant changes, and five of them required the presence of EsrB, while others exhibited higher levels in the esrB mutant. The diverse functions of these proteins were identified, including amino acid metabolism, oxidative stress defense and energy production. Interestingly, superoxidase dismutase and thiol peroxidase were the most significantly down-regulated by EsrB. Furthermore, other reported reactive oxygen species (ROS) resistance-related genes were also down-regulated by EsrB as revealed by quantitative real-time. Compared with the wild-type and the complement strain esrB+, ΔesrB displayed a significantly enhanced ROS resistance. These results demonstrated that EsrB plays important roles in the ROS resistance pathway in E. piscicida grown in LB conditions.

It is an attractive strategy to develop a recombinant bacterial vector vaccine by expressing exogenous protective antigen to induce the immune response, and the main concern is how to enhance the cellular internalization of antigen produced by bacterial vector. Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides which facilitate cellular uptake of various molecular cargoes and therefore have great potentials in vector vaccine design. In this work, eleven different CPPs were fused to the C-terminus of EGFP respectively, and the resultant EGFP-CPP fusion proteins were expressed and purified to assay their cross-membrane transport in macrophage J774 A.1 cells. Among the tested CPPs, TAT showed an excellent capability to deliver the cargo protein EGFP into cytoplasm. In order to establish an efficient antigen delivery system in Escherichia coli, the EGFP-TAT synthesis circuit was combined with an in vivo inducible lysis circuit PviuA-E in E. coli to form an integrated antigen delivery system, the resultant E. coli was proved to be able to lyse upon the induction of a mimic in vivo signal and thus release intracellular EGFP-TAT intensively, which were assumed to undergo a more efficient intracellular delivery by CPP to evoke protective immune responses. Based on the established antigen delivery system, the protective antigen gene flgD from an invasive intracellular fish pathogen Edwardsiella tarda EIB202, was applied to establish an E. coli recombinant vector vaccine. This E. coli vector vaccine presented superior immune protection (RPS = 63%) under the challenge with E. tarda EIB202, suggesting that the novel antigen delivery system had great potential in bacterial vector vaccine applications.

Edwardsiella tarda is an intractable Gram-negative pathogen in many fish species to cause edwardsiellosis. Its infection leads to extensive losses in a diverse array of commercially important fish. The type III secretion system (T3SS) has been considered as one of the major virulence factors and plays important roles in its intracellular lifestyle. In this study, an E. tarda EIB202 mutant WED with deletions in the T3SS genes for EseB, EseC, EseD and EscA, along with the aroC gene for the biosynthesis of chorismic acid, as well as the curing of endogenous plasmid pEIB202 was constructed by allelic exchange strategy. Compared to the wild-type EIB202 which was highly virulent towards turbot (Scophthamus maximus) via intraperitoneal (i.p.), intramuscular (i.m.) injection or immersion and caused systemic infection in turbot as well as the unexpected red mouth symptom when immersion challenged, WED was highly attenuated when inoculated into turbot via i.m., i.p. and immersion routes, and exhibited significantly impaired capacity to survive in fish tissues. WED showed 5700-fold higher 50% lethal dose (LD50) than that of the wild type when i.m. or i.p. challenged. Inoculation with WED by i.p. or immersion injection routes elicited significant protection against the challenge of the wild-type E. tarda after 5 weeks of vaccination. The vaccinated fish produced low while significant level of specific antibody and showed increased expression of immune-related factors including IL-1β, IFN-γ, MHC II, MHC-I and CD8, indicating that WED possesses significant immunoprotective potential. Furthermore, our data indicated that a single dose of i.p. and immersion vaccination with WED could produce significant protection as long as 12 and 6 months, respectively. These results demonstrated the feasibility of WED as a live attenuated vaccine in turbot against edwardsiellosis by immersion or i.p. injection routes.

Background Zebrafish (Danio rerio) is a prominent vertebrate model of human development and pathogenic disease and has recently been utilized to study teleost immune responses to infectious agents threatening the aquaculture industry. In this work, to clarify the host immune mechanisms underlying the protective effects of a putative vaccine and improve its immunogenicity in the future efforts, high-throughput RNA sequencing technology was used to investigate the immunization-related gene expression patterns of zebrafish immunized with Edwardsiella tarda live attenuated vaccine. Results Average reads of 18.13 million and 14.27 million were obtained from livers of zebrafish immunized with phosphate buffered saline (mock) and E. tarda vaccine (WED), respectively. The reads were annotated with the Ensembl zebrafish database before differential expressed genes sequencing (DESeq) comparative analysis, which identified 4565 significantly differentially expressed genes (2186 up-regulated and 2379 down-regulated in WED; p Conclusion These data provided insights into the molecular mechanisms underlying zebrafish immune response to WED immunization and might aid future studies to develop a highly immunogenic vaccine against gram-negative bacteria in teleosts.

The aims of this study were to reveal the roles of the gene locus qseEGF in the pathogenesis of Edwardsiella tarda.Genome sequencing of fish pathogen E. tarda EIB202 reveals that the gene locus qseEGF, which encodes a novel two-component system QseEF, were located in E. tarda. The transcription of qseE, qseF and qseG was firstly characterized to be cotranscribed by reverse-transcribed PCR (RT-PCR). The mutant strains ΔqseE, ΔqseF and ΔqseG were constructed with in-frame deletion strategy. Compared with the wild type, all of the mutants showed attenuated virulence and impaired intracellular survival capabilities. Deletion in qseE, qseF and qseG resulted in different effects on hemolysin production in E. tarda. qRT-PCR results indicated that QseEF played a role in regulation of secretion systems, which in turn affected the virulence of E. tarda.The results manifested that QseEF system affected the virulence in E. tarda EIB202 by controlling the secretion system and hemolysin production. QseE, QseG and QseF in E. tarda serve for the physiological fitness and pathogenesis related to the bacterial survival in macrophage and in vivo of fish.The present results suggested that the important role of two-component system QseEF in regulation of E. tarda pathogenesis and its potential for attenuated live vaccine construction.

Edwardsiella bacteria are leading fish pathogens causing huge losses to aquaculture industries worldwide. E. tarda is a broad-host range pathogen that infects more than 20 species of fish and other animals including humans while E. ictaluri is host-adapted to channel catfish causing enteric septicemia of catfish (ESC). Thus, these two species consist of a useful comparative system for studying the intricacies of pathogen evolution. Here we present for the first time the phylogenomic comparisons of 8 genomes of E. tarda and E. ictaluri isolates. Genome-based phylogenetic analysis revealed that E. tarda could be separate into two kinds of genotypes (genotype I, EdwGI and genotype II, EdwGII) based on the sequence similarity. E. tarda strains of EdwGI were clustered together with the E. ictaluri lineage and showed low sequence conservation to E. tarda strains of EdwGII. Multilocus sequence analysis (MLSA) of 48 distinct Edwardsiella strains also supports the new taxonomic relationship of the lineages. We identified the type III and VI secretion systems (T3SS and T6SS) as well as iron scavenging related genes that fulfilled the criteria of a key evolutionary factor likely facilitating the virulence evolution and adaptation to a broad range of hosts in EdwGI E. tarda. The surface structure-related genes may underlie the adaptive evolution of E. ictaluri in the host specification processes. Virulence and competition assays of the null mutants of the representative genes experimentally confirmed their contributive roles in the evolution/niche adaptive processes. We also reconstructed the hypothetical evolutionary pathway to highlight the virulence evolution and niche adaptation mechanisms of Edwardsiella. This study may facilitate the development of diagnostics, vaccines, and therapeutics for this under-studied pathogen.

A fish vaccine candidate, live attenuated Vibrio anguillarum, which can protect fish from vibriosis, was established in our laboratory. In this study, the protective immunological mechanism of live attenuated V. anguillarum was investigated in zebrafish as a model animal. After bath-vaccinated with the live attenuated strain, zebrafish were challenged with wild pathogenic strain to test the immunoprotection of the live attenuated strain. As the results, specific antibody response of fish against V. anguillarum was found to gradually increase during 28 days post-vaccination, and remarkable protection was showed with a high relative protection survival (RPS) of about 90%. Moreover, the vaccination changed the expressions of several immune-related genes in the spleens and livers of zebrafish. Among them, the expressions of pro-inflammatory factors such as IL-1 and IL-8 were tenderly up-regulated with about 3-4 fold in 1-7 days post-vaccination, while MHC II rose to a peak level of 4-fold in 7th day post-vaccination. These results gave some important messages about the mechanism of specific protection induced by live attenuated V. anguillarum and showed the availability of zebrafish model in the evaluation of the vaccine candidate.