Chatterjee Abhishek, Caballero-Franco Celia, Bakker Dannika, Totten Stephanie, Jardim Armando
From the Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, Ste-Anne-de-Bellevue, Québec H9X 3V9, Canada.
From the Institute of Parasitology and Centre for Host-Parasite Interactions, McGill University, Ste-Anne-de-Bellevue, Québec H9X 3V9, Canada
J Biol Chem. 2015 Oct 16;290(42):25579-94. doi: 10.1074/jbc.M115.648204. Epub 2015 Aug 31.
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 ∼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 ∼280-320-kDa oligomeric structure consisting of ∼6-7 subunits.
肠出血性大肠杆菌是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一种称为III型分泌系统的注射器样细胞器将细菌的毒力因子直接递送到上皮细胞胞质溶胶中。III型分泌系统蛋白EspD是在宿主细胞膜上形成转运孔所需的关键因子。在这里，我们表明重组EspD自发整合到大型单层囊泡（LUV）脂质双层中；然而，孔的形成需要掺入阴离子磷脂，如磷脂酰丝氨酸和酸性pH值。用荧光葡聚糖进行的泄漏试验证实，EspD形成了内径约为2.5 nm的结构。蛋白酶图谱表明，EspD的两个跨膜螺旋发夹穿透脂质层，将N端和C端结构域定位在LUVs的腔外表面。最后，戊二醛交联和速率区带离心相结合表明，LUV膜中的EspD形成了一种约280-320 kDa的寡聚结构，由约6-7个亚基组成。
