da Cunha Maria, Pais Sara V, Bugalhão Joana N, Mota Luís Jaime
UCIBIO-REQUIMTE, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal.
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.
PLoS One. 2017 Jun 16;12(6):e0178856. doi: 10.1371/journal.pone.0178856. eCollection 2017.
Chlamydia trachomatis is a human bacterial pathogen causing ocular and genital infections. It multiplies exclusively within an intracellular membrane-bound vacuole, the inclusion, and uses a type III secretion system to manipulate host cells by injecting them with bacterially-encoded effector proteins. In this work, we characterized the expression and subcellular localization in infected host cells of the C. trachomatis CT142, CT143, and CT144 proteins, which we previously showed to be type III secretion substrates. Transcriptional analyses in C. trachomatis confirmed the prediction that ct142, ct143 and ct144 are organized in an operon and revealed that their expression is likely driven by the main σ factor, σ66. In host cells infected by C. trachomatis, production of CT142 and CT143 could be detected by immunoblotting from 20-26 h post-infection. Immunofluorescence microscopy of infected cells revealed that from 20 h post-infection CT143 appeared mostly as globular structures outside of the bacterial cells but within the lumen of the inclusion. Furthermore, immunofluorescence microscopy of cells infected by C. trachomatis strains carrying plasmids producing CT142, CT143, or CT144 under the control of the ct142 promoter and with a C-terminal double hemagglutinin (2HA) epitope tag revealed that CT142-2HA, CT143-2HA or CT144-2HA showed an identical localization to chromosomally-encoded CT143. Moreover, CT142-2HA or CT144-2HA and CT143 produced by the same bacteria co-localized in the lumen of the inclusion. Overall, these data suggest that the CT142, CT143, and CT144 type III secretion substrates are secreted into the lumen of the inclusion where they might form a protein complex.
沙眼衣原体是一种引起眼部和生殖器感染的人类细菌病原体。它仅在细胞内膜结合的液泡（包涵体）内繁殖，并利用III型分泌系统通过向宿主细胞注射细菌编码的效应蛋白来操纵宿主细胞。在这项研究中，我们对沙眼衣原体CT142、CT143和CT144蛋白在感染宿主细胞中的表达和亚细胞定位进行了表征，我们之前证明这些蛋白是III型分泌底物。沙眼衣原体的转录分析证实了ct142、ct143和ct144组成一个操纵子的预测，并表明它们的表达可能由主要的σ因子σ66驱动。在沙眼衣原体感染的宿主细胞中，感染后20-26小时通过免疫印迹可检测到CT142和CT143的产生。感染细胞的免疫荧光显微镜检查显示，感染后20小时，CT143主要表现为细菌细胞外但包涵体腔内的球状结构。此外，对携带在ct142启动子控制下产生CT142、CT143或CT144的质粒且带有C末端双血凝素（2HA）表位标签的沙眼衣原体菌株感染的细胞进行免疫荧光显微镜检查，结果显示CT142-2HA、CT143-2HA或CT144-2HA与染色体编码的CT143具有相同的定位。此外，同一细菌产生的CT142-2HA或CT144-2HA与CT143在包涵体腔内共定位。总体而言，这些数据表明CT142、CT143和CT144这三种III型分泌底物被分泌到包涵体腔内，在那里它们可能形成一种蛋白质复合物。
