Functional analyses of the type III effectors in pathogenic E.coli

致病性大肠埃希氏菌通过III型分泌系统将一组效应物输送到宿主细胞中，这一步骤是疾病发展所必需的。在这项研究中，我们证明了III型效应器，ESPG及其同系物ESPG2，触发了肌动蛋白应激纤维的形成和黏附细菌下微管网络的破坏。这两种效应器都被证明具有与微管蛋白相互作用的能力，并在体外刺激微管失稳。最近的一项研究表明，微管结合的全球环境基金-H1，一种RhoA特异性的鸟嘌呤核苷酸交换因子，通过微管失稳转化为其活性形式，这一系列事件导致了RhoA的刺激。事实上，ESPG和EspG2诱导的应力纤维的形成被显性负性形式的gef-H1和RhoA抑制，而不是rac1和cdc42的表达。这些结果表明，EspG/EspG2对微管网络的影响触发了…的激活更多的RhoA-ROCK信号通路是通过环境基金-H1的活性实现的。此外，我们还发现ESPG/EspG2改变了上皮细胞旁细胞的通透性。当野生型EPEC感染MDCK细胞时，RhoA被激活，这一事件依赖于ESPG或EspG2进入宿主细胞。相反，与WT EPEC一样，感染ESPG/espG2双敲除突变体可导致跨上皮细胞电阻丧失和ZO-1断裂，表明ESPG/EspG2不参与EPEC感染过程中紧密连接的破坏。尽管表达ESPG和EspG2的MDCK细胞整体形态正常，并保持了完全组装的紧密连接，但细胞旁对4-kDa葡聚糖的通透性显著增加，但对500-kDa葡聚糖的胞外通透性没有显著增加。这份报告首次揭示了病原体可以调节上皮细胞的大小选择性细胞旁通透性，以引发疾病过程。较少

Enteropathogenic Escherichia coli delivers a subset of effectors into host cells via a type III secretion system, and this step is required for the progression of disease. In this study, we demonstrated that the type III effectors, EspG and its homolog EspG2, trigger actin stress fiber formation and the destruction of the microtubule networks beneath adherent bacteria. Both effectors were shown to possess the ability to interact with tubulins, and to stimulate microtubule destabilization in vitro. A recent study showed that microtubule-bound GEF-H1, a RhoA-specific guanine nucleotide exchange factor, was converted to its active form by microtubule destabilization, and this sequence of events resulted in RhoA stimulation. Indeed, EspG- and EspG2-induced stress fiber formation was inhibited by the expression of dominant-negative forms of GEF-H1 and RhoA, but not of Rac1 and Cdc42. These results indicate that the impact of EspG/EspG2 on microtubule networks triggers the activation of the … More RhoA-ROCK signaling pathway via GEF-H1 activity. In addition, we revealed that the EspG/EspG2 alter epithelial paracellular permeability. When MDCK cells were infected with wild-type (WT) EPEC, RhoA was activated, and this event was dependent on the delivery of either EspG or EspG2 into host cells. In contrast, a loss of transepithelial electrical resistance and ZO-1 disruption were induced by infection with an espG/espG2 double-knockout mutant, as was the case with the WT EPEC, indicating that EspG/EspG2 is not involved in the disruption of tight junctions during EPEC infection. Although EspG- and EspG2-expressing MDCK cells exhibited normal overall morphology and maintained fully assembled tight junctions, the paracellular permeability to 4-kDa dextran, but not the paracellular permeability to 500-kDa dextran, was greatly increased. This report reveals for the first time that a pathogen can regulate the size-selective paracellular permeability of epithelial cells in order to elicit a disease process. Less