Program in Infectious Diseases, School of Public Health, University of California, 51 Koshland Hall, Berkeley, CA 94720, USA.
Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, 750 Republican St Seattle, WA 98109, USA.
Pathog Dis. 2018 Mar 1;76(2). doi: 10.1093/femspd/fty011.
Chlamydia secrete into host cells a diverse array of effector proteins, but progress in characterizing the spatiotemporal localization of these proteins has been hindered by a paucity of genetic approaches in Chlamydia and also by the challenge of studying these proteins within the live cellular environment. We adapted a split-green fluorescent protein (GFP) system for use in Chlamydia to label chlamydial effector proteins and track their localization in host cells under native environment. The efficacy of this system was demonstrated by detecting several known Chlamydia proteins including IncA, CT005 and CT694. We further used this approach to detect two chlamydial deubiquitinases (CT867 and CT868) within live cells during the infection. CT868 localized only to the inclusion membrane at early and late developmental stages. CT867 localized to the chlamydial inclusion membrane at an early developmental stage and was concomitantly localized to the host plasma membrane at a late stage during the infection. These data suggest that chlamydial deubiquitinase play important roles for chlamydial pathogenesis by targeting proteins at both the plasma membrane and the chlamydial inclusion membrane. The split-GFP technology was demonstrated to be a robust and efficient approach to identify the secretion and cellular localization of important chlamydial virulence factors.
沙眼衣原体向宿主细胞分泌多种效应蛋白，但由于在沙眼衣原体中缺乏遗传方法，以及在活细胞环境中研究这些蛋白的挑战性，在描述这些蛋白的时空定位方面的进展受到了阻碍。我们改编了一种分裂绿色荧光蛋白（GFP）系统，用于标记沙眼衣原体效应蛋白，并在天然环境下追踪它们在宿主细胞中的定位。该系统的功效通过检测几种已知的沙眼衣原体蛋白，包括 IncA、CT005 和 CT694 得到了证明。我们进一步在感染过程中使用这种方法在活细胞中检测到两种衣原体去泛素化酶（CT867 和 CT868）。CT868 仅在早期和晚期发育阶段定位于包涵体膜。CT867 在早期发育阶段定位于衣原体包涵体膜，在感染后期同时定位于宿主质膜。这些数据表明，衣原体去泛素化酶通过靶向质膜和衣原体包涵体膜上的蛋白，在衣原体发病机制中发挥重要作用。分裂 GFP 技术被证明是一种强大而有效的方法，可用于鉴定重要的衣原体毒力因子的分泌和细胞定位。
