Identification and analysis of three virulence-associated TonB-dependent outer membrane receptors of Pseudomonas fluorescens.
Identification and analysis of three virulence-associated TonB-dependent outer membrane receptors of Pseudomonas fluorescens.
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DOI：
10.3354/dao02771
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发表时间：
2014-08
影响因子：
1.4
通讯作者：
Shu-ren Zhang;Lu Zhang;Li Sun
中科院分区：
文献类型：
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作者：
Shu-ren Zhang;Lu Zhang;Li Sun
荧光假单胞菌是一种革兰氏阴性细菌，可以感染广泛的养殖鱼类。然而，荧光假单胞菌作为一种鱼类病原菌，其致病机制却知之甚少。在这项研究中，我们确定并分析了3 TonB依赖性外膜受体（TDRs）从致病性荧光假单胞菌菌株从鱼分离。计算机模拟分析显示，所有3种蛋白质（命名为Tdr 1至3）都具有典型的TDR结构域。定量真实的时间RT-PCR分析表明，tdr 1，tdr 2，和tdr 3表达上调铁缺乏条件下。与野生型相比，缺失tdr 1、tdr 2和tdr 3的突变体在生长上有不同程度的延迟。在大菱鲆模型中的感染表明，所有3个突变体的能力，desseminate到宿主组织和定殖受损。此外，tdr 1和tdr 3突变体表现出显着降低的毒力。当用作亚单位疫苗时，纯化的重组蛋白Tdr 1，Tdr 2，特别是Tdr 3在大菱鲆中引起对致死性荧光假单胞菌攻击的显著保护。接种疫苗的鱼产生特异性血清抗体，当与荧光假单胞菌孵育时，其阻断荧光假单胞菌在鱼细胞中的感染。总之，这些结果表明，Tdr 1，Tdr 2和Tdr 3是铁调节因子，参与细菌毒力和诱导保护性免疫作为亚单位疫苗。
Pseudomonas fluorescens is a Gram-negative bacterium that can infect a wide range of farmed fish. However, very little is known about the virulence mechanism of P. fluorescens as a fish pathogen. In this study, we identified and analyzed 3 TonB-dependent outer membrane receptors (TDRs) from a pathogenic P. fluorescens strain isolated from fish. In silico analysis revealed that all 3 proteins (named Tdr1 to 3) possess structural domains typical of TDRs. Quantitative real time RT-PCR analysis showed that tdr1, tdr2, and tdr3 expressions were upregulated under iron-depleted conditions. Compared to the wild type, mutants defective in tdr1, tdr2, and tdr3 were retarded in growth to different extents. Infection in a turbot Scophthalmus maximus model showed that all 3 mutants were impaired in their ability to desseminate into and colonize host tissues. In addition, the tdr1 and tdr3 mutants exhibited significantly reduced virulence. When used as subunit vaccines, purified recombinant proteins of Tdr1, Tdr2, and, in particular, Tdr3 elicited significant protection in turbot against lethal P. fluorescens challenge. The vaccinated fish produced specific serum antibodies, which, when incubated with P. fluorescens, blocked infection of P. fluorescens in fish cells. Together these results indicate that Tdr1, Tdr2, and Tdr3 are iron-regulated factors that participate in bacterial virulence and induce protective immunity as subunit vaccines.