Structural and functional studies of the enteropathogenic Escherichia coli type III needle complex protein EscJ
Structural and functional studies of the enteropathogenic Escherichia coli type III needle complex protein EscJ
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DOI：
10.1111/j.1365-2958.2005.04508.x
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发表时间：
2005-03-01
影响因子：
3.6
通讯作者：
Matthews, S
中科院分区：
文献类型：
--
作者：
Crepin, VF;Prasannan, S;Matthews, S
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传递到宿主真核细胞的膜和细胞质中。TTSS由保守针复合物（NC）组成，该复合物由一组内膜和外膜环由质周棒连接而成。肠致病性大肠杆菌（EPEC）是一种细胞外腹泻病原体，它利用TTSS诱导肌动蛋白聚合并定植于肠上皮。在EPEC中，预计EscJ以一种依赖的方式靶向周质，并架起TTSS膜相关环的桥梁。在这项研究中，我们用核磁共振波谱法确定了EscJ的整体褶皱。我们发现EscJ包含两个亚结构域（D1 -成熟蛋白中的1 - 55个氨基酸残基，D2 - 90 - 170个氨基酸残基），每个亚结构域由两个a -螺旋两侧的三链β片组成。一个柔性区（残基60 - 85）耦合了结构区D1和D2。在单个EscJ突变菌中，EscJ D1和EscJ D2的周质过表达未能恢复蛋白分泌活性，这表明柔性连接体对杆功能至关重要。相比之下，在同一野生型细菌中，EscJ D1和EscJ D2的质周过表达呈显性阴性表型，表明TTSS组装缺陷和蛋白质易位。
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 a - 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.