Rockey D D, Heinzen R A, Hackstadt T
Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Disease, Hamilton, Montana 59840, USA.
Mol Microbiol. 1995 Feb;15(4):617-26. doi: 10.1111/j.1365-2958.1995.tb02371.x.
Chlamydiae are obligate intracellular bacteria which occupy a non-acidified vacuole (the inclusion) throughout their developmental cycle. Little is known about events leading to the establishment and maintenance of the chlamydial inclusion membrane. To identify chlamydial proteins which are unique to the intracellular phase of the life cycle, an expression library of Chlamydia psittaci DNA was screened with convalescent antisera from infected animals and hyperimmune antisera generated against formalin-killed purified chlamydiae. Overlapping genomic clones were identified which expressed a 39 kDa protein only recognized by the convalescent sera. Sequence analysis of the clones identified two open reading frames (ORFs), one of which (ORF1) coded for a predicted 39 kDa gene product. The ORF1 sequence was amplified and fused to the malE gene of Escherichia coli and antisera were raised against the resulting fusion protein. Immunoblotting with these antisera demonstrated that the 39 kDa protein was present in lysates of infected cells and in reticulate bodies (RBs), but was at the limit of detection in lysates of purified C. psittaci elementary bodies. Fluorescence microscopy experiments demonstrated that this protein was localized in the inclusion membrane of infected HeLa cells, but was not detected on the developmental forms within the inclusion. Because the protein produced by ORF1 is deposited on the inclusion membrane of infected cells, this gene has been designated incA, (inclusion membrane protein A) and its gene product, IncA. In addition to the inclusion membrane, these antisera labelled structures that extended from the inclusion over the nucleus or into the cytoplasm of infected cells. Immunoblotting also demonstrated that IncA, in lysates of infected cells, had a migration pattern that seemed indicative of post-translational modification. This pattern was not observed in immunoblots of RBs or in the E. coli expressing IncA. Collectively, these data identify a chlamydial gene which codes for a protein that is released from RB and is localized in the inclusion membrane of infected cells.
衣原体是专性细胞内细菌，在其整个发育周期中占据一个非酸化的液泡（包涵体）。关于导致衣原体包涵体膜形成和维持的事件，我们了解甚少。为了鉴定衣原体生命周期细胞内阶段所特有的蛋白质，用感染动物的恢复期抗血清和针对福尔马林灭活的纯化衣原体产生的超免疫抗血清筛选了鹦鹉热衣原体DNA的表达文库。鉴定出重叠的基因组克隆，其表达一种仅被恢复期血清识别的39 kDa蛋白质。对这些克隆进行序列分析，鉴定出两个开放阅读框（ORF），其中一个（ORF1）编码一个预测的39 kDa基因产物。扩增ORF1序列并将其与大肠杆菌的malE基因融合，针对所得融合蛋白制备抗血清。用这些抗血清进行免疫印迹表明，39 kDa蛋白质存在于感染细胞的裂解物和网状体（RB）中，但在纯化的鹦鹉热衣原体原体裂解物中的检测限处。荧光显微镜实验表明，该蛋白质定位于感染的HeLa细胞的包涵体膜中，但在包涵体内的发育形式上未检测到。由于ORF1产生的蛋白质沉积在感染细胞的包涵体膜上，该基因被命名为incA（包涵体膜蛋白A）及其基因产物IncA。除了包涵体膜外，这些抗血清还标记了从包涵体延伸到感染细胞核上或细胞质中的结构。免疫印迹还表明，感染细胞裂解物中的IncA具有一种似乎表明翻译后修饰的迁移模式。在RB的免疫印迹或表达IncA的大肠杆菌中未观察到这种模式。总体而言，这些数据鉴定出一个衣原体基因，该基因编码一种从RB释放并定位于感染细胞包涵体膜中的蛋白质。
