Crepin Valerie F, Martinez Eric, Shaw Robert K, Frankel Gad, Daniell Sarah J
Division of Cell and Molecular Biology, Flowers Building, Imperial College London, London SW7 2AZ, UK.
J Mol Biol. 2008 Apr 18;378(1):243-50. doi: 10.1016/j.jmb.2008.02.042. Epub 2008 Mar 4.
Enteropathogenic Escherichia coli utilise a filamentous type III secretion system to translocate effector proteins into host gut epithelial cells. The primary constituent of the extracellular component of the filamentous type III secretion system is EspA. This forms a long flexible helical conduit between the bacterium and host and has a structure almost identical to that of the flagella filament. We have inserted the D3 domain of FliCi (from Salmonella typhimurium) into the outer domain of EspA and have studied the structure and function of modified filaments when expressed in an enteropathogenic E. coli espA mutant. We found that the chimeric protein EspA-FliCi filaments were biologically active as they supported protein secretion and translocation [assessed by their ability to trigger actin polymerisation beneath adherent bacteria (fluorescent actin staining test)]. The expressed filaments were recognised by both EspA and FliCi antisera. Visualisation and analysis of the chimeric filaments by electron microscopy after negative staining showed that, remarkably, EspA filaments are able to tolerate a large protein insertion without a significant effect on their helical architecture.
肠道致病性大肠杆菌利用丝状III型分泌系统将效应蛋白转运到宿主肠道上皮细胞中。丝状III型分泌系统细胞外成分的主要组成部分是EspA。它在细菌和宿主之间形成一条长而灵活的螺旋管道，其结构与鞭毛丝几乎相同。我们将来自鼠伤寒沙门氏菌的FliCi的D3结构域插入到EspA的外部结构域中，并研究了在肠道致病性大肠杆菌espA突变体中表达时修饰后的细丝的结构和功能。我们发现嵌合蛋白EspA-FliCi细丝具有生物活性，因为它们支持蛋白质分泌和转运[通过它们在粘附细菌下方引发肌动蛋白聚合的能力（荧光肌动蛋白染色试验）来评估]。表达的细丝被EspA和FliCi抗血清识别。负染后通过电子显微镜对嵌合细丝进行可视化和分析表明，值得注意的是，EspA细丝能够耐受大量蛋白质插入，而对其螺旋结构没有显著影响。
