杀香鱼假单胞菌3株毒力相关基因缺失突变株的构建及其生物学特性

Construction and biological characterization of three virulence-related gene deletion mutants of Pseudomonas plecoglossicida

摘要:

杀香鱼假单胞菌(Pseudomonas plecoglossicida)是大黄鱼内脏白点病的致病菌，目前对该菌的致病机制已有一定了解，并筛选出部分有效疫苗，但尚无商品化疫苗问世。目的 深入研究该菌的致病机制，开发高效的杀香鱼假单胞菌弱毒活疫苗。方法 以亲本株杀香鱼假单胞菌NB2011为基础，采用双同源重组法，选择与群体感应系统(quorum sensing, QS)相关的luxR基因、σ因子rpoE基因进行敲除，构建缺失突变株；同时构建主要毒力因子六型分泌系统(T6SS1)与luxR基因联合缺失株ΔT6SS1ΔluxR。对缺失突变株进行生物学特征检测、毒力特性分析及对金鱼的免疫保护率评估。结果 成功构建缺失突变菌株ΔluxR、ΔT6SS1ΔluxR及ΔrpoE。与野生株相比，3株缺失突变株的生长速度、泳动能力以及游泳能力未表现出显著差异；ΔluxR、ΔT6SS1ΔluxR两突变株的生物膜形成能力显著降低。观察不同菌株对小鼠巨噬细胞J774A.1的内化、吸附和胞内增殖现象，发现3株缺失突变株均能在巨噬细胞中存活，但增殖能力明显低于野生株。以1.0×10⁷ cells/mL的浓度腹腔注射人工感染草金鱼，与野生株相比，2株单突变株对鱼体的毒力表现了明显的降低；相同剂量的双突变株ΔT6SS1ΔluxR攻击草金鱼未表现出毒力，其对草金鱼的LD₅₀>10⁸ cells/mL。选择双突变株ΔT6SS1ΔluxR注射接种草金鱼，28 d后用野生株进行人工攻击，免疫保护率为78.60%，达到有效保护水平。结论 本研究成功构建了3株毒力基因缺失突变株，突变株的毒力明显下降，其中ΔT6SS1ΔluxR株有望作为开发杀香鱼假单胞菌弱毒活疫苗的候选株。

Abstract:

Pseudomonas plecoglossicida is the pathogen of visceral white spot disease in large yellow croaker (Larimichthys crocea). At present, the pathogenic mechanism of P. plecoglossicida has been partially understood, and some effective vaccines have been screened, whereas no commercial vaccine has been developed.Objective To deepen the understanding about the pathogenic mechanism and develop efficient attenuated live vaccines of P. plecoglossicida.Methods We targeted luxR and the RNA polymerase σ factor gene rpoE associated with quorum sensing to construct deletion mutants of P. plecoglossicida NB2011 by double homologous recombination. In addition, the strain ΔT6SS1ΔluxR with deletion of both the type VI secretion system 1 (T6SS1) gene and luxR was constructed. The biological characteristics and virulence of the mutants were analyzed. The relative percent of survival of fish after vaccination with the double deletion mutant was investigated.Results We successfully constructed ΔluxR, ΔT6SS1ΔluxR, and ΔrpoE. Compared with the wild type, none of the three mutants showed significant changes in the growth rate, swarming ability or swimming ability, while ΔluxR and ΔT6SS1ΔluxR showed significant decreases in biofilm formation. The internalization, adsorption, and intracellular proliferation of the mutants in mouse macrophages were observed. All the three mutants could survive in J774A.1 macrophages and showed lower proliferation capacities than the wild type. Compared with the wild type, the two single mutants showed significantly reduced virulence to goldfish (Carassius auratus) after intraperitoneal injection at a concentration of 1.0×10⁷ cells/mL. The double deletion mutant ΔT6SS1ΔluxR showed no virulence when challenging goldfish at the same dose, with the LD₅₀>10⁸ cells/mL. In the immune protection experiment, the goldfish was vaccinated with ΔT6SS1ΔluxR, and the fish was artificially challenged with the wild type 28 days later. The relative percent of survival reached 78.60%, which indicated effective protection.Conclusion In this study, three gene deletion mutants were successfully constructed, and the virulence of the mutants was significantly decreased. Among them, ΔT6SS1ΔluxR is expected to be a candidate strain for the development of the attenuated live vaccine against P. plecoglossicida infection.