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

沙眼衣原体是人类眼部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细胞质中的膜状空泡(包涵体)中。与其他含有病原体的空泡不同，衣原体-包涵体缺乏细胞内运输的标记，但从高尔基体后胞外囊泡途径获得成分。衣原体通过裂解高尔基体基质蛋白Golgin-84诱导高尔基体碎裂，这与包涵体获得脂质有关。我们最近已经证明，衣原体蛋白水解酶CPAF裂解Golgin-84，并且异位表达的CPAF足以诱导高尔基体碎裂。此外，人工合成的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的这一功能的机制和重要性。CPAF要么在感染期间被特异性抑制，要么在异位表达。这些实验操作对囊泡运输、向包裹体的运输以及包裹体和细菌的发育的影响将使用细胞生物学以及蛋白质组和脂肪组学方法进行监测。这一机制的进化保守性将通过对衣原体样生物物种的CPAF的分析进一步验证。这些研究将加深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体后胞外途径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.