Phenotypic traits,virulence‐associated gene profile and genetic relatedness of Edwardsiella tarda isolates from Japanese eel Anguilla japonica in Korea

Edwardsiella tarda is the predominant bacterium in farm‐cultured eel in Korea. Here, we evaluated the heterogeneity of 37 E. tarda isolates derived from Japanese eel with various origins (olive flounder, common carp and ornamental fish) between 2003 and 2010. Regardless of origins, the biochemical characteristics of E. tarda isolates were homogenous except hydrogen sulfide production, citrate utilization and mannitol fermentation. Based on the phylogenetic analysis of 16S rRNA, E. tarda isolates could be classified into two subgroups and displayed a close relation with Edwardsiella ictaluri and Edwardsiella hosinae lineages, suggesting that the subgroup I has been a predominant type in the Jeonnam and Jeonbuk provinces. I‐CeuI‐based pulsed‐field gel electrophoresis (PFGE) typing showed that the isolates from Japanese eels belonged to 11 pulsotypes, indicating that the presence of highly genomic diversity. Additionally, two isolates, ET‐060 and ET‐191, showed a high frequency of virulence genes (100%) and caused 90% and 60% mortality in Japanese eel, respectively. This finding suggests a substantial congruence of virulence gene profiles and pathogenicity. Our results demonstrate that the intraspecific diversity within E. tarda strains from Japanese eel has been in prior existence.

Significance and Impact of the Study

Based on the biochemical characteristics, the phylogenetic property of the 16S rRNA gene and PFGE types of Edwardsiella tarda, we could identify the intraspecific diversity of isolates from Japanese eel, Anguilla japonica in Korea. In addition, this study describes the strong congruence of virulence‐related genes and pathogenicity, suggesting that the virulence profile may be useful tool for prediction of pathogenicity.     

A Streptococcus iniae DNA vaccine delivered by a live attenuated Edwardsiella tarda via natural infection induces cross‐genus protection

Edwardsiella tarda and Streptococcus iniae are important fish pathogens. We have reported previously a live E. tarda vaccine based on the attenuated strain TX5RM and a S. iniae DNA vaccine based on the antigen Sia10. In this study, we examined the possibility of constructing a cross‐genus vaccine by taking advantage of the residual infectivity of TX5RM and using it as a carrier host for the natural delivery of a S. iniae DNA vaccine. For this purpose, the recombinant TX5RM, TX5RMS10, was created, which harbours and retains stably the DNA vaccine plasmid pCS10 that expresses Sia10. When flounder were vaccinated with TX5RMS10 via oral and immersion routes, TX5RMS10 was detected in multiple tissues within 12–14 days postvaccination (p.v.). At 7 and 14 days p.v., expression of the DNA vaccine was detected in spleen, kidney and liver. Following E. tarda and S. iniae challenge at one and 2 months p.v., the vaccinated fish exhibited relative per cent survival rates of 69–83%. Immunological analysis indicated that TX5RMS10‐vaccinated fish produced specific serum antibodies and exhibited enhanced expression of a wide range of immune genes.     

Overexpression of mltA in Edwardsiella tarda reduces resistance to antibiotics and enhances lethality in zebra fish

Aims: The aim of this study was to investigate the role of membrane‐bound lytic murein transglycosylase A (MltA) in a bacterial fish pathogen Edwardsiella tarda. Methods and Results: An mltA in‐frame deletion mutant (ΔmltA) and an mltA overexpression strain (mltA⁺) of Edw. tarda were constructed through double‐crossover allelic exchange and by transformation of a low‐copy plasmid carrying the intact mltA into the ΔmltA mutant, respectively. Either inactivation or overexpression of MltA in Edw. tarda resulted in elevated sensitivity to β‐lactam antibiotics and lower viability in oligotrophic or high osmotic environment than wild‐type strain. Autolysis induced by EDTA was reduced in ΔmltA strain, while mltA⁺ strain was virtually flimsy, indicating that MltA is responsible for the lysis effect. Moreover, mltA⁺ strain exhibited significant increases in lipopolysaccharide (LPS) biosynthesis and virulence to zebra fish compared with wild‐type strain. Conclusions: The results indicated that MltA plays essential roles in β‐lactam antibiotics and environmental stresses resistance, autolysis, LPS biosynthesis and pathogenicity of Edw. tarda. This is the first report that MltA has a virulence‐related function in Edw. tarda. Significance and Impact of the Study: This study provided useful information for further studies on pathogenesis of Edw. tarda.     

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

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 < 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. Conclusions: 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. Significance and Impact: The mutant ΔaroCΔeseBCDΔesaC might serve as an effective live attenuated vaccine against edwardsiellosis.     

