Department of Veterinary Biosciences, College of Veterinary Medicine, Infectious Diseases Institute, The Ohio State University, Columbus, OH 43210.
Proc Natl Acad Sci U S A. 2024 Aug 13;121(33):e2405209121. doi: 10.1073/pnas.2405209121. Epub 2024 Aug 6.
The obligatory intracellular bacterium causes human granulocytic anaplasmosis, an emerging zoonosis. has limited biosynthetic and metabolic capacities, yet it effectively replicates inside of inclusions/vacuoles of eukaryotic host cells. Here, we describe a unique Type IV secretion system (T4SS) effector, R-olgi xit site protein of (EgeA). In cells infected by , secreted native EgeA, EgeA-GFP, and the C-terminal half of EgeA (EgeA-C)-GFP localized to containing inclusions. In uninfected cells, EgeA-C-GFP localized to cis-Golgi, whereas the N-terminal half of EgeA-GFP localized to the ER. Pull-down assays identified EgeA-GFP binding to a transmembrane protein in the ER, Transport and Golgi organization protein 1 (TANGO1). By yeast two-hybrid analysis, EgeA-C directly bound Sec1 family domain-containing protein 1 (SCFD1), a host protein of the cis-Golgi network that binds TANGO1 at ER-Golgi exit sites (ERES). Both TANGO1 and SCFD1 localized to the inclusion surface. Furthermore, knockdown of EgeA or either host TANGO1 or SCFD1 significantly reduced infection. TANGO1 and SCFD1 prevent ER congestion and stress by facilitating transport of bulky or unfolded proteins at ERES. A bulky cargo collagen and the ER-resident chaperon BiP were transported into inclusions, and several ER stress marker genes were not up-regulated in infected cells. Furthermore, EgeA transfection reduced collagen overexpression-induced BiP upregulation. These results suggest that by binding to the two ERES proteins, EgeA redirects the cargo-adapted ERES to pathogen-occupied inclusions and reduces ERES congestion, which facilitates nutrient acquisition and reduces ER stress for survival and proliferation.
必需的细胞内细菌导致人类粒细胞无形体病，一种新兴的人畜共患病。它的生物合成和代谢能力有限，但它能有效地在真核宿主细胞的包含体/空泡内复制。在这里，我们描述了一种独特的 IV 型分泌系统（T4SS）效应子，即（EgeA）的 R-olgixit 站点蛋白。在感染的细胞中，分泌天然的 EgeA、EgeA-GFP 和 EgeA 的 C 末端一半（EgeA-C）-GFP 定位于包含体。在未感染的细胞中，EgeA-C-GFP 定位于顺式高尔基体，而 EgeA-GFP 的 N 末端一半定位于内质网。下拉实验鉴定出 EgeA-GFP 与内质网中一种跨膜蛋白，即运输和高尔基体组织蛋白 1（TANGO1）结合。通过酵母双杂交分析，EgeA-C 直接与 Sec1 家族结构域蛋白 1（SCFD1）结合，SCFD1 是顺式高尔基体网络的一种宿主蛋白，在 ER-Golgi 出口部位（ERES）与 TANGO1 结合。TANGO1 和 SCFD1 都定位于包涵体表面。此外，敲低 EgeA 或任何一种宿主 TANGO1 或 SCFD1 都会显著降低的感染。TANGO1 和 SCFD1 通过在 ERES 处促进大体积或未折叠蛋白的运输，防止 ER 拥挤和应激。大体积货物胶原蛋白和内质网驻留分子伴侣 BiP 被运入包涵体，感染细胞中几个 ER 应激标记基因没有上调。此外，EgeA 转染降低了胶原蛋白过表达诱导的 BiP 上调。这些结果表明，通过与两个 ERES 蛋白结合，EgeA 将货物适应的 ERES 重定向到被病原体占据的包涵体，并减少 ERES 拥挤，从而促进营养物质的获取并减少 ER 应激，以利于生存和增殖。
