Pathogenicity and immunogenicity of Edwardsiella piscicida ferric uptake regulator (fur) mutations in zebrafish

Edwardsiella piscicida is the etiological agent of edwardsiellosis in fish and causes severe economic losses in global aquaculture. Vaccination would be the most effective method to prevent infectious diseases and their associated economic losses. The ferric uptake regulator (Fur) is an important transcriptional global regulator of Gramnegative bacteria. In this study, we examined the regulatory function of Fur in E. piscicida. We designed a strain that displays features of the wild-type virulent strain of E. piscicida at the time of immunization to enable strains first to effectively colonize lymphoid tissues and then to exhibit a regulated delayed attenuation in vivo to preclude inducing disease symptoms. Regulated delayed attenuation in vivo is based on the substitution of a tightly regulated araC P-araBAD cassette for the promoter of the fur gene such that expression of this gene is dependent on arabinose provided during growth. Thus, following E. piscicida mutant colonization of lymphoid tissues, the Fur protein ceases to be synthesized due to the absence of arabinose such that attenuation is gradually manifest in vivo to preclude induction of diseases symptoms. We deleted the promoter, including all sequences that interact with activator or repressor proteins, for the fur gene, and substituted the improved araC P-araBAD cassette to yield an E. piscicida strain with the Delta P-fur170:TT araC P-araBAD fur deletion-insertion mutation (chi 16012). Compared to the wild-type strain J118, chi 16012 exhibited retarded growth and enhanced siderophore production in the absence of arabinose. mRNA levels of Fur-regulated genes were analyzed in iron deplete or replete condition in wild-type and fur mutant strains. We observed zebrafish immunized with chi 16012 showed better colonization and protection compared to the Delta fur (chi 16001). Studies showed that E. piscicida strain chi 16012 is attenuated and induces systemic and mucosal IgM titer in zebrafish. In addition, we found an increase in transcript levels of tnf-alpha, il-1 beta, il-8 and ifn-gamma in different tissues of zebrafish immunized with chi 16012 compared to the unimmunized group. We conclude that, E. piscicida with regulated delayed attenuation could be an effective immersion vaccine for the aquaculture industry.