Evidence for the secretion of Chlamydia trachomatis CopN by a type III secretion mechanism
Evidence for the secretion of Chlamydia trachomatis CopN by a type III secretion mechanism
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DOI：
10.1046/j.1365-2958.2000.02212.x
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发表时间：
2000-12-01
影响因子：
3.6
通讯作者：
Hackstadt, T
中科院分区：
文献类型：
--
作者：
Fields, KA;Hackstadt, T
沙眼衣原体是一种具有医学重要性的专性细胞内病原体，在被称为包涵体的空泡内进行复制。进入宿主细胞后便启动一个发育周期，其表现为具有感染性的原体（EBs）转变为更大的、无感染性的网状体（RBs）。对沙眼衣原体基因组的分析表明，衣原体拥有可能编码Ⅲ型分泌系统的基因。在其他革兰氏阴性病原体中，Ⅲ型分泌机制用于将毒力因子直接靶向宿主细胞胞质，并且对完全毒力至关重要。为了评估沙眼衣原体中功能性Ⅲ型分泌机制的可能性，我们最初聚焦于一个基因座，该基因座包含的基因所编码的产物与其他Ⅲ型系统中的分子伴侣（Scc1）、分泌孔组分（Cds1和Cds2）以及分泌蛋白（CopN）具有相似性。通过对感染后2、6、12和20小时从感染的HeLa细胞单层中提取的总RNA进行逆转录聚合酶链反应（RT - PCR）来检测基因表达，并根据存在的沙眼衣原体基因组数量进行标准化。在所有时间点都检测到了Scc1的mRNA，而所有其他测试基因的mRNA在12小时和20小时被大量检测到。用Scc1和CopN特异性抗体进行的免疫印迹分析显示，CopN和Scc1存在于感染20小时后收获的原体、网状体和全培养物提取物中。CopN与耶尔森菌属的分泌蛋白YopN同源，通过间接免疫荧光对感染20小时后的单层细胞进行分析，当用CopN特异性抗体探测时，包涵体膜有特异性标记，但用Scc1特异性抗体则没有。带有组氨酸标签的CopN和一种衣原体胞质对照蛋白（NrdB）在含有或不含毒性质粒pYV的小肠结肠炎耶尔森氏菌中表达。CopN（而非NrdB）以一种依赖Ca²⁺和pYV的方式由小肠结肠炎耶尔森氏菌分泌。这些数据表明，沙眼衣原体假定的Ⅲ型分泌系统的组分得到表达，并且这些产物中至少有一种由衣原体分泌到包涵体膜。CopN也可由耶尔森菌Ⅲ型分泌系统分泌这一观察结果为衣原体通过Ⅲ型机制分泌蛋白质这一观点提供了支持。
The medically significant, obligate intracellular pathogen Chlamydia trachomatis replicates within vacuoles termed inclusions. A developmental cycle is initiated after entry into a host cell and is manifested by the transformation of infectious elementary bodies (EBs) to larger, non-infectious reticulate bodies (RBs). Analysis of the C. trachomatis genome has revealed that chlamydiae possess genes that may encode a type III secretion apparatus. In other Gram-negative pathogens, the type III secretion mechanism is used to target virulence factors directly to the host cell cytoplasm and is essential for full virulence. To evaluate the possibility of a functional type III secretion mechanism in C. trachomatis, we initially focused on a locus containing genes encoding products with similarity to chaperones (Scc1), secretion pore components (Cds1 and Cds2) and secreted proteins (CopN) from other type III systems. Gene expression was tested by reverse transcriptase-polymerase chain reaction (RT-PCR) of total RNA extracted from infected HeLa cell monolayers at 2, 6, 12 and 20 h after infection and normalized for the number of C. trachomatis genomes present. Message was detected for Scc1 at all times, whereas message for all other tested genes was detected in significant amounts at 12 h and 20 h. Immunoblot analysis with Scc1- and CopN-specific antibodies revealed that CopN and Scc1 were present in EBs, RBs and whole-culture extracts harvested 20 h after infection. CopN is homologous to the secreted protein YopN of Yersinia sp., and analysis of monolayers 20 h after infection via indirect immunofluorescence showed specific labelling of inclusion membranes when probed with CopN-specific antibodies but not with Scc1-specific antibodies. His-tagged CopN and a chlamydial cytoplasmic control protein (NrdB) were expressed in Yersinia enterocolitica containing or lacking the virulence plasmid pYV. CopN, but not NrdB, was secreted by Y. enterocolitica in a Ca2+- and pYV-dependent fashion. These data indicate that components of the putative type III apparatus of C. trachomatis are expressed and that at least one of these products is secreted by chlamydiae to the inclusion membrane. The observation that CopN is also secreted by the Yersinia type III apparatus provides support for the notion that chlamydiae secrete proteins via a type III mechanism.