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

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.     

Determination of the heterogeneous interactome between Edwardsiella tarda and fish gills

Edwardsiellosis caused by Edwardsiella tarda is a frequent occurrence throughout the world and has resulted in extensive losses in aquaculture. However, information regarding to protein-protein interaction between the pathogenic cells and host is not available although the portal of entry of the pathogen is determined. In this study, fish gill and bacterial pull-down approaches were used to isolate both bacterial outer membrane proteins that bind to gills and fish gill proteins that interact with bacterial cells, respectively. Eight interacting bacterial proteins and twelve interacting fish proteins were obtained. The genes of seven bacterial proteins were cloned and expressed for preparation of antibodies. The prepared antibodies were used to investigate protein-protein interactions between bacterial cells and fish gills. Five heterogeneous protein-protein interactions were determined. Moreover, the protective ability of three of the bacterial recombinant proteins, selected at random, was investigated in a mouse model where they showed significant protection. The gill proteins were highly homologous proteins with from humans and other animals where they are known to be involved in host immunity. These findings indicate that the heterogeneous interactome has significantly biological significance. Our results demonstrate a way to determine and understand the heterogeneous interaction between of E. tarda and gills.     

Edwardsiella tarda DnaK: expression, activity, and the basis for the construction of a bivalent live vaccine against E. tarda and Streptococcus iniae

Edwardsiella tarda and Streptococcus iniae are important aquaculture pathogens that affect many species of farmed fish. In this study, we analyzed the expression, activity, and immunoprotective potential of E. tarda heat shock protein DnaK. We found that dnaK expression was upregulated under conditions of heat shock, oxidative stress, and infection of host cells. Recombinant DnaK (rDnaK) purified from Escherichia coli exhibited ATPase activity and induced protection in Japanese flounder (Paralichthys olivaceus) against lethal E. tarda challenge. On the basis of these results and our previous observation that a protective S. iniae antigen Sia10 which, when expressed heterogeneously in E. coli DH5α, is secreted into the extracellular milieu, we constructed a chimeric antigen by fusing DnaK to Sia10. The resulting fusion protein Sia10-DnaK was expressed in DH5α via the plasmid pTDK. Western blot analysis indicated that Sia10-DnaK was detected in the culture supernatant of DH5α/pTDK. When flounder were vaccinated with live DH5α/pTDK, strong protection was observed against both E. tarda and S. iniae. ELISA analysis detected specific serum antibody production in fish vaccinated with rDnaK and DH5α/pTDK. Taken together, these results indicate that rDnaK is an intrinsic ATPase with immunoprotective property and that Sia10-DnaK delivered by a live bacterial host is an effective bivalent vaccine candidate against E. tarda and S. iniae infection.     

Inv1: an Edwardsiella tarda invasin and a protective immunogen that is required for host infection

Invasin is an outer membrane protein that is known to mediate entry of enteric bacteria into mammalian cells. In this study, we analyzed the function and immunoprotective potential of the invasin Inv1 from Edwardsiella tarda, a serious fish pathogen that can also infect humans. In silico analysis indicated that Inv1 possesses a conserved N-terminal DUF3442 domain and a C-terminal group 1 bacterial Ig-like domain. Subcellular localization analysis showed that Inv1 is exposed on cell surface and could be recognized by specific antibodies. Mutation of inv1 had no effect on bacterial growth but attenuates overall bacterial virulence and impaired the ability of E. tarda to attach and invade into host cells. Consistent with these observations, antibody blocking of Inv1 inhibited E. tarda infection of host cells. To examine the immunoprotective potential of Inv1, recombinant Inv1 (rInv1) corresponding to the DUF3442 domain was purified and used to vaccinate Japanese flounder (Paralichthys olivaceus). The results showed that rInv1 induced strong protection against lethal-dose challenge of E. tarda. ELISA analysis showed that rInv1-vaccinated fish produced specific serum antibodies that could enhance the serum bactericidal activity against E. tarda. Taken together, these results indicate that Inv1 is a surface-localized virulence factor that is involved in host infection and can induce effective immunoprotection when used as a subunit vaccine.     

Identification of novel putative virulence factors, adhesin AIDA and type VI secretion system, in atypical strains of fish pathogenic Edwardsiella tarda by genomic subtractive hybridization

Edwardsiella tarda, which is known to be the causative agent of edwardsiellosis in freshwater and marine fish, has two motility phenotypes. Typical strains exhibiting motility are isolated mainly from freshwater fish and Japanese flounder. Atypical strains exhibiting non-motility are isolated mainly from marine fish, with the exception of Japanese flounder. Subtractive hybridization was performed to identify genomic differences between these two phenotypes. Two fragments which showed homology to potential virulence factors were isolated from atypical strains: the autotransporter adhesin AIDA and a component of T6SS. We analysed DNA sequences of about 5 kbp containing these fragments and identified two partial ORF, and ORF encoding for other components of T6SS. The predicted amino acid sequences showed remarkably low homology to components of T6SS reported in the typical E. tarda strain PPD130/91. Furthermore, the organization of these ORF was different from the gene cluster of the typical E. tarda strain. AIDA and T6SS may therefore be associated with different pathogenicity in typical and atypical E. tarda hosts.     

Probiotic Lactobacillus sp. inhibit growth,biofilm formation and gene expression of caries‐inducing Streptococcus mutans

Streptococcus mutans contributes significantly to dental caries, which arises from homoeostasic imbalance between host and microbiota. We hypothesized that Lactobacillus sp. inhibits growth, biofilm formation and gene expression of Streptococcus mutans. Antibacterial (agar diffusion method) and antibiofilm (crystal violet assay) characteristics of probiotic Lactobacillus sp. against Streptococcus mutans (ATCC 25175) were evaluated. We investigated whether Lactobacillus casei (ATCC 393), Lactobacillus reuteri (ATCC 23272), Lactobacillus plantarum (ATCC 14917) or Lactobacillus salivarius (ATCC 11741) inhibit expression of Streptococcus mutans genes involved in biofilm formation, quorum sensing or stress survival using quantitative real‐time polymerase chain reaction (qPCR). Growth changes (OD600) in the presence of pH‐neutralized, catalase‐treated or trypsin‐treated Lactobacillus sp. supernatants were assessed to identify roles of organic acids, peroxides and bacteriocin. Susceptibility testing indicated antibacterial (pH‐dependent) and antibiofilm activities of Lactobacillus sp. against Streptococcus mutans. Scanning electron microscopy revealed reduction in microcolony formation and exopolysaccharide structural changes. Of the oral normal flora, L. salivarius exhibited the highest antibiofilm and peroxide‐dependent antimicrobial activities. All biofilm‐forming cells treated with Lactobacillus sp. supernatants showed reduced expression of genes involved in exopolysaccharide production, acid tolerance and quorum sensing. Thus, Lactobacillus sp. can inhibit tooth decay by limiting growth and virulence properties of Streptococcus mutans.     

Effects of quorum sensing autoinducer degradation gene on virulence and biofilm formation of Pseudomonas aeruginosa

The aiiA gene from Bacillus thuringiensis was cloned into the Pseudomonas/E. coli shuttle vector and transformed into Pseudomonas aeruginosa strain PAO1. Western blotting showed that the AiiA protein was expressed in PAO1. After induction by IPTG for 6 h and 18 h, expression of the aiiA gene in PAO1 completely degraded the quorum sensing autoinducers N-acylhomoserine lactones (AHLs): N-oxododecanoyl-L-homoserine lactone (OdDHL) and N-butyryl-L-homoserine lactone (BHL). The reduced amount of AHLs in PAO1 was also correlated with decreased expression and production of several virulence factors such as elastase and pyocyanin. AiiA expression also influenced bacterial swarming motility. Most importantly, our studies indicated that aiiA played significant roles in P. aeruginosa biofilm formation and dispersion, as observed by the differences of the biofilm formation on liquid and solid surfaces, and biofilm structures under a scanning electron microscope. These authors contributed equally to this work Supported by the National Natural Science Foundation of China (Grant No. 30570020) and Natural Science Foundation of Hubei Province of China (Grant No. 2004ABA120)     

Recombinant outer membrane protein A (OmpA) of Edwardsiella tarda, a potential vaccine candidate for fish, common carp

Outer membrane protein A (OmpA) is a component of the outer membrane of Edwardsiella tarda and is wildly distributed in Enterobacteriaceae family. The gene encoding the OmpA protein was cloned from E. tarda and expressed in Escherichia coli M15 cells. The recombinant OmpA protein containing His₆ residues was estimated to have a molecular weight of ∼38 kDa. In Western blot the native protein showed expression at ∼36 kDa molecular weight which was within the range of major outer membrane proteins (36–44 kDa) observed in this study. All E. tarda isolates tested harbored the ompA gene and the antibody raised to this protein was seen to cross react with other Gram negative bacteria. The OmpA protein characterized in this study was observed to be highly immunogenic in both rabbit and fish. In Enzyme linked immunosorbent assay, rabbit antisera showed an antibody titer of 1: 128,000. Common carp vaccinated with recombinant OmpA protein elicited high antibody production and immunized fish showed a relative percentage survival of 54.3 on challenge.