The coiled-coil domain of EspA is essential for the assembly of the type III secretion translocon on the surface of enteropathogenic Escherichia coli
The coiled-coil domain of EspA is essential for the assembly of the type III secretion translocon on the surface of enteropathogenic Escherichia coli
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DOI：
10.1074/jbc.274.50.35969
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发表时间：
1999-12-10
影响因子：
4.8
通讯作者：
Frankel, G
中科院分区：
文献类型：
--
作者：
Delahay, RM;Knutton, S;Frankel, G
致肠性大肠杆菌(EPEC)利用III型分泌系统将毒力相关的效应蛋白输送到宿主细胞。已知有四种蛋白质EspA、EspE、EspD和TIR是形成特征性的“附着和消失”(A/E)肠道病变所必需的，它们通过EPEC III型分泌系统输出。最近的工作表明，EspA是丝状结构的主要组成部分，在EPEC的表面阐述了EspB和TIR的易位所需的，预测EspA的羧基末端包括一个α螺旋区域，这表明七肽重复单元ABCDEFG中的a和d位置被疏水残基占据，这表明有卷曲-螺旋相互作用的倾向。在这里，我们展示了EPEC培养上清液中的多聚体EspA亚型以及EspA：EspA在固相上的相互作用。特定EspA七肽残基的非保守氨基酸替换产生了EPEC突变体，该突变体在细丝组装方面存在缺陷，但仍具有诱发A/E病变的能力；额外的突变完全取消了EspA细丝组装和A/E病变的形成。这些结果证明了与鞭毛生物合成的相似性，并表明EspA的盘绕线圈结构域是组装EspA细丝相关的III型分泌易位子所必需的。
Enteropathogenic E. coli (EPEC) utilize a type III secretion system to deliver virulence-associated effector proteins to the host cell. Four proteins, EspA, EspE, EspD, and Tir, which are integral to the formation of characteristic "attaching and effacing" (A/E) intestinal lesions, are known to be exported via the EPEC type III secretion system. Recent work demonstrated that EspA is a major component of a filamentous structure, elaborated on the surface of EPEC, which is required for translocation of EspB and Tir, The carboxyl terminus of EspA is predicted to comprise an alpha-helical region, which demonstrates heptad periodicity whereby positions a and d in the heptad repeat unit abcdefg are occupied by hydrophobic residues, indicating a propensity for coiled-coil interactions. Here we demonstrate multimeric EspA isoforms in EPEC culture supernatants and EspA:EspA interaction on solid phase. Non-conservative amino acid substitution of specific EspA heptad residues generated EPEC mutants defective in filament assembly but which retained the ability to induce A/E lesions; additional mutation totally abolished EspA filament assembly and A/E lesion formation. These results demonstrate a similarity to flagellar biosynthesis and indicate that the coiled-coil domain of EspA is required for assembly of the EspA filament-associated type III secretion translocon.