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 Gram-negative 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 ΔP_fur170:TT araC P_araBAD fur deletion-insertion mutation (χ16012). Compared to the wild-type strain J118, χ16012 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 χ16012 showed better colonization and protection compared to the Δfur (χ16001). Studies showed that E. piscicida strain χ16012 is attenuated and induces systemic and mucosal IgM titer in zebrafish. In addition, we found an increase in transcript levels of tnf-α, il-1β, il-8 and ifn-γ in different tissues of zebrafish immunized with χ16012 compared to the unimmunized group. We conclude that, E. piscicida with regulated delayed attenuation could be an effective immersion vaccine for the aquaculture industry.

爱德华氏菌是鱼类爱德华氏菌的病原体，在全球水产养殖中造成严重的经济损失。接种疫苗将是预防传染病及其相关经济损失的最有效方法。铁摄取调节剂（Fur）是革兰氏阴性细菌的重要转录全局调节剂。在这项研究中，我们检查了毛皮在大肠杆菌中的调控功能。我们设计了一种菌株，该菌株显示了大肠杆菌的野生型强毒菌株的特征在免疫时，首先使菌株能够有效地定居淋巴组织，然后在体内表现出受控的延迟减毒作用，从而避免诱发疾病症状。体内受控的延迟减毒是基于严格调控的araC P _araBAD盒替代fur基因的启动子，使得该基因的表达依赖于生长过程中提供的阿拉伯糖。因此，继E. piscicida突变的淋巴组织定居，由于缺乏阿拉伯糖，Fur蛋白停止合成，从而在体内逐渐表现出衰减，从而阻止了疾病症状的诱导。我们删除的启动子，包括所有这样的序列与活化剂或抑制蛋白相互作用，为对毛皮基因，和取代的改进的阿糖胞苷P _araBAD启动盒以产生E. piscicida应变与ΔP _fur170：TT阿糖胞苷P _araBAD启动 毛皮缺失插入突变（χ16012）。与野生型菌株J118相比，在没有阿拉伯糖的情况下，χ16012的生长受到抑制，铁载体的生成增强。在野生型和毛皮突变菌株中，在贫铁或富铁条件下分析了Fur调节基因的mRNA水平。我们观察到斑马鱼与χ16012免疫相比Δ表现出较好的殖民统治和保护毛皮（χ16001）。研究表明，斑马鱼大肠杆菌χ16012减毒并诱导斑马鱼体内和粘膜的IgM滴度。另外，我们发现tnf-α，il-1β，il-8和ifn-γ的转录水平增加与未免疫组相比，用χ16012免疫的斑马鱼不同组织中的免疫组织化学表达。我们的结论是，具有受控的延迟衰减的E. piscicida可能对水产养殖业是一种有效的浸没疫苗。