Janakiraman Anuradha, Fixen Kathryn R, Gray Andrew N, Niki Hironori, Goldberg Marcia B
Division of Infectious Diseases, Massachusetts General Hospital, Cambridge, Massachusetts 02139, USA.
J Bacteriol. 2009 Oct;191(20):6300-11. doi: 10.1128/JB.00833-09. Epub 2009 Aug 14.
Autotransporters are outer membrane proteins that are widely distributed among gram-negative bacteria. Like other autotransporters, the Shigella autotransporter IcsA, which is required for actin assembly during infection, is secreted at the bacterial pole. In the bacterial cytoplasm, IcsA localizes to poles and potential cell division sites independent of the cell division protein FtsZ. To identify bacterial proteins involved in the targeting of IcsA to the pole in the bacterial cytoplasm, we screened a genome-scale library of Escherichia coli proteins tagged with green fluorescent protein (GFP) for those that displayed a localization pattern similar to that of IcsA-GFP in cells that lack functional FtsZ using a strain carrying a temperature-sensitive ftsZ allele. For each protein that mimicked the localization of IcsA-GFP, we tested whether IcsA localization was dependent on the presence of the protein. Although these approaches did not identify a polar receptor for IcsA, the cytoplasmic chaperone DnaK both mimicked IcsA localization at elevated temperatures as a GFP fusion and was required for the localization of IcsA to the pole in the cytoplasm of E. coli. DnaK was also required for IcsA secretion at the pole in Shigella flexneri. The localization of DnaK-GFP to poles and potential cell division sites was dependent on elevated growth temperature and independent of the presence of IcsA or functional FtsZ; native DnaK was found to be enhanced at midcell and the poles. A second Shigella autotransporter, SepA, also required DnaK for secretion, consistent with a role of DnaK more generally in the chaperoning of autotransporter proteins in the bacterial cytoplasm.
自转运蛋白是外膜蛋白，广泛分布于革兰氏阴性菌中。与其他自转运蛋白一样，志贺氏菌自转运蛋白IcsA在感染过程中是肌动蛋白组装所必需的，它在细菌极处分泌。在细菌细胞质中，IcsA定位于极和潜在的细胞分裂位点，与细胞分裂蛋白FtsZ无关。为了鉴定参与将IcsA靶向细菌细胞质中极的细菌蛋白，我们使用携带温度敏感ftsZ等位基因的菌株，筛选了一个用绿色荧光蛋白（GFP）标记的大肠杆菌蛋白基因组规模文库，寻找那些在缺乏功能性FtsZ的细胞中显示出与IcsA-GFP相似定位模式的蛋白。对于每个模拟IcsA-GFP定位的蛋白，我们测试了IcsA定位是否依赖于该蛋白的存在。尽管这些方法没有鉴定出IcsA的极性受体，但细胞质伴侣蛋白DnaK在高温下作为GFP融合蛋白模拟了IcsA的定位，并且是IcsA在大肠杆菌细胞质中定位于极所必需的。弗氏志贺氏菌中IcsA在极处的分泌也需要DnaK。DnaK-GFP定位于极和潜在的细胞分裂位点依赖于较高的生长温度，且与IcsA或功能性FtsZ的存在无关；天然DnaK在细胞中部和极处增强。志贺氏菌的另一种自转运蛋白SepA的分泌也需要DnaK，这与DnaK在细菌细胞质中更普遍地陪伴自转运蛋白的作用一致。
