Multiple domains are required for the toxic activity of Pseudomonas aeruginosa ExoU
Multiple domains are required for the toxic activity of Pseudomonas aeruginosa ExoU
复制标题
DOI：
10.1128/jb.183.14.4330-4344.2001
复制
发表时间：
2001-07-01
影响因子：
3.2
通讯作者：
Frank, DW
中科院分区：
文献类型：
--
作者：
Finck-Barbançon, V;Frank, DW
铜绿假单胞菌的ExoU表达与许多上皮细胞和巨噬细胞系中的急性细胞毒性相关。在体内，ExoU负责上皮损伤。缺乏已知的基序或与其他蛋白质的显著同源性表明ExoU可能具有新的毒性机制。为了研究ExoU的细胞内效应，我们在中国仓鼠卵巢细胞中开发了瞬时转染系统。用全长但不截短形式的ExoU转染抑制报告基因表达。在与编码ExoU的构建体共转染后，报告基因活性的抑制与细胞渗透性和死亡相关。截短版本的ExoU的毒性可以通过共表达的分子的其余部分从单独的质粒中的反式。这种策略被用来映射N-和C-末端区域的ExoU是必要的，但不足以毒性。蛋白质中间区域的破坏降低了毒性。假定分子的这一部分允许N-和C-末端区域在功能上彼此互补。与ExoS和ExoT相反，天然和重组ExoU分子不寡聚化或形成聚集体。ExoU的复杂结构域结构表明，与其他铜绿假单胞菌编码的III型效应物（ExoS和ExoT）一样，ExoU毒性可能来自具有一种以上活性的分子。
Expression of ExoU by Pseudomonas aeruginosa is correlated with acute cytotoxicity in a number of epithelial and macrophage cell lines. In vivo, ExoU is responsible for epithelial injury. The absence of a known motif or significant homology with other proteins suggests that ExoU may possess a new mechanism of toxicity. To study the intracellular effects of ExoU, we developed a transient-transfection system in Chinese hamster ovary cells. Transfection with full-length but not truncated forms of ExoU inhibited reporter gene expression. Inhibition of reporter activity after cotransfection with ExoU-encoding constructs was correlated with cellular permeability and death. The toxicity of truncated versions of ExoU could be restored by coexpression of the remainder of the molecule from separate plasmids in trans. This strategy was used to map N- and C-terminal regions of ExoU that are necessary but not sufficient for toxicity. Disruption of a middle region of the protein reduces toxicity. This portion of the molecule is postulated to allow the N- and C-terminal regions to functionally complement one another. In contrast to ExoS and ExoT, native and recombinant ExoU molecules do not oligomerize or form aggregates. The complex domain structure of ExoU suggests that, like other P. aeruginosa-encoded type III effecters (ExoS and ExoT), ExoU toxicity may result from a molecule that possesses more than one activity.