Identification and characterization of a Chlamydia trachomatis early operon encoding four novel inclusion membrane proteins
Identification and characterization of a Chlamydia trachomatis early operon encoding four novel inclusion membrane proteins
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DOI：
10.1046/j.1365-2958.1999.01523.x
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发表时间：
1999-08-01
影响因子：
3.6
通讯作者：
Hackstadt, T
中科院分区：
文献类型：
--
作者：
Scidmore-Carlson, MA;Shaw, EI;Hackstadt, T
沙眼衣原体是一种细菌专性细胞内寄生虫，在称为包涵体的空泡内复制，不与溶酶体融合。内化后2 h内，C.沙眼包涵体停止与内吞途径相互作用，而是与含有外源合成的NBD-鞘磷脂的胞吐囊泡融合。胞吐囊泡的融合和溶酶体融合的长期避免都需要衣原体基因的早期表达。衣原体包涵体的修饰可能通过衣原体蛋白的表达和插入包涵体膜而发生。为了鉴定候选包涵体膜蛋白，针对从C.沙眼感染的HeLa细胞。通过间接免疫荧光，这种抗血清识别的包涵体膜，并通过免疫印迹分析，识别三个衣原体特异性抗原的分子量约为15，18和21 kDa。IncG编码18 kDa和21 kDa的衣原体抗原。沙眼，血清型L2，基因组表达文库。另外三个基因incD、因斯和incF与incG共转录。针对该操纵子的四个基因中的每一个的单特异性抗血清证明基因产物定位于衣原体包涵体膜。紧接着含有incD-G的操纵子下游是C。沙眼衣原体印加同源物。比如IncD E F和G C沙眼衣原体印加也定位于包涵体膜。逆转录-聚合酶链反应（RT-PCR）分析表明，IncD-G，但不是印加，在第一个2小时内转录后内化，使他们的候选人衣原体因子所需的修改新生衣原体列入。
Chlamydia trachomatis is a bacterial obligate intracellular parasite that replicates within a vacuole, termed an inclusion, that does not fuse with lysosomes. Within 2 h after internalization, the C. trachomatis inclusion ceases to interact with the endocytic pathway and, instead, becomes fusogenic with exocytic vesicles containing exogenously synthesized NBD-sphingomyelin. Both fusion of exocytic vesicles and longterm avoidance of lysosomal fusion require early chlamydial gene expression. Modification of the chlamydial inclusion probably occurs through the expression and insertion of chlamydial protein(s) into the inclusion membrane. To identify candidate inclusion membrane proteins, antisera were raised against a total membrane fraction purified from C. trachomatis-infected HeLa cells. By indirect immunofluorescence, this antisera recognized the inclusion membrane and, by immunoblot analysis, recognized three chlamydial-specific antigens of approximate molecular weights 15, 18 and 21 kDa. IncG, encoding an 18 kDa and 21 kDa doublet chlamydial antigen, was identified by screening a C. trachomatis, serovar L2, genomic expression library. Three additional genes, incD, incE and incF, were co-transcribed with incG. Monospecific antisera against each of the four genes of this operon demonstrated that the gene products were localized to the chlamydial inclusion membrane. Immediately downstream from the operon containing incD-G was the C. trachomatis homologue of incA. Like IncD, E, F and G, C. trachomatis IncA is also localized to the inclusion membrane. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that IncD-G, but not incA, are transcribed within the first 2 h after internalization, making them candidates for chlamydial factors required for the modification of the nascent chlamydial inclusion.