Construction Of An Attenuated Two-component Regulatory System KdpDE Strains Of Vibrio Alginolyticus And Evaluation Of Their Immunogenic And Cross-protective Effects As Vaccines

The two-component regulatory systems play an important role in the growth and virulence of bacteria. In this study, the phosphate regulatory sensor protein(KdpD) gene and phosphate transcriptional regulatory protein(KdpE) gene of Vibrio alginolyticus strain HY9901 were cloned. Sequence analysis revealed that the length of kdpD gene is1374 bp, encoding a putative protein of 457 amino acids. The length of kdpE gene is 732 bp and encodes a putative protein of 243 amino acids. A phylogenetic tree was constructed by MEGA 5.0 software, indicating that the KdpD and KdpE of V.alginolyticus had high genetic relationship with Vibrio campbellii and Vibrio Parahaemolyticus. Using SWISS-MODEL work-space, the three-dimensional structures of HATPase_c and REC conserved domain in the KdpD and KdpE were determined.Combining with chloramphenicol(Cm) and sucrose screening, we constructed three in-frame deletion mutants of kdpDE, kdpD and kdpE using overlap PCR and homologous recombination technology. We then investigated the effect of the mutant on its growth,ECPase activity and virulence in fish. The mutants showed no differences of ECPase activity and growth from the wild-type strain. But a decrease in swarming ability and biofilm formation was found. Moreover, virulence assays with zebrafish model confirmed that virulence of ΔkdpDE declined by 78.5 times, and the ΔkdpD and ΔkdpE decreased 8.84-fold and 19.6-fold compared with the wild-type strain. These results confirmed that the attenuated strains were constructed successfully.When the initiated bacteria density was 106CFU/mL, there was no bacteria detected after 14 days in water. No bacteria survived in zebrafish 7 days later after injection vaccination. The attenuated strains of Vibrio alginolyticus are safe.Furthermore, the mutants were used as antigens to immunize zebrafish. To estimate immunoprotection and immune routes of the mutants, the zebrafish were challenged by V.alginolyticus and Vibrio harveyi at 28 days after immersed and injected vaccination. The relative percentage survival(RPS) values of injection group were 73.3%, 76.7% and83.3% against V. alginolyticus, while the RPS values of immersion group were 60%,63.3% and 66.7%. The results showed that the ΔkdpDE of V. alginolyticus has a highimmunogenicity. In addition, the vaccination led to cross-protection to V. harveyi and the RPS values of injection group and immersion group were 55.2% to 65.5% and 44.8% to55.2% respectively. These results indicate that the mutants are effective vaccines candidate against V. alginolyticus infection.