Crepin Valérie F, Prasannan Sunil, Shaw Rob K, Wilson Rebecca K, Creasey Elizabeth, Abe Cecilia M, Knutton Stuart, Frankel Gad, Matthews Steve
Centres for Molecular Microbiology and Infection, Imperial College London, London SW7 2AZ, UK.
Mol Microbiol. 2005 Mar;55(6):1658-70. doi: 10.1111/j.1365-2958.2005.04508.x.
The type III secretion system (TTSS) is a macromolecular structure that spans the cell wall of Gram-negative bacterial pathogens, enabling delivery of virulence effector proteins directly to the membranes and cytosol of host eukaryotic cells. TTSS consists of a conserved needle complex (NC) that is composed of sets of inner and outer membranes rings connected by a periplasmic rod. Enteropathogenic Escherichia coli (EPEC) is an extracellular diarrhoeagenic pathogen that uses TTSS to induce actin polymerization and colonizes the intestinal epithelium. In EPEC, EscJ is predicted to be targeted to the periplasm, in a sec-dependent manner, and to bridge the TTSS membrane-associated rings. In this study we determined the global fold of EscJ using Nuclear Magnetic Resonance spectroscopy. We show that EscJ comprises two subdomains (D1 - amino acid residues 1-55 in the mature protein, and D2 - amino acid residues 90-170), each comprising a three-stranded beta-sheet flanked by two alpha-helices. A flexible region (residues 60-85) couples the structured regions D1 and D2. Periplasmic overexpression of EscJ(D1) and EscJ(D2) in a single escJ mutant bacterium failed to restore protein secretion activity, suggesting that the flexible linker is essential for the rod function. In contrast, periplasmic overexpression of EscJ(D1) and EscJ(D2) in the same wild-type bacterium had a dominant-negative phenotype suggesting defective assembly of the TTSS and protein translocation.
III型分泌系统（TTSS）是一种跨越革兰氏阴性细菌病原体细胞壁的大分子结构，能够将毒力效应蛋白直接递送至宿主真核细胞的膜和胞质溶胶中。TTSS由一个保守的针状复合物（NC）组成，该复合物由通过周质杆连接的内膜环和外膜环组成。肠致病性大肠杆菌（EPEC）是一种细胞外致腹泻病原体，它利用TTSS诱导肌动蛋白聚合并在肠道上皮定植。在EPEC中，EscJ预计以sec依赖性方式靶向周质，并连接TTSS膜相关环。在本研究中，我们使用核磁共振光谱法确定了EscJ的整体折叠结构。我们发现EscJ由两个亚结构域组成（成熟蛋白中的D1 - 氨基酸残基1 - 55，以及D2 - 氨基酸残基90 - 170），每个亚结构域都包含一个由两条α螺旋侧翼环绕的三链β折叠。一个柔性区域（残基60 - 85）连接结构区域D1和D2。在单个escJ突变细菌中周质过表达EscJ（D1）和EscJ（D2）未能恢复蛋白质分泌活性，这表明柔性连接子对于杆状功能至关重要。相比之下，在同一野生型细菌中周质过表达EscJ（D1）和EscJ（D2）具有显性负性表型，表明TTSS组装缺陷和蛋白质转运存在问题。
