荧光假单胞菌TonB依赖型外膜受体P698的研究

Study of Pseudomonas Fluorescens Ton B-dependent outer Membrane Receptors P698

目的:鱼类病原菌荧光假单胞菌是一种革兰氏阴性菌,在水产上可以引起多种经济鱼类的疾病,作为一种鱼类病原菌目前对其致病机理还知之甚少。本研究从鱼类病原菌荧光假单胞菌TSS中克隆得到了一个Ton B依赖型的外膜受体(命名为P698),分析了其与细菌致病性的关系,研究了P698作为亚单位疫苗的免疫保护效应。方法:通过序列分析、荧光定量PCR、基因敲除、体外蛋白重组等方法研究P698的蛋白结构、表达情况以及其与细菌毒力的联系和免疫原性。结果:研究发现P698具有Ton B依赖型外膜受体家族的典型结构。与正常培养条件相比,在缺铁条件下培养的TSS中p698基因的表达没有明显变化。通过插入失活获得p698缺失的突变株TSSP,生长分析发现跟野生株相比,突变株TSSP的生长能力有明显降低。在以大菱鲆为模型的活体侵染实验中发现,与野生株相比,突变株的体内侵染复制能力都有明显降低。为检测P698的免疫保护性,我们对P698进行了体外重组表达,获得了重组蛋白。用重组P698蛋白作亚单位疫苗免疫大菱鲆,在免疫后一个月的鱼体检测到了特异抗体的存在,并且受免疫鱼对于致死剂量的TSS攻毒表现出了显著的保护效应。结论:我们的研究结果表明P698与细菌的侵染有一定相关性,并且作为亚单位疫具有一定的免疫保护效应。

Objective： 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 one Ton B-dependent outer membrane receptors（TDRs） named P698 from a pathogenic P. fluorescens strain isolated from fish. Subsequently,its relevance in bacteria virulence and its ability in the induction of protective immunity were detected in our study. Methods： Silico analysis, quantitative real time RT-PCR analysis and purification of recombinant protein were involved for the study of structure, gene expression, pathogenicity relevance and immunogenicity of P698. Results： Our study found that the P698 possess structural domains typical of TDRs and p698 expressions were not changed under iron-depleted conditions. Compared to the wild type, the mutants that are defective in p698 were retarded in growth to different extents. Live infection in a turbot（Scophthalmus maximus） model showed that mutant strain was impaired in the ability to disseminate into and colonize host tissues. When used as subunit vaccines, the vaccinated fish produced specific serum antibodies and elicited significant protections in turbot against lethal P. fluorescens challenge. Conclusions： Together these results indicate that the P698 is participate in bacterial virulence and can induce protective immunity as subunit vaccines.