Establishment and maintenance of the chlamydial inclusion: requirement for septins and the inhibition of host cell translation

沙眼衣原体是人眼感染和性传播疾病的常见病原体，是一种专性细胞内细菌，生长在人类宿主细胞质的膜结合液泡（包涵体）中。与其他含病原体的液泡不同，衣原体包涵体缺乏内吞运输的标记，但从高尔基胞外泡途径获得成分。衣原体通过切割高尔基基质蛋白golgin-84诱导高尔基体断裂，这与包涵体获得脂质有关。我们最近的研究表明，衣原体蛋白酶CPAF可裂解高尔基蛋白84，并且异位表达的CPAF足以诱导高尔基断裂。进一步，合成的caspase-1抑制剂WEHDfmk被鉴定为CPAF的抑制剂。在本项目中，我们旨在了解CPAF功能的机制和重要性。CPAF在感染过程中被特异性抑制或异位表达。这些实验操作对囊泡转运、转运到包涵体以及包涵体和细菌的发展的影响将使用细胞生物学、蛋白质组学和脂质组学方法进行监测。该机制的进化保守性将进一步通过对衣原体样生物物种的CPAF分析来验证。这些研究将增强我们对衣原体包涵体的维持和发展，以及细胞内病原体劫持高尔基后胞外通路的机制和重要性的理解。

Chlamydia trachomatis, a frequent agent of human eye infection and sexually transmitted disease, is an obligate intracellular bacterium that grows in a membrane-bounded vacuole (the inclusion) in the cytoplasm of the human host cell. Unlike other pathogen-containing vacuoles, the Chlamydia-inclusion lacks markers of endocytic transport but acquires components from the post-Golgi exocytic vesicular pathway. Chlamydia induces fragmentation of the Golgi apparatus through cleavage of the Golgi-matrix protein golgin-84, and this has been linked to lipid acquisition by the inclusion. We have recently shown that the Chlamydia protease CPAF cleaves golgin-84 and that ectopically expressed CPAF is sufficient to induce Golgi-fragmentation. Further, the synthetic caspase-1-inhibitor WEHDfmk was identified as an inhibitor of CPAF. In this project we aim at understanding mechanism and importance of this function of CPAF. CPAF will be either specifically inhibited during infection or ectopically expressed. The effect of these experimental manipulations on vesicular transport, transport to the inclusion and the development of inclusion and bacteria will be monitored using cell biological as well as proteomic and lipidomic approaches. The evolutionary conservation of this mechanism will further be tested by the analysis of CPAF from species of Chlamydia-like organisms. These studies will enhance our understanding of maintenance and development of the chlamydial inclusion and the mechanisms and importance of the hijacking of the post-Golgi exocytic pathway by an intracellular pathogen.