Tarda EIB202 Research Articles

Global transcriptional regulator FNR regulates the pyruvate cycle and proton motive force to play a role in aminoglycosides resistance of Edwardsiella tarda.

Bacterial metabolism is related to resistance and susceptibility to antibiotics. Fumarate and nitrate reduction regulatory protein (FNR) is a global transcriptional regulator that regulates metabolism. However, the role of FNR in antibiotic resistance is elusive. Here, fnr deletion mutant was constructed and used to test the role in Edwardsiella tarda EIB202 (EIB202). Δfnr exhibited elevated sensitivity to aminoglycosides. The mutant had a globally enhanced metabolome, with activated alanine, aspartate, and glutamate metabolism and increased abundance of glutamic acid as the most impacted pathway and crucial biomarker, respectively. Glutamate provides a source for the pyruvate cycle (the P cycle) and thereby relationship between exogenous glutamate-activated P cycle and gentamicin-mediated killing was investigated. The activated P cycle elevated proton motive force (PMF). Consistently, exogenous glutamate potentiated gentamicin-mediated killing to EIB202 as the similarity as the loss of FNR did. These findings reveal a previously unknown regulation by which FNR downregulates glutamate and in turn inactivates the P cycle, which inhibits PMF and thereby exhibits the resistance to aminoglycosides.

Edwardsiella tarda Tunes Tricarboxylic Acid Cycle to Evade Complement-Mediated Killing.

Evasion of complement-mediated killing is a common phenotype for many different types of pathogens, but the mechanism is still poorly understood. Most of the clinic isolates of Edwardsiella tarda, an important pathogen infecting both of human and fish, are commonly found serum-resistant. To explore the potential mechanisms, we applied gas chromatography-mass spectrometry (GC-MS)-based metabolomics approaches to profile the metabolomes of E. tarda EIB202 in the presence or absence of serum stress. We found that tricarboxylic acid (TCA) cycle was greatly enhanced in the presence of serum. The quantitative real-time PCR (qRT-PCR) and enzyme activity assays validated this result. Furthermore, exogenous succinate that promotes the TCA cycle increased serum resistance, while TCA cycle inhibitors (bromopyruvate and propanedioic acid) that inhibit TCA cycle, attenuated serum resistance. Moreover, the enhanced TCA cycle increased membrane potential, thus decreased the formation of membrane attack complex at cell surface, resulting serum resistance. These evidences suggested a previously unknown membrane potential-dependent mechanism of serum resistance. Therefore, our findings reveal that pathogen mounts a metabolic trick to cope with the serum complement-mediated killing.

Nuevos genes relacionados con el sistema secretor tipo 4 de Edwardsiella tarda

Edwardsiella tarda es una bacteria Gram-negativa responsable de edwardsiellosis, una enfermedad de peces y mamíferos incluido el humano, la cual se caracteriza por la formación de múltiples abscesos. Distintas cepas de E. tarda poseen un número de genes asociados con virulencia, resistencia a múltiples antibióticos y sistemas secretores de toxinas que explican en cierto grado su capacidad de sobrevivir dentro de células fagocíticas y de infectar a diversos hospederos. En estudios previos, nuestro grupo secuencio parcialmente el genoma de una cepa virulenta de E. tarda (ETSJ54) aislada de lenguado japonés (Paralichthys olivaceus) con edwardsiellosis y reportó un número de genes asociados a su virulencia. En este estudio se ha re-analizado los datos de secuenciación y en este proceso se identificaron varios genes que codifican para la estructura de superficie Pili y el sistema secretor tipo IV, la mayoría de los cuales estuvieron rodeados por genes codificadores de transposasas y otros genes de origen plasmídico. La secuencia de nucleótidos de dichos genes no tuvieron identidad con la de los genes previamente reportados en E. tarda EIB202, una cepa virulenta aislada de turbot (Scophthalmus maximus) en China. Los resultados sugieren diferencias en el contenido genético de cepas de E. tarda de distinto origen geográfico y la necesidad de desarrollar nuevos proyectos de secuenciamiento de genomas de E. tarda.

An immunochromatographic test strip for rapid detection of fish pathogen Edwardsiella tarda

Abstract Background Edwardsiella tarda, the etiologic agent of edwardsiellosis, is a devastating fish pathogen prevailing in worldwide aquaculture industries and accounting for severe economic losses. There is a raising concern about E. tarda being a significant zoonotic pathogen, and it is urgent to develop a rapid detection of this pathogen. This is the first study to develop a test strip for rapid detection of E. tarda in turbot. Results Mouse monoclonal antibodies (MAbs) and rabbit polyclonal antibody (PAb) against E. tarda were generated from immunization of mice and rabbits with a virulent isolate of E. tarda EIB202. Two MAbs specific to isolates of E. tarda were obtained, and one of them (25C1) was selected to conjugate with colloidal gold as the detector antibody. Rabbit PAb was used as the capture antibody. It was found the strip had no cross-reactivity with non-E. tarda bacterial microbes and the limit of detection (LOD) was 1 × 105 colony-forming units (CFU)/ml. The detection could be visually observed by the naked eye within 5 min. This test strip was verified with a similar detection limit and much less analysis time compared with a dot blot immunoassay (1 × 105 CFU/ml for LOD and 120 min for reaction time). When the samples were mixed with turbot tissue homogenates, strong immunoreactivity was observed over 105 CFU/ml, which suggested that the turbot tissue homogenates did not affect the detection of the strip. Pre-enrichment with homogenized turbot tissue for 12 h could increase the detection limit of the E. tarda present in the sample up to 1 to 10 CFU/ml. In practice, in detecting 20 turbot ascite samples infected by E. tarda, the immunochromatographic test strip showed a high accuracy (100% positive). Conclusions The immunochromatographic test strip offers great promise for a rapid, simple, and economical method of E. tarda on-site detection, and with different antibodies, it might be used to detect other aquatic pathogens.

A quorum sensing-based in vivo expression system and its application in multivalent bacterial vaccine

BackgroundDelivery 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.ResultsIn 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%).ConclusionsQuorum 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.

WcaJ, the initiating enzyme for colanic acid synthesis, is required for lipopolysaccharide production, biofilm formation and virulence in Edwardsiella tarda

Abstract Edwardsiella tarda , a Gram-negative bacterium, is the causative agent of edwardsiellosis, which is a major problem for the aquaculture industry worldwide. Undecaprenyl-phosphate glycosyltransferase, WcaJ, as the initiating enzyme for colanic acid synthesis, is hypothesized to be involved in the pathogenesis of infection. In this study, a wcaJ in-frame deletion mutant (Δ wcaJ ) of a virulent isolate of E. tarda EIB202 was constructed through double crossover allelic exchange by means of the suicide vector pRE118. A complementary strain (Δ wcaJ (pACYC184K- wcaJ )) was obtained by electrotransformation of a low-copy plasmid pACYC184K carrying the intact wcaJ into the Δ wcaJ mutant. Several virulence-associated phenotypes of the wild-type, the wcaJ mutant and complementary strains were tested. It was found that the deletion of wcaJ reduced biofilm formation, lipopolysaccharide (LPS) production, adherence and internalization to Epithelioma papulosum cyprini (EPC) cells and pathogenicity to zebrafish embryos. All the phenotypes displayed by the deletion mutant were restored in the complementary strain. These results revealed that WcaJ is required for pathogenicity in E. tarda.

GC/MS-based metabolomics approach to identify biomarkers differentiating survivals from death in crucian carps infected by Edwardsiella tarda

Microbial disease problems constitute the largest single cause of economic losses in aquaculture. An understanding of immune system in aquaculture animals how to function in defense against bacterial infections is especially important to control these diseases and improve food quality and safety. In the present study, we use a crucian carp model to explore which pathways and metabolites are crucial for the defense against infection caused by Edwardsiella tarda EIB202. We establish the metabolic profile of crucian carps and then compare the metabolic difference between survivals and dead fish by self-control. We identify elevating unsaturated fatty acid biosynthesis and decreasing fructose and mannose metabolism as the most key pathways and increasing palmitic acid and decreasing d-mannose as the most crucial metabolites differentiating survivals from death in these fish infected by E. tarda. Our findings highlight the importance of metabolic strategy against bacterial infections.

Cell-penetrating peptides mediated protein cross-membrane delivery and its use in bacterial vector vaccine

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.

HU-induced polymorphous filamentation in fish pathogen Edwardsiella tarda leading to reduced invasion and virulence in zebrafish

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.

Edwardsiella tarda mutant disrupted in type III secretion system and chorismic acid synthesis and cured of a plasmid as a live attenuated vaccine in turbot

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.

Comparative proteomic analysis reveals new components of the PhoP regulon and highlights a role for PhoP in the regulation of genes encoding the F1F0 ATP synthase in Edwardsiella tarda

Edwardsiella tarda is an important cause of haemorrhagic septicaemia in fish and also of gastro- and extra-intestinal infections in humans. We have recently demonstrated that the PhoP-PhoQ two-component regulatory system plays important roles in both virulence and stress tolerance in E. tarda. In this study, the proteomes of the WT and phoP mutant strains were compared to define components of the PhoP regulon in E. tarda EIB202. Overall, 18 proteins whose expression levels exhibited a twofold or greater change were identified; 13 of these proteins were found to require the presence of PhoP for full expression, while five were expressed at a higher level in the phoP mutant background. Identified proteins represented diverse functional categories, including energy production, amino acid metabolism and oxidative stress defence. Quantitative real-time PCR analysis of the mRNA levels for the identified proteins confirmed the proteomics data. Interestingly, the β subunit of the F1F0 ATP synthase, playing an important role in growth and virulence of E. tarda, was listed as one of the proteins whose expression was greatly dependent on PhoP. The F1F0 ATP synthase was encoded in a gene cluster (atpIBEFHAGDC) and the nine genes were transcribed as an operon. PhoP positively regulated the transcription of the nine ATP synthase genes and exerted this effect through direct binding to the promoter of atpI. Overall, the results provide new insights into the PhoP regulon and unravel a novel role for PhoP in the regulation of the F1F0 ATP synthase.

Proteomic analysis of a twin-arginine translocation-deficient mutant unravel its functions involved in stress adaptation and virulence in fish pathogenEdwardsiella tarda

Edwardsiella tarda is a Gram-negative, facultative aerobic pathogen which infects multifarious hosts including fish, amphibians and human beings. A twin-arginine translocation (Tat) gene cluster important for high-salt tolerance in E.tarda was identified previously. Here the genetic structure and pleiotropic roles of the Tat system in physiological adaptation of the bacterium were further characterized. Functional analysis indicated that tatD was not required for Tat export process and tatE might be an allelic gene of tatA in the bacterium. The results showed that disruption in the Tat system did not affect the morphology and biofilm formation in E.tarda, but did affect motility, hemagglutination, cell aggregation and infection of eukaryotic cells (e.g. macrophage J774a). Comparative proteomics analysis of subcellular proteins using two-dimensional gel electrophoresis and a qualitative shotgun protein sequencing method were implemented to identify proteins differentially expressed in E.tarda EIB202 vs. ∆tatABCD. The results revealed a large repertoire of differentially expressed proteins (n=61), shedding light on the Tat system associated with virulence and stress-associated processes in E.tarda.

Immune response of turbot (Scophthalmus maximus L.) to a broad spectrum vaccine candidate, recombinant glyceraldehyde-3-phosphate dehydrogenase of Edwardsiella tarda

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.

Identification of qseEGF genetic locus and its roles in controlling hemolytic activity and invasion in fish pathogen Edwardsiella tarda

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 tarda flagellar protein FlgD: A protective immunogen against edwardsiellosis

Edwardsiella tarda is a gram-negative bacterium and a causative agent of edwardsiellosis, resulting to severe loss of the aquaculture industry. In this study, based on the reverse vaccinology, sixteen flagellar proteins were selected from highly pathogenic E. tarda EIB202 genome information and in silico analyzed as potential vaccine candidates. Among them, ten recombinant proteins were highly expressed in Escherichia coli and successfully purified. The immunoprotective potentials of these purified recombinant proteins were evaluated in zebrafish model. And recombinant FlgD and FliD were found to lead to a high relative percent survival (RPS, about 70%) against E. tarda EIB202. Furthermore, FlgD required in flagellum hook assembly brought about the similar immune protection in turbot. The immune responses of zebrafish and turbot to recombinant FlgD were also investigated, and the results indicated that its high protection was mainly involved in cellular mediated immune response, corresponding to the intracellular pathogenicity of E. tarda.

Comparative analysis of the roles of catalases KatB and KatG in the physiological fitness and pathogenesis of fish pathogen Edwardsiella tarda

The aim of this study was to reveal functional redundancy and variation of the two catalases KatB and KatG in Edwardsiella tarda. 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 H(2) O(2) . katG mutant exhibited much more elevated sensitivity to exogenous H(2) O(2) 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 LD(50) values for zebrafish. KatB and KatG in Edw. tarda serve for the physiological fitness, and pathogenesis related the bacterial survival in macrophage and in vivo of fish. Counteracting ROS for systemic infection, Edw. tarda catalase KatG and KatB merits as potential targets for attenuated live vaccine construction.

Characterization of Edwardsiella tarda rpoN: roles in σ70 family regulation, growth, stress adaption and virulence toward fish

Edwardsiella tarda EIB202, a Gram-negative pathogen with strong virulence, is an opportunistic pathogen capable of causing edwardsiellosis with high mortality to fish. Alternative sigma factor 54 (RpoN) is an important regulator of virulence and stress resistance genes in many bacterial species and mainly responsible for transcription of genes in nitrogen utilization. In this study, the in-frame rpoN deletion mutant was constructed to analyze the function of RpoN in Edwardsiella tarda firstly. Compared to the wild-type and complemented strain rpoN (+), the ΔrpoN was impaired in terms of the ability to survive under oxidative stress, osmotic stress and acid resistance, as well as the growth in Luria-Bertani medium, demonstrating essential roles of RpoN in stress resistance and nitrogen utilization. In addition, the ΔrpoN displayed markedly decreased biofilm formation and chondroitinase activity and was attenuated in virulence reflected in the increased median lethal dose value and extended infection cycle. Real-time polymerase chain reaction revealed that the expression levels of σ(70) class changed in varying degrees in the rpoN mutant. Especially, the expression levels of rpoS and fliA were down-regulated 4.1-fold and 7.9-fold in stationary phase in comparison with the wild type, respectively. Furthermore, two differential expression genes, znuA and flhC, were detected in the wild type and ΔrpoN using the method of differential display reverse transcription PCR.

Secreted glyceraldehyde-3-phosphate dehydrogenase as a broad spectrum vaccine candidate against microbial infection in aquaculture

Subcellullar localizations and cross-immunities of GAPDHs from six common pathogenic bacteria in aquaculture were investigated. 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%. 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. Recombinant GAPDH could be designed as a broad spectrum vaccine candidate against multiple microbial infections in aquaculture.

A novel in vivo inducible expression system in Edwardsiella tarda for potential application in bacterial polyvalence vaccine

Recombinant bacterial vector vaccine is an attractive vaccination strategy to induce the immune response to a carried protective antigen, and the main concern of bacterial vector vaccine is to establish a stable antigen expression system in vector bacteria. Edwardsiella tarda is an important facultative intracellular pathogen of both animals and humans, and its attenuated derivates are excellent bacterial vectors for use in recombinant vaccine design. In this study, we design an in vivo inducible expression system in E. tarda and establish potential recombinant E. tarda vector vaccines. With wild type strain E. tarda EIB202 as a vector, 53 different bacteria-originated promoters were examined for iron-responsive transcription in vitro, and the promoters P dps and P yncE showed high transcription activity. The transcription profiles in vivo of two promoters were further assayed, and P dps revealed an enhanced in vivo inducible transcription in macrophage, larvae and adult zebra fish. The gapA34 gene, encoding the protective antigen GAPDH from the fish pathogen Aeromonas hydrophila LSA34, was introduced into the P dps-based protein expression system, and transformed into attenuated E. tarda strains. The resultant recombinant vector vaccine WED/pUTDgap was evaluated in turbot ( Scophtalmus maximus). Over 60% of the vaccinated fish survived under the challenge with A. hydrophila LSA34 and E. tarda EIB202, suggesting that the P dps-based antigen delivery system had great potential in bacterial vector vaccine application.

Search for live attenuated vaccine candidate against edwardsiellosis by mutating virulence-related genes of fish pathogen Edwardsiella tarda

The aims of this study were to construct and evaluate the live attenuated vaccine against edwardsiellosis on zebra fish model. 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<0.05). The zebra fish vaccinated with ΔaroC, ΔaroCΔesrC, ΔaroCΔslyA and ΔaroCΔeseBCDΔesaC via i.m. injection showed ideal protection, resulting in relative per cent survival (RPS) of 68.3, 71.3, 80.1 and 81% against subsequent challenge with the wild-type Edw. tarda EIB202. ΔaroCΔeseBCDΔesaC behaved a low virulence and the highest RPS on zebra fish model. When the zebra fish were vaccinated with ΔaroCΔeseBCDΔesaC via injection, the expression of immune-related factors including IgM and MHC II was up-regulated. The mutant ΔaroCΔeseBCDΔesaC might serve as an effective live attenuated vaccine against edwardsiellosis.