[Show abstract][Hide abstract] ABSTRACT: 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.
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%).
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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).
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.
[Show abstract][Hide abstract] ABSTRACT: In this study, expressions of some immune parameters among embryos during different development stages from immunized and mock-immunized female fish were compared at day 14 post immunization of the zebrafish brood stock vaccinated with the live attenuated Vibrio anguillarum vaccine MVAV6203. It was found that transcriptional levels of innate immune cell gene markers including spi.1, l-plastin, mpx and gata-1 as well as the myeloid transcription factor c/ebpβ were up-regulated and expressed earlier in embryos of immunized female zebrafish than naive embryos during early development stage. Investigation through ELISA revealed an increase in antibody level in egg cytosol of the female zebrafish following immunization prior to breeding, which was transferred to larvae through eggs. Rise of specific antibody against wild-type V. anguillarumMVM425 was detected at hour 24 post fertilization (p.f.) eggs from immunized brood stock. Extracts of the newly fertilized eggs from immunized zebrafish also showed a higher capability of killing V. anguillarum MVM425. Moreover, larvae from immunized zebrafish also showed a higher capability of resisting the V. anguillarum MVM425 from proliferation after immersion challenge. Interestingly, the higher content of total IgM and specific antibody in eggs and larvae at 24, 48 and 72 h p.f. were positively correlated with their higher antibacterial activity against V. anguillarum MVAV425. Our results showed that, the development of immune cells was enhanced and the maternally derived antibody could protect early embryos and larvae from attack of specific pathogens via vaccination with a live attenuated vaccine.
Aquaculture Research 06/2015; DOI:10.1111/are.12821 · 1.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of the World Aquaculture Society 06/2015; 46(3). DOI:10.1111/jwas.12188 · 0.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Edwardsiella tarda is a leading fish pathogen haunting worldwide aquaculture industry. In E. tarda, two-component system EsrA-EsrB positively regulates type III and VI secretion systems (T3SS and T6SS) and negatively regulates hemolysin EthA, which has been demonstrated to be essential for the invasion processes in fish. In order to develop a live attenuated vaccine (LAV) with high invasiveness to be practically and economically used as immersion-administered vaccine in aquaculture, here, we generated a random mutation library of esrB sequences by error-prone PCR and introduced them into the E. tarda esrB deletion mutant. The mutant YWZ47 with significantly increased hemolytic activity and low T3SS and T6SS secretion was screened. Phenotypes including extracellular protein profiles, invasion in macrophages, lethality toward fish, and infection kinetics were investigated in the wild-type strain EIB202 and the mutants ΔesrB, ΔT3SS, ΔT6SS, ΔT3SS/ΔT6SS, and YWZ47. Compared to the documented LAV strain ΔesrB, YWZ47 showed higher invasive capability and low in vivo virulence toward fish. Significantly higher relative percent survival (RPS) could be generated in turbot (Scophthalmus maximus) against the challenge of the wild-type EIB202 when inoculated through immersion route, and the RPS was comparable with that of ΔesrB through intraperitoneal (i.p.) injection inoculation. Two mutated points, K167M and H197L, were found by sequence analysis of EsrBYWZ47 variant. These structural modifications underpin the variations in the regulatory functions of the mutant and wild-type EsrB. This study promoted understanding of virulence regulation by EsrB in E. tarda and presented a promising candidate of invasive attenuated vaccine used in aquaculture industries.
[Show abstract][Hide abstract] ABSTRACT: 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 ET080813T (= DSM27202T = CCUG 64215T = CCTCC AB2013118T = MCCC 1K00238T). 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.
[Show abstract][Hide abstract] ABSTRACT: Edwardsiella tarda, an enteric opportunistic pathogen, is associated with acute to chronic edwardsiellosis in cultured fish, resulting in heavy losses in aquaculture. To date, the pathogenesis of E. tarda has been extensively studied and a great deal of vaccine candidates have been attempted. However, the research on the predisposition of E. tarda infection is poorly reported. In this study, the effects of intestinal inflammation on E. tarda infection were investigated using a zebrafish model that influenced by perturbation of intestinal microbiota. Featured symptoms of edwardsiellosis were observed in intestinal inflammatory zebrafish compared with healthy fish. Higher bacterial numbers were detected in both mucosal tissues (intestine, skin and gills) and lymphoid tissues (liver, spleen and kidney) of inflammatory zebrafish while the bacterial loads in healthy zebrafish appeared to be relatively lower by 10-100 folds. Moreover, significant up-regulation of IL-1β, TNF-α and iNOS was noticed in multiple tissues of zebrafish with intestinal inflammation between 6 and 72 h post infection. However, only moderate elevation was observed in the gills and liver of healthy fish. Furthermore, the expression of genes involved in neutrophil recruitment (mpx, IL-8 and LECT2) and antimicrobial response (β-defensin and hepcidin) showed notable up-regulation in the intestine of inflammatory zebrafish. These results demonstrate that fish with intestinal inflammation is more susceptible to E. tarda and the antimicrobial response during E. tarda infection might inhibit the growth of intestinal microbiota that E. tarda could resist. Our results suggest that maintaining good management to avoid intestinal inflammation is a feasible prevention measure against edwardsiellosis.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
The evpP gene in fish pathogen Edwardsiella tarda, coding the T6SS secretory protein EvpP and carrying an evpA-evpO independent promoter region, was crucial for host cell invasion. The transcription of evpP was positively regulated by either the two-component system EsrA-EsrB or iron concentration, and its overexpression was known to enhance the invasion ability in our previous study. This work demonstrated that the H-NS protein, a pleiotropic regulator of gene expression, was a new transcriptional modulator of evpP gene. The results showed that in vivo the transcriptional level of evpP was downregulated by H-NS and in vitro this global regulator interacted directly with evpP promoter region. Moreover, DNase I footprinting experiments mapping the interaction regions of H-NS and evpP revealed that this global regulator bound to evpP promoter and neighbouring areas at multiple sites. We provided a new insight into evpP regulation network and demonstrated the repression of H-NS to the transcription of evpP gene.
Significance and impact of the study:
Recently, the devastating fish disease edwardsiellosis caused by Edwardsiella tarda has been widely concerned. The xenogeneic silencing of the classic regulator H-NS to the T6SS secretory protein EvpP, which played an important role in the virulence of Edw. tarda, was firstly reported in this study. It raised a better understanding of the virulence regulation of EvpP and provided more information about the complex infection mechanism of this pathogen. Our findings would contribute to the development of live attenuated vaccines against edwardsiellosis thus reducing the economic losses caused by this bacterium.
[Show abstract][Hide abstract] ABSTRACT: In recent years, increasing diseases especially bacterial diseases have brought a host of losses with the expansive cultivation of turbot (Scophthalmus maximus). In order to do more research about the immune system of turbot for better understanding the mechanism of resisting diseases, the immunoglobulin genes related to secretory and membrane-bound IgM (s-IgM and m-IgM) of turbot were cloned using homology sequences cloning and SMART RACE PCR method. The heavy chain of s-IgM cDNA is 1900 bp in length including a leader region, a variable region, four constant regions (CH1, CH2, CH3 and CH4) and a C-terminal while the cDNA of m-IgM is 1795 bp with the same leader region, variable region, three constant regions (CH1, CH2 and CH3) and two transmembrane regions (TM1 and TM2). The sequence of IgM gene was also obtained and the structure consisted of V-CH1-CH2-CH3-CH4-TM1-TM2 is similar to other fishes. The highest level of s-IgM expression was observed in spleen, followed by kidney, gills, eyes, skin of the healthy turbot whereas the same profile of m-IgM expression is found with low level. And s-IgM takes up dominant proportion of total IgM expression. Also the relative expressions of s-IgM and m-IgM were analyzed in turbot vaccinated with the live attenuated vaccine Vibrio anguillarum. Not only the transcriptions of both s-IgM and m-IgM in liver, spleen and kidney of turbot injected with V. anguillarum MVAV6203 were up-regulated but also the expressions of s-IgM and m-IgM in spleen, kidney, gut, skin and gills of bath-vaccinated turbot were increased. Comparing the ratio changes of relative expression of m-IgM and s-IgM in vaccinated turbot, we found that the proportion of m-IgM were increasing in both administration routes, which probably indicated that the increasing expression of m-IgM strengthen the phagocytic ability of B cells.
[Show abstract][Hide abstract] ABSTRACT: Live attenuated vaccine is one of the efficient vaccine candidates in aquaculture, which can be easily delivered to fish via bath-vaccination. An outstanding advantage of bath-vaccination is that vaccine delivery is through the same route as that utilized by many fish pathogens, generating specific mucosal immune responses. In this work, we investigated the mucosal immune responses induced by a live attenuated Vibrio anguillarum vaccine in zebrafish via bath-vaccination. Bacteria proliferated rapidly in 3 hours after vaccination and maintained at a high level until 6 hours in the intestine. Besides, bacteria persisted in the intestine for a longer time whereas decreased rapidly in the skin and gills. Moreover, a significant up-regulation of TLR5 triggering a MyD88-dependent signaling pathway was observed in the intestine, which implied that flagella were the crucial antigenic component of the live attenuated vaccine. And macrophages and neutrophils showed active responses participating in antigen recognition and sampling after vaccination. Furthermore, an inflammation was observed with plenty of lymphocytes in the intestine at 24 h post vaccination but eliminated within 7 days. In conclusion, the live attenuated V. anguillarum vaccine induced notable mucosal immune responses in the intestine which could be used as a mucosal vaccine vector in the future.
[Show abstract][Hide abstract] ABSTRACT: Edwardsiella tarda the etiological agent for edwardsiellosis, a devastating fish disease prevailing in worldwide aquaculture industries was subjected to a molecular genetic study. To research into the influence when RpoN (σ54) and RpoS (σ38) were deleted simultaneously, the double deletion mutant of RpoN (σ54) and RpoS (σ38), namely rnrs, was constructed. Firstly, RpoN and RpoS are both essential for H2O2, starvation, high osmotic pressure and acid resistance, which have synergistic effect. Secondly, virulence of rnrs reduces significantly compared to E. tarda EIB 202 WT, ΔrpoN mutant and ΔrpoS mutant. Furthermore, transcriptional control of rpoS by rpoN in stationary phase was observed through qRT-PCR, while rpoS had no influence on rpoN in the level of transcription. Meanwhile, regulation of flagellar sigma factor σF (FliA) and other flagella-related genes including flgA, flgK, flgL, motA, and motB by rpoS, and rpoN was found. fliA and other flagella-related genes were controlled positively by rpoN, while negatively by rpoS. At last, two differential expression genes in transcriptional level of rnrs strain were detected by DD-RT-PCR, namely cheY and narK. This study therefore indicated interaction between sigma factors RpoN and RpoS, which modulates stress response, virulence, motility, and provides new insights into the regulatory networks of E. tarda.
[Show abstract][Hide abstract] ABSTRACT: Insulin precursor fusion protein expressed in Pichia pastoris is a single-chain protein with a spacer peptide (EEAEAEAEPK) localized at its N-terminal. Currently, the one-step transpeptidation reaction with low yield and high cost is generally employed to convert the insulin precursor fusion protein into human insulin ester. In this study, a two-step transpeptidation reaction was proposed that separating cleavage step from coupling step so that each reaction was performed under its optimal conditions. Using this method, the total efficiency doubled and the reaction time was shortened compared to the one-step method. In addition, the amount of O-t-butyl-L-threonine t-butyl ester and trypsin dosages were reduced by 50% and 75%, respectively. This two-step transpeptidation strategy was simple and efficient and could be used for the pharmaceutical production of human insulin. This article is protected by copyright. All rights reserved.