Knockout of two alanine racemase genes of Vibrio anguillarum and its potential as an attenuated vaccine in olive flounder (Paralichthys olivaceus)

Abstract

Vibriosis caused by Vibrio anguillarum is one of the most common bacterial diseases in cultured fish worldwide. Alanine racemases (Alrs) catalyze conversion of l-alanine to d-alanine. As d-alanine is an essential component of the peptidoglycan layer of bacteria, the alanine racemase gene (alr) knockout bacteria cannot grow without d-alanine supplementation. In the genome of V. anguillarum, two alanine racemase genes (alr1 and alr2) are present. In the present study, we produced auxotrophic V. anguillarum mutants by knockout of alr and/or alr2 genes using the allelic exchange method, and analyzed in vitro & in vivo replication ability and virulence in olive flounder (Paralichthys olivaceus) fingerlings. Furthermore, the potential of the mutant V. anguillarum (Δalr1Δalr2 V. anguillarum) as an attenuated live vaccine was evaluated. In vitro growth of Δalr1 V. anguillarum stopped in the absence of d-alanine. In the virulence experiment, the LD50 of Δalr1 V. anguillarum in olive flounder fingerlings was approximately 1000-fold lower than that of wild-type V. anguillarum. However, there were no differences between Δalr2 V. anguillarum and wild-type V. anguillarum in in vitro replication and in vivo virulence to olive flounder. These results suggest that the alr1 is the main source for the production of alanine racemase in V. anguillarum, and knockout of alr1 alone is sufficient to stop the bacterial replication. The amino acid sequence of the alr2 showed high homology to V. cholerae's broad spectrum racemase (BsrV) that was previously known as alr2 (recently redesignated as bsrV). The survival rates of V. anguillarum in low salinities were significantly reduced by the knockout of alr2, which is one of the properties of V. cholerae BsrV, suggesting that the alr2 of V. anguillarum is a bsr gene that is corresponding to bsrV of V. cholerae. Although the alr2 of V. anguillarum was involved in the resistance to osmotic challenge, the alr2 knockout was not accompanied by attenuation of the bacteria in fish. In the vaccine experiment, the survival rate and serum agglutination titer of fish immunized with 1×105CFU/fish of Δalr1Δalr2 V. anguillarum were significantly higher than fish immunized with 1×105CFU/fish of FKC V. anguillarum, suggesting that Δalr1Δalr2 V. anguillarum can elicit protective immune responses more efficiently than FKC V. anguillarum. Statement of relevanceIn this paper, we generated auxotrophic Vibrio anguillarum mutants by knockout of alanine racemase genes, and evaluated their potential as a prophylactic vaccine in olive flounder. The results of the vaccine experiment showed that the survival rate and serum agglutination titer of fish immunized with 1×105CFU/fish of Δalr1Δalr2 V. anguillarum were significantly higher than fish immunized with 1×105CFU/fish of FKC V. anguillarum, suggesting that Δalr1Δalr2 V. anguillarum can elicit protective immune responses more efficiently than FKC V. anguillarum.