Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD
Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD
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DOI：
10.1074/jbc.m115.648204
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发表时间：
2015-10-16
影响因子：
4.8
通讯作者：
Jardim, Armando
中科院分区：
文献类型：
--
作者：
Chatterjee, Abhishek;Caballero-Franco, Celia;Jardim, Armando
肠出血性大肠埃希菌是引起胃肠道和腹泻疾病的病原体。与肠出血性大肠杆菌相关的发病机制涉及通过称为III型分泌系统的注射器状细胞器将毒力因子从细菌直接输送到上皮细胞胞浆中。III型分泌系统蛋白EspD是在宿主细胞膜上形成易位孔所必需的关键因素。在这里，我们证明了重组ESPD自发地整合到大的单层囊泡(LUV)脂双层中；然而，孔的形成需要加入阴离子磷脂，如磷脂酰丝氨酸和酸性pH。荧光右旋糖苷的渗漏分析证实，ESPD形成了内径类似于2.5 nm的结构。蛋白酶谱分析表明，EspD的两个跨膜螺旋发夹穿透脂层，定位于狼疮肾外表面的N-末端和C-末端结构域。最后，戊二醛交联和速率区带离心法的结合表明，EspD在LUV膜中形成了类似于280-320 kDa的低聚结构，由类似的6-7个亚基组成。
Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of similar to 2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an similar to 280-320-kDa oligomeric structure consisting of similar to 6-7 subunits.