Scott M E, Dossani Z Y, Sandkvist M
Department of Biochemistry, American Red Cross, Jerome H. Holland Laboratory, 15601 Crabbs Branch Way, Rockville, MD 20855, USA.
Proc Natl Acad Sci U S A. 2001 Nov 20;98(24):13978-83. doi: 10.1073/pnas.241411198. Epub 2001 Nov 6.
Bacteria have long been thought of as little more than sacks of homogeneously distributed enzymes. However, recent cytological studies indicate that bacteria are compartmentalized with proteins involved in processes such as cell division, motility, chemotaxis, and development located at distinct sites. We have used the green fluorescent protein as a reporter to determine the cellular distribution of the extracellular protein secretion (eps)-encoded type II secretion complex responsible for extracellular secretion of cholera toxin and hemagglutinin/protease in Vibrio cholerae. Real-time monitoring of green fluorescent protein fused to EpsM in living cells indicated that, like the single polar flagellum, the Eps complex is located at the old pole after cell division. Eps-dependent protease secretion was also visualized in single cells by fluorescence microscopy by using intramolecularly quenched casein. This analysis demonstrated that active protease secretion is focused at the poles and colocalizes with the site of the polar Eps apparatus. These results suggest that the type II secretion complex is responsible for directed delivery of virulence factors during cholera pathogenesis.
长期以来，细菌一直被认为只不过是均匀分布着酶的袋子。然而，最近的细胞学研究表明，细菌是分区化的，参与细胞分裂、运动性、趋化性和发育等过程的蛋白质位于不同的位点。我们使用绿色荧光蛋白作为报告分子，来确定负责霍乱弧菌中霍乱毒素和血凝素/蛋白酶细胞外分泌的胞外蛋白分泌（eps）编码的II型分泌复合物的细胞分布。对活细胞中与EpsM融合的绿色荧光蛋白进行实时监测表明，与单极鞭毛一样，Eps复合物在细胞分裂后位于旧极。通过使用分子内淬灭的酪蛋白，荧光显微镜也在单细胞中观察到了Eps依赖性蛋白酶的分泌。该分析表明，活性蛋白酶分泌集中在极部，并与极性Eps装置的位点共定位。这些结果表明，II型分泌复合物在霍乱发病机制中负责毒力因子的定向传递。
