Identification of Protective Immunogens From Extracellular Secretome of Edwardsiella Tarda

Edwardsiella tarda is a gram negative bacterium of the Enterobacteriaceae. The bacterium has a strong virulence to many aquatic animal species, which brings a great economic loss in aquaculture. It is also an opportunistic pathogen to humans. Therefore, it is quite important to find out an efficient way to control and prevent the infectious diseases caused by this bacterium.

Vaccination has been known to be the most effective method to prevent infectious diseases caused by pathogenic bacteria for a long time. In view of this, to find out the vaccine candidates with high protective ability against E. tarda is increasingly valued by researchers. Until now, only some outer membrane and cytoplasm have been demonstrated to be effective protective antigens in E. tarda. Recently, increasing interests have been paid on extracellular secretory proteins. The secretory proteins are bacterial extracellular components and may activate host’s immune response. In E. tarda, studies related to extracellular secretory proteins includes the mechanisms of type III and type IV secretion system and the secretory proteome of E. tarda, but whether the proteins secreted out of the bacterium showed immune protection is largely unknown. Thus, investigation on protective immunogens from extracellular secretory proteins may provide efficient candidates for development of vaccines.

Determination of E. tarda extracellular secretory proteins  Out of 3575 proteins of E. tarda EIB202, 29 proteins were predicted as extracellular secretory proteins and 9 proteins existed in multi-locations with over 1.00 of extracellular scores using PSORTb software. Thus, total 38 proteins were annotated to be extracellular secretory proteins in the present study.

Gene cloning and expression and purification of their recombinant proteins

Then, 20 out of the 38 secretory protein genes were randomly selected and successfully amplified in the present study. They were ETAE_2467, FlgD, ETAE_2130, ETAE_2088, ETAE_2845, ETAE_2132, ETAE_2916, ETAE_1033, PapH, ETAE_2496, ETAE_3382, ETAE_1902, FlgK, FlgL, GcP, FlgG, ETAE_1030, EseC, FlgE, EvpC.  Their cDNAs were located in the place with their corresponding molecular mass and then recycled by 1% agorose electrophoresis. The amplification reliability was checked by digestion with restriction endonucleases and sequencing. All of the gene fragments were validated.  Then the gene fragments were cloned into expression vector pET-32a (+) plasmid (Novagen). The recombinant plasmids were transformed into E. coli BL21 (DH3) and screened by grown on LB plate containing ampicillin. Recombinants were picked and cultured in LB medium, and then IPTG was added to induce the expression of recombinant proteins. The recombinant proteins were purified and checked by SDS-PAGE. Only single bands were detected in all of these samples, which indicate the purity of these proteins. These data show that these recombinant proteins may be used for functional investigation.

Investigation of protective immunogens using active immunization 

To find out the candidates of vaccines against E. tarda, the twenty recombinant proteins were used to confer immune protection against infection caused by E. tarda EIB202 in zebrafish model using the active immunization approach. The zebrafish were grouped and then separately immunized with the 20 recombinant proteins and control was only injected with adjuvent. Bacterial challenge was carried out post 28 days of immunization. Among the 20 secretory proteins, ten, EseC, ETAE_2088, FlgD, ETAE_2130, EvpC, ETAE_2845, ETAE_1902, PapH, ETAE_1030, FlgE, showed significant immune protection, compared with the control group. Very importantly, the first four showed approximately 60% RPS and thus were potential candidates of vaccine against E.tarda infections.

Identification of extracellular secretory proteins with highly immune protection

Moreover, we wanted to know whether the four proteins with highly immune protection were detected in extracellular production of the bacterium.  Antisera to EseC, ETAE_1902, ETAE_2088, FlgD were commercially from Chengxue Biotech. Corp. Guangzhou and were used as primary antibodies in Western blotting for detection of the four proteins in extracellular fraction and whole-cell lysate fraction. The whole cell lysate fraction was used in this detection because it might be a positive control for the detection of these proteins in the extracellular fraction as well as be indictor of antibody specificity. The antisera separately recognized EseC, ETAE_1902, ETAE_2088, FlgD in the places with corresponding molecular mass using whole-cell lysate fraction, whereas they only recognized EseC and ETAE_2088 in the respective places using the extracellular fraction. These results indicate that EseC and ETAE_2088 were extracellular secretary proteins. ETAE_1902 and FlgD were not detected in the extracellular production in this study, which might be related to the following reasons that they may be low abundance proteins or induced proteins.

In summary, identification of highly protective immunogens is the most key step in the development of efficient subunit vaccines. Here we present a systematic investigation on protective immunogens of extracellular secretory proteins in E. tarda.  Half out of 20 secretory proteins is verified to be protective immunogens, in which four show approximately 60% of RPS in zebrafish. These results may have significant implications in not only providing efficiently protective immunogens for development of vaccine, but also understanding bacterial stimulation from extracellular secretory proteins to host immune systems.