Department of Cell and Developmental Biology, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
Bio-imaging Unit, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
PLoS Pathog. 2019 Jun 26;15(6):e1007851. doi: 10.1371/journal.ppat.1007851. eCollection 2019 Jun.
Enteropathogenic E. coli (EPEC) is an extracellular diarrheagenic human pathogen which infects the apical plasma membrane of the small intestinal enterocytes. EPEC utilizes a type III secretion system to translocate bacterial effector proteins into its epithelial hosts. This activity, which subverts numerous signaling and membrane trafficking pathways in the infected cells, is thought to contribute to pathogen virulence. The molecular and cellular mechanisms underlying these events are not well understood. We investigated the mode by which EPEC effectors hijack endosomes to modulate endocytosis, recycling and transcytosis in epithelial host cells. To this end, we developed a flow cytometry-based assay and imaging techniques to track endosomal dynamics and membrane cargo trafficking in the infected cells. We show that type-III secreted components prompt the recruitment of clathrin (clathrin and AP2), early (Rab5a and EEA1) and recycling (Rab4a, Rab11a, Rab11b, FIP2, Myo5b) endocytic machineries to peripheral plasma membrane infection sites. Protein cargoes, e.g. transferrin receptors, β1 integrins and aquaporins, which exploit the endocytic pathways mediated by these machineries, were also found to be recruited to these sites. Moreover, the endosomes and cargo recruitment to infection sites correlated with an increase in cargo endocytic turnover (i.e. endocytosis and recycling) and transcytosis to the infected plasma membrane. The hijacking of endosomes and associated endocytic activities depended on the translocated EspF and Map effectors in non-polarized epithelial cells, and mostly on EspF in polarized epithelial cells. These data suggest a model whereby EPEC effectors hijack endosomal recycling mechanisms to mislocalize and concentrate host plasma membrane proteins in endosomes and in the apically infected plasma membrane. We hypothesize that these activities contribute to bacterial colonization and virulence.
肠致病性大肠杆菌（EPEC）是一种细胞外致腹泻性人类病原体，感染小肠上皮细胞的顶浆膜。EPEC 利用 III 型分泌系统将细菌效应蛋白转运到其上皮宿主中。这种活动颠覆了感染细胞中许多信号和膜运输途径，被认为有助于病原体的毒力。这些事件的分子和细胞机制尚未得到很好的理解。我们研究了 EPEC 效应蛋白劫持内体的方式，以调节上皮宿主细胞中的内吞作用、回收和转胞吐作用。为此，我们开发了一种基于流式细胞术的测定法和成像技术来跟踪感染细胞中的内体动力学和膜货物运输。我们表明，III 型分泌成分促使网格蛋白（网格蛋白和 AP2）、早期（Rab5a 和 EEA1）和回收（Rab4a、Rab11a、Rab11b、FIP2、Myo5b）内吞机制募集到周边质膜感染部位。还发现，利用这些机制介导的内吞途径的蛋白货物，例如转铁蛋白受体、β1 整合素和水通道蛋白，也被募集到这些部位。此外，内体和货物募集到感染部位与货物内吞周转率（即内吞和回收）和向感染质膜的转胞吐作用增加相关。内体和相关内吞活动的劫持依赖于非极化上皮细胞中的易位 EspF 和 Map 效应蛋白，并且在极化上皮细胞中主要依赖于 EspF。这些数据表明了一种模型，即 EPEC 效应蛋白劫持内体回收机制，将宿主质膜蛋白错误定位并集中在内体和感染的质膜顶侧。我们假设这些活动有助于细菌定植和毒力。
