Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain
Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain
复制标题
DOI：
10.1128/aem.00159-09
复制
发表时间：
2009-07-01
影响因子：
4.4
通讯作者：
Sun, Li
中科院分区：
文献类型：
--
作者：
Hu, Yong-hua;Liu, Chun-sheng;Sun, Li
从从病鱼中分离的致病性荧光假单胞菌TSS中克隆了编码自转运蛋白的基因pfa1。pfa1的表达在感染过程中增强，并受生长期和生长条件的调节。pfa1的突变显著减弱TSS的总体细菌毒力，并损害TSS在生物膜产生、与宿主细胞相互作用、调节宿主免疫应答以及在宿主血液中传播的能力。pfa1编码的推定蛋白质长度为1，242个氨基酸，其特征在于存在三个典型的自转运蛋白功能结构域。PfaI的乘客结构域包含一个推定的丝氨酸蛋白酶（Pap），当在大肠杆菌中表达并纯化为重组蛋白时，该蛋白酶表现出明显的蛋白水解活性。与PfaI在细菌毒力中所起的重要作用一致，纯化的重组Pap对培养的鱼细胞具有深刻的细胞毒性作用。酶分析表明，重组Pap是相对热稳定的，并具有最佳温度和pH值为50 ℃和pH 8.0。PfaI的结构域是必不可少的自动转运活动的本地化，并在此基础上，PfaI为基础的自动展示系统（命名为AT 1）的工程，以促进插入和运输的异源蛋白。当在E.在大肠杆菌中，AT1能够将整合的迟缓爱德华氏菌免疫原（Et18）递送到细菌细胞表面。与纯化的重组Et18相比，E.大肠杆菌通过AT1诱导的免疫保护作用显著增强。
A gene, pfa1, encoding an autotransporter was cloned from a pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded by pfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified from Escherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50 degrees C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